Chapter 2: Systemic Response to Injury and Metabolic Support

*This chapter is dedicated to its previous author, Dr. Stephen Lowry, my mentor and friend.

1. Endogenous damage-associated molecular patterns (DAMPs) are produced following tissue and cellular injury. These molecules interact with immune and nonimmune cell receptors to initiate a “sterile” systemic inflammatory response following severe traumatic injury.

2. In many cases, DAMP molecules are sensed by pattern recognition receptors (PRRs), which are the same receptors that cells use to sense invading pathogens. This explains, in part, the similar clinical picture of systemic inflammation observed in injured and/or septic patients.

3. The central nervous system receives information with regard to injury-induced inflammation via soluble mediators as well as direct neural projections that transmit information to regulatory areas in the brain. The resulting neuroendocrine reflex plays an important modulatory role in the immune response.

4. Inflammatory signals activate key cellular stress responses (the oxidative stress response, the heat shock protein response, the unfolded protein response, autophagy, and programmed cell death), which serve to mobilize cellular defenses and resources in an attempt to restore homeostasis.

5. The cells, mediators, signaling mechanisms, and pathways that compose and regulate the systemic inflammatory response are closely networked and tightly regulated by transcriptional events as well as by epigenetic mechanisms, posttranslational modification, and microRNA synthesis.

6. Nutritional assessments, whether clinical or laboratory guided, and intervention should be considered at an early juncture in all surgical and critically ill patients.

7. Management of critically ill and injured patients is optimized with the use of evidence-based and algorithm-driven therapy.

The inflammatory response to injury or infection occurs as a consequence of the local or systemic release of “pathogen-associated” or “damage-associated” molecules, which use similar signaling pathways to mobilize the necessary resources required for the restoration of homeostasis. Minor host insults result in a localized inflammatory response that is transient and in most cases beneficial. Major host insults, however, may lead to amplified reactions, resulting in systemic inflammation, remote organ damage, and multiple organ failure in as many as 30% of those who are severely injured. Recent data support this idea and suggest that severely injured patients who are destined to die from their injuries differ from survivors only in the degree and duration of their dysregulated acute inflammatory response.^1,2

This topic is highly relevant because systemic inflammation is a central feature³ of both sepsis and severe trauma. Understanding the complex pathways that regulate local and systemic inflammation is necessary to develop therapies to intervene during overwhelming sepsis or after severe injury. Sepsis, defined by a systemic inflammatory response to infection, is a disease process with an incidence of over 900,000 cases per year. Further, trauma is the leading cause of mortality and morbidity for individuals under age 45.

In this chapter, we will review what is known about the soluble and cellular effectors of the injury-induced inflammatory response; how the signals are sensed, transduced, and modulated; and how their dysregulation is associated with immune suppression. We will also discuss how these events are monitored and regulated by the central nervous system. Finally, we will review how injury reprograms cellular metabolism, in an attempt to mobilize energy and structural stores to meet the challenge of restoring homeostasis.

The Detection of Injury is Mediated by Members of the Damage-Associated Molecular Pattern Family

Traumatic injury activates the innate immune system to produce a systemic inflammatory response in an attempt to limit damage and to restore homeostasis. It includes two general responses: (a) an acute proinflammatory response resulting from innate immune system recognition of ligands, and (b) an anti-inflammatory response that may serve to modulate the proinflammatory phase and direct a return to homeostasis (Fig. 2-1). This is accompanied by a suppression of adaptive immunity.⁴ Rather than occurring sequentially, recent data indicate that all three responses are simultaneously and rapidly induced following severe traumatic injury.²

The degree of the systemic inflammatory response following trauma is proportional to injury severity and is an independent predictor of subsequent organ dysfunction and resultant mortality. Recent work has provided insight into the mechanisms by which immune activation in this setting is triggered. The clinical features of the injury-mediated systemic inflammatory response, characterized by increased body temperature, heart rate, respirations, and white blood cellcount, are similar to those observed with infection (Table 2-1). While significant efforts have been devoted to establishing a microbial etiology for this response, it is now widely accepted that systemic inflammation following trauma is sterile. Although the mechanisms for the sterile response are less well understood, it is likely to result from endogenous molecules that are produced as a consequence of tissue damage or cellular stress, as may occur with hemorrhagic shock and resuscitation.⁵ Termed alarmins or damage-associated molecular patterns (DAMPs), these effectors, along with the pathogen-associated molecular patterns (PAMPs), interact with specific cell receptors that are located both on the cell surface and intracellularly.⁶ The best described of these receptors are members of the toll-like receptor family.

Trauma DAMPs are structurally diverse endogenous molecules that are immunologically active. Table 2-2 includes a partial list of DAMPs that are released either passively from necrotic/damaged cells or actively from physiologically “stressed” cells by upregulation or overexpression. Once they are outside the cell, DAMPs promote the activation of innate immune cells, as well as the recruitment and activation of antigen-presenting cells, which are engaged in host defense.⁷ The best-characterized DAMP with significant preclinical evidence for its release after trauma and with a direct link to the systemic inflammatory response is high-mobility group protein B1 (HMGB1). Additional evidence for the role of DAMP molecules in postinjury inflammation, including mitochondrial proteins and DNA, as well as extracellular matrix molecules, is also presented.

High-Mobility Group Protein B1

The best-characterized DAMP in the context of the injury-associated inflammatory response is HMGB1 protein, which is rapidly released into the circulation within 30 minutes following trauma. HMGB1 is highly evolutionarily conserved across species. It was first described as a constitutively expressed, nonhistone chromosomal protein that participated in a variety of nuclear events, including DNA repair and transcription. HMGB1 was also detected in the cytosol and extracellular fluids at low levels, although its function outside the cell was not clear. Subsequent studies have proven, however, that HMGB1 is actively secreted from immune-competent cells stimulated by PAMPs (e.g., endotoxin) or by inflammatory cytokines (e.g., tumor necrosis factor and interleukin-1). This process occurs outside the classic secretory pathway via a mechanism that is independent of endoplasmic reticulum and the Golgi complex. Moreover, recent data indicate that HMGB1 release can be regulated by the inflammasome.⁸ Stressed nonimmune cells such as endothelial cells and platelet also actively secrete HMGB1. Finally, passive release of HMGB1 can occur following cell death, whether it is programmed or uncontrolled (necrosis).

Once outside the cell, HMGB1 interacts with its putative receptors either alone or in concert with pathogenic molecules to activate the immune response, and in this way, functions as a proinflammatory cytokine. HMGB1 has been shown to signal via the toll-like receptors (TLR2, TLR4, TLR9), the receptor for advanced glycosylation end products (RAGE), CD24, and others. The activation of TLRs mainly occurs in myeloid cells, whereas RAGE is thought to be the receptor target in endothelial and somatic cells. The diverse proinflammatory biologic responses that result from HMGB1 signaling include: (a) the release of cytokines and chemokines from macrophages/monocytes and dendritic cells; (b) neutrophil activation and chemotaxis; (c) alterations in epithelial barrier function, including increased permeability; and (d) increased procoagulant activity on platelet surfaces, among others.⁹ In particular, HMGB1 binding to TLR4 triggers the proinflammatory cytokine release that mediates “sickness behavior.” This effect is dependent on the highly conserved domain structure of HMGB1 that can be recapitulated by a synthetic 20-amino acid peptide containing a critical cysteine residue at position 106.¹⁰

Recent data have explored the role of this cysteine residue, as well as two others that are highly conserved, in the biologic function of HMGB1. They demonstrate that the redox state of the three residues regulates the receptor binding ability of HMGB1 to influence its activity, including cytokine production. For example, a thiol at C106 is required for HMGB1 to promote macrophage tumor necrosis factor (TNF) release. In addition, a disulfide bond between C23 and C45 is also required for cytokine release because reduction of the disulfide linkage or further oxidation will reduce the ability of HMGB1 to function as a cytokine. Therefore, if all three cysteine residues are in reduced form, HMGB1 lacks the ability to bind and signal through TLR4, but gains the capacity to bind to CXCL12 to activate CXCR4 and serve as a chemotactic mediator. Importantly, shifts between the redox states have been demonstrated and indicate that redox state dynamics are important regulators of HMGB1.¹¹

Importantly, HMGB1 levels in human subjects following injury correlate with the Injury Severity Score, complement activation, and an increase in circulating inflammatory mediators such as TNF.¹² Unchecked, excessive HMGB1 has the capacity to promote a self-injurious innate immune response. In fact, exogenous administration of HMGB1 to normal animals produces fever, weight loss, epithelial barrier dysfunction, and even death.

A Role for Mitochondrial DAMPs in the Injury-Mediated Inflammatory Response

Mitochondrial proteins and/or DNA can act as DAMPs by triggering an inflammatory response to necrosis and cellular stress. Specifically, the release of mitochondrial DNA (mtDNA) and formyl peptides from damaged or dysfunctional mitochondria has been implicated in activation of the macrophage inflammasome, a cytosolic signaling complex that responds to cellular stress. In support of this idea, plasma mtDNA has been shown to be thousands of times higher in both trauma patients and patients undergoing femoral fracture repair when compared to normal volunteers. Further, direct injection of mitochondria lysates in an animal model caused remote organ damage, including liver and lung inflammation.¹³ These data suggest that with stress or tissue injury, mtDNA and peptides are released from damaged mitochondria where they can contribute to a sterile inflammatory response. From an evolutionary perspective, given that eukaryotic mitochondria derive from bacterial origin, it would make sense that they retain bacterial features capable of eliciting a strong response that is typically associated with a pathogen trigger. For example, mtDNA is circular and contains hypomethylated CpG motifs that resemble bacterial CpG DNA. It is thus capable of producing formylated peptides, which potently induce an inflammatory phenotype in neutrophils, by increasing chemotaxis, oxidative burst, and cytokine secretion. In addition, the mitochondrial transcription factor A (TFAM), a highly abundant mitochondrial protein, is functionally and structurally homologous to HMGB1. It has also been shown be released in high amounts from damaged cells where it acts in conjunction with mtDNA to activate TLR9 signaling.¹⁴

Extracellular Matrix Molecules Act as DAMPs

Recent work has explored the role of extracellular matrix (ECM) proteins in the TLR-mediated inflammatory response that follows tissue injury. These molecules, which are sequestered under normal conditions, can be released in a soluble form with proteolytic digestion of the ECM. Proteoglycans, glycosaminoglycans, and glycoproteins such as fibronectin have all been implicated as key players in the DAMP/TLR interaction. Proteoglycans, in particular, have also been shown to activate the intracellular inflammasomes that trigger sterile inflammation. These molecules, which consist of a protein core with one or more covalently attached glycosaminoglycan chains, can be membrane-bound, secreted, or proteolytically cleaved and shed from the cell surface.

Biglycan is one of the first proteoglycans to be described as a TLR ligand.¹⁵ It consists of a protein core containing leucine-rich repeat regions, with two glycosaminoglycan (GAG) side chains (chondroitin sulfate or dermatan sulfate). Although biglycan typically exists in a matrix-bound form, with tissue injury, it is released from the ECM in a soluble form where it interacts with TLR2 or TLR4 to generate an immediate inflammatory response.

Various proinflammatory cytokines and chemokines, including TNF-α and interleukin (IL)-1β, are downstream effector molecules of biglycan/TLR2/4 signaling. Among these, the mechanism of biglycan-mediated autonomous synthesis and secretion of mature IL-1β is unique. Usually, release of mature IL-1β from the cell requires two signals, one which is needed to initiate synthesis (TLR2/4-mediated) and the other to process pro-IL-1β to its mature form (inflammasome-mediated). How is it possible for biglycan to provide both signals? Current evidence indicates that when soluble biglycan binds to the TLR, it simultaneously serves as a ligand for a purinergic receptor, which facilitates the inflammasome activation required for IL-1β processing.¹⁶ These data support the idea that DAMP-mediated signals can initiate a robust inflammatory response.

DAMPs Are Ligands for Pattern Recognition Receptors

The inflammatory response that occurs following traumatic injury is similar to that observed with pathogen exposure. Not surprising, surface and cytoplasmic receptors that mediate the innate immune response to microbial infection have been implicated in the activation of sterile inflammation. In support of this idea, genes have been identified that are dysregulated acutely both in response to a microbial ligand administered to human volunteers and in response to traumatic injury in a large patient population.¹⁷ The classes of receptors that are important for sensing damaged cells and cell debris are part of the larger group of germline encoded pattern recognition receptors (PRRs). The best-described ligands for these receptors are microbial components, the PAMPs. The PRRs of the innate immune system fall into at least four distinct classes: TLRs, calcium-dependent (C-type) lectin receptors (CLRs), retinoic acid–inducible gene (RIG)-I-like receptors (RLRs), and the nucleotide-binding domain, leucine-rich repeat–containing (NBD-LRR) proteins (NLRs; also nucleotide-binding and oligomerization domain [NOD]-like receptors). Following receptor ligation, intracellular signaling modulates transcriptional and posttranslational events necessary for host defense by coordinating the synthesis and release of cytokines and chemokines to either initiate or suppress the inflammatory response. The best described of these, the TLRs, NLRs, and CLRs, are discussed in the following sections.

Toll-Like Receptors

The TLRs are evolutionarily conserved type 1 transmembrane proteins that are the best-characterized PRRs in mammalian cells. They were first identified in Drosophila, where a mutation in the Toll gene led to its identification as a key component in their immune defense against fungal infection. The first human TLR, TLR4, was identified shortly thereafter. Now, more than 10 human TLR family members have been identified, with distinct ligands that include lipid, carbohydrate, peptide, and nucleic acid components of various pathogens. TLRs are expressed on both immune and nonimmune cells. At first, the expression of TLR was thought to be isolated to professional antigen-presenting cells such as dendritic cells and macrophages. However, mRNA for TLR family members have been detected in most cells of myeloid lineage, as well as natural killer (NK) cells.¹⁸ In addition, activation of T cells increases their TLR expression and induces their survival and clonal expansion. Direct engagement of TLR in T-regulatory (Treg) cells promotes their expansion and reprograms them to differentiate into T helper cells, which in turn provides help to effector cells. In addition, B cells express a distinct subset of the TLR family that determines their ability to respond to DAMPs; however, the significance of restricted TLR expression in these cells is not yet clear.

All TLRs consist of an extracellular domain, characterized by multiple leucine-rich repeats (LRRs), and a carboxy-terminal, intracellular toll/IL-1 receptor (TIR) domain. The LRR domains recognize bacterial and viral PAMPs in the extracellular environment (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11) or in the endolysosomes (TLR3, TLR7, TLR8, TLR9, and TLR10). Although the role of TLRs in sepsis has been well described, more recent data indicate that a subset of the TLRs, TLR4 in particular, also recognizes DAMPs released from injured cells and tissues.¹⁹ Signal transduction occurs with receptor dimerization and recruitment of cytoplasmic adaptor proteins. These adaptor molecules initiate and amplify downstream signals, resulting in the activation of transcription. The transcription factors, which include nuclear factor-κB (NF-κB), activator protein (AP)-1, and interferon regulatory factor (IRF), bind to regulatory elements in promoters and/or enhancers of target genes leading to the upregulation of a large cohort of genes that include interferon (IFN)-α and IFN-β, nitric oxide synthase 2 (NOS2A), and TNF, which play critical roles in initiating innate immune responses to cellular injury and stress. Given the importance of TLR triggering of the innate immune response to immune homeostasis, it is no surprise that the process is tightly regulated. TLR expression is significantly increased following blunt traumatic injury. Further, TLR signaling is controlled at multiple levels, both posttranscriptionally via ubiquitination, phosphorylation, and microRNA actions that affect mRNA stability, as well as by the localization of the TLRs and their signaling complexes within the cell.

Nucleotide-Binding Oligomerization Domain-Like Receptor Family

The NLRs are a large family of proteins composed of intracellular PRRs that sense both endogenous (DAMPs) and exogenous (PAMPs) molecules to trigger innate immune activation. The best characterized of the NLRs is the NLR family pyrin domain-containing 3 (NLRP3), which is highly expressed in peripheral blood leukocytes. It forms the key “sensing” component of the larger, multiprotein inflammasome complex, which is composed of NLRP3; the adapter protein apoptosis-associated speck-like protein containing a CARD (ASC); and the effector protein, caspase 1.²⁰ In the cytoplasm, the receptor resides in an inactive form due to an internal interaction between two adjacent and highly conserved domains. In conjunction with a priming event, such as mitochondrial stress, phagocytosed DAMPs can be sensed by NLRP3, resulting in the removal of the self-repression. The protein can then oligomerize and recruit other complex members. The net result is the autoactivation of pro-caspase 1 to caspase 1. The NLRP3 inflammasome plays a central role in immune regulation by initiating the caspase 1–dependent processing and secretion of the proinflammatory cytokines IL-1β and IL-18. In fact, NLRP3 is the key protein in the mechanism by which IL-1β production is regulated in macrophages. NLRP3 inflammasome activity is tightly regulated by cell-cell interactions, cellular ion flux, and oxidative stress in order to maintain a balanced immune response to danger signals.

While the role of the NLRP3 inflammasome in the sterile inflammatory response following trauma has not been well described, recent evidence suggests that genetic variations in the NLRP3 gene might affect the magnitude of immune inflammatory responses following trauma. Single nucleotide polymorphisms within the NLRP3 gene were found to be associated with increased risk of sepsis and multiple organ dysfunction syndrome in patients with major trauma.²¹ In an animal model of burn injury, early inflammasome activation has been detected in a variety of immune cells (NK cells, CD4/CD8 T cells, and B cells), as determined by the assessment of caspase 1 cleavage by flow cytometry.²² Further, inhibition of caspase 1 activity in vivo results in increased burn mortality, suggesting that inflammasome activation may play an unanticipated protective role in the host response to injury that may be linked to increased production of specific cytokines. In addition to the NLRP3 inflammasome, there are numerous other NLRP sensors that are capable of detecting a diverse range of molecular targets. Among them are those endogenous molecules that are released as a consequence of tissue injury and cellular stress (hypoxia/hypoperfusion).

C-Type Lectin Receptors

Macrophages and dendritic cells possess receptors that detect molecules released from damaged or dying cells in order to retrieve and process antigens from cell corpses for T-cell presentation. A key family of receptors that directs this process is the CLR family that includes the selectin and the mannose receptor families and that binds carbohydrates in a calcium-dependent fashion. Best described for their sensing of PAMPs, particularly fungal antigens, the CLRs can also act to promote the endocytosis and clearance of cell corpses. More recent work has demonstrated, however, that a subset of CLR receptors such as dendritic cell-NK lectin group receptor-1 (DNGR-1) and macrophage-inducible C-type lectin receptor (Mincle) recognize DAMPS of intracellular origin, such as F-actin and the ribonucleoprotein SAP-130.²³ Ligation and activation of Mincle promotes its interaction with an Fcγ receptor, which contains immunoreceptor tyrosine-based activation motifs. This leads to proinflammatory cytokine, chemokine, and nitric oxide production, in addition to neutrophil recruitment. In this way, Mincle may contribute to local inflammation at sites of tissue injury.

Soluble Pattern Recognition Molecules: The Pentraxins

Soluble pattern recognition molecules (PRMs) are a molecularly diverse group of molecules that share a conserved mode of action that is defined by complement activation, agglutination and neutralization, and opsonization. The best described of the PRMs are the pentraxins. PRMs can be synthesized at sites of injury and inflammation by macrophages and dendritic cells, while neutrophils can store PRMs and can release them rapidly following activation. In addition, epithelial tissues (the liver in particular) serve as a reservoir source for systemic mass release. The short pentraxin, C-reactive protein (CRP), was the first PRM to be identified. Serum amyloid protein (SAP), which has 51% sequence similarity to human CRP, also contains the pentraxin molecular signature. CRP and SAP plasma levels are low (≤3 mg/L) under normal circumstances. However, CRP is synthesized by the liver in response to IL-6, increasing serum levels more than a 1000-fold. Thus, CRP is considered part of the acute-phase protein response in humans. For this reason, CRP has been studied as a marker of the proinflammatory response in many clinical settings, including appendicitis, vasculitis, and ulcerative colitis. CRP and SAP are ancient immune molecules that share many functional properties with antibodies: they bind bacterial polysaccharides, ECM components, apoptotic cells, and nuclear materials, as well as all three classes of Fcγ receptors (FcγR). Both molecules also participate in the activation and regulation of complement pathways. In this way, short pentraxins can link immune cells to the complement system.²⁴

Finally, significant data support a role for pentraxin 3 (PTX3), a long pentraxin family member, in the “sterile” inflammatory response associated with cellular stress. While CRP is produced solely in the liver, PTX3 is produced by various cells in peripheral tissues, including immune cells. PTX3 plasma concentrations increase rapidly in various inflammatory conditions, including sepsis. Further, in a recent prospective study of polytraumatized patients, serum PTX3 concentrations were highly elevated, peaking at 24 hours. In addition, PTX3 concentrations at admission were associated with injury severity, whereas higher PTX3 serum concentrations 24 hours after admission correlated with lower probability for survival.²⁵

Pattern Recognition Receptor Signaling: Toll-Like Receptors and the Inflammasome

As noted earlier, members of the TLR family respond to endogenous molecules released from damaged or stressed cells. In animal models, activation of TLRs in the absence of bacterial pathogens correlates with the development of critical illness including “sterile inflammation.” What we know about TLR signaling events has largely been derived from the TLR-mediated response to bacterial pathogens. However, it is likely that the intracellular adaptors required for signal transmission by TLRs in response to exogenous ligands are conserved and used for “damage” sensing of endogenous (“self”) ligands as well. The intracellular domain structure of TLRs is highly conserved and is characterized by a cytoplasmic toll/IL-1R homology (TIR) domain. Binding of ligand to the receptor results in a receptor dimer, either a homodimer (e.g., TLR4/TLR4) or heterodimer (e.g., TLR2/TLR1), which recruits a number of adaptor proteins to the TIR domains, through TIR-TIR interaction.²⁶ With one exception (TLR3), the universal adaptor protein central to the TLR signaling complex is myeloid differentiation factor 88 (MyD88), a member of the IL-1 receptor subfamily. MyD88 works through the recruitment of a second TIR-containing adaptor, MyD88 adaptor-like protein (Mal), in the context of TLR4 and TLR2 signaling, which serves as a bridge between MyD88 and activated TLRs to initiate signal transduction. It is interesting that Mal’s adaptor function requires cleavage of the carboxy-terminal portion of the protein by caspase 1, a key effector of the inflammasome.²⁷ This finding suggests an important synergy between TLRs and NLRs that may potentiate TLR-mediated signaling. There are three other TIR domain-containing adaptor proteins that are also important to TLR-signaling events; these are TIR-domain-containing adapter-inducing INF-β (TRIF), TRIF-related adaptor molecule (TRAM), and sterile α- (SAM) and HEAT/armadillo (ARM) motif-containing protein (SARM). Two of these, TRIF and TRAM, are involved in the MyD88-independent signaling pathways, which are activated by TLR3 and TLR4.

Signaling through the MyD88-dependent pathway results in the activation of numerous cytoplasmic protein kinases including IL-1 receptor–associated kinases (IRAK-1 and IRAK-4), resulting in an interaction with TNF receptor–associated factor 6 (TRAF6). TRAF6, an E3 ubiquitin ligase, forms a complex with two other proteins, which together activate the complex that subsequently phosphorylates IκB kinase (IKK)-β and the MAP kinases (MAPKs). Ultimately, the phosphorylation of IκB by the IKK complex and NEMO (NF-κB essential modulator) leads to its degradation, which frees NF-κB and allows its translocation to the nucleus and the transcription of NF-κB target genes. Simultaneously, MAPK activation is critical for activation of the activator protein-1 (AP-1) transcription factor, and thus production of inflammatory cytokines. The MyD88-independent pathway acts through TRIF to activate NF-κB, similar to the MyD88-dependent pathway. However, TRIF can also recruit other signaling molecules to phosphorylate interferon-regulatory factor 3 (IRF3), which induces expression of type I IFN genes.²⁶

Signaling from the Inflammasome

As discussed earlier, activation and assembly of the inflammasome in response to DAMP sensing result in the cleavage of pro-caspase 1 into two products. This event is pivotal to all known inflammasome signaling pathways. The caspase 1 products assemble to form the IL-1 converting enzyme (ICE), which cleaves the IL-1 cytokines, IL-1β, IL-18, and IL-33. This final step is required for activation and secretion of the cytokines from the cell.²⁰ IL-1β and IL-18 are potent proinflammatory cytokines that promote key immune responses that are essential to host defense. Thus, the synthesis, processing, and secretion of these cytokines are tightly regulated, as successful cytokine release requires a two-step process. The first signal, which is typically TLR-mediated, initiates the synthesis and storage of the inactive cytokine precursors in the cytoplasm. The second signal, which is inflammasome-mediated, initiates proteolytic cleavage of the procytokine, which is a requirement for its activation and secretion from the cell. Of further interest, evidence has demonstrated that both IL-1β and IL-18 lack a signal sequence, which is usually necessary for those proteins that are destined for cellular export. These signal peptides target proteins to the endoplasmic reticulum (ER) and to the Golgi complex, where they are packaged for secretion from the cell through the classical secretory pathway. More than 20 proteins in addition to IL-1β and IL-18 undergo unconventional protein secretion independent of the ER and Golgi complex.²⁸ The list includes signaling molecules involved in inflammatory, cell survival, and repair responses, such as HMGB1, IL-1α, galectins 1 and 3, and FGF2. Currently, the mechanisms responsible for unconventional protein secretion are not understood; however, the process is also evident in yeast under conditions of cellular stress. It makes evolutionary sense that a mechanism for rapid secretion of stored proteins essential to the stress response is highly conserved.

The central nervous system (CNS) communicates with the body through ordered systems of sensory and motor neurons, which receive and integrate information to generate a coordinated response. Rather than being an immune-privileged organ, recent work indicates that the CNS receives information with regard to injury-induced inflammation both via soluble mediators as well as direct neural projections that transmit information to regulatory areas in the brain (Fig. 2-2). How does the CNS sense inflammation? DAMPs and inflammatory molecules convey stimulatory signals to the CNS via multiples routes. For example, soluble inflammatory signaling molecules from the periphery can reach neurons and glial cells directly through the fenestrated endothelium of the circumventricular organs (CVO) or via a leaky blood brain barrier in pathologic settings such as may occur following a traumatic brain injury.²⁹ In addition, inflammatory stimuli can interact with receptors located on the brain endothelial cells to generate a variety of proinflammatory mediators (cytokines, chemokines, adhesion molecules, proteins of the complement system, and immune receptors) that directly impact the brain parenchyma. Not surprising, this response is countered by potent anti-inflammatory signaling, a portion of which is provided by the hypothalamic-pituitary-adrenal (HPA) axis and the release of systemic glucocorticoids. Inflammatory stimuli in the CNS result in behavioral changes, such as increased sleep, lethargy, reduced appetite, and the most common feature of infection, fever.

Information regarding peripheral inflammation and tissue damage can also be signaled to the brain via afferent neural fibers, particularly those of the vagus nerve.³⁰ These afferent fibers can interconnect with neurons that project to the hypothalamus to modulate the HPA axis. In addition, afferent vagal nerve impulses modulate cells in the brain stem, at the dorsal motor nucleus of the vagus, from which efferent preganglionic parasympathetic impulses originate. Axons from these cells, which comprise the visceromotor component of the vagus nerve, form an “inflammatory reflex” that feeds back to the periphery to regulate inflammatory signaling events.³¹ Although the mechanisms by which cholinergic signals from the CNS regulate immune cells in the periphery are incompletely understood, recent evidence has provided some mechanistic insight. The first line of evidence to support this idea is the observation that vagal stimulation reduces proinflammatory cytokine production from the spleen in several experimental models systems.³² This effect is dependent on both the vagal efferent signals and, in part, splenic catecholaminergic nerve fibers that originate in the celiac plexus and that terminate in a T-cell–rich area of the spleen. Interestingly, these signals propagated by adrenergic nerves result in measurable increases in acetylcholine (ACh) levels in the spleen. In addition, the resident immune cells in the spleen require the expression of cholinergic receptors, specifically α7 nicotinic acetylcholine receptors (α7nAChR), for the suppression of cytokine synthesis.³³ How is this effect mediated? The apparent source of ACh is choline-acetyltransferase–expressing T cells, which compose 2% to 3% of CD4⁺ T cells in the spleen and are capable of ACh production. Data also indicate that the vagus nerve may regulate inflammation in tissues that it directly innervates.

Neuroendocrine Response to Injury

Traumatic injury results in complex neuroendocrine signaling from the brain that serves to enhance immune defense and rapidly mobilize substrates necessary to meet essential energy and structural needs. The two principle neuroendocrine pathways that orchestrate the host response are the hypothalamic-pituitary-adrenal (HPA) axis, which results in the release of glucocorticoid hormones, and the sympathetic nervous system, which results in release of the catecholamines, epinephrine, and norepinephrine. Virtually every hormone of the HPA axis influences the physiologic response to injury and stress (Table 2-3), but some with direct influence on the inflammatory response or immediate clinical impact are highlighted here, including growth hormone (GH), macrophage inhibitory factor (MIF), aldosterone, and insulin.

The Hypothalamic-Pituitary-Adrenal Axis

One of the main mechanisms by which the brain responds to injury-associated stress is through activation of the HPA axis. Following injury, corticotrophin-releasing hormone (CRH) is secreted from the paraventricular nucleus (PVN) of the hypothalamus. This action is mediated in part by circulating cytokines produced as a result of the innate immune response to injury. These include TNF-α, IL-1β, IL-6, and the type I IFNs (IFN-α/β). Cytokines that are produced as a result of the adaptive immune response (IL-2 and IFN-γ) are also capable of increasing cortisol release. Direct neural input via afferent vagal fibers that interconnect with neurons projecting to the hypothalamus can also trigger CRH release. CRH acts on the anterior pituitary to stimulate the secretion of adrenocorticotropin hormone (ACTH) into the systemic circulation. Interestingly, the cytokines that act on the hypothalamus are also capable of stimulating ACTH release from the anterior pituitary so that marked elevations in ACTH and in cortisol can occur that are proportional in magnitude to the injury severity. Additionally, pain, anxiety, vasopressin, angiotensin II, cholecystokinin, vasoactive intestinal peptide, and catecholamines all contribute to ACTH release in the injured patient.

ACTH acts on the zona fasciculata of the adrenal glands to synthesize and secrete glucocorticoids (Fig. 2-3). Cortisol is the major glucocorticoid in humans and is essential for survival during significant physiologic stress. The resulting increase in cortisol levels following trauma have several important anti-inflammatory actions.

Cortisol elicits its many actions through a cytosolic receptor, the glucocorticoid receptor (GR). Because it is lipid soluble, cortisol can diffuse through the plasma membrane to interact with its receptor, which is sequestered in the cytoplasm in a complex with heat shock proteins (Fig. 2-4). Upon ligand binding, the GR is activated and can employ a number of mechanisms to modulate proinflammatory gene transcription and signaling events, with a “net” anti-inflammatory effect.³⁴ For example, the activated GR complex can interact with transcription factors to sequester them in the cytoplasm, promote their degradation, or inhibit them through other mechanisms. Affected target genes include proinflammatory cytokines, growth factors, adhesion molecules, and nitric oxide. In addition, glucocorticoids can negatively affect the access of the transcription factor, NF-κB, to the promoter regions of its target genes via a mechanism that involves histone deacetylase 2. In this way, glucocorticoids can inhibit a major mechanism by which TLR ligation induces proinflammatory gene expression.³⁵ The GR complex can also bind to specific nucleotide sequences (termed glucocorticoid response elements) to promote the transcription of genes that have anti-inflammatory functions. These include IL-10 and IL-1 receptor antagonist. Further, GR complex activation can indirectly influence TLR activity via an interaction with signaling pathways such as the mitogen-activated protein kinase and transforming growth factor–activated kinase-1 (TAK1) pathways. Finally, a recent report demonstrated that the GR complex can target both suppressor of cytokine signaling 1 (SOCS1) and type 1 IFNs to regulate TLR-induced STAT1 activation.³⁶

Adrenal insufficiency represents a clinical syndrome highlighted largely by inadequate amounts of circulating cortisol and aldosterone. Classically, adrenal insufficiency is described in patients with atrophic adrenal glands caused by exogenous steroid administration who undergo a stressor such as surgery. These patients subsequently manifest signs and symptoms such as tachycardia, hypotension, weakness, nausea, vomiting, and fever. Critical illness may be associated with a relative adrenal insufficiency such that the adrenal gland cannot mount an effective cortisol response to match the degree of injury. More recently, investigators have determined that critical illness-associated cortisol insufficiency in trauma patients occurs more frequently than previously thought.³⁷ It has a bimodal presentation in which the patient is at increased risk both early following the injury-associated inflammatory response and in a delayed fashion, with sepsis being the initiating event. Laboratory findings in adrenal insufficiency include hypoglycemia from decreased gluconeogenesis, hyponatremia from impaired renal tubular sodium resorption, and hyperkalemia from diminished kaliuresis. Rigorous testing to establish the diagnosis includes monitoring of basal and ACTH-stimulated cortisol levels, both of which are lower than normal during adrenal insufficiency. Treatment strategies remain controversial; however, they include low-dose steroid supplementation.³⁸

Macrophage Inhibitory Factor Modulates Cortisol Function

Macrophage inhibitory factor (MIF) is a proinflammatory cytokine expressed by a variety of cells and tissues, including the anterior pituitary, macrophages, and T lymphocytes. Several important functions of MIF in innate and adaptive immune responses and in inflammation have been described, supporting the idea that MIF may function to counteract the anti-inflammatory activity of glucocorticoids.³⁹ For example, MIF has been reported to play a central role in the exacerbation of inflammation associated with acute lung injury, where it has been detected in the affected lungs and in alveolar macrophages. MIF has also been reported to upregulate the expression of TLR4 in macrophages.⁴⁰ Finally, an early increase in plasma MIF has been detected in severely injured patients and was found to correlate with NF-κB translocation and respiratory burst in polymorphonuclear lymphocytes (PMNs) derived from severely injured patients. Further, nonsurvivors were shown to have higher serum MIF concentrations early after injury than survivors.⁴¹ These data suggest that targeting MIF after injury may be beneficial in preventing early PMN activation and subsequent organ failure in severely injured patients.

Growth Hormone, Insulin-Like Growth Factor, and Ghrelin

Growth hormone (GH) is a neurohormone expressed primarily by the pituitary gland that has both metabolic and immunomodulatory effects. GH promotes protein synthesis and insulin resistance and enhances the mobilization of fat stores. GH secretion is upregulated by hypothalamic GH-releasing hormone and downregulated by somatostatin. GH primarily exerts its downstream effects through direct interaction with GH receptors and through the enhanced hepatic synthesis of insulin-like growth factor (IGF)-1, an anabolic growth factor that is known to improve the metabolic rate, gut mucosal function, and protein loss after traumatic injury. Less than 5% of IGF-1 circulates free in the plasma, with the remainder bound principally to one of six IGF-binding proteins (IGFBPs), the majority to IGFBP-3. In the liver, IGF stimulates protein synthesis and glycogenesis; in adipose tissue, it increases glucose uptake and lipid utilization; and in skeletal muscles, it mediates glucose uptake and protein synthesis. In addition to its effects on cellular metabolism, GH enhances phagocytic activity of immunocytes through increased lysosomal superoxide production. It also increases the proliferation of T-cell populations.⁴² The catabolic state that follows severe injury has been linked to the suppression of the GH-IGF-IGFBP axis, as critical illness is associated with decreased circulating IGF levels. Not surprising, the administration of exogenous recombinant human GH (rhGH) has been studied in a prospective, randomized trial of critically ill patients where it was associated with increased mortality, prolonged ventilator dependence, and increased susceptibility to infection.⁴³ More recently, circulating GH levels were examined on admission in 103 consecutive critically ill adult patients. In this study, circulating GH levels were about seven-fold increased in the 24 nonsurvivors when compared with survivors, and GH level was an independent predictor of mortality, along with the APACHE II/SAPS II scores. In distinct contrast, the effect of rhGH administration in severely burned children, both acutely and following prolonged treatment, has been proven to be beneficial. Pediatric burn patients receiving rhGH demonstrated markedly improved growth and lean body mass, whereas hypermetabolism was significantly attenuated.⁴⁴ This finding was associated with significant increases in serum GH, IGF-1, and IGFBP-3.

Ghrelin, a natural ligand for the GH-secretagogue receptor 1a (GHS-R1a), is an appetite stimulant that is secreted by the stomach. GHS-R1a is expressed in a variety of tissues in different concentrations including the immune cells, B and T cells, and neutrophils. Ghrelin seems to play a role in promoting GH secretion and in glucose homeostasis, lipid metabolism, and immune function. In a rodent gut ischemia/reperfusion model, ghrelin administration inhibited proinflammatory cytokine release, reduced neutrophil infiltration, ameliorated intestinal barrier dysfunction, attenuated organ injury, and improved survival. It is interesting that this effect was dependent on an intact vagus nerve and that intracerebroventricular injection of ghrelin was also protective.⁴⁵ These data suggest that the effect of ghrelin is mediated via the CNS, most likely through the “cholinergic anti-inflammatory pathway.” More recently, high ghrelin levels were demonstrated in critically ill patients as compared to healthy controls, independent of the presence of inflammatory markers. Moreover, the high ghrelin levels were a positive predictor of intensive care unit survival in septic patients, matching previous results from animal models.

The Role of Catecholamines in Postinjury Inflammation

Injury-induced activation of the sympathetic nervous system results in secretion of ACh from the preganglionic sympathetic fibers innervating the adrenal medulla. The adrenal medulla is a special case of autonomic innervation and is considered a modified postganglionic neuron. Thus, ACh signaling to the resident chromaffin cells ensures that a surge of epinephrine (EPI) and norepinephrine (NE) release into the circulation takes place in a ratio that is tightly regulated by both central and peripheral mechanisms. Circulating levels of EPI and NE are three- to four-fold elevated, an effect that persists for an extended time. The release of EPI can be modulated by transcriptional regulation of phenylethanolamine N-methyltransferase (PNMT), which catalyzes the last step of the catecholamine biosynthesis pathway methylating NE to form EPI. PNMT transcription, a key step in the regulation of EPI production, is activated in response to stress and tissue hypoxia by hypoxia-inducible factor 1α (HIF1A).

Catecholamine release almost immediately prepares the body for the “fight or flight” response with well-described effects on the cardiovascular and pulmonary systems and on metabolism. These include increased heart rate, myocardial contractility, conduction velocity, and blood pressure; the redirection of blood flow to skeletal muscle; increased cellular metabolism throughout the body; and mobilization of glucose from the liver via glycogenolysis, gluconeogenesis, lipolysis, and ketogenesis. To compound the resulting hyperglycemia, insulin release is decreased mainly through the stimulation of α-adrenergic pancreatic receptors. Hyperglycemia, as will be discussed later, contributes to the proinflammatory response and to further mitochondrial dysfunction.

The goal of this well-orchestrated catecholamine response is to re-establish and maintain the systems’ homeostasis, including the innate immune system. Circulating catecholamines can directly influence inflammatory cytokine production.⁴⁶ Data indicate that basal EPI levels condition the activity and responsiveness of cytokine-secreting cells, which may explain large interindividual variability in innate cytokine profiles observed following injury. Epinephrine infusion at higher doses has been found to inhibit production of TNF-α in vivo and to enhance the production of the anti-inflammatory cytokine IL-10.⁴⁷ Additionally, in vitro studies indicate that stress levels of glucocorticoids and EPI, acting in concert, can inhibit production of IL-12, a potent stimulator of Th1 responses. Further, they have been shown in vitro to decrease Th1 cytokine production and increase Th2 cytokine production to a significantly greater degree compared to either adrenal hormone alone. Thus, catecholamines secreted from the adrenal gland, specifically EPI, play a role in both innate proinflammatory cytokine regulation and adaptive Th responses, and may act in concert with cortisol during the injury response to modulate cytokine activity.⁴⁸

How are these effects explained? It is well established that a variety of human immune cells (e.g., mononuclear cells, macrophages, granulocytes) express adrenergic receptors that are members of the family of G-protein–coupled receptors that act through the activation of intracellular second messengers such as cyclic adenosine monophosphate (cAMP) and calcium ion influx (discussed in more detail later). These second messengers can regulate a variety of immune cell functions, including the release of inflammatory cytokines and chemokines.

The sympathetic nervous system also has direct immune-modulatory properties via its innervation of lymphoid tissues that contain resting and activated immune cells. With stimulation of these postganglionic nerves, NE is released where it can interact with β₂-adrenergic receptors expressed by CD4⁺ T and B lymphocytes, many of which also express α₂-adrenergic receptors. Additionally, endogenous catecholamine expression has been detected in these cells, as has the machinery for catecholamine synthesis. For example, human peripheral blood mononuclear cells contain inducible mRNA for the catecholamine-generating enzymes, tyrosine-hydroxylase and dopamine-β-hydroxylase, and data suggest that cells can regulate their own catecholamine synthesis in response to extracellular cues. Exposure of peripheral blood mononuclear cells to NE triggers a distinct genetic profile that indicates a modulation of Th cell function. What the net effect of dopamine, NE, and EPI synthesis by circulating and resident immune cells may be relative to that secreted by the adrenal medulla is not clear and is an area that would certainly benefit from ongoing research efforts to identify novel therapeutic targets.

Aldosterone

Aldosterone is a mineralocorticoid released by the zona glomerulosa of the adrenal cortex. It binds to the mineralocorticoid receptor (MR) of principal cells in the collecting duct of the kidney where it can stimulate expression of genes involved in sodium reabsorption and potassium excretion to regulate extracellular volume and blood pressure. MRs have also been shown to have effects on cell metabolism and immunity. For example, recent studies show aldosterone interferes with insulin signaling pathways and reduces expression of the insulin-sensitizing factors, adiponectin and peroxisome proliferator activated receptor-γ (PPAR-γ), which contribute to insulin resistance. In the immune system, mononuclear cells, such as monocytes and lymphocytes, have been shown to possess an MR that binds aldosterone with high specificity, regulating sodium and potassium flux, as well as plasminogen activator inhibitor-1 and p22 phox expression, in these cells.⁴⁹ Further, aldosterone inhibits cytokine-mediated NF-κB activation in neutrophils, which also possess a functional MR.

Insulin

Hyperglycemia and insulin resistance are hallmarks of injury and critical illness due to the catabolic effects of circulating mediators, including catecholamines, cortisol, glucagon, and GH. The increase in these circulating proglycemic factors, particularly EPI, induces glycogenolysis, lipolysis, and increased lactate production independent of available oxygen in a process that is termed “aerobic glycolysis.” Although there is an increase in insulin production at the same time, severe stress is frequently associated with insulin resistance, leading to decreased glucose uptake in the liver and the periphery contributing to acute hyperglycemia. Insulin is a hormone secreted by the pancreas, which mediates an overall host anabolic state through hepatic glycogenesis and glycolysis, peripheral glucose uptake, lipogenesis, and protein synthesis.⁵⁰

The insulin receptor (IR) is widely expressed and consists of two isoforms, which can form homo- or heterodimers with insulin binding. Dimerization leads to receptor autophosphorylation and activation of intrinsic tyrosine kinase activity. Downstream signaling events are dependent on the recruitment of the adaptor proteins, insulin receptor substrate (IRS-1), and Shc to the IR. Systemic insulin resistance likely results from proinflammatory signals, which modulate the phosphorylation of IRS-1 to affect its function.

Hyperglycemia during critical illness is predictive of increased mortality in critically ill trauma patients.⁵¹ It can modulate the inflammatory response by altering leukocyte functions, and the resulting decreases in phagocytosis, chemotaxis, adhesion, and respiratory burst activities are associated with an increased risk for infection. In addition, glucose administration results in a rapid increase in NF-κB activation and proinflammatory cytokine production. Insulin therapy to manage hyperglycemia has grown in favor and has been shown to be associated with both decreased mortality and a reduction in infectious complications in select patient populations. However, the trend toward tight glycemic control in the intensive care unit failed to show benefit when examined in several reviews.⁵² Thus, the ideal blood glucose range within which to maintain critically ill patients and to avoid hypoglycemia has yet to be determined.

Reactive Oxygen Species and the Oxidative Stress Response

Reactive oxygen and nitrogen species (ROS and RNS, respectively) are small molecules that are highly reactive due to the presence of unpaired outer orbit electrons. They can cause cellular injury to both host cells and invading pathogens through the oxidation of cell membrane substrates. Oxygen radicals are produced as a by-product of oxygen metabolism in the mitochondria as well as by processes mediated by cyclooxygenases, NADPH oxidase (NOX), and xanthine oxidase. The main areas of ROS production include mitochondrial respiratory chain, peroxisomal fatty acid metabolism, cytochrome P450 reactions, and the respiratory burst of phagocytic cells. In addition, protein folding in the endoplasmic reticulum can also result in the formation of ROS.⁵³ Potent oxygen radicals include oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals. RNS include NO and nitrite. The synthesis of ROS is regulated at several checkpoints and via several signaling mechanisms, including Ca²⁺ signaling, phosphorylation, and small G protein activation, which influence both the recruitment of the molecules required for NOX function and the synthesis of ROS in the mitochondria. NOX activation is triggered by a number of inflammatory mediators (e.g., TNF, chemokines, lysophospholipids, complement, and leukotrienes). Host cells are protected from the damaging effects of ROS through a number of mechanisms. The best described of these is via the upregulation and/or activation of endogenous antioxidant proteins. However, pyruvate kinase also provides negative feedback for ROS synthesis, as do molecules that react nonenzymatically with ROS. Under normal physiologic conditions, ROS production is balanced by these antioxidative strategies. In this context, ROS can act effectively as signaling molecules through their ability to modulate cysteine residues by oxidation and thus influence the functionality of target proteins.⁵⁴ This has recently been described as a mechanism in the regulation of phosphatases. ROS can also contribute to transcription activity both indirectly, through its effects on transcription factor lifespan, and directly, through the oxidation of DNA. An important role for ROS has been well described in phagocytes, which use these small molecules for pathogen killing. Recent data, however, indicate that ROS may mediate inflammasome activation by diverse agonists.⁵⁵ In addition, ROS appear to be involved in adaptive immunity. They have been described as a prime source of phosphatase activation in both B and T lymphocytes, which can regulate the function of key receptors and intracellular signaling molecules in these cells by affecting phosphorylation events.

The Heat Shock Response

Heat shock proteins (HSPs) are a group of intracellular proteins that are increasingly expressed during times of stress, such as burn injury, inflammation, oxidative stress, and infection. HSPs are expressed in the cytoplasm, nucleus, endoplasmic reticulum, and mitochondria, where they function as molecular chaperones that help monitor and maintain appropriate protein folding.⁵⁶ HSPs accomplish this task through the promotion of protein refolding, the targeting of misfolded proteins for degradation, and the assistance of partially folded proteins to appropriate membrane compartments. HSPs bind also bind foreign proteins and thereby function as intracellular chaperones for ligands such as bacterial DNA and endotoxin. HSPs are presumed to protect cells from the effects of traumatic stress and, when released by damaged cells, alert the immune system of the tissue damage. However, depending on their location and the type of immune cell in which they are expressed, HSPs may exert proinflammatory immune activating signals or anti-inflammatory immune dampening signals (Table 2-4).⁵⁷

The Unfolded Protein Response

Secreted, membrane-bound, and organelle-specific proteins fold in the lumen of the endoplasmic reticulum (ER) where they also receive their posttranslational modifications. Millimolar calcium concentrations are required to maintain the normal cellular protein folding capacity. Cellular stress decreases calcium concentration in the ER, disrupting the machinery required for this process and leading to the accumulation of misfolded or unfolded proteins. These occurrences are sensed by a highly conserved array of signaling proteins in the ER, including inositol requiring enzyme 1 (IRE1), protein kinase RNA (PKR)–like ER kinase (PERK), and activating transcription factor 6 (ATF6). Together, this complex generates the unfolded protein response (UPR), a mechanism by which ER distress signals are sent to the nucleus to modulate transcription in an attempt to restore homeostasis. Prolongation of the UPR, indicative of irreversible cellular damage, can result in cell death. Genes activated in the UPR result not only in the inhibition of translation, but also other potentially immunomodulatory events including induction of the acute-phase response, activation of NF-κB, and the generation of antibody-producing B cells.⁵⁸

Burn injury leads to the marked reduction in ER calcium levels and activation of UPR sensing proteins. Moreover, recent data in a series of burn patients strongly link the UPR to insulin resistance and hyperglycemia in these patients.⁵⁹ Thus, a better understanding of the UPR, which is triggered by severe inflammation, may allow the identification of novel therapeutic targets for injury-associated insulin resistance.

Autophagy

Under normal circumstances, cells need to have a way of disposing of damaged organelles and debris aggregates that are too large to be managed by proteasomal degradation. In order to accomplish this housekeeping task, cells use a process referred to as “macroautophagy” (autophagy), which is thought to have originated as a stress response.⁶⁰ The steps of autophagy include the engulfment of cytoplasm/organelle by an “isolation membrane,” which is also called a phagophore. The edges of the phagophore then fuse to form the autophagosome, a double-membraned vesicle that sequesters the cytoplasmic material and that is a characteristic feature of autophagy. The autophagosome then fuses with a lysosome to form an autolysosome where the contents, together with the inner membrane, are degraded. This process is controlled by numerous autophagy-specific genes and by the specific kinase, mammalian target of rapamycin (mTOR).

As noted earlier, autophagy is a normal cellular process that occurs in quiescent cells for cellular maintenance. However, under conditions of hypoxia and low cellular energy, autophagy is induced in an attempt to provide additional nutrients for energy production. The induction of autophagy promotes a shift from aerobic respiration to glycolysis and allows cellular components of the autophagosome to be hydrolyzed to energy substrates. Increased levels of autophagy are typical in activated immune cells and are a mechanism for the disposal of ROS and phagocytosed debris.

Recent data support the idea that autophagy may also play an important role in the immune response.⁶¹ Autophagy is stimulated by Th1 cytokines and with activation of TLR in macrophages, but is inhibited by Th2 cytokines. It is also recognized as an important regulator of cytokine secretion, particularly those cytokines of the IL-1 family that are dependent on inflammasome processing for activation. For example, autophagosomes can sequester and degrade pro-IL-1β and inflammasome components. In animal models of sepsis, inhibition of autophagy results in increased proinflammatory cytokine levels that correlate with increased mortality.⁶² These data suggest that autophagy is a protective mechanism whereby the cell can regulate the levels of cytokine production.

Apoptosis

Apoptosis (regulated cell death) is an energy-dependent, organized mechanism for clearing senescent or dysfunctional cells, including macrophages, neutrophils, and lymphocytes, without promoting an inflammatory response. This contrasts with cellular necrosis that results in disorganized intracellular molecule release with subsequent immune activation and inflammatory response. Systemic inflammation modulates apoptotic signaling in active immunocytes, which subsequently influences the inflammatory response through the loss of effector cells.

Apoptosis proceeds primarily through two pathways: the extrinsic pathway and the intrinsic pathway. The extrinsic pathway is activated through the binding of death receptors (e.g., Fas, TNFR), which leads to the recruitment of Fas-associated death domain protein and subsequent activation of caspase 3 (Fig. 2-5). On activation, caspases are the effectors of apoptotic signaling because they mediate the organized breakdown of nuclear DNA. The intrinsic pathway proceeds through protein mediators (e.g., Bcl-2, Bcl-2–associated death promoter, Bcl-2–associated X protein, Bim) that influence mitochondrial membrane permeability. Increased membrane permeability leads to the release of mitochondrial cytochrome C, which ultimately activates caspase 3 and thus induces apoptosis. These pathways do not function in a completely autonomous manner, because there is significant interaction and crosstalk between mediators of both extrinsic and intrinsic pathways. Apoptosis is modulated by several regulatory factors, including inhibitor of apoptosis proteins and regulatory caspases (e.g., caspases 1, 8, 10).

Apoptosis during sepsis may influence the ultimate competency of the acquired immune response. In a murine model of peritoneal sepsis, increased lymphocyte apoptosis was associated with mortality, which may be due to a resultant decrease in IFN-γ release. In postmortem analysis of patients who expired from overwhelming sepsis, there was an increase in lymphocyte apoptosis, whereas macrophage apoptosis did not appear to be affected. Clinical trials have observed an association between the degree of lymphopenia and disease severity in sepsis. In addition, after the phagocytosis of apoptotic cells by macrophages, anti-inflammatory mediators such as IL-10 are released that may exacerbate immune suppression during sepsis. Neutrophil apoptosis is inhibited by inflammatory products, including TNF, IL-1, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IFN-γ. This retardation in regulated cell death may prolong and exacerbate secondary injury through neutrophil free radical release as the clearance of senescent cells is delayed.⁶³

Necroptosis

Cellular necrosis refers to the premature uncontrolled death of cells in living tissue typically caused by accidental exposure to external factors, such as ischemia, inflammation, or trauma, which result in extreme cellular stress. Necrosis is characterized by the loss of plasma membrane integrity and cellular collapse with extrusion of cytoplasmic contents, but the cell nuclei typically remain intact. Recent data have defined a process by which necrosis occurs through a series of well-described steps that are dependent on a signaling pathway that involves the receptor-interacting protein kinase (RIPK) complex. Termed “necroptosis,” it occurs in response to specific stimuli, such as TNF- and TLR-mediated signals.⁶⁴ For example, ligation of the TNF receptor 1 (TNFR1) under conditions in which caspase 8 is inactivated (e.g., by pharmacologic agents) results in the overgeneration of ROS and a metabolic collapse. The net result is programmed necrosis (necroptosis). The effect of cell death by necroptosis on the immune response is not yet known. However, it is likely that the “DAMP” signature that occurs in response to necroptotic cell death is an important contributor to the systemic inflammatory response. Evidence to support this concept was provided by investigators who examined the role of necroptosis in murine models of sepsis. They demonstrated that Ripk3^−/− mice were capable of recovering body temperature better, exhibited lower circulating DAMP levels, and survived at higher rates than their wild-type littermates.⁶⁵ These data suggest that the cellular damage that occurs with programmed necrosis exacerbates the sepsis-associated systemic inflammatory response.

Cytokines

Cytokines are a class of protein signaling compounds that are essential for both innate and adaptive immune responses. Cytokines mediate a broad sequence of cellular responses, including cell migration, DNA replication, cell turnover, and immunocyte proliferation (Table 2-5). When functioning locally at the site of injury and infection, cytokines mediate the eradication of invading microorganisms and also promote wound healing. However, an exaggerated proinflammatory cytokine response to inflammatory stimuli may result in hemodynamic instability (i.e., septic shock) and metabolic derangements (i.e., muscle wasting). Anti-inflammatory cytokines also are released, at least in part, as an opposing influence to the proinflammatory cascade. These anti-inflammatory mediators may also result in immunocyte dysfunction and host immunosuppression. Cytokine signaling after an inflammatory stimulus can best be represented as a finely tuned balance of opposing influences and should not be oversimplified as a “black and white” proinflammatory/anti-inflammatory response. A brief discussion of the important cytokine molecules is included.

Tumor Necrosis Factor-α

TNF-α is a cytokine that is rapidly mobilized in response to stressors such as injury and infection and is a potent mediator of the subsequent inflammatory response. TNF is primarily synthesized by immune cells, such as macrophages, dendritic cells, and T lymphocytes, but nonimmune cells have also been reported to secrete low amounts of the cytokine.

TNF is generated in a precursor form called transmembrane TNF that is expressed as a trimer on the surface of activated cells. After being processed by the metalloproteinase TNF-α–converting enzyme (TACE; also known as ADAM-17), a smaller, soluble form of TNF is released, which mediates its biologic activities through type 1 and 2 TNF receptors (TNFR1; TNFR2).⁶⁶ Transmembrane TNF-α also binds to TNFR1 and TNFR2, but its biologic activities are likely mediated through TNFR2. While the two receptors share homology in their ligand binding regions, there are distinct differences that regulate their biologic function. For example, TNFR1 is expressed by a wide variety of cells but is typically sequestered in the Golgi complex. Following appropriate cell signaling, TNFR1 is mobilized to the cell surface, where it sensitizes cells to TNF, or it can be cleaved from the surface in the form of a soluble receptor that can neutralize TNF.⁶⁷ In contrast, TNFR2 expression is confined principally to immune cells where it resides in the plasma membrane. Both TNF receptors are capable of binding intracellular adaptor proteins that lead to activation of complex signaling processes and mediate the effects of TNF.

Although the circulating half-life of soluble TNF is brief, it acts upon almost every differentiated cell type, eliciting a wide range of important cellular responses. In particular, TNF elicits many metabolic and immunomodulatory activities. It stimulates muscle breakdown and cachexia through increased catabolism, insulin resistance, and redistribution of amino acids to hepatic circulation as fuel substrates. TNF also mediates coagulation activation, cell migration, and macrophage phagocytosis, and enhances the expression of adhesion molecules, prostaglandin E₂, platelet-activating factor, glucocorticoids, and eicosanoids. Recent studies indicate that a significant early TNF response after trauma may be associated with improved survival in these patients.⁶⁸

Interleukin-1

IL-1α and IL-1β, which are encoded by two distinct IL-1 genes, were the first described members of the IL-1 cytokine family. Currently, the family has expanded to 11 members, with the three major forms being IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1Rα). IL-1α and IL-1β share similar biologic functions, but have limited sequence homology. They use the same cell surface receptor, termed IL-1 receptor type 1 (IL-1R1), which is present on nearly all cells. Although IL-1Rα is synthesized and released in response to the same stimuli that lead to IL-1 production, it lacks the necessary domain to form a bioactive complex with the IL-1 receptor when bound. Thus, IL-1Rα serves as a competitive antagonist for the receptor. IL-1R activation initiates signaling events, which result in the synthesis and release of a variety of inflammatory mediators.

The IL-1α precursor is constitutively expressed and stored in a variety of healthy cells, including epithelium, endothelium, and platelets. Both the precursor and mature forms of IL-1α are active. With appropriate signals, IL-1α moves to the cell membrane where it can act on adjacent cells bearing the IL-1 receptor. It can also be released directly from injured cells. In this way, IL-1α is believed to function as a DAMP, which promotes the synthesis of inflammatory mediators, such as chemokines and eicosanoids. These mediators attract neutrophils to the injured site, facilitate their exit from the vasculature, and promote their activation. Once they have reached their target, neutrophil lifespan is extended by the presence of IL-1α.⁶⁹

IL-1β, a multifunctional proinflammatory cytokine, is not detectable in healthy cells. Rather, its expression and synthesis occur in a more limited number of cells, such as monocytes, tissue macrophages, and dendritic cells, following their activation. IL-1β expression is tightly regulated at multiple levels (e.g., transcription, translation, and secretion), although the rate-limiting step is its transcription. IL-1β is synthesized and released in response to inflammatory stimuli, including cytokines (TNF, IL-18) and foreign pathogens. IL-1α or IL-1β itself can also induce IL-1β transcription. In contrast to IL-1α, IL-1β is synthesized as an inactive precursor molecule. The formation of mature IL-1β requires the assembly of the inflammasome complex by the cell and the activation of caspase 1, which is required for the processing of stored pro-IL-1β. Mature IL-1β is then released from the cell via an unconventional secretory pathway. IL-1β has a spectrum of proinflammatory effects that are largely similar to those induced by TNF, and injection of IL-1β alone is sufficient to induce inflammation. High doses of either IL-1β or TNF are associated with profound hemodynamic compromise. Interestingly, low doses of both IL-1β and TNF combined elicit hemodynamic events similar to those elicited by high doses of either mediator, which suggests a synergistic effect.

There are two primary receptor types for IL-1: IL-1R1 and IL-1R2. IL-1R1 is widely expressed and mediates inflammatory signaling on ligand binding. IL-1R2 is proteolytically cleaved from the membrane surface to soluble form on activation and thus serves as another mechanism for competition and regulation of IL-1 activity. IL-1α or IL-1β binds first to IL-1R1. This is followed by recruitment of a transmembrane coreceptor, termed the IL-1R accessory protein (IL-1RAcP). A complex is formed of IL-1R1 plus IL-1 plus the coreceptor. The signal is initiated with recruitment of the adaptor protein MyD88 to the toll–IL-1 receptor (TIR) domains of the receptor complex and signal transduction then occurs via intermediates, which are homologous to the signal cascade initiated by TLRs. These events culminate in the activation of NF-κB and its nuclear translocation.⁷⁰

Interleukin-2

IL-2 is a multifunctional cytokine produced primarily by CD4⁺ T cells after antigen activation, which plays a pivotal role in the immune response. Other cellular sources for IL-2 include CD8⁺ and NK T cells, mast cells, and activated dendritic cells. Discovered as a T-cell growth factor, IL-2 also promotes CD8⁺ T-cell and NK cell cytolytic activity and modulates T-cell differentiation programs in response to antigen. Thus, IL-2 promotes naïve CD4⁺ T-cell differentiation into T helper 1 (Th1) and T helper 2 (Th2) cells while inhibiting T helper 17 (Th17) and T follicular helper (Tfh) cell differentiation. Moreover, IL-2 is essential for the development and maintenance of T regulatory (Treg) cells and for activation-induced cell death, thereby mediating tolerance and limiting inappropriate immune reactions. The upregulation of IL-2 requires calcium as well as protein kinase C signaling, which leads to the activation of transcription factors such as nuclear factor of activated T cells (NFAT) and NF-κB. MicroRNAs also play a role in the regulation of IL-2 expression.⁷¹

IL-2 binds to IL-2 receptors (IL-2R), which are expressed on leukocytes. IL-2Rs are formed from various combinations of three receptor subunits: IL-2Rα, IL-2Rβ, and IL-2Rγ; these form low-, medium-, and high-affinity forms of the receptor depending on the subunit combination. IL-2Rγ has been renamed the common cytokine receptor γ chain (γ_c), which is now known to be shared by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Constitutive IL-2 receptor expression is low and is inducible by T-cell receptor ligation and cytokine stimulation. Importantly, the transcription of each receptor subunit is individually regulated via a complex process to effect tight control of surface expression. Once the receptor is ligated, the major IL-2 signaling pathways that are engaged include Janus kinase (JAK) signal transducer and activator of transcription (STAT), Shc-Ras-MAPK, and phosphoinositol-3-kinase (PI3K)-AKT. Partly due to its short half-life of <10 minutes, IL-2 is not readily detectable after acute injury. IL-2 receptor blockade induces immunosuppressive effects and can be pharmacologically used for organ transplantation. Attenuated IL-2 expression observed during major injury or blood transfusion may contribute to the relatively immunosuppressed state of the surgical patient.⁷²

Interleukin-6

Following burn or traumatic injury, DAMPs from damaged or dying cells stimulate TLRs to produce IL-6, a pleiotropic cytokine that plays a central role in host defense. IL-6 levels in the circulation are detectable by 60 minutes, peak between 4 and 6 hours, and can persist for as long as 10 days. Further, plasma levels of IL-6 are proportional to the degree of injury. In the liver, IL-6 strongly induces a broad spectrum of acute-phase proteins such as CRP and fibrinogen, among others, whereas it reduces expression of albumin, cytochrome P450, and transferrin. In lymphocytes, IL-6 induces B-cell maturation into immunoglobulin-producing cells and regulates Th17/Treg balance. IL-6 modulates T-cell behavior by inducing the development of Th17 cells and inhibiting Treg cell differentiation in conjunction with transforming growth factor-β. IL-6 also promotes angiogenesis and increased vascular permeability, which are associated with local inflammatory responses. To date, 10 IL-6 family cytokines have been identified, including IL-6, oncostatin M, neuropoietin, IL-11, IL-27, and IL-31, all of which use trans signaling.⁷³

The IL-6 receptor (IL-6R, gp80) is expressed on hepatocytes, monocytes, B cells, and neutrophils in humans. However, many other cells respond to IL-6 through a process known as trans signaling.⁷⁴ In this case, soluble IL-6Rs (sIL-6R) exist in the serum and bind to IL-6, forming an IL-6/sIL-6R complex. The soluble receptor is produced by proteolytic cleavage from the surface of neutrophils in a process that is stimulated by CRP, complement factors, and leukotrienes. The IL-6/sIL-6R complex can then bind to the gp130 receptor, which is expressed ubiquitously on cells. Upon IL-6 stimulation, gp130 transduces two major signaling pathways: the JAK-STAT3 pathway and the SHP2-Gab-Ras-Erk-MAPK pathway, which is regulated by cytoplasmic suppressor of cytokine signaling (SOCS3). These signaling events can lead to increased expression of adhesion molecules as well as proinflammatory chemokines and cytokines. High plasma IL-6 levels have been associated with mortality during intra-abdominal sepsis.⁷⁵

Interleukin-10

We have talked almost exclusively about the factors that initiate the inflammatory response following cellular stress or injury. The re-establishment of immune homeostasis following these events requires the resolution of inflammation and the initiation of tissue repair processes. IL-10 plays a central role in this anti-inflammatory response by regulating the duration and magnitude of inflammation in the host.

The IL-10 family currently has six members including IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26. IL-10 is produced by a variety of immune cells of both myeloid and lymphoid origin. Its synthesis is upregulated during times of stress and systemic inflammation; however, each cell type that produces IL-10 does so in response to different stimuli, allowing for tight control of its expression. IL-10 exerts effects by binding to the IL-10 receptor (IL-10R), which is a tetramer formed from two distinct subunits, IL-10R1 and IL-10R2. Specifically, IL-10 binds first to the IL-10R1 subunit, which then recruits IL-10R2, allowing the receptor complex to form. Whereas IL-10R2 is widely expressed, IL-10R1 expression is confined to leukocytes so that this differential expression of the receptor confines the effects of IL-10 to the immune system. Once receptor ligation occurs, signaling proceeds by the activation of JAK1 and STAT3. In particular, STAT3 in conjunction with IL-10 is absolutely required for the transcription of genes responsible for the anti-inflammatory response. IL-10 inhibits the secretion of proinflammatory cytokines, including TNF and IL-1, partly through the downregulation of NF-κB, and thereby functions as a negative feedback regulator of the inflammatory cascade.⁷⁶ In macrophages, IL-10 suppresses the transcription of 20% of all lipopolysaccharide (LPS)-induced genes. Further, experimental models of inflammation have shown that neutralization of IL-10 increases TNF production and mortality, whereas restitution of circulating IL-10 reduces TNF levels and subsequent deleterious effects. Increased plasma levels of IL-10 also have been associated with mortality and disease severity after traumatic injury. IL-10 may significantly contribute to the underlying immunosuppressed state during sepsis through the inhibition and subsequent anergy of immunocytes. For example, IL-10 produced by Th2 cells directly suppresses Th1 cells and can feedback to suppress Th2 cell activity.⁷⁷

Interleukin-12

IL-12 is unique among the cytokines in being the only heterodimeric cytokine. This family, which includes IL-12, IL-23, IL-27, and IL-35, consists of an α-chain that is structurally similar to the IL-6 cytokine and a β-chain that is similar to the class I receptor for cytokines. The individual IL-12 family members are formed from various combinations of the α and β subunits. Despite the sharing of individual subunits and the similarities of their receptors, the IL-12 cytokines have different biologic functions. IL-12 and IL-23 are considered proinflammatory, stimulatory cytokines with key roles in the development of Th1 and Th17 subsets of helper T cells. In contrast, both IL-27 and IL-35 appear to have immunoregulatory functions that are associated with cytokine inhibition in specific Treg cell populations, particularly the Th17 cells.⁷⁸ The effects of these cytokines require specific receptor chains that are also shared among the cytokines. The complexity of signaling is evidenced by the fact that these receptor chains can function both as dimers and as monomers. Ligation of the IL-12 receptors initiates signaling events mediated by the JAK-STAT pathway. IL-12 synthesis and release are increased during endotoxemia and sepsis.⁷⁹ IL-12 stimulates lymphocytes to increase secretion of IFN-γ with the costimulus of IL-18 and also stimulates NK cell cytotoxicity and helper T-cell differentiation in this setting. IL-12 release is inhibited by IL-10. IL-12 deficiency inhibits phagocytosis in neutrophils. In experimental models of inflammatory stress, IL-12 neutralization conferred a mortality benefit in mice during endotoxemia. However, in a cecal ligation and puncture model of intraperitoneal sepsis, IL-12 blockade was associated with increased mortality. Furthermore, later studies of intraperitoneal sepsis observed no difference in mortality with IL-12 administration; however, IL-12 knockout mice exhibited increased bacterial counts and inflammatory cytokine release, which suggests that IL-12 may contribute to an antibacterial response. IL-12 administration in chimpanzees is capable of stimulating the release of proinflammatory mediators such as IFN-γ and also anti-inflammatory mediators, including IL-10, soluble TNFR, and IL-1 receptor antagonists. In addition, IL-12 enhances coagulation as well as fibrinolysis.

Interleukin-18

IL-18 is a member of the IL-1 superfamily of cytokines. First noted as an IFN-γ–inducing factor produced by LPS-stimulated macrophages, IL-18 expression is found both in immune cells and nonimmune cells at low to intermediate levels. However, activated macrophages and Kupffer cells produce large amounts of mature IL-18. Similar to IL-1β, IL-18 is synthesized and stored as an inactive precursor form (pro-IL-18), and activation requires processing by caspase 1 in response to the appropriate signaling. It then exits the cell through a nontraditional secretory pathway. The IL-18 receptor (IL-18R) is composed of two subunits, IL-18Rα and IL-18Rβ, and is a member of the IL-1R superfamily, which is structurally similar in its cytoplasmic domains to the TLR.

One unique biologic property of IL-18 is the potential, in conjunction with IL-12, to promote the Th1 response to bacterial infection. At the same time, exogenous IL-18 can also enhance the Th2 response and Ig-mediated humoral immunity, as well as augment neutrophil function. Recent studies suggest that IL-18 therapy may hold promise as effective therapy in promoting immune recovery after severe surgical stress.⁸⁰

Interferons

Interferons were first recognized as soluble mediators that inhibited viral replication through the activation of specific antiviral genes in infected cells. Interferons are categorized into three types based on receptor specificity and sequence homology. The two major types, type I and type II, are discussed here.

Type I interferons, of which there are 20, include IFN-α, IFN-β, and IFN-ω, which are structurally related and bind to a common receptor, IFN-α receptor. They are likely produced by most cell types and tissues in response to appropriate pathogens or DAMP signaling. Type I interferons are expressed in response to many stimuli, including viral antigens, double-stranded DNA, bacteria, tumor cells, and LPS. Type I interferons influence adaptive immune responses by inducing the maturation of dendritic cells and by stimulating class I major histocompatibility complex (MHC) expression. IFN-α and IFN-β also enhance immune responses by increasing the cytotoxicity of NK cells both in culture and in vivo. Further, they have been implicated in the enhancement of chemokine synthesis, particularly those that recruit myeloid cells and lymphoid cells. Thus, IFN/STAT signaling has important effects on the mobilization, tissue recruitment, and activation of immune cells that compose the inflammatory infiltrate. In contrast, IFN-I appears to inhibit inflammasome activity, possibly via IL-10.⁸¹

Many of the physiologic effects observed with increased levels of IL-12 and IL-18 are mediated through IFN-γ. IFN-γ is a type II interferon that is secreted by various T cells, NK cells, and antigen-presenting cells in response to bacterial antigens, IL-2, IL-12, and IL-18. IFN-γ stimulates the release of IL-12 and IL-18. Negative regulators of IFN-γ include IL-4, IL-10, and glucocorticoids. IFN-γ binding with a cognate receptor activates the JAK-STAT pathway, leading to subsequent induction of biologic responses. Macrophages stimulated by IFN-γ demonstrate enhanced phagocytosis and microbial killing and increased release of oxygen radicals, partly through an NADP-dependent phagocyte oxidase. IFN-γ mediates macrophage stimulation and thus may contribute to acute lung injury after major surgery or trauma. Diminished IFN-γ level, as seen in knockout mice, is associated with increased susceptibility to both viral and bacterial pathogens. In addition, IFN-γ promotes differentiation of T cells to the helper T-cell subtype 1 and also enhances B-cell isotype switching to immunoglobulin G.⁸²

Receptors of all IFN subtypes belong to the class II of cytokine receptors and use the JAK-STAT signaling pathway for nuclear signaling, although different STAT activation (e.g., STAT1 and STAT2) is favored by individual receptors.

Granulocyte-Macrophage Colony-Stimulating Factor/Interleukin-3/Interleukin-5

GM-CSF, IL-3, and IL-5 compose a small family of cytokines that regulate the growth and activation of immune cells. They are largely the products of activated T cells, which when released stimulate the behavior of myeloid cells by inducing cytokine expression and antigen presentation. In this way, GM-CSF, IL-3, and IL-5 are able to link the innate and acquired immune responses. With the exception of eosinophils, GM-CSF, IL-3, and IL-5 are not essential for constitutive hematopoietic cell function. Rather, they play an important role when the host is stressed, by serving to increase the numbers of activated and sensitized cells required to bolster host defense.⁸³ Currently, GM-CSF is in clinical trials for administration to children with an Injury Severity Score >10 following blunt or penetrating trauma. The goal of the study is to provide evidence of the effectiveness of GM-CSF as an agent that can ameliorate posttraumatic immune suppression.

Receptors for the GM-CSF/IL-3/IL-5 family of cytokines are expressed at very low levels on hematopoietic cells. Similar to the other cytokine receptors discussed, they are heterodimers composed of a cytokine-specific α subunit and a common β subunit (βc), which is shared by all three receptors and is required for high-affinity signal transduction. The binding of cytokine to its receptor activates JAK2-STAT–, MAPK-, and PI3K-mediated signaling events to regulate a variety of important cell behaviors including effector function in mature cells.

Eicosanoids

Omega-6 Polyunsaturated Fat Metabolites: Arachidonic Acid

Eicosanoids are derived primarily by oxidation of the membrane phospholipid, arachidonic acid [all-cis-5,8,11,14-eicosatetraenoic acid; 20:4(ω-6) eicosatetraenoic acid], which is relatively abundant in the membrane lipids of inflammatory cells. They are composed of three families, which include prostaglandins, thromboxanes, and leukotrienes. Arachidonic acid is not stored free in the cell but in an esterified form in phospholipids and neutral lipids. When a cell senses the proper stimulus, arachidonic acid is released from phospholipids or diacylglycerols by the enzymatic activation of phospholipase A2 (Fig. 2-6A). Prostanoids, which include all of the prostaglandins and the thromboxanes, result from the sequential action of the cyclooxygenase (COX) enzyme and terminal synthetases on arachidonic acid. In contrast, arachidonic acid may be oxidized along the lipoxygenase pathway via the central enzyme 5-lipoxygenase, to produce several classes of leukotrienes and lipoxins. In general, the effects of eicosanoids are mediated via specific receptors, which are members of a superfamily of G-protein–coupled receptors.

Eicosanoids are not stored within cells but are instead generated rapidly in response to many stimuli, including hypoxic injury, direct tissue injury, endotoxin (lipopolysaccharide), NE, vasopressin, angiotensin II, bradykinin, serotonin, ACh, cytokines, and histamine. Eicosanoid pathway activation also leads to the formation of the anti-inflammatory compound lipoxin, which inhibits chemotaxis and NF-κB activation. Glucocorticoids, nonsteroidal anti-inflammatory drugs, and leukotriene inhibitors block the end products of eicosanoid pathways. Eicosanoids have a broad range of physiologic roles, including neurotransmission and vasomotor regulation. They are also involved in immune cell regulation (Table 2-6) by modulating the intensity and duration of inflammatory responses. The production of eicosanoids is cell- and stimulus-specific. Therefore, the signaling events that are initiated will depend on the concentrations and types of eicosanoids generated, as well as the unique complement of receptors expressed by their target cells. For example, prostaglandin E₂ (PGE₂) suppresses the effector function of macrophages (i.e., phagocytosis and intracellular pathogen killing) via a mechanism that is dependent on increased cAMP levels. PGE₂ also modulates chemokine production and enhances local accumulation of regulatory T cells and myeloid-derived suppressor cells. Prostacyclin (PGI₂) has an inhibitory effect on Th1- and Th2-mediated immune responses, while enhancing Th17 differentiation and cytokine production. Leukotrienes are potent mediators of capillary leakage as well as leukocyte adherence, neutrophil activation, bronchoconstriction, and vasoconstriction. Leukotriene B₄ is synthesized from arachidonic acid in response to acute Ca²⁺ signaling induced by inflammatory mediators.⁸⁴ High-affinity leukotriene receptors (BLT1) are expressed primarily in leukocytes, including granulocytes, eosinophils, macrophages, and differentiated T cells, whereas the low-affinity receptor is expressed in many cell types. Activation of BLT1 results in inhibition of adenylate cyclase and reduced production of cAMP. Not surprisingly, a role for leukotriene B₄ signaling in abrogating the effects of prostaglandins on macrophage effector function has recently been shown.⁸⁵

Recent evidence supports a role for lipid droplets (LDs) as an important intracellular source of arachidonic acid. LDs are neutral lipid storage organelles ubiquitous to eukaryotic cells that are a rich source of esterified arachidonic acid especially in leukocytes. Accumulation of LDs in response to TLR signaling has been reported with an associated increase in the generation of eicosanoid metabolites.⁸⁶

While experimental models of sepsis have shown a benefit to inhibiting eicosanoid production, human sepsis trials have failed to show a mortality benefit.⁸⁷ Eicosanoids also have several recognized metabolic effects. COX pathway products inhibit pancreatic β-cell release of insulin, whereas lipoxygenase pathway products stimulate β-cell activity. Prostaglandins such as PGE₂ can inhibit gluconeogenesis through the binding of hepatic receptors and also can inhibit hormone-stimulated lipolysis.⁸⁸

Omega-3 Polyunsaturated Fat Metabolites: All-cis-5,8,11,14,17-Eicosapentaenoic Acid [20:5(ω-3) Eicosapentaenoic Acid]

As noted earlier, polyunsaturated fatty acid (PUFA) metabolites of endogenous arachidonic acid function as inflammatory mediators and have significant roles in the inflammatory response. The major direct dietary source of arachidonic acid is from meat. However, a much larger quantity of ω-6 PUFAs is ingested as linoleic acid, which is found in many vegetable oils, including corn, sunflower, and soybean oils, and in products made from such oils, such as margarines. Linolenic acid is not synthesized in mammals; however, it can be converted to arachidonic acid through lengthening of the carbon chain and the addition of double bonds. The second major family of PUFAs is the ω-3 fatty acid. They can also be derived from shorter chain ω-3 fatty acids of plant origin such as α-linolenic acid, which can be converted after ingestion to eicosapentaenoic acid (EPA) and to docosahexaenoic acid (DHA). ω-3 fatty acids are found in cold water fish, especially tuna, salmon, mackerel, herring, and sardine, which can provide between 1.5 and 3.5 g of these long-chain ω-3 PUFAs per serving. EPA and DHA are also substrates for the COX and lipoxygenase (LOX) enzymes that produce eicosanoids, but the mediators produced have a different structure from the arachidonic acid–derived mediators, and this influences their potency (Fig. 2-6B). In addition, ω-3 fatty acids are reported to have specific anti-inflammatory effects, including inhibition of NF-κB activity, TNF release from hepatic Kupffer cells, and leukocyte adhesion and migration. These are achieved via two purported mechanisms: (a) by decreasing the production of arachidonic acid (ω-6)–derived proinflammatory mediators (by competition for the same enzymes) and (b) by generation of proresolving bioactive lipid mediators. In fact, key derivatives of ω-3 PUFAs, termed resolvins, have been identified and synthesized. Resolvins are now categorized as either E-series (from EPA) or D-series (from DHA). In a variety of model systems, resolvins have been shown to attenuate the inflammatory phenotypes of a number of immune cells.⁸⁹

The ratio of dietary ω-6 to ω-3 PUFAs is reflected in the membrane composition of various cells, including cells of the immune system, which has potential implications for the inflammatory response. For example, a diet that is rich in ω-6 PUFAs will result in cells whose membranes are “ω-6 PUFA rich.” When ω-6 PUFAs are the main plasma membrane lipid available for phospholipase activity, more proinflammatory PUFAs (i.e., two-series prostaglandins) are generated. Many lipid preparations are soy-based and thus primarily composed of ω-6 fatty acids. These are thought to be “inflammation enhancing.” Nutritional supplementation with ω-3 fatty acid has the potential to dampen inflammation by shifting the cell membrane composition in favor of ω-3 PUFAs.

In experimental models of sepsis, ω-3 fatty acids inhibit inflammation, ameliorate weight loss, increase small-bowel perfusion, and may increase gut barrier protection. In human studies, ω-3 supplementation is associated with decreased production of TNF, IL-1β, and IL-6 by endotoxin-stimulated monocytes. In a study of surgical patients, preoperative supplementation with ω-3 fatty acid was associated with reduced need for mechanical ventilation, decreased hospital length of stay, and decreased mortality with a good safety profile.⁹⁰

Plasma Contact System

Complement

Following traumatic injury, there is almost immediate activation of the complement system, which is a major effector mechanism of the innate immune system. The complement system was thought to act initially as the required “first line of defense” for the host against pathogens, by binding and clearing them from the circulation. Recent data indicate that complement also participates in the elimination of immune complexes as well as damaged and dead cells. In addition, complement is recognized as contributing to mobilization of hematopoietic stem/progenitor cells and lipid metabolism.⁹¹ Although complement activation is typically depicted as a linear process in which parallel pathways are activated, it actually functions more like a central node that is tightly networked with other systems. Then, depending on the activating signal, several initiation and regulatory events act in concert to heighten immune surveillance.

Complement activation proceeds via three different pathways. Initiation of these pathways occurs by the binding and activation of the recognition unit of each pathway to its designated ligand. The classical pathway, which is often referred to as “antibody dependent,” is initiated by direct binding of C1q to its common ligands, which include immunoglobulin (Ig) M/IgG aggregates. Alternately C1q can activate complement signaling by binding to soluble pattern recognition molecules such as pentraxins (e.g., CRP). In a series of subsequent activation and amplification steps, the pathway ultimately leads to the assembly of the C3 convertase, which cleaves C3 into C3a and C3b. As C3b then complexes with C3 convertase, the C5 convertase is activated, cleaving C5 into C5a and C5b. C3a and C5a are potent anaphylatoxins. C3b acts as an opsonin, whereas C5b initiates the formation of the membrane attack complex. When C5b associates with C6 and C7, the complex becomes inserted into cell membrane and interacts with C8, inducing the binding of several units of C9 to form a lytic pore.

The lectin pathway of complement activation is initiated by mannose-binding lectins or ficolins, which act as the soluble PRM by binding specific carbohydrate structures that are often present on pathogens. The alternative pathway also includes a PRM-based initiation mechanism that resembles those found in the lectin pathway but involves properdin. The latter recognizes several PAMPs and DAMPs on foreign and apoptotic cells. Once bound, it initiates and propagates the complement response by attracting fluid-phase C3b to recognized surfaces and by stabilizing C3 convertase complexes. Despite its name, the alternative pathway may account for up to 80% to 90% of total complement activation.⁹²

The major source of the circulating complement components is the liver. Complement proteins can also be produced locally where they have been implicated in the regulation of adaptive immune processes. Complement protein synthesis has been demonstrated in immune cells, including T cells, which when surface bound, interact with C3 and C4 receptors. Also, complement synergistically enhances TLR-induced production of proinflammatory cytokines through convergence of their signaling pathways.

Kallikrein-Kinin System

The kallikrein-kinin system is a group of proteins that contribute to inflammation, blood pressure control, coagulation, and pain responses. Prekallikrein is synthesized in the liver and circulates in the plasma bound to high molecular weight kininogen (HK). A variety of stimuli lead to the binding of prekallikrein-HK complex to Hageman factor, (factor XII) followed by its activation, to produce the serine protease kallikrein, which plays a role in the coagulation cascade. HK, produced by the liver, is cleaved by kallikrein to form bradykinin (BK). The kinins (e.g., BK) mediate several physiologic processes, including vasodilation, increased capillary permeability, tissue edema, pain pathway activation, inhibition of gluconeogenesis, and increased bronchoconstriction. They also increase renal vasodilation and consequently reduce renal perfusion pressure. Kinin receptors are members of the rhodopsin family of G-protein–coupled receptors and are located on vascular endothelium and smooth muscle cells. Kinin receptors are rapidly upregulated following TLR4 signaling and in response to cytokines and appear to have important effects on both immune cell behavior and on immune mediators.⁹³ For example, B1 activation results in increased neutrophil chemotaxis, while increased B2 receptor expression causes activation of arachidonic-prostaglandin pathways. Bradykinin and kallikrein levels are increased during gram-negative bacteremia, hypotension, hemorrhage, endotoxemia, and tissue injury. The degree of elevation in the levels of these mediators has been associated with the magnitude of injury and mortality. Clinical trials using bradykinin antagonists have shown some benefit in patients with gram-negative sepsis.⁹⁴

Serotonin

Serotonin is a monoamine neurotransmitter (5-hydroxytryptamine [5-HT]) derived from tryptophan. Serotonin is synthesized by neurons in the CNS as well as by intestinal enterochromaffin cells, which are the major source of plasma 5-HT. Once in the plasma, 5-HT is taken up rapidly into platelets via the serotonin transporter (SERT) where it is either stored in the dense granules in millimolar concentrations or targeted for degradation. It is interesting that the surface expression of SERT on platelets is sensitive to plasma 5-HT levels, which in turn modulates platelet 5-HT content. Receptors for serotonin are widely distributed in the periphery and are found in the gastrointestinal tract, cardiovascular system, and some immune cells.⁹⁵ Serotonin is a potent vasoconstrictor and also modulates cardiac inotropy and chronotropy through nonadrenergic cAMP pathways. Serotonin is released at sites of injury, primarily by platelets. Recent work has demonstrated an important role for platelet 5-HT in the local inflammatory response to injury. Using mice that lack the nonneuronal isoform of tryptophan hydroxylase (Tph1), the rate-limiting step for 5-HT synthesis in the periphery, investigators demonstrated fewer neutrophils rolling on mesenteric venules.⁹⁶ Tph1^–/– mice, in response to an inflammatory stimulus, also showed decreased neutrophil extravasation. Finally, survival of lipopolysaccharide-induced endotoxic shock was reduced in Tph1^–/– mice. Together, these data indicate an important role for nonneuronal 5-HT in neutrophil recruitment to sites inflammation and injury.

Histamine

Histamine is a short-acting endogenous amine that is widely distributed throughout the body. It is synthesized by histidine decarboxylase (HDC), which decarboxylates the amino acid histidine. Histamine is either rapidly released or stored in neurons, skin, gastric mucosa, mast cells, basophils, and platelets, and plasma levels are increased with hemorrhagic shock, trauma, thermal injury, and sepsis.⁹⁷ Not surprisingly, circulating cytokines can increase immune cell expression of HDC to further contribute to histamine synthesis. There are four histamine receptor (HR) subtypes with varying physiologic roles, but they are all members of the rhodopsin family of G-protein–coupled receptors. H1R binding mediates vasodilation, bronchoconstriction, intestinal motility, and myocardial contractility. H1R knockout mice demonstrate significant immunologic defects, including impaired B- and T-cell responses. H2R binding is best described for its stimulation of gastric parietal cell acid secretion. However, H2R can also modulate a range of immune system activities, such as mast cell degranulation, antibody synthesis, Th1 cytokine production, and T-cell proliferation. H3R was initially classified as a presynaptic autoreceptor in the peripheral nervous system and CNS. However, data using H3R knockout mice demonstrate that it also participates in inflammation in the CNS. H3R knockout mice display increased severity of neuroinflammatory diseases, which correlates with dysregulation of blood-brain barrier permeability and increased expression of macrophage inflammatory protein 2, IFN-inducible protein 10, and CXCR3 by peripheral T cells. H4R is expressed primarily in bone marrow but has also been detected in leukocytes, including neutrophils, eosinophils, mast cells, dendritic cells, T cells, and basophils. H4R is emerging as an important modulator of chemoattraction and cytokine production in these cells. Thus, it is clear that cells of both the innate and adaptive immune response can be regulated by histamine, which is upregulated following injury.⁹⁸

Cytokine Receptor Families and Their Signaling Pathways

Cytokines act on their target cells by binding to specific membrane receptors. These receptor families have been organized by structural motifs and include: type I cytokine receptors, type II cytokine receptors, chemokine receptors, TNF receptors (TNFRs), and transforming growth factor receptors (TGFRs). In addition, there are cytokine receptors that belong to the immunoglobulin receptor superfamilies. Several of these receptors have characteristic signaling pathways that are associated with them. These will be reviewed in the following sections.

JAK-STAT Signaling

A major subgroup of cytokines, comprising roughly 60 factors, bind to receptors termed type I/II cytokine receptors. Cytokines that bind these receptors include type I IFNs, IFN-γ, many ILs (e.g., IL-6, IL-10, IL-12, and IL-13), and hematopoietic growth factors. These cytokines play essential roles in the initiation, maintenance, and modulation of innate and adaptive immunity for host defense. All type I/II cytokine receptors selectively associate with the Janus kinases (JAKs), which represent a family of tyrosine kinases that mediate the signal transduction for these receptors. JAKs are constitutively bound to the cytokine receptors, and on ligand binding and receptor dimerization, activated JAKs phosphorylate the receptor to recruit signal transducer and activator of transcription (STAT) molecules (Fig. 2-7). Activated STAT proteins further dimerize and translocate into the nucleus where they modulate the transcription of target genes. Rather than being a strictly linear pathway, it is likely that individual cytokines activate more than one STAT. The molecular implications for this in terms of cytokine signaling are still being unraveled. Interestingly, STAT-DNA binding can be observed within minutes of cytokine binding. STATs have also been shown to modulate gene transcription via epigenetic mechanisms. Thus, JAKs and STATs are central players in the regulation of key immune cell function, by providing a signaling platform for proinflammatory cytokines (IL-6 via JAK1 and STAT3) and anti-inflammatory cytokines (IL-10 via STAT3) and integrating signals required for helper and regulatory T-cell development and differentiation. The JAK/STAT pathway is inhibited by the action of phosphatase, the export of STATs from the nucleus, and the interaction of antagonistic proteins.⁹⁹

Suppressors of Cytokine Signaling

Suppressor of cytokine signaling (SOCS) molecules are a family of proteins that function as a negative feedback loop for type I and II cytokine receptors by terminating JAK-STAT signaling. There are currently eight family members; SOCS1-3 are typically associated with cytokine receptor signaling, whereas SOCS4-8 are associated with growth factor receptor signaling. PRRs, including both TLR and C-type lectin receptors, have also been shown to activate SOCS. Interestingly, induction of SOCS proteins is also achieved through activators of JAK-STAT signaling, creating an inhibitory feedback loop through which cytokines can effectively self-regulate by extinguishing their own signal. SOCS molecules can positively and negatively influence the activation of macrophages and dendritic cells and are crucial for T-cell development and differentiation. All SOCS proteins are able to regulate receptor signaling through the recruitment of proteasomal degradation components to their target proteins, whether the target is a specific receptor or an associated adaptor molecule. Once associated with the SOCS complex, target proteins are readily ubiquinated and targeted to the proteasome for degradation. SOCS1 and SOCS3 can also exert an inhibitory effect on JAK-STAT signaling via their N-terminal kinase inhibitory region (KIR) domain, which acts as a pseudosubstrate for JAK. The KIR domain binds with high affinity to the JAK kinase domain to inhibit its activity. SOCS3 has been shown to be a positive regulator of TLR4 responses in macrophages via inhibition of IL-6 receptor–mediated STAT3 activation.¹⁰⁰ A deficiency of SOCS activity may render a cell hypersensitive to certain stimuli, such as inflammatory cytokines and GHs. Interestingly, in a murine model, SOCS knockout resulted in a lethal phenotype in part because of unregulated interferon signaling.

Chemokine Receptors Are Members of the G-Protein–Coupled Receptor Family

All chemokine receptors are members of the G-protein–coupled seven-transmembrane family of receptors (GPCR), which is one of the largest and most diverse of the membrane protein families. GPCRs function by detecting a wide spectrum of extracellular signals, including photons, ions, small organic molecules, and entire proteins. After ligand binding, GPCRs undergo conformational changes, causing the recruitment of heterotrimeric G proteins to the cytoplasmic surface (Fig. 2-8). Heterotrimeric G proteins are composed of three subunits, Gα, Gβ, and Gγ, each of which has numerous members, adding to the complexity of the signaling. When signaling however, G proteins perform functionally as dimers because the signal is communicated either by the Gα subunit or the Gβγ complex. The GPCR family includes the receptors for catecholamines, bradykinins, and leukotrienes, in addition to a variety of other ligands important to the inflammatory response.¹⁰¹ In general, GPCRs can be classified according to their pharmacologic properties into four main families: class A rhodopsin-like, class B secretin-like, class C metabotropic glutamate/pheromone, and class D frizzled receptors. As noted earlier, GPCR activation by ligand binding results in an extracellular domain shift, which is then transmitted to cytoplasmic portion of the receptor to facilitate coupling to its principle effector molecules, the heterotrimeric G proteins. Although there are more than 20 known Gα subunits, they have been divided into four families based on sequence similarity, which has served to define both receptor and effector coupling. These include Gα_s and Gα_i, which signal through the activation (Gα_s) or inhibition (Gα_i) of adenylate cyclase to increase or decrease cAMP levels, respectively. Increased intracellular cAMP can activate gene transcription through the activity of intracellular signal transducers such as protein kinase A. The Ga subunits also include the Gq pathway, which stimulates phospholipase C-β to produce the intracellular messengers inositol trisphosphate and diacylglycerol. Inositol triphosphate triggers the release of calcium from intracellular stores, whereas diacylglycerol recruits protein kinase C to the plasma membrane for activation. Finally, Gα_12/13 appears to act through Rho- and Ras-mediated signaling.

Tumor Necrosis Factor Superfamily

The signaling pathway for TNFR1 (55 kDa) and TNFR2 (75 kDa) occurs by the recruitment of several adapter proteins to the intracellular receptor complex. Optimal signaling activity requires receptor trimerization. TNFR1 initially recruits TNFR-associated death domain (TRADD) and induces apoptosis through the actions of proteolytic enzymes known as caspases, a pathway shared by another receptor known as CD95 (Fas). CD95 and TNFR1 possess similar intracellular sequences known as death domains (DDs), and both recruit the same adapter proteins known as Fas-associated death domains (FADDs) before activating caspase 8. TNFR1 also induces apoptosis by activating caspase 2 through the recruitment of receptor-interacting protein (RIP). RIP also has a functional component that can initiate NF-κB and c-Jun activation, both favoring cell survival and proinflammatory functions. TNFR2 lacks a DD component but recruits adapter proteins known as TNFR-associated factors 1 and 2 (TRAF1, TRAF2) that interact with RIP to mediate NF-κB and c-Jun activation. TRAF2 also recruits additional proteins that are antiapoptotic, known as inhibitor of apoptosis proteins (IAPs).

Transforming Growth Factor-β Family of Receptors

Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine expressed by immune cells that has potent immunoregulatory activities. Specifically, recent data indicate that TGF-β is essential for T-cell homeostasis, as mice deficient in TGF-β1 develop a multiorgan autoimmune inflammatory disease and die a few weeks after birth, an effect that is dependent on the presence of mature T cells. The receptors for TGF-β ligands are the TGF-β superfamily of receptors, which are type I transmembrane proteins that contain intrinsic serine/threonine kinase activity. These receptors comprise two subfamilies, the type I and the type II receptors, which are distinguished by the presence of a glycine/serine-rich membrane domain found in the type I receptors. Each TGF-β ligand binds a characteristic combination of type I and type II receptors, both of which are required for signaling. Whether the type I or the type II receptor binds first is ligand-dependent, and the second type I or type II receptor is then recruited to form a heteromeric signaling complex. When TGF-β binds to the TGF-β receptor, heterodimerization activates the receptor, which then directly recruits and activates a receptor-associated Smad (Smad2 or Smad3) through phosphorylation. An additional “common” Smad is then recruited. The activated Smad complex translocates into the nucleus and, with other nuclear cofactors, regulates the transcription of target genes. TGF-β can also induce the rapid activation of the Ras-extracellular signal-regulated kinase (ERK) signaling pathway in addition to other MAPK pathways (JNK, p38MAPK). How does TGF-β inhibit immune responses? One of the most important effects is the suppression of IL-2 production by T cells. It also inhibits T-cell proliferation.¹⁰² More recently, it was noted that TGF-β can regulate the maturation of differentiated dendritic cells and dendritic cell–mediated T-cell responses. Importantly, TGF-β can induce “alternative activation” macrophages, designated M2 macrophages, which express a wide array of anti-inflammatory molecules, including IL-10 and arginase-1.

Transcriptional Events Following Blunt Trauma

Recent data have examined the transcriptional response in circulating leukocytes in a large series of patients who suffered severe blunt trauma. This work identified an overwhelming shift in the leukocyte transcriptome, with more than 80% of the cellular functions and pathways demonstrating some alteration in gene expression. In particular, changes in gene expression for pathways involved in the systemic inflammatory, innate immune, compensatory anti-inflammatory, and adaptive immune responses were simultaneous and marked. Moreover, they occurred rapidly (within 4 to 12 hours) and were prolonged for days and weeks. When different injuries (i.e., blunt trauma, burn injury, human model of endotoxemia) were compared, the patterns of gene expression were surprisingly similar, suggesting that the stress response to both injury and inflammation is highly conserved and may follow a universal pathway that includes common denominators. Finally, delayed clinical recovery and organ injury were not associated with a distinct pattern of transcriptional response elements.² These data describe a new paradigm based on the observation of a rapid and coordinated transcriptional response to severe traumatic injury that involves both the innate and adaptive immune systems. Further, the data support the idea that individuals who are destined to die from their injuries are characterized primarily by the degree and duration of their dysregulated inflammatory response rather than a “unique signature” indicative of a “second hit.”

Transcriptional Regulation of Gene Expression

Many genes are regulated at the point of DNA transcription and thus influence whether messenger RNA (mRNA) and its subsequent product are expressed (Fig. 2-9). Gene expression relies on the coordinated action of transcription factors and coactivators (i.e., regulatory proteins), which are complexes that bind to highly specific DNA sequences upstream of the target gene known as the promoter region. Enhancer sequences of DNA mediate gene expression, whereas repressor sequences are noncoding regions that bind proteins to inhibit gene expression. For example, NF-κB is one of the best-described transcription factors, which has a central role in regulating the gene products expressed after inflammatory stimuli (Fig. 2-10). NF-κB is composed of two smaller polypeptides, p50 and p65. NF-κB resides in the cytosol in the resting state primarily through the inhibitory binding of inhibitor of κB (I-κB). In response to an inflammatory stimulus such as TNF, IL-1, or endotoxin, a sequence of intracellular mediator phosphorylation reactions leads to the degradation of I-κB and subsequent release of NF-κB. On release, NF-κB travels to the nucleus and promotes gene expression. NF-κB also stimulates the gene expression forI-κB, which results in negative feedback regulation. In clinical appendicitis, for example, increased NF-κB activity was associated with initial disease severity, and levels returned to baseline within 18 hours after appendectomy in concert with resolution of the inflammatory response.³⁰

Epigenetic Regulation of Transcription

The DNA access of protein machineries involved in transcription processes is tightly regulated by histones, which are a family of basic proteins that associate with DNA in the nucleus. Histone proteins help to condense the DNA into tightly packed nucleosomes that limit transcription. Emerging evidence indicates that transcriptional activation of many proinflammatory genes requires nucleosome remodeling that is modulated by the posttranslational modification of histone proteins through the recruitment of histone-modifying enzymes.¹⁰³ There are at least seven identified chromatin modifications including acetylation, methylation, phosphorylation, ubiquitinylation, sumoylation, ADP ribosylation, deimination, and proline isomerization. Recently, the development of chromatin immunoprecipitation (ChIP) coupled to massively parallel DNA sequencing technology (ChIP-Seq) has enabled the mapping of histone modifications in living cells in response to TLR signaling. In this way, it has allowed the identification of the large number of posttranslational histone modifications that are “written” and “erased” by histone-modifying enzymes. The role of histone modifications in the regulation of gene expression is referred to as “epigenetic” control.

Addition of an acetyl group to lysine residues on histones is an epigenetic mark associated with gene activation. These acetyl groups are reversibly maintained by histone acetyltransferases (HATs) and histone deacetylases. Ultimately, histone acetylation is monitored by bromodomain-containing proteins such as the bromodomain and extraterminal domain (BET) family of proteins, which can regulate a number of important epigenetically controlled processes.

Upon TLR4 activation, HATs are recruited to proinflammatory gene promoters where acetylation of specific histone residues serves as an organizing node for a complex of proteins that ultimately phosphorylate the large subunit of RNA polymerase II, promoting the elongation of inflammatory gene transcripts.¹⁰⁴ Recently, investigators used a novel pharmacologic approach that targeted inflammatory gene expression by interfering with the recognition of acetylated histones by BET proteins. A synthetic compound (I-BET) that “mimicked” acetylated histones functioned as a BET antagonist.¹⁰⁵ In this way, pretreatment decreased overall histone acetylation to reduce the expression of select inflammatory genes in LPS-activated macrophages. Additionally, I-BET conferred protection against bacteria-induced sepsis. Recent studies have also demonstrated a role for histone methyltransferases in proinflammatory gene programs.

Translation Regulation of Inflammatory Gene Expression

Once mRNA transcripts are generated, they can also be regulated by a variety of mechanisms, including (a) splicing, which can cleave mRNA and remove noncoding regions; (b) capping, which modifies the 5′ ends of the mRNA sequence to inhibit breakdown by exonucleases; and (c) the addition of a polyadenylated tail, which adds a noncoding sequence to the mRNA, to regulate the half-life of the transcript. Recent data have identified microRNAs (miRNAs) as important translational regulators of gene expression via their binding to partially complementary sequences in the 3′-untranslated region (3′-UTR) of target mRNA transcripts.¹⁰⁶ Binding of miRNA to the mRNA usually results in gene silencing. MicroRNAs are endogenous, single-stranded RNAs of approximately 22 nucleotides in length that are highly conserved in eukaryotes. miRNAs are encoded either singly or can be transcribed in “polycistronic” clusters and produced by an elaborate expression and processing mechanism. After a primary miRNA transcript is generated by RNA polymerase II or III, it is processed in the nucleus to produce a short hairpin precursor miRNA transcript. The precursor is then transported into the cytoplasm where the final mature miRNA is generated by a protein termed Dicer. The mature double-stranded miRNA is then incorporated into the RNA-induced silencing complex (RISC) in the cytoplasm. Once programmed with a small RNA, RISC can silence targeted genes by one of several distinct mechanisms, working at (a) the level of protein synthesis through translation inhibition, (b) the transcript level through mRNA degradation, or (c) the level of the genome itself through the formation of heterochromatin or by DNA elimination. Recent data indicate that miRNAs are involved in TLR signaling in the innate immune system by targeting multiple molecules in the TLR signaling pathways.¹⁰⁷ For example, evidence has shown that miR-146a can inhibit the expression of IRAK1 and TRAF6, impair NF-κB activity, and suppress the expression of NF-κB target genes such as IL-6, IL-8, IL-1β, and TNF-α.

Platelets

Platelets are small (2 μm), circulating fragments of a larger cell precursor, the megakaryocyte, that is located chiefly within the bone marrow. Although platelets lack a nucleus, they contain both mRNA and a large number of cytoplasmic and surface proteins that equip them for diverse functionality. While their role in hemostasis is well described, more recent work suggests that platelets play a role in both local and systemic inflammatory responses, particularly following ischemia reperfusion. Platelets express functional scavenger and TLRs that are important detectors of both pathogens and “damage”-associated molecules.¹⁰⁸ At the site of tissue injury, complex interactions between platelets, endothelial cells, and circulating leukocytes facilitate cellular activation by the numerous local alarmins and immune mediators. For example, platelet-specific TLR4 activation can cause thrombocytes to bind to and activate neutrophils to extrude their DNA to form neutrophil extracellular traps (NETs), an action that facilitates the capacity of the innate immune system to trap bacteria, but also leads to local endothelial cell damage.¹⁰⁹

Once activated, platelets adopt an initial proinflammatory phenotype by expressing and releasing a variety of adhesion molecules, cytokines, and other immune modulators, including HMGB1, IL-1β, and CD40 ligand (CD40L; CD154). However, activated platelets also express large amounts of the immunosuppressive factor TGF-β, which has been implicated in Treg cell homeostasis. Recently, in a large animal model of hemorrhage, TGF-β levels were shown to be significantly increased 2 hours after injury, suggesting a possible mechanism for injury-related immune dysfunction.¹¹⁰ And although soluble CD154 was not increased following hemorrhage and traumatic brain injury in that study, in a murine model of mesenteric ischemia-reperfusion injury platelet expression of CD40 and CD154 was linked to remote organ damage.

Lymphocytes and T-Cell Immunity

The expression of genes associated with the adaptive immune response is rapidly altered following severe blunt trauma.² In fact, significant injury is associated with adaptive immune suppression that is characterized by altered cell-mediated immunity, specifically the balance between the major populations of Th cells. In fact, Th lymphocytes are functionally divided into subsets, which principally include Th1 and Th2 cells, as well as Th17 and inducible Treg cells. Derived from precursor CD4⁺ Th cells, each of these groups produces specific effector cytokines that are under unique transcriptional control. CD4 T cells play central roles in the function of the immune system through their effects on B-cell antibody production and their enhancement of specific Treg cell functions and macrophage activation. The specific functions of these cells include the recognition and killing of intracellular pathogens (cellular immunity; Th1 cells), regulation of antibody production (humoral immunity; Th2 cells), and maintenance of mucosal immunity and barrier integrity (Th17 cells). These activities have been characterized as proinflammatory (Th1) and anti-inflammatory (Th2), respectively, as determined by their distinct cytokine signatures (Fig. 2-11). Activation of Th1 cytokine-producing cells following injury has been linked to signaling events triggered by endogenous ligands, often composed of intracellular proteins (e.g., mitochondrial and nuclear-binding proteins) or ECM fragments released with cellular damage. As discussed earlier, these DAMPs are recognized by members of the TLR superfamily, including TLR2, TLR4, and TLR9, and can activate innate immune pathways.

A healthy immune response depends on a balanced Th1/Th2 response. Following injury, however, there is a reduction in Th1 cell differentiation and cytokine production in favor of an increased population of Th2 lymphocytes and their signaling products. As a consequence, both macrophage activation and proinflammatory cytokine synthesis are inhibited. This imbalance, which may be associated with decreased IL-12 production by activated monocytes/macrophages, has been associated with increased risk of infectious complications following surgery and trauma. What are the systemic mechanisms responsible for this shift? Several events have been implicated, including the direct effect of glucocorticoids on monocyte IL-12 production and T-cell IL-12 receptor expression. In addition, sympathoadrenal catecholamine production has also been demonstrated to reduce IL-12 production and proinflammatory cytokine synthesis.¹¹¹ Finally, more recent work has implicated circulating immature myeloid cells, termed myeloid-derived suppressor cells, that have immune suppressive activity particularly through their increased expression of arginase.¹¹² These cells have the potential to deplete the microenvironment of arginine, leading to further T-cell dysfunction.

Recent evidence suggests that Th17 cells and their effector cytokines, IL-17, IL-21, and IL-22, regulate mucosal immunity and barrier function. While their specific role in the inflammatory response following trauma is not well understood, both murine and human studies indicate that normal Th17 effector functions are disordered following burn injury, due to the inhibition of normal Th17 cell development by IL-10.¹¹³ These changes may contribute to remote organ damage and further susceptibility to infection in this setting.

Dendritic Cells

Recent studies have focused on the cellular components of the immune system in the context of polytrauma. While the activation of granulocytes and monocyte/macrophages following trauma has been well described, more recent work has also demonstrated that dendritic cells (DCs) are also activated in response to damage signals, to stimulate both the innate and the adaptive immune responses. For example, primary “danger signals” that are recognized and activated by DCs include debris from damaged or dying cells (e.g., HMGB1, nucleic acids including single nucleotides, and degradation products of the ECM). DCs are specialized antigen-presenting cells (APCs) that have three major functions. They are frequently referred to as “professional APCs” since their principal function is to capture, process, and present both endogenous and exogenous antigens, which, along with their costimulatory molecules, are capable of inducing a primary immune response in resting naïve T lymphocytes. In addition, they have the capacity to further regulate the immune response, both positively and negatively, through the upregulation and release of immunomodulatory molecules such as the chemokine CCL5 and the CXC chemokine CXCL5. Finally, they have been implicated both in the induction and maintenance of immune tolerance as well as in the acquisition of immune memory.¹¹⁴ There are distinct classes and subsets of DC, which are functionally heterogeneous. Further, subsets of DC at distinct locations have been shown to express different levels damage-sensing receptors (e.g., TLR) that dictate a preferential response to DAMP at that site. While relatively small in number relative to the total leukocyte population, the diverse distribution of DC in virtually all body tissues underlines their potential for a collaborative role in the initiation of the trauma-induced sterile systemic inflammatory response.

Eosinophils

Eosinophils are immunocytes whose primary functions are antihelminthic. Eosinophils are found mostly in tissues such as the lung and gastrointestinal tract, which may suggest a role in immune surveillance. Eosinophils can be activated by IL-3, IL-5, GM-CSF, chemoattractants, and platelet-activating factor. Eosinophil activation can lead to subsequent release of toxic mediators, including ROSs, histamine, and peroxidase.¹¹⁵

Mast Cells

Mast cells are important in the primary response to injury because they are located in tissues. TNF release from mast cells has been found to be crucial for neutrophil recruitment and pathogen clearance. Mast cells are also known to play an important role in the anaphylactic response to allergens. On activation from stimuli including allergen binding, infection, and trauma, mast cells produce histamine, cytokines, eicosanoids, proteases, and chemokines, which leads to vasodilatation, capillary leakage, and immunocyte recruitment. Mast cells are thought to be important cosignaling effector cells of the immune system via the release of IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, and IL-14, as well as macrophage migration–inhibiting factor.¹¹⁶

Monocyte/Macrophages

Monocytes are mononuclear phagocytes that circulate in the bloodstream and can differentiate into macrophages, osteoclasts, and DCs on migrating into tissues. Macrophages are the main effector cells of the immune response to infection and injury, primarily through mechanisms that include phagocytosis of microbial pathogens, release of inflammatory mediators, and clearance of apoptotic cells. Moreover, these cells fulfill homeostatic roles beyond host defense by performing important functions in the remodeling of tissues, both during development and in the adult animal.

In tissues, mononuclear phagocytes are quiescent. However, they respond to external cues (e.g., PAMPs, DAMPs, activated lymphocytes) by changing their phenotype. In response to various signals, macrophages may undergo classical M1 activation (stimulated by TLR ligands and IFN-γ) or alternative M2 activation (stimulated by type II cytokines IL-4/IL-13); these states mirror the Th1-Th2 polarization of T cells. The M1 phenotype is characterized by the expression of high levels of proinflammatory cytokines, like TNF-α, IL-1, and IL-6, in addition to the synthesis of ROS and RNS. M1 macrophages promote a strong Th1 response. In contrast, M2 macrophages are considered to be involved in the promotion of wound repair and the restoration of immune homeostasis through their expression of arginase-1 and IL-10, in addition to a variety of PRRs (e.g., scavenging molecules).¹¹⁷

In humans, downregulation of monocyte TNFR expression has been demonstrated experimentally and clinically during systemic inflammation. In clinical sepsis, nonsurviving patients with severe sepsis have an immediate reduction in monocyte surface TNFR expression with failure to recover, whereas surviving patients have normal or near-normal receptor levels from the onset of clinically defined sepsis. In patients with congestive heart failure, there is also a significant decrease in the amount of monocyte surface TNFR expression compared with control patients. In experimental models, endotoxin has been shown to differentially regulate over 1000 genes in murine macrophages with approximately 25% of these corresponding to cytokines and chemokines. During sepsis, macrophages undergo phenotypic reprogramming highlighted by decreased surface human leukocyte antigen DR (a critical receptor in antigen presentation), which also may contribute to host immunocompromise during sepsis.¹¹⁸

Neutrophils

Neutrophils are among the first responders to sites of infection and injury and, as such, are potent mediators of acute inflammation. Chemotactic mediators from a site of injury induce neutrophil adherence to the vascular endothelium and promote eventual cell migration into the injured tissue. Neutrophils are circulating immunocytes with short half-lives (4 to 10 hours). However, inflammatory signals may promote the longevity of neutrophils in target tissues, which can contribute to their potential detrimental effects and bystander injury. Once primed and activated by inflammatory stimuli, including TNF, IL-1, and microbial pathogens, neutrophils are able to enlist a variety of killing mechanisms to manage invading pathogens. Phagocytosed bacteria are killed using NADPH oxygenase-dependent generation of ROS or by releasing lytic enzymes and antibacterial proteins into the phagosome. Neutrophils can also dump their granule contents into the extracellular space, and many of these proteins also have important effects on the innate and adaptive immune responses. When highly activated, neutrophils can also extrude a meshwork of chromatin fibers, composed of DNA and histones that are decorated with granule contents. Termed neutrophil extracellular traps (NETs), this is an effective mechanism whereby neutrophils can immobilize bacteria to facilitate their killing.¹¹⁹ NETs may also serve to prime T cells, making their threshold for activation lower.

Neutrophils do facilitate the recruitment of monocytes into inflamed tissues. These recruited cells are capable of phagocytosing apoptotic neutrophils to contribute to resolution of the inflammatory response.¹²⁰

Vascular Endothelium

Under physiologic conditions, vascular endothelium has overall anticoagulant properties mediated via the production and cell surface expression of heparin sulfate, dermatan sulfate, tissue factor pathway inhibitor, protein S, thrombomodulin, plasminogen, and tissue plasminogen activator. Endothelial cells also perform a critical function as barriers that regulate tissue migration of circulating cells. During sepsis, endothelial cells are differentially modulated, which results in an overall procoagulant shift via decreased production of anticoagulant factors, which may lead to microthrombosis and organ injury.

Neutrophil-Endothelium Interaction

The regulated inflammatory response to infection facilitates neutrophil and other immunocyte migration to compromised regions through the actions of increased vascular permeability, chemoattractants, and increased endothelial adhesion factors referred to as selectins that are elaborated on cell surfaces (Table 2-7). In response to inflammatory stimuli released from sentinel leukocytes in the tissues, including chemokines, thrombin, leukotrienes, histamine, and TNF, vascular endothelium are activated and their surface protein expression is altered. Within 10 to 20 minutes, prestored reservoirs of the adhesion molecule P-selectin are mobilized to the cell surface where it can mediate neutrophil recruitment (Fig. 2-12). After 2 hours, endothelial cell transcriptional processes provide additional surface expression of E-selectin. E-selectin and P-selectin bind P-selectin glycoprotein ligand-1 (PSGL-1) on the neutrophils to orchestrate the capture and rolling of these leukocytes and allow targeted immunocyte extravasation. Immobilized chemokines on the endothelial surface create a chemotactic gradient to further enhance immune cell recruitment.¹²¹ Also important are secondary leukocyte-leukocyte interactions in which PGSL-1 and L-selectin binding facilitates further leukocyte tethering. Although there are distinguishable properties among individual selectins in leukocyte rolling, effective rolling most likely involves a significant degree of functional overlap.¹²²

Chemokines

Chemokines are a family of small proteins (8 to 13 kDa) that were first identified through their chemotactic and activating effects on inflammatory cells. They are produced at high levels following nearly all forms of injury in all tissues, where they are key attractants for immune cell extravasation. There are more than 50 different chemokines and 20 chemokine receptors that have been identified. Chemokines are released from endothelial cells, mast cells, platelets, macrophages, and lymphocytes. They are soluble proteins, which when secreted, bind to glycosaminoglycans on the cell surface or in the ECM. In this way, the chemokines can form a fixed chemical gradient that promotes immune cell exit to target areas. Chemokines are distinguished (in general) from cytokines by virtue of their receptors, which are members of the G-protein–coupled receptor superfamily. Most chemokine receptors recognize more than one chemokine ligand, leading to redundancy in chemokine signaling.

The chemokines are subdivided into families based on their amino acid sequences at their N-terminus. For example, CC chemokines contain two N-terminus cysteine residues that are immediately adjacent (hence the “C-C” designation), whereas the N-terminal cysteines in CXC chemokines are separated by a single amino acid. The CXC chemokines are particularly important for neutrophil (PMN) proinflammatory function. Members of the CXC chemokine family, which include IL-8, induce neutrophil migration and secretion of cytotoxic granular contents and metabolites. Additional chemokine families include the C and CX3C chemokines.¹²¹

Nitric Oxide

Nitric oxide (NO) was initially known as endothelium-derived relaxing factor due to its effect on vascular smooth muscle. Normal vascular smooth muscle cell relaxation is maintained by a constant output of NO that is regulated in the endothelium by both flow- and receptor-mediated events. NO can also reduce microthrombosis by reducing platelet adhesion and aggregation (Fig. 2-13) and interfering with leukocyte adhesion to the endothelium. NO easily traverses cell membranes, has a short half-life of a few seconds, and is oxidized into nitrate and nitrite.

Endogenous NO formation is derived largely from the action of NO synthase (NOS), which is constitutively expressed in endothelial cells (NOS3). NOS generates NO by catalyzing the degradation of L-arginine to L-citrulline and NO, in the presence of oxygen and NADPH. There are two additional isoforms of NOS: neuronal NOS (NOS1) and inducible NOS (iNOS/NOS2). The vasodilatory effects of NO are mediated by guanylyl cyclase, an enzyme that is found in vascular smooth muscle cells and most other cells of the body. When NO is formed by endothelium, it rapidly diffuses into adjacent cells where it binds to and activates guanylyl cyclase. This enzyme catalyzes the dephosphorylation of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), which serves as a second messenger for many important cellular functions, particularly for signaling smooth muscle relaxation.

NO synthesis is increased in response to proinflammatory mediators such as TNF-α and IL-1β, as well as microbial products, due to the upregulation of iNOS expression.¹²³ In fact, studies in both animal models and humans have shown that severe systemic injury and associated hemorrhage produce an early upregulation of iNOS in the liver, lung, spleen, and vascular system. In these circumstances, NO is reported to function as an immunoregulator, which is capable of modulating cytokine production and immune cell development. In particular, recent data support a role for iNOS in the regulation of T-cell dysfunction in the setting of trauma as evidenced by suppressed proliferative and Th1 cytokine release.¹²⁴

Increased NO is also detectable in septic shock, where it is associated with low peripheral vascular resistance and hypotension. Increased production of NO in this setting correlates with changes in vascular permeability and inhibition of noradrenergic nerve transmission. While the increased NO in sepsis is largely attributed to greater iNOS activity and expression, cytokines are reported to modulate NO release by increasing arginine availability through the expression of the cationic amino acid transporter (CAT) or by increasing tetrahydrobiopterin levels, a key cofactor in NO synthesis. Additional effects associated with excess NO include protein and membrane phospholipid alterations by nitrosylation and the inhibition of mitochondrial respiration. Inhibition of NO production seemed initially to be a promising strategy in patients with severe sepsis. However, a randomized clinical trial in patients with septic shock determined that treatment with a nonselective NOS inhibitor was associated with an increase in mortality compared with placebo.¹²⁵ More recent data using an ovine model of peritonitis demonstrated that selective iNOS inhibition reduced pulmonary artery hypertension and gas exchange impairment and promoted higher visceral organ blood flow, coinciding with lower plasma cytokine concentrations.¹²⁶ These data suggest that specific targeting of iNOS in the setting of sepsis may remain a viable therapeutic option.

Prostacyclin

The immune effects of prostacyclin (PGI₂) were discussed earlier. The best described effects of PGI₂ are in the cardiovascular system, however, where it is produced by vascular endothelial cells. Prostacyclin is a potent vasodilator that also inhibits platelet aggregation. In the pulmonary system, PGI₂ reduces pulmonary blood pressure and bronchial hyperresponsiveness. In the kidneys, PGI₂ modulates renal blood flow and glomerular filtration rate. Prostacyclin acts through its receptor (a G-protein–coupled receptor of the rhodopsin family) to stimulate the enzyme adenylate cyclase, allowing the synthesis of cAMP from adenosine triphosphate (ATP). This leads to a cAMP-mediated decrease in intracellular calcium and subsequent smooth muscle relaxation.

During systemic inflammation, endothelial prostacyclin expression is impaired, and thus the endothelium favors a more procoagulant profile. Exogenous prostacyclin analogues, both intravenous and inhaled, have been used to improve oxygenation in patients with acute lung injury. Early clinical studies with prostacyclin have delivered some encouraging results, showing that infusion of prostacyclin improved cardiac index, splanchnic blood flow as measured by intestinal tonometry, and oxygen delivery in patients with sepsis. Importantly, there was no significant decrease in mean arterial pressure.¹²⁷

Endothelins

Endothelins (ETs) are potent mediators of vasoconstriction and are composed of three members: ET-1, ET-2, and ET-3. ETs are 21-amino-acid peptides derived from a 38-amino-acid precursor molecule. ET-1, synthesized primarily by endothelial cells, is the most potent endogenous vasoconstrictor and is estimated to be 10 times more potent than angiotensin II. ET release is upregulated in response to hypotension, LPS, injury, thrombin, TGF-β, IL-1, angiotensin II, vasopressin, catecholamines, and anoxia. ETs are primarily released to the abluminal side of endothelial cells, and very little is stored in cells; thus a plasma increase in ET is associated with a marked increase in production. The half-life of plasma ET is between 4 and 7 minutes, which suggests that ET release is primarily regulated at the transcriptional level. Three ET receptors, referred to as ET_A, ET_B, and ET_C, have been identified and function via the G-protein–coupled receptor mechanism. ET_B receptors are associated with increased NO and prostacyclin production, which may serve as a feedback mechanism. Atrial ET_A receptor activation has been associated with increased inotropy and chronotropy. ET-1 infusion is associated with increased pulmonary vascular resistance and pulmonary edema and may contribute to pulmonary abnormalities during sepsis. At low levels, in conjunction with NO, ETs regulate vascular tone. However, at increased concentrations, ETs can disrupt the normal blood flow and distribution and may compromise oxygen delivery to the tissue. Recent data link ET expression in pulmonary vasculature with persistent inflammation associated with the development of pulmonary hypertension.¹²⁸ ET expression is linked to posttranslational and transcriptional initiation of the unfolded protein response in the affected cells, which results in the production of inflammatory cytokines. Finally, ET-1 levels correlate with levels of brain natriuretic peptide and CRP, as well as the Sequential Organ Failure Assessment score in septic patients.¹²⁹

Platelet-Activating Factor

Phosphatidylcholine is a major lipid constituent of the plasma membrane. Its enzymatic processing by cytosolic phospholipase A₂ (cPLA₂) or calcium-independent phospholipase A₂ (iPLA₂) generates powerful small lipid molecules, which function as intracellular second messengers. One of these is arachidonic acid, the precursor molecule for eicosanoids. Another is platelet-activating factor (PAF). During acute inflammation, PAF is released by immune cells following the activation of PLA₂. The receptor for PAF (PAFR), which is constitutively expressed by platelets, leukocytes, and endothelial cells, is a G-protein–coupled receptor of the rhodopsin family. Ligand binding to the PAFR promotes the activation and aggregation of platelets and leukocytes, leukocyte adherence, motility, chemotaxis, and invasion, as well as ROS generation.¹³⁰ Additionally, PAF activation of human PMNs induces extrusion of NETs, while platelet activation induces IL-1 via a novel posttranscriptional mechanism. Finally, PAFR ligation results not only in the upregulation of numerous proinflammatory genes including COX-2, iNOS, and IL-6, but also in the generation of lipid intermediates such as arachidonic acid and lysophospholipids through the activation of PLA₂. Antagonists to PAF receptors have been experimentally shown to mitigate the effects of ischemia and reperfusion injury. Of note, human sepsis is associated with a reduction in the levels of PAF-acetylhydrolase, which inactivates PAF by removing an acetyl group. Indeed, PAF-acetylhydrolase administration in patients with severe sepsis has yielded some reduction in multiple organ dysfunction and mortality¹³¹; however, larger phase III clinical trials failed to show benefit.

Natriuretic Peptides

The natriuretic peptides, atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), are a family of peptides that are released primarily by atrial tissue but are also synthesized by the gut, kidney, brain, adrenal glands, and endothelium. The functionally active forms of the peptides are C-terminal fragments of a larger prohormone, and both N- and C-terminal fragments are detectable in the blood (referred to a N-terminal pro-BNP and pro-ANF, respectively). ANF and BNP share most biologic properties including diuretic, natriuretic, vasorelaxant, and cardiac remodeling properties that are effected by signaling through a common receptor: the guanylyl cyclase-A (GC-A) receptor. They are both increased in the setting of cardiac disorders; however, recent evidence indicates some distinctions in the setting of inflammation. For example, endotoxemia in healthy volunteers increased plasma N-terminal pro-BNP without changing heart rate and blood pressure. Also, elevated pro-BNP has been detected in septic patients in the absence of myocardial dysfunction and appears to have prognostic significance.¹³²

The initial hours after surgical or traumatic injury are metabolically associated with a reduced total body energy expenditure and urinary nitrogen wasting. On adequate resuscitation and stabilization of the injured patient, a reprioritization of substrate use ensues to preserve vital organ function and to support repair of injured tissue. This phase of recovery also is characterized by functions that participate in the restoration of homeostasis, such as augmented metabolic rates and oxygen consumption, enzymatic preference for readily oxidizable substrates such as glucose, and stimulation of the immune system. Understanding of the collective alterations in amino acid (protein), carbohydrate, and lipid metabolism characteristic of the surgical patient lays the foundation upon which metabolic and nutritional support can be implemented.

Metabolism during Fasting

Fuel metabolism during unstressed fasting states has historically served as the standard to which metabolic alterations after acute injury and critical illness are compared (Fig. 2-14). To maintain basal metabolic needs (i.e., at rest and fasting), a normal healthy adult requires approximately 22 to 25 kcal/kg per day drawn from carbohydrate, lipid, and protein sources. This requirement can be as high as 40 kcal/kg per day in severe stress states, such as those seen in patients with burn injuries.

In the healthy adult, principal sources of fuel during short-term fasting (<5 days) are derived from muscle protein and body fat, with fat being the most abundant source of energy (Table 2-8). The normal adult body contains 300 to 400 g of carbohydrates in the form of glycogen, of which 75 to 100 g are stored in the liver. Approximately 200 to 250 g of glycogen are stored within skeletal, cardiac, and smooth muscle cells. The greater glycogen stores within the muscle are not readily available for systemic use due to a deficiency in glucose-6-phosphatase but are available for the energy needs of muscle cells. Therefore, in the fasting state, hepatic glycogen stores are rapidly and preferentially depleted, which results in a fall of serum glucose concentration within hours (<16 hours).

During fasting, a healthy 70-kg adult will use 180 g of glucose per day to support the metabolism of obligate glycolytic cells such as neurons, leukocytes, erythrocytes, and the renal medullae. Other tissues that use glucose for fuel are skeletal muscle, intestinal mucosa, fetal tissues, and solid tumors.

Glucagon, NE, vasopressin, and angiotensin II can promote the utilization of glycogen stores (glycogenolysis) during fasting. Although glucagon, EPI, and cortisol directly promote gluconeogenesis, EPI and cortisol also promote pyruvate shuttling to the liver for gluconeogenesis. Precursors for hepatic gluconeogenesis include lactate, glycerol, and amino acids such as alanine and glutamine. Lactate is released by glycolysis within skeletal muscles, as well as by erythrocytes and leukocytes. The recycling of lactate and pyruvate for gluconeogenesis is commonly referred to as the Cori cycle, which can provide up to 40% of plasma glucose during starvation (Fig. 2-15).

Lactate production from skeletal muscle is insufficient to maintain systemic glucose needs during short-term fasting (simple starvation). Therefore, significant amounts of protein must be degraded daily (75 g/d for a 70-kg adult) to provide the amino acid substrate for hepatic gluconeogenesis. Proteolysis during starvation, which results primarily from decreased insulin and increased cortisol release, is associated with elevated urinary nitrogen excretion from the normal 7 to 10 g per day up to 30 g or more per day.¹³³ Although proteolysis during starvation occurs mainly within skeletal muscles, protein degradation in solid organs also occurs.

In prolonged starvation, systemic proteolysis is reduced to approximately 20 g/d, and urinary nitrogen excretion stabilizes at 2 to 5 g/d (Fig. 2-16). This reduction in proteolysis reflects the adaptation by vital organs (e.g., myocardium, brain, renal cortex, and skeletal muscle) to using ketone bodies as their principal fuel source. In extended fasting, ketone bodies become an important fuel source for the brain after 2 days and gradually become the principal fuel source by 24 days.

Enhanced deamination of amino acids for gluconeogenesis during starvation consequently increases renal excretion of ammonium ions. The kidneys also participate in gluconeogenesis by the use of glutamine and glutamate, and can become the primary source of gluconeogenesis during prolonged starvation, accounting for up to one half of systemic glucose production.

Lipid stores within adipose tissue provide 40% or more of caloric expenditure during starvation. Energy requirements for basal enzymatic and muscular functions (e.g., gluconeogenesis, neural transmission, and cardiac contraction) are met by the mobilization of triglycerides from adipose tissue. In a resting, fasting, 70-kg person, approximately 160 g of free fatty acids and glycerol can be mobilized from adipose tissue per day. Free fatty acid release is stimulated in part by a reduction in serum insulin levels and in part by the increase in circulating glucagon and catecholamine. Such free fatty acids, like ketone bodies, are used as fuel by tissues such as the heart, kidney (renal cortex), muscle, and liver. The mobilization of lipid stores for energy importantly decreases the rate of glycolysis, gluconeogenesis, and proteolysis, as well as the overall glucose requirement to sustain the host. Furthermore, ketone bodies spare glucose utilization by inhibiting the enzyme pyruvate dehydrogenase.

Metabolism after Injury

Injuries or infections induce unique neuroendocrine and immunologic responses that differentiate injury metabolism from that of unstressed fasting (Fig. 2-17). The magnitude of metabolic expenditure appears to be directly proportional to the severity of insult, with thermal injuries and severe infections having the highest energy demands (Fig. 2-18). The increase in energy expenditure is mediated in part by sympathetic activation and catecholamine release, which has been replicated by the administration of catecholamines to healthy human subjects. Lipid metabolism after injury is intentionally discussed first, because this macronutrient becomes the primary source of energy during stressed states.¹³⁴

Lipid Metabolism after Injury

Lipids are not merely nonprotein, noncarbohydrate fuel sources that minimize protein catabolism in the injured patient. Lipid metabolism potentially influences the structural integrity of cell membranes as well as the immune response during systemic inflammation. Adipose stores within the body (triglycerides) are the predominant energy source (50% to 80%) during critical illness and after injury. Fat mobilization (lipolysis) occurs mainly in response to catecholamine stimulus of the hormone-sensitive triglyceride lipase. Other hormonal influences that potentiate lipolysis include adrenocorticotropic hormone (ACTH), catecholamines, thyroid hormone, cortisol, glucagon, GH release, and reduction in insulin levels.¹³⁵

Lipid Absorption

Although the process is poorly understood, adipose tissue provides fuel for the host in the form of free fatty acids and glycerol during critical illness and injury. Oxidation of 1 g of fat yields approximately 9 kcal of energy. Although the liver is capable of synthesizing triglycerides from carbohydrates and amino acids, dietary and exogenous sources provide the major source of triglycerides. Dietary lipids are not readily absorbable in the gut but require pancreatic lipase and phospholipase within the duodenum to hydrolyze the triglycerides into free fatty acids and monoglycerides. The free fatty acids and monoglycerides are then readily absorbed by gut enterocytes, which resynthesize triglycerides by esterification of the monoglycerides with fatty acyl coenzyme A (acyl-CoA) (Fig. 2-19). Long-chain triglycerides (LCTs), defined as those with 12 carbons or more, generally undergo this process of esterification and enter the circulation through the lymphatic system as chylomicrons. Shorter fatty acid chains directly enter the portal circulation and are transported to the liver by albumin carriers. Hepatocytes use free fatty acids as a fuel source during stress states but also can synthesize phospholipids or triglycerides (i.e., very-low-density lipoproteins) during fed states. Systemic tissue (e.g., muscle and the heart) can use chylomicrons and triglycerides as fuel by hydrolysis with lipoprotein lipase at the luminal surface of capillary endothelium.¹³⁶ Trauma or sepsis suppresses lipoprotein lipase activity in both adipose tissue and muscle, presumably mediated by TNF.

Lipolysis and Fatty Acid Oxidation

Periods of energy demand are accompanied by free fatty acid mobilization from adipose stores. This is mediated by hormonal influences (e.g., catecholamines, ACTH, thyroid hormones, GH, and glucagon) on triglyceride lipase through a cAMP pathway (Fig. 2-20). In adipose tissues, triglyceride lipase hydrolyzes triglycerides into free fatty acids and glycerol. Free fatty acids enter the capillary circulation and are transported by albumin to tissues requiring this fuel source (e.g., heart and skeletal muscle). Insulin inhibits lipolysis and favors triglyceride synthesis by augmenting lipoprotein lipase activity as well as intracellular levels of glycerol-3-phosphate. The use of glycerol for fuel depends on the availability of tissue glycerokinase, which is abundant in the liver and kidneys.

Free fatty acids absorbed by cells conjugate with acyl-CoA within the cytoplasm. The transport of fatty acyl-CoA from the outer mitochondrial membrane across the inner mitochondrial membrane occurs via the carnitine shuttle (Fig. 2-21). Medium-chain triglycerides (MCTs), defined as those 6 to 12 carbons in length, bypass the carnitine shuttle and readily cross the mitochondrial membranes. This accounts in part for the fact that MCTs are more efficiently oxidized than LCTs. Ideally, the rapid oxidation of MCTs makes them less prone to fat deposition, particularly within immune cells and the reticuloendothelial system—a common finding with lipid infusion in parenteral nutrition.¹³⁷ However, exclusive use of MCTs as fuel in animal studies has been associated with higher metabolic demands and toxicity, as well as essential fatty acid deficiency.

Within the mitochondria, fatty acyl-CoA undergoes beta oxidation, which produces acetyl-CoA with each pass through the cycle. Each acetyl-CoA molecule subsequently enters the tricarboxylic acid (TCA) cycle for further oxidation to yield 12 ATP molecules, carbon dioxide, and water. Excess acetyl-CoA molecules serve as precursors for ketogenesis. Unlike glucose metabolism, oxidation of fatty acids requires proportionally less oxygen and produces less carbon dioxide. This is frequently quantified as the ratio of carbon dioxide produced to oxygen consumed for the reaction and is known as the respiratory quotient (RQ). An RQ of 0.7 would imply greater fatty acid oxidation for fuel, whereas an RQ of 1 indicates greater carbohydrate oxidation (overfeeding). An RQ of 0.85 suggests the oxidation of equal amounts of fatty acids and glucose.

Ketogenesis

Carbohydrate depletion slows the entry of acetyl-CoA into the TCA cycle secondary to depleted TCA intermediates and enzyme activity. Increased lipolysis and reduced systemic carbohydrate availability during starvation diverts excess acetyl-CoA toward hepatic ketogenesis. A number of extrahepatic tissues, but not the liver itself, are capable of using ketones for fuel. Ketosis represents a state in which hepatic ketone production exceeds extrahepatic ketone utilization.

The rate of ketogenesis appears to be inversely related to the severity of injury. Major trauma, severe shock, and sepsis attenuate ketogenesis by increasing insulin levels and by causing rapid tissue oxidation of free fatty acids. Minor injuries and infections are associated with modest elevations in plasma free fatty acid concentrations and ketogenesis. However, in minor stress states ketogenesis does not exceed that in nonstressed starvation.

Carbohydrate Metabolism

Ingested and enteral carbohydrates are primarily digested in the small intestine, where pancreatic and intestinal enzymes reduce the complex carbohydrates to dimeric units. Disaccharidases (e.g., sucrase, lactase, and maltase) within intestinal brush borders dismantle the complex carbohydrates into simple hexose units, which are transported into the intestinal mucosa. Glucose and galactose are primarily absorbed by energy-dependent active transport coupled to the sodium pump. Fructose absorption, however, occurs by concentration-dependent facilitated diffusion. Neither fructose or galactose within the circulation nor exogenous mannitol (for neurologic injury) evokes an insulin response. Intravenous administration of low-dose fructose in fasting humans has been associated with nitrogen conservation, but the clinical utility of fructose administration in human injury remains to be demonstrated.

Discussion of carbohydrate metabolism primarily refers to the utilization of glucose. The oxidation of 1 g of carbohydrate yields 4 kcal, but sugar solutions such as those found in intravenous fluids or parenteral nutrition provide only 3.4 kcal/g of dextrose. In starvation, glucose production occurs at the expense of protein stores (i.e., skeletal muscle). Hence, the primary goal for maintenance glucose administration in surgical patients is to minimize muscle wasting. The exogenous administration of small amounts of glucose (approximately 50 g/d) facilitates fat entry into the TCA cycle and reduces ketosis. Unlike in starvation in healthy subjects, in septic and trauma patients, provision of exogenous glucose never has been shown to fully suppress amino acid degradation for gluconeogenesis. This suggests that during periods of stress, other hormonal and proinflammatory mediators have a profound influence on the rate of protein degradation and that some degree of muscle wasting is inevitable. The administration of insulin, however, has been shown to reverse protein catabolism during severe stress by stimulating protein synthesis in skeletal muscles and by inhibiting hepatocyte protein degradation. Insulin also stimulates the incorporation of elemental precursors into nucleic acids in association with RNA synthesis in muscle cells.

In cells, glucose is phosphorylated to form glucose-6-phosphate. Glucose-6-phosphate can be polymerized during glycogenesis or catabolized in glycogenolysis. Glucose catabolism occurs by cleavage to pyruvate or lactate (pyruvic acid pathway) or by decarboxylation to pentoses (pentose shunt) (Fig. 2-22).

Excess glucose from overfeeding, as reflected by RQs >1.0, can result in conditions such as glucosuria, thermogenesis, and conversion to fat (lipogenesis). Excessive glucose administration results in elevated carbon dioxide production, which may be deleterious in patients with suboptimal pulmonary function, as well as hyperglycemia, which may contribute to infectious risk and immune suppression.

Injury and severe infections acutely induce a state of peripheral glucose intolerance, despite ample insulin production at levels several fold above baseline. This may occur in part due to reduced skeletal muscle pyruvate dehydrogenase activity after injury, which diminishes the conversion of pyruvate to acetyl-CoA and subsequent entry into the TCA cycle. The three-carbon structures (e.g., pyruvate and lactate) that consequently accumulate are shunted to the liver as substrate for gluconeogenesis. Furthermore, regional tissue catheterization and isotope dilution studies have shown an increase in net splanchnic glucose production by 50% to 60% in septic patients and a 50% to 100% increase in burn patients.¹³⁷ The increase in plasma glucose levels is proportional to the severity of injury, and this net hepatic gluconeogenic response is believed to be under the influence of glucagon. Unlike in the nonstressed subject, in the hypermetabolic, critically ill patient, the hepatic gluconeogenic response to injury or sepsis cannot be suppressed by exogenous or excess glucose administration but rather persists. Hepatic gluconeogenesis, arising primarily from alanine and glutamine catabolism, provides a ready fuel source for tissues such as those of the nervous system, wounds, and erythrocytes, which do not require insulin for glucose transport. The elevated glucose concentrations also provide a necessary energy source for leukocytes in inflamed tissues and in sites of microbial invasions.

The shunting of glucose away from nonessential organs such as skeletal muscle and adipose tissues is mediated by catecholamines. Experiments with infusing catecholamines and glucagon in animals have demonstrated elevated plasma glucose levels as a result of increased hepatic gluconeogenesis and peripheral insulin resistance. Interestingly, although glucocorticoid infusion alone does not increase glucose levels, it does prolong and augment the hyperglycemic effects of catecholamines and glucagon when glucocorticoid is administered concurrently with the latter.

Glycogen stores within skeletal muscles can be mobilized by EPI activation of β-adrenergic receptors, GTP-binding proteins (G proteins), which subsequently activates the second messenger, cAMP. The cAMP activates phosphorylase kinase, which in turn leads to conversion of glycogen to glucose-1-phosphate. Phosphorylase kinase also can be activated by the second messenger, calcium, through the breakdown of phosphatidylinositol phosphate, which is the case in vasopressin-mediated hepatic glycogenolysis.¹³⁸

Glucose Transport and Signaling

Hydrophobic cell membranes are relatively impermeable to hydrophilic glucose molecules. There are two distinct classes of membrane glucose transporters in human systems. These are the facilitated diffusion glucose transporters (GLUTs) that permit the transport of glucose down a concentration gradient (Table 2-9) and the Na⁺/glucose secondary active transport system (SGLT), which transports glucose molecules against concentration gradients by active transport.

Numerous functional human GLUTs have been cloned since 1985. GLUT1 is expressed at its highest level in human erythrocytes, where it may function to increase the glucose carrying capacity of the blood. It is expressed on several other tissues, but little is found in the liver and skeletal muscle. GLUT1 plays a critical role in cerebral glucose uptake as the major GLUT isoform that is constitutively expressed by the endothelium in the blood-brain barrier. GLUT2 is the major glucose transporter of hepatocytes. It is also expressed by intestinal absorptive cells, pancreatic β-cells, renal tubule cells, and insulin-secreting β-cells of the pancreas. GLUT2 is important for glucose uptake and release in the fed and fasted states. GLUT3 is highly expressed in neuronal tissue of the brain and appears to be important to neuronal glucose uptake. GLUT4 is significant to human metabolism because it is the primary glucose transporter of insulin-sensitive tissues, adipose tissue, and skeletal and cardiac muscle. Under basal conditions, these transporters are usually packaged as intracellular vesicles, but when insulin levels rise, rapid translocation of these vesicles to the cell surface occurs, increasing glucose uptake and metabolism in these tissues and preventing chronic elevations in blood glucose levels. A defect in this insulin-mediated translocation of GLUT4 to the plasma membrane causes peripheral insulin resistance. GLUT4 therefore plays a critical role in the regulation of whole-body glucose homeostasis. GLUT5 has been identified in several tissues but is primarily expressed in the jejunum. Although it possesses some capacity for glucose transport, it is predominantly a fructose transporter.¹³⁹

SGLTs are distinct glucose transport systems found in the intestinal epithelium and in the proximal renal tubules. These systems transport both sodium and glucose intracellularly, and glucose affinity for this transporter increases when sodium ions are attached. SGLT1 is prevalent on brush borders of small intestine enterocytes and primarily mediates the active uptake of luminal glucose. In addition, SGLT1 within the intestinal lumen also enhances gut retention of water through osmotic absorption. SGLT1 and SGLT2 are both associated with glucose reabsorption at proximal renal tubules.

Protein and Amino Acid Metabolism

The average protein intake in healthy young adults ranges from 80 to 120 g/d, and every 6 g of protein yields approximately 1 g of nitrogen. The degradation of 1 g of protein yields approximately 4 kcal of energy, similar to the yield in carbohydrate metabolism.

After injury, the initial systemic proteolysis, mediated primarily by glucocorticoids, increases urinary nitrogen excretion to levels in excess of 30 g/d, which roughly corresponds to a loss in lean body mass of 1.5% per day. An injured individual who does not receive nutrition for 10 days can theoretically lose 15% lean body mass. Therefore, amino acids cannot be considered a long-term fuel reserve, and indeed excessive protein depletion (i.e., 25% to 30% of lean body weight) is not compatible with sustaining life.¹⁴⁰

Protein catabolism after injury provides substrates for gluconeogenesis and for the synthesis of acute-phase proteins. Radiolabeled amino acid incorporation studies and protein analyses confirm that skeletal muscles are preferentially depleted acutely after injury, whereas visceral tissues (e.g., the liver and kidney) remain relatively preserved. The accelerated urea excretion after injury also is associated with the excretion of intracellular elements such as sulfur, phosphorus, potassium, magnesium, and creatinine. Conversely, the rapid utilization of elements such as potassium and magnesium during recovery from major injury may indicate a period of tissue healing.

The net changes in protein catabolism and synthesis correspond to the severity and duration of injury (Fig. 2-23). Elective operations and minor injuries result in lower protein synthesis and moderate protein breakdown. Severe trauma, burns, and sepsis are associated with increased protein catabolism. The rise in urinary nitrogen and negative nitrogen balance can be detected early after injury and peak by 7 days. This state of protein catabolism may persist for as long as 3 to 7 weeks. The patient’s prior physical status and age appear to influence the degree of proteolysis after injury or sepsis. Activation of the ubiquitin-proteasome system in muscle cells is one of the major pathways for protein degradation during acute injury. This response is accentuated by tissue hypoxia, acidosis, insulin resistance, and elevated glucocorticoid levels.

The goal of nutritional support in the surgical patient is to prevent or reverse the catabolic effects of disease or injury. Although several important biologic parameters have been used to measure the efficacy of nutritional regimens, the ultimate validation for nutritional support in surgical patients should be improvement in clinical outcome and restoration of function.

Estimation of Energy Requirements

Overall nutritional assessment is undertaken to determine the severity of nutrient deficiencies or excess and to aid in predicting nutritional requirements. Pertinent information is obtained by determining the presence of weight loss, chronic illnesses, or dietary habits that influence the quantity and quality of food intake. Social habits predisposing to malnutrition and the use of medications that may influence food intake or urination should also be investigated. Physical examination seeks to assess loss of muscle and adipose tissues, organ dysfunction, and subtle changes in skin, hair, or neuromuscular function reflecting frank or impending nutritional deficiency. Anthropometric data (i.e., weight change, skinfold thickness, and arm circumference muscle area) and biochemical determinations (i.e., creatinine excretion, albumin level, prealbumin level, total lymphocyte count, and transferrin level) may be used to substantiate the patient’s history and physical findings. However, it is imprecise to rely on any single or fixed combination of the findings to accurately assess nutritional status or morbidity. Appreciation for the stresses and natural history of the disease process, in combination with nutritional assessment, remains the basis for identifying patients in acute or anticipated need of nutritional support.

A fundamental goal of nutritional support is to meet the energy requirements for essential metabolic processes and tissue repair. Failure to provide adequate nonprotein energy sources will lead to consumption of lean tissue stores. The requirement for energy may be measured by indirect calorimetry and trends in serum markers (e.g., prealbumin level) and estimated from urinary nitrogen excretion, which is proportional to resting energy expenditure.¹³⁸ However, the use of indirect calorimetry, particularly in the critically ill patient, is labor intensive and often leads to overestimation of caloric requirements.

Basal energy expenditure (BEE) may also be estimated using the Harris-Benedict equations:

where W = weight in kilograms; H = height in centimeters; and A = age in years.

These equations, adjusted for the type of surgical stress, are suitable for estimating energy requirements in the majority of hospitalized patients. It has been demonstrated that the provision of 30 kcal/kg per day will adequately meet energy requirements in most postsurgical patients, with a low risk of overfeeding. After trauma or sepsis, energy substrate demands are increased, necessitating greater nonprotein calories beyond calculated energy expenditure (Table 2-10). These additional nonprotein calories provided after injury are usually 1.2 to 2.0 times greater than calculated resting energy expenditure, depending on the type of injury. It is seldom appropriate to exceed this level of nonprotein energy intake during the height of the catabolic phase.

The second objective of nutritional support is to meet the substrate requirements for protein synthesis. An appropriate nonprotein-calorie:nitrogen ratio of 150:1 (e.g., 1 g N = 6.25 g protein) should be maintained, which is the basal calorie requirement provided to limit the use of protein as an energy source. There is now greater evidence suggesting that increased protein intake and a lower calorie:nitrogen ratio of 80:1 to 100:1 may benefit healing in selected hypermetabolic or critically ill patients. In the absence of severe renal or hepatic dysfunction precluding the use of standard nutritional regimens, approximately 0.25 to 0.35 g of nitrogen per kilogram of body weight should be provided daily.¹⁴¹

Vitamins and Minerals

The requirements for vitamins and essential trace minerals usually can be met easily in the average patient with an uncomplicated postoperative course. Therefore, vitamins usually are not given in the absence of preoperative deficiencies. Patients maintained on elemental diets or parenteral hyperalimentation require complete vitamin and mineral supplementation. Commercial enteral diets contain varying amounts of essential minerals and vitamins. It is necessary to ensure that adequate replacement is available in the diet or by supplementation. Numerous commercial vitamin preparations are available for intravenous or intramuscular use, although most do not contain vitamin K and some do not contain vitamin B₁₂ or folic acid. Supplemental trace minerals may be given intravenously via commercial preparations. Essential fatty acid supplementation also may be necessary, especially in patients with depletion of adipose stores.

Overfeeding

Overfeeding usually results from overestimation of caloric needs, as occurs when actual body weight is used to calculate the BEE in patient populations such as the critically ill with significant fluid overload and the obese. Indirect calorimetry can be used to quantify energy requirements but frequently overestimates BEE by 10% to 15% in stressed patients, particularly if they are receiving ventilatory support. In these instances, estimated dry weight should be obtained from preinjury records or family members. Adjusted lean body weight also can be calculated. Overfeeding may contribute to clinical deterioration via increased oxygen consumption, increased carbon dioxide production and prolonged need for ventilatory support, fatty liver, suppression of leukocyte function, hyperglycemia, and increased risk of infection.

Rationale for Enteral Nutrition

Enteral nutrition generally is preferred over parenteral nutrition based on the lower cost of enteral feeding and the associated risks of the intravenous route, including vascular access complications.¹⁴² Of further consideration are the consequences of gastrointestinal tract disuse, which include diminished secretory IgA production and cytokine production as well as bacterial overgrowth and altered mucosal defenses. For example, laboratory models have long demonstrated that luminal nutrient contact reduces intestinal mucosal atrophy compared with parenteral or no nutritional support.

The benefits of enteral feeding in patients undergoing elective surgery appear to be linked to their preoperative nutritional status. Studies comparing postoperative enteral and parenteral nutrition in patients undergoing gastrointestinal surgery have demonstrated reduced infectious complications and acute-phase protein production in those fed by the enteral route. Yet prospectively randomized studies of patients with adequate nutritional status (albumin ≥4 g/dL) undergoing gastrointestinal surgery demonstrate no differences in outcome and complications between those administered enteral nutrition and those given maintenance intravenous fluids alone in the initial days after surgery.¹⁴³ Furthermore, intestinal permeability studies in well-nourished patients undergoing upper gastrointestinal cancer surgery demonstrated normalization of intestinal permeability and barrier function by the fifth postoperative day.¹⁴⁴ The data for critically ill or injured patients are more definitive as to the benefits of enteral nutrition. Meta-analysis of studies involving critically ill patients demonstrates a 44% reduction in infectious complications in those receiving enteral nutritional support compared with those receiving parenteral nutrition. Most prospectively randomized studies in patients with severe abdominal and thoracic trauma demonstrate significant reductions in infectious complications in patients given early enteral nutrition compared with those who were unfed or received parenteral nutrition. In critically ill patients, prospective studies have also demonstrated that early enteral nutrition is associated with better small-intestinal carbohydrate absorption, shorter duration of mechanical ventilation, and shorter time in the intensive care unit. The exception has been in studies of patients with closed-head injury, in whom no significant differences in outcome were demonstrated between early jejunal feeding and other nutritional support modalities. Moreover, early gastric feeding after closed-head injury was frequently associated with underfeeding and calorie deficiency due to the difficulties in overcoming gastroparesis and the high risk of aspiration. While current evidence remains inconclusive about the benefits of “early” (as defined by feeding in the first 24 hours) versus “late” (as defined by feeding >24 hours after burn) enteral nutrition in burn patients as to its impact on mortality rates, there is reason to believe that early enteral nutrition may positively modulate the initial hypermetabolic response and help to maintain mucosal immunity.

In summary, enteral nutrition is preferred for most critically ill patients—an evidence-based practice supported by clinical data involving a variety of critically ill patient populations, including those with trauma, burns, head injury, major surgery, and acute pancreatitis. For intensive care unit patients who are hemodynamically stable and have a functioning gastrointestinal tract, early enteral feeding (within 24 to 48 hours of arrival in the intensive care unit) has become a recommended standard of care.¹⁴⁵ For patients undergoing elective surgery, healthy patients without malnutrition who are undergoing uncomplicated surgery can tolerate 10 days of partial starvation (i.e., maintenance intravenous fluids only) before any clinically significant protein catabolism occurs. Earlier intervention is likely indicated for patients in whom preoperative protein-calorie malnutrition has been identified. Other clinical scenarios for which the benefits of enteral nutritional support have been substantiated include permanent neurologic impairment, oropharyngeal dysfunction, short-bowel syndrome, and bone marrow transplantation.

Initiation of enteral nutrition should occur immediately after adequate resuscitation, most readily determined by adequate urine output. The presence of bowel sounds and the passage of flatus or stool are not absolute prerequisites for initiation of enteral nutrition, but in the setting of gastroparesis, feedings should be administered distal to the pylorus. Gastric residuals of 200 mL or more in a 4- to 6-hour period or abdominal distention requires cessation of feeding and adjustment of the infusion rate. Concomitant gastric decompression with distal small-bowel feedings may be appropriate in certain patients such as closed-head injury patients with gastroparesis. There is no evidence to support withholding enteric feedings for patients after bowel resection or for those with low-output enterocutaneous fistulas of <500 mL/d. In fact, a recent systematic review of studies of early enteral feeding (within 24 hours of gastrointestinal surgery) showed no effect on anastomotic leak and a reduction in mortality. Early enteral feeding is also associated with reduced incidence of fistula formation in patients with open abdomen. Enteral feeding should also be offered to patients with short-bowel syndrome or clinical malabsorption, but necessary calories, essential minerals, and vitamins should be supplemented using parenteral modalities.

Hypocaloric Enteral Nutrition

As noted earlier, critically ill and/or injured patients demonstrate increased resting energy expenditure associated with altered metabolism. While several methods exist to predict the energy requirement, the recommended caloric dose for critically ill patients varies, ranging from 25 to 30 kcal/kg. The perceived benefit of achieving the caloric target is to meet the patient’s energy needs and to avoid the loss of lean body mass. However, recent evidence supports the idea of caloric restriction, attributing its benefits to improved cellular function in terms of effects on mitochondrial free radical generation, the plasma membrane redox system, and insulin sensitivity. Further support was offered by a single-center, randomized controlled trial that compared permissive underfeeding with target enteral feeding (caloric goal: 60% to 70% compared with 90% to 100% of calculated requirement) in critically ill medical and surgical patients.¹⁴⁶ This study demonstrated that permissive underfeeding was associated with lower mortality and morbidity than was target feeding. However, current guidelines do not recommend hypocaloric feeding without confirmation of these data from the multicenter trial that is currently ongoing. A recent study examined the use of trophic feedings in patients with acute lung injury. Trophic feedings refer to providing a minimal amount of enteral feedings, which are presumed to have beneficial effects despite not meeting daily caloric needs. When the trophic feeding group (enteral feeding at 10 mL/h) was compared with the full-feeding group (25 mL/h) over the first 6 days of feeding, there was no improvement in ventilator-free days, 60-day mortality, or infectious complications.¹⁴⁷

Enteral Formulas

For most critically ill patients, the choice of enteral formula will be determined by a number of factors and will include a clinical judgment as to the “best fit” for the patients’ needs. In general, feeding formulas to consider are gastrointestinal tolerance-promoting, anti-inflammatory, immune-modulating, organ supportive, and standard enteral nutrition. In addition, guidelines from professional nutrition societies identify certain populations of patients who can benefit from formulations with specific pharmaconutrients.¹⁴⁸ For many others, each physician must use his or her own clinical judgment about what formula will best meet the patient’s needs.

The functional status of the gastrointestinal tract determines the type of enteral solutions to be used. Patients with an intact gastrointestinal tract will tolerate complex solutions, but patients who have not been fed via the gastrointestinal tract for prolonged periods are less likely to tolerate complex carbohydrates. In those patients who are having difficulty tolerating standard enteral formulas, peptide- and MCT-based formulas with prebiotics can lessen gastrointestinal tolerance problems. Additionally, in patients with demonstrated malabsorption issues, such as with inflammatory bowel diseases or short-bowel syndrome, current guidelines endorse the provision of hydrolyzed protein formulas to improve absorption. Guidelines have not yet been made with regard to the fiber content of enteral formulas. However, recent evidence indicates that supplementation of enteral formulas with soluble dietary fiber may be beneficial for improving stool consistency in patients suffering from diarrhea.

Factors that influence the choice of enteral formula also include the extent of organ dysfunction (e.g., renal, pulmonary, hepatic, or gastrointestinal), the nutrients needed to restore optimal function and healing, and the cost of specific products. There are still no conclusive data to recommend one category of product over another, and nutritional support committees typically develop the most cost-efficient enteral formulary for the most commonly encountered disease categories within the institution.

As discussed extensively in the first sections of this chapter, surgery and trauma result in a significant “sterile” inflammatory response that impacts the innate and adaptive immune systems. The provision of immune-modulating nutrients, termed “immunonutrition,” is one mechanism by which the immune response can be supported and an attempt made to lower infectious risk. At present, the best-studied immunonutrients are glutamine, arginine, and ω-3 PUFAs.

“Immunonutrients.” Glutamine

is the most abundant amino acid in the human body, comprising nearly two thirds of the free intracellular amino acid pool. Of this, 75% is found within the skeletal muscles. In healthy individuals, glutamine is considered a nonessential amino acid, because it is synthesized within the skeletal muscles and the lungs. Glutamine is a necessary substrate for nucleotide synthesis in most dividing cells and hence provides a major fuel source for enterocytes. It also serves as an important fuel source for immunocytes such as lymphocytes and macrophages and is a precursor for glutathione, a major intracellular antioxidant. During stress states such as sepsis, or in tumor-bearing hosts, peripheral glutamine stores are rapidly depleted, and the amino acid is preferentially shunted as a fuel source toward the visceral organs and tumors, respectively.¹⁴⁹ These situations create, at least experimentally, a glutamine-depleted environment, with consequences including enterocyte and immunocyte starvation. Glutamine metabolism during stress in humans, however, may be more complex than is indicated in previously reported animal data. Although it is hypothesized that provision of glutamine may preserve immune cell and enterocyte function and enhance nitrogen balance during injury or sepsis, the clinical outcome is very strongly dependent on the patient population, as will be discussed later.

Arginine, also a nonessential amino acid in healthy subjects, first attracted attention for its immunoenhancing properties, wound-healing benefits, and association with improved survival in animal models of sepsis and injury.¹⁵⁰ As with glutamine, the benefits of experimental arginine supplementation during stress states are diverse. In clinical studies involving critically ill and injured patients and patients who have undergone surgery for certain malignancies, enteral administration of arginine has led to net nitrogen retention and protein synthesis, whereas isonitrogenous diets have not. Some of these studies also provide in vitro evidence of enhanced immunocyte function. The clinical utility of arginine supplementation in improving overall patient outcome remains an area of investigation.

As previously discussed, ω-3 PUFAs (canola oil or fish oil) displace ω-6 fatty acids in cell membranes, which theoretically reduces the proinflammatory response from prostaglandin production. Hence, there has been significant interest in reducing the ratio of ω-6 to ω-3 fatty acids.

Low-Residue Isotonic Formulas

Most low-residue isotonic formulas provide a caloric density of 1.0 kcal/mL, and approximately 1500 to 1800 mL are required to meet daily requirements. These low-osmolarity compositions provide baseline carbohydrates, protein, electrolytes, water, fat, and fat-soluble vitamins (some do not have vitamin K) and typically have a nonprotein-calorie:nitrogen ratio of 150:1. These contain no fiber bulk and therefore leave minimum residue. These solutions usually are considered to be the standard or first-line formulas for stable patients with an intact gastrointestinal tract.

Isotonic Formulas with Fiber

Isotonic formulas with fiber contain soluble and insoluble fiber, which is most often soy based. Physiologically, fiber-based solutions delay intestinal transit time and may reduce the incidence of diarrhea compared with nonfiber solutions. Fiber stimulates pancreatic lipase activity and is degraded by gut bacteria into short-chain fatty acids (SCFAs), an important fuel for colonocytes. Recent data have also demonstrated the expression of SCFA receptors on leukocytes, suggesting that fiber fermentation by the colonic microbiome may indirectly regulate immune cell function. Future work in this area is likely to demonstrate important links between fiber type, microbiome composition, and immune health.

Immune-Enhancing Formulas

Immune-enhancing formulas are fortified with special nutrients that are purported to enhance various aspects of immune or solid organ function. Such additives include glutamine, arginine, ω-3 fatty acids, and nucleotides.¹⁵¹ Although several trials have proposed that one or more of these additives reduce surgical complications and improve outcome, these results have not been uniformly corroborated by other trials. The Canadian Clinical Practice Guidelines currently do not recommend the addition of arginine supplements for critically ill patients due to the potential for harm when used in septic patients.¹⁵² With regard to ω-3 PUFAs, results from the EDEN-Omega study demonstrated that twice-daily enteral supplementation of ω-3 fatty acids, α-linolenic acid, and antioxidants did not improve the primary endpoint of ventilator-free days or other clinical outcomes in patients with acute lung injury and may be harmful.¹⁵³ Glutamine supplementation should be strictly guided by the individual patient condition. Enteral and parenteral supplementation with glutamine appears to have a harmful effect in critically ill patients with multiorgan failure as evidenced by significantly increased mortality (REDOXS study). However, for burn or trauma patients who are hemodynamically stable and without evidence of organ dysfunction, glutamine supplementation has been shown to be beneficial in terms of decreased LOS and infectious complications.

Calorie-Dense Formulas

The primary distinction of calorie-dense formulas is a greater caloric value for the same volume. Most commercial products of this variety provide 1.5 to 2 kcal/mL and therefore are suitable for patients requiring fluid restriction or those unable to tolerate large-volume infusions. As expected, these solutions have higher osmolality than standard formulas and are suitable for intragastric feedings.

High-Protein Formulas

High-protein formulas are available in isotonic and nonisotonic mixtures and are proposed for critically ill or trauma patients with high protein requirements. These formulas have nonprotein-calorie:nitrogen ratios between 80:1 and 120:1. While some observational studies show improved outcomes with higher protein intakes in critically ill patients, there are limited data from randomized trials, which prevents making strong conclusions about the dose of protein in critically ill patients.

Elemental Formulas

Elemental formulas contain predigested nutrients and provide proteins in the form of small peptides. Complex carbohydrates are limited, and fat content, in the form of MCTs and LCTs, is minimal. The primary advantage of such a formula is ease of absorption, but the inherent scarcity of fat, associated vitamins, and trace elements limits its long-term use as a primary source of nutrients. Due to its high osmolarity, dilution or slow infusion rates usually are necessary, particularly in critically ill patients. These formulas have been used frequently in patients with malabsorption, gut impairment, and pancreatitis, but their cost is significantly higher than that of standard formulas. To date, there has been no evidence of their benefit in routine use.

Renal Failure Formulas

The primary benefits of renal formulas are the lower fluid volume and concentrations of potassium, phosphorus, and magnesium needed to meet daily calorie requirements. This type of formulation almost exclusively contains essential amino acids and has a high nonprotein-calorie:nitrogen ratio; however, it does not contain trace elements or vitamins.

Pulmonary Failure Formulas

In pulmonary failure formulas, fat content is usually increased to 50% of the total calories, with a corresponding reduction in carbohydrate content. The goal is to reduce carbon dioxide production and alleviate ventilation burden for failing lungs.

Hepatic Failure Formulas

Close to 50% of the proteins in hepatic failure formulas are branched-chain amino acids (e.g., leucine, isoleucine, and valine). The goal of such a formula is to reduce aromatic amino acid levels and increase the levels of branched-chain amino acids, which can potentially reverse encephalopathy in patients with hepatic failure.¹⁵⁴ The use of these formulas is controversial, however, because no clear benefits have been proven by clinical trials. Protein restriction should be avoided in patients with end-stage liver disease, because such patients have significant protein-energy malnutrition that predisposes them to additional morbidity and mortality.¹⁵⁵

Access for Enteral Nutritional Support

The available techniques and repertoire for enteral access have provided multiple options for feeding the gut. Presently used methods and preferred indications are summarized in Table 2-11.¹⁵⁶

Nasoenteric Tubes

Nasogastric feeding should be reserved for those with intact mentation and protective laryngeal reflexes to minimize risks of aspiration. Even in intubated patients, nasogastric feedings often can be recovered from tracheal suction. Nasojejunal feedings are associated with fewer pulmonary complications including risk of pneumonia, but access past the pylorus requires greater effort to accomplish. Therefore, routine use of small-bowel feedings is preferred in units where small-bowel access is readily feasible. Where there may be difficulties obtaining access, small-bowel feedings may be considered a priority for those patients at high risk for intolerance to enteral nutrition (e.g., high gastric residuals).

Blind insertion of nasogastric feeding tubes is fraught with misplacement, and air instillation with auscultation is inaccurate for ascertaining proper positioning. Radiographic confirmation is usually required to verify the position of the nasogastric feeding tube.

Several methods have been recommended for the passage of nasoenteric feeding tubes into the small bowel, including use of prokinetic agents, right lateral decubitus positioning, gastric insufflation, tube angulation, and application of clockwise torque. However, the successful placement of feeding tubes by these methods is highly variable and operator dependent. Furthermore, it is time consuming, and success rates for intubation past the duodenum into the jejunum by these methods are <20%. Fluoroscopy-guided intubation past the pylorus has a >90% success rate, and more than half of these intubations result in jejunal placement. Similarly, endoscopy-guided placement past the pylorus has high success rates, but attempts to advance the tube beyond the second portion of the duodenum using a standard gastroduodenoscope are unlikely to be successful.

Small-bowel feeding is more reliable for delivering nutrition than nasogastric feeding. Furthermore, the risks of aspiration pneumonia can be reduced by 25% with small-bowel feeding compared with nasogastric feeding. The disadvantages of the use of nasoenteric feeding tubes are clogging, kinking, and inadvertent displacement or removal of the tube and nasopharyngeal complications. If nasoenteric feeding will be required for longer than 30 days, access should be converted to a percutaneous one.¹⁵⁷

Percutaneous Endoscopic Gastrostomy

The most common indications for percutaneous endoscopic gastrostomy (PEG) include impaired swallowing mechanisms, oropharyngeal or esophageal obstruction, and major facial trauma. It is frequently used for debilitated patients requiring caloric supplementation, hydration, or frequent medication dosing. It is also appropriate for patients requiring passive gastric decompression. Relative contraindications for PEG placement include ascites, coagulopathy, gastric varices, gastric neoplasm, and lack of a suitable abdominal site. Most tubes are 18F to 28F in size and may be used for 12 to 24 months.

Identification of the PEG site requires endoscopic transillumination of the anterior stomach against the abdominal wall. A 14-gauge angiocatheter is passed through the abdominal wall into the fully insufflated stomach. A guidewire is threaded through the angiocatheter, grasped by snares or forceps, and pulled out through the mouth. The tapered end of the PEG tube is secured to the guidewire and is pulled into position out of the abdominal wall. The PEG tube is secured without tension against the abdominal wall, and many have reported using the tube within hours of placement. It has been the practice of some to connect the PEG tube to a drainage bag for passive decompression for 24 hours before use, allowing more time for the stomach to seal against the peritoneum.

If endoscopy is not available or technical obstacles preclude PEG placement, the interventional radiologist can attempt the procedure percutaneously under fluoroscopic guidance by first insufflating the stomach against the abdominal wall with a nasogastric tube. If this also is unsuccessful, surgical gastrostomy tube placement can be considered, particularly with minimally invasive methods. When surgery is contemplated, it may be wise to consider directly accessing the small bowel for nutrition delivery.

Although PEG tubes enhance nutritional delivery, facilitate nursing care, and are superior to nasogastric tubes, serious complications occur in approximately 3% of patients. These complications include wound infection, necrotizing fasciitis, peritonitis, aspiration, leaks, dislodgment, bowel perforation, enteric fistulas, bleeding, and aspiration pneumonia.¹⁵⁸ For patients with significant gastroparesis or gastric outlet obstruction, feedings through PEG tubes are hazardous. In such cases, the PEG tube can be used for decompression and allow access for converting the PEG tube to a transpyloric feeding tube.

Percutaneous Endoscopic Gastrostomy-Jejunostomy and Direct Percutaneous Endoscopic Jejunostomy

Although gastric bolus feedings are more physiologic, patients who cannot tolerate gastric feedings or who have significant aspiration risks should be fed directly past the pylorus. In the percutaneous endoscopic gastrostomy-jejunostomy (PEG-J) method, a 9F to 12F tube is passed through an existing PEG tube, past the pylorus, and into the duodenum. This can be achieved by endoscopic or fluoroscopic guidance. With weighted catheter tips and guidewires, the tube can be further advanced past the ligament of Treitz. However, the incidence of long-term PEG-J tube malfunction has been reported to be >50% as a result of retrograde tube migration into the stomach, kinking, or clogging.

Direct percutaneous endoscopic jejunostomy (DPEJ) tube placement uses the same techniques as PEG tube placement but requires an enteroscope or colonoscope to reach the jejunum. DPEJ tube malfunctions are probably less frequent than PEG-J tube malfunctions, and kinking or clogging is usually averted by placement of larger-caliber catheters. The success rate of DPEJ tube placement is variable because of the complexity of endoscopic skills required to locate a suitable jejunal site. In such cases where endoscopic means are not feasible, surgical jejunostomy tube placement is more appropriate, especially when minimally invasive techniques are available.

Surgical Gastrostomy and Jejunostomy

For a patient undergoing complex abdominal or trauma surgery, thought should be given during surgery to the possible routes for subsequent nutritional support, because laparotomy affords direct access to the stomach or small bowel. The only absolute contraindication to feeding jejunostomy is distal intestinal obstruction. Relative contraindications include severe edema of the intestinal wall, radiation enteritis, inflammatory bowel disease, ascites, severe immunodeficiency, and bowel ischemia. Needle-catheter jejunostomies also can be done with a minimal learning curve. The biggest drawback usually is possible clogging and knotting of the 6F catheter.¹⁵⁹

Abdominal distention and cramps are common adverse effects of early enteral nutrition. Some have also reported impaired respiratory mechanics as a result of intolerance to enteral feedings. These are mostly correctable by temporarily discontinuing feedings and resuming at a lower infusion rate.

Pneumatosis intestinalis and small-bowel necrosis are infrequent but significant problems in patients receiving jejunal tube feedings. Several contributing factors have been proposed, including the hyperosmolarity of enteral solutions, bacterial overgrowth, fermentation, and accumulation of metabolic breakdown products. The common pathophysiology is believed to be bowel distention and consequent reduction in bowel wall perfusion. Risk factors for these complications include cardiogenic and circulatory shock, vasopressor use, diabetes mellitus, and chronic obstructive pulmonary disease. Therefore, enteral feedings in the critically ill patient should be delayed until adequate resuscitation has been achieved. As alternatives, diluting standard enteral formula, delaying the progression to goal infusion rates, or using monomeric solutions with low osmolality requiring less digestion by the gastrointestinal tract all have been successfully used.

Parenteral nutrition is the continuous infusion of a hyperosmolar solution containing carbohydrates, proteins, fat, and other necessary nutrients through an indwelling catheter inserted into the superior vena cava. To obtain the maximum benefit, the calorie:protein ratio must be adequate (at least 100 to 150 kcal/g nitrogen), and both carbohydrates and proteins must be infused simultaneously. When the sources of calories and nitrogen are given at different times, there is a significant decrease in nitrogen utilization. These nutrients can be given in quantities considerably greater than the basic caloric and nitrogen requirements, and this method has proved to be highly successful in achieving growth and development, positive nitrogen balance, and weight gain in a variety of clinical situations. Clinical trials and meta-analysis of studies of parenteral feeding in the perioperative period have suggested that preoperative nutritional support may benefit some surgical patients, particularly those with extensive malnutrition. Short-term use of parenteral nutrition in critically ill patients (i.e., duration of <7 days) when enteral nutrition may have been instituted is associated with higher rates of infectious complications. After severe injury, parenteral nutrition is associated with higher rates of infectious risks than is enteral feeding (Table 2-12). Clinical studies have demonstrated that parenteral feeding with complete bowel rest results in augmented stress hormone and inflammatory mediator response to an antigenic challenge. However, parenteral feeding still is associated with fewer infectious complications than no feeding at all. In cancer patients, delivery of parenteral nutrition has not been shown to benefit clinical response, prolong survival, or ameliorate the toxic effects of chemotherapy, and infectious complications are increased.

Rationale for Parenteral Nutrition

The principal indications for parenteral nutrition are malnutrition, sepsis, or surgical or traumatic injury in seriously ill patients for whom use of the gastrointestinal tract for feedings is not possible. In some instances, intravenous nutrition may be used to supplement inadequate oral intake. The safe and successful use of parenteral nutrition requires proper selection of patients with specific nutritional needs, experience with the technique, and an awareness of the associated complications. In patients with significant malnutrition, parenteral nutrition can rapidly improve nitrogen balance, which may enhance immune function. Routine postoperative use of parenteral nutrition is not shown to have clinical benefit and may be associated with a significant increase in complication rate. As with enteral nutrition, the fundamental goals are to provide sufficient calories and nitrogen substrate to promote tissue repair and to maintain the integrity or growth of lean tissue mass. The following are patient groups for whom parenteral nutrition has been used in an effort to achieve these goals:

1. Newborn infants with catastrophic gastrointestinal anomalies, such as tracheoesophageal fistula, gastroschisis, omphalocele, or massive intestinal atresia

2. Infants who fail to thrive due to gastrointestinal insufficiency associated with short-bowel syndrome, malabsorption, enzyme deficiency, meconium ileus, or idiopathic diarrhea

3. Adult patients with short-bowel syndrome secondary to massive small-bowel resection (<100 cm without colon or ileocecal valve or <50 cm with intact ileocecal valve and colon)

4. Patients with enteroenteric, enterocolic, enterovesical, or high-output enterocutaneous fistulas (>500 mL/d)

5. Surgical patients with prolonged paralytic ileus after major operations (>7 to 10 days), multiple injuries, or blunt or open abdominal trauma, or patients with reflex ileus complicating various medical diseases

6. Patients with normal bowel length but with malabsorption secondary to sprue, hypoproteinemia, enzyme or pancreatic insufficiency, regional enteritis, or ulcerative colitis

7. Adult patients with functional gastrointestinal disorders such as esophageal dyskinesia after cerebrovascular accident, idiopathic diarrhea, psychogenic vomiting, or anorexia nervosa

8. Patients with granulomatous colitis, ulcerative colitis, or tuberculous enteritis in whom major portions of the absorptive mucosa are diseased

9. Patients with malignancy, with or without cachexia, in whom malnutrition might jeopardize successful use of a therapeutic option

10. Patients in whom attempts to provide adequate calories by enteral tube feedings or high residuals have failed

11. Critically ill patients who are hypermetabolic for >5 days or for whom enteral nutrition is not feasible

Patients in whom hyperalimentation is contraindicated include the following:

1. Patients for whom a specific goal for patient management is lacking or for whom, instead of extending a meaningful life, inevitable dying would be delayed

2. Patients experiencing hemodynamic instability or severe metabolic derangement (e.g., severe hyperglycemia, azotemia, encephalopathy, hyperosmolality, and fluid-electrolyte disturbances) requiring control or correction before hypertonic intravenous feeding is attempted

3.  Patients for whom gastrointestinal tract feeding is feasible; in the vast majority of instances, this is the best route by which to provide nutrition

4.  Patients with good nutritional status

5.  Infants with <8 cm of small bowel, because virtually all have been unable to adapt sufficiently despite prolonged periods of parenteral nutrition

6.  Patients who are irreversibly decerebrate or otherwise dehumanized

Total Parenteral Nutrition

Total parenteral nutrition (TPN), also referred to as central parenteral nutrition, requires access to a large-diameter vein to deliver the entire nutritional requirements of the individual. Dextrose content of the solution is high (15% to 25%), and all other macronutrients and micronutrients are deliverable by this route.

Peripheral Parenteral Nutrition

The lower osmolarity of the solution used for peripheral parenteral nutrition (PPN), secondary to reduced levels of dextrose (5% to 10%) and protein (3%), allows its administration via peripheral veins. Some nutrients cannot be supplemented because they cannot be concentrated into small volumes. Therefore, PPN is not appropriate for repleting patients with severe malnutrition. It can be considered if central routes are not available or if supplemental nutritional support is required. Typically, PPN is used for short periods (<2 weeks). Beyond this time, TPN should be instituted.

Initiation of Parenteral Nutrition

The basic solution for parenteral nutrition contains a final concentration of 15% to 25% dextrose and 3% to 5% crystalline amino acids. The solutions usually are prepared in sterile conditions in the pharmacy from commercially available kits containing the component solutions and transfer apparatus. Preparation in the pharmacy under laminar flow hoods reduces the incidence of bacterial contamination of the solution. Proper preparation with suitable quality control is absolutely essential to avoid septic complications.

The proper provision of electrolytes and amino acids must take into account routes of fluid and electrolyte loss, renal function, metabolic rate, cardiac function, and the underlying disease state.

Intravenous vitamin preparations also should be added to parenteral formulas. Vitamin deficiencies are rare occurrences if such preparations are used. In addition, because vitamin K is not part of any commercially prepared vitamin solution, it should be supplemented on a weekly basis. During prolonged parenteral nutrition with fat-free solutions, essential fatty acid deficiency may become clinically apparent and manifests as dry, scaly dermatitis and loss of hair. The syndrome may be prevented by periodic infusion of a fat emulsion at a rate equivalent to 10% to 15% of total calories. Essential trace minerals may be required after prolonged TPN and may be supplied by direct addition of commercial preparations. The most frequent presentation of trace mineral deficiencies is the eczematoid rash developing both diffusely and at intertriginous areas in zinc-deficient patients. Other rare trace mineral deficiencies include a microcytic anemia associated with copper deficiency and glucose intolerance presumably related to chromium deficiency. The latter complications are seldom seen except in patients receiving parenteral nutrition for extended periods. The daily administration of commercially available trace mineral supplements will obviate most such problems.

Depending on fluid and nitrogen tolerance, parenteral nutrition solutions generally can be increased over 2 to 3 days to achieve the desired infusion rate. Insulin may be supplemented as necessary to ensure glucose tolerance. Administration of additional intravenous fluids and electrolytes may occasionally be necessary in patients with persistently high fluid losses. The patient should be carefully monitored for development of electrolyte, volume, acid-base, and septic complications. Vital signs and urinary output should be measured regularly, and the patient should be weighed regularly. Frequent adjustments of the volume and composition of the solutions are necessary during the course of therapy. Samples for measurement of electrolytes are drawn daily until levels are stable and every 2 or 3 days thereafter. Blood counts, blood urea nitrogen level, levels of liver function indicators, and phosphate and magnesium levels are determined at least weekly.

The urine or capillary blood glucose level is checked every 6 hours, and serum glucose concentration is checked at least once daily during the first few days of the infusion and at frequent intervals thereafter. Relative glucose intolerance, which often manifests as glycosuria, may occur after initiation of parenteral nutrition. If blood glucose levels remain elevated or glycosuria persists, the dextrose concentration may be decreased, the infusion rate slowed, or regular insulin added to each bottle. The rise in blood glucose concentration observed after initiating parenteral nutrition may be temporary, as the normal pancreas increases its output of insulin in response to the continuous carbohydrate infusion. In patients with diabetes mellitus, additional insulin may be required.

Potassium is essential to achieve positive nitrogen balance and replace depleted intracellular stores. In addition, a significant shift of potassium ion from the extracellular to the intracellular space may take place because of the large glucose infusion, with resultant hypokalemia, metabolic alkalosis, and poor glucose utilization. In some cases as much as 240 mEq of potassium ion daily may be required. Hypokalemia may cause glycosuria, which would be treated with potassium, not insulin. Thus, before giving insulin, the serum potassium level must be checked to avoid exacerbating the hypokalemia.

Patients with insulin-dependent diabetes mellitus may exhibit wide fluctuations in blood glucose levels while receiving parenteral nutrition. This may require protocol-driven intravenous insulin therapy. In addition, partial replacement of dextrose calories with lipid emulsions may alleviate these problems in selected patients.

Lipid emulsions derived from soybean or safflower oils are widely used as an adjunctive nutrient to prevent the development of essential fatty acid deficiency, although recent data support reducing the overall ω-6 PUFA load in favor of ω-3 PUFAs or MCTs. There is no evidence of enhanced metabolic benefit when >10% to 15% of calories are provided as lipid emulsions. Although the administration of 500 mL of 20% fat emulsion one to three times a week is sufficient to prevent essential fatty acid deficiency, it is common to provide fat emulsions on a daily basis to provide additional calories. The triple mix of carbohydrate, fat, and amino acids is infused at a constant rate during a 24-hour period. The theoretical advantages of a constant fat infusion rate include increased efficiency of lipid utilization and reduction in the impairment of reticuloendothelial function normally identified with bolus lipid infusions. The addition of lipids to an infusion bag may alter the stability of some micronutrients in a dextrose–amino acid preparation.

The delivery of parenteral nutrition requires central intravenous access. Temporary or short-term access can be achieved with a 16-gauge percutaneous catheter inserted into a subclavian or internal jugular vein and threaded into the superior vena cava. More permanent access with the intention of providing long-term or home parenteral nutrition can be achieved by placement of a catheter with a subcutaneous port for access by tunneling a catheter with a substantial subcutaneous length or threading a long catheter through the basilic or cephalic vein into the superior vena cava.

Complications of Parenteral Nutrition

Technical Complications

One of the more common and serious complications associated with long-term parenteral feeding is sepsis secondary to contamination of the central venous catheter. Contamination of solutions should be also considered but is rare when proper pharmacy protocols have been followed. Central line–associated bloodstream infections (CLA-BSI) occur as a consequence of hematogenous seeding of the catheter with bacteria. One of the earliest signs of systemic sepsis from CLA-BSI may be the sudden development of glucose intolerance (with or without temperature increase) in a patient who previously has been maintained on parenteral alimentation without difficulty. When this occurs, or if high fever (>38.5°C [101.3°F]) develops without obvious cause, a diligent search for a potential septic focus is indicated. Other causes of fever should also be investigated. If fever persists, the infusion catheter should be removed and submitted for culture. If the catheter is the cause of the fever, removal of the infectious source is usually followed by rapid defervescence. Some centers are now replacing catheters considered at low risk for infection over a guidewire. However, if blood cultures are positive and the catheter tip is also positive, then the catheter should be removed and placed in a new site. Should evidence of infection persist over 24 to 48 hours without a definable source, the catheter should be replaced into the opposite subclavian vein or into one of the internal jugular veins and the infusion restarted.¹⁶⁰

The use of multilumen catheters may be associated with a slightly increased risk of infection. This is most likely associated with greater catheter manipulation and intensive use. The rate of catheter infection is highest for those placed in the femoral vein, lower for those in the jugular vein, and lowest for those in the subclavian vein. When catheters are indwelling for <3 days, infection risks are negligible. If indwelling time is 3 to 7 days, the infection risk is 3% to 5%. Indwelling times of >7 days are associated with a catheter infection risk of 5% to 10%. Strict adherence to barrier precautions also reduces the rate of infection, as can the implementation of procedure checklists to ensure compliance with evidence-based guidelines shown to reduce infectious risk.¹⁶¹

Other complications related to catheter placement include the development of pneumothorax, hemothorax, hydrothorax, subclavian artery injury, thoracic duct injury, cardiac arrhythmia, air embolism, catheter embolism, and cardiac perforation with tamponade. All of these complications may be avoided by strict adherence to proper techniques. Further, the use of ultrasonographic guidance during central venous line placement has been demonstrated to significantly decrease the failure rate, complication rate, and number of attempts required for successful access.¹⁶²

Metabolic Complications

Hyperglycemia may develop with normal rates of infusion in patients with impaired glucose tolerance or in any patient if the hypertonic solutions are administered too rapidly. This is a particularly common complication in patients with latent diabetes and in patients subjected to severe surgical stress or trauma. Treatment of the condition consists of volume replacement with correction of electrolyte abnormalities and the administration of insulin. This complication can be avoided with careful attention to daily fluid balance and frequent monitoring of blood glucose levels and serum electrolytes.

Increasing experience has emphasized the importance of not overfeeding the parenterally nourished patient. This is particularly true for the depleted patient in whom excess calorie infusion may result in carbon dioxide retention and respiratory insufficiency. In addition, excess feeding also has been related to the development of hepatic steatosis or marked glycogen deposition in selected patients. Cholestasis and formation of gallstones are common in patients receiving long-term parenteral nutrition. Mild but transient abnormalities of serum transaminase, alkaline phosphatase, and bilirubin levels occur in many parenterally nourished patients. Failure of the liver enzymes to plateau or return to normal over 7 to 14 days should suggest another etiology.

Intestinal Atrophy

Lack of intestinal stimulation is associated with intestinal mucosal atrophy, diminished villous height, bacterial overgrowth, reduced lymphoid tissue size, reduced IgA production, and impaired gut immunity. The full clinical implications of these changes are not well realized, although bacterial translocation has been demonstrated in animal models. The most efficacious method to prevent these changes is to provide at least some nutrients enterally. In patients requiring TPN, it may be feasible to infuse small amounts of feedings via the gastrointestinal tract.