1297 e1 • eBOX 110 2 Common Features of Cytokines • Cytokine secretion is relatively brief and self limited • Secretion of many cytokines requires new mRNA transcription and new protein translation •[.]
1297.e1 • eBOX 110.2 Common Features of Cytokines • Cytokine secretion is relatively brief and self-limited • Secretion of many cytokines requires new mRNA transcription and new protein translation • Expression and secretion is regulated by specific cellular signals • A given cytokine can have multiple cellular sources • A given cytokine can have multiple cellular targets • A given cytokine can have multiple functions regarding cellular function or activation • Cytokines can have redundant activities/functions with other cytokines • Many cytokines regulate the activity and expression of other cytokines mRNA, Messenger ribonucleic acid 1298 S E C T I O N X I Pediatric Critical Care: Immunity and Infection IL-18 has the ability to skew naïve T cells toward either a Th1 or Th2 phenotype In addition, it appears that IL-18 may serve as an early biomarker to distinguish between gram-positive and gramnegative sepsis IL-10 is the best studied and most well-known antiinflammatory cytokine.48,49 As an antiinflammatory cytokine, IL-10 antagonizes the proinflammatory effects of other cytokines and can thereby keep inflammation in check IL-10 inhibits expression of cytokines such as TNF-a, IL-1b, and IL-8 and can inhibit expression of adhesion molecules In addition, IL-10 can “deactivate” monocytes by downregulating the expression of major histocompatibility complex (MHC) surface molecules Thus, IL-10 has a number of interesting properties that could potentially be leveraged therapeutically to limit excessive inflammation during sepsis This theoretical consideration must be tempered by the ability of IL-10 to deactivate monocytes and thereby potentially impair the ability to adequately clear infection (i.e., the immunosuppression paradigm depicted in Fig 110.1) Indeed, it has been reported that in children with MODS, higher plasma IL-10 levels correlate with higher mortality and that higher monocyte messenger ribonucleic acid (mRNA) levels of IL-10 correlate with increased length of stay in the ICU.50 Similar observations have been reported in adult patients with septic shock.51 High-mobility group box (HMGB-1) has long been known as a nonhistone DNA binding protein More recently, it has been recognized that HMGB-1 also exists in the extracellular compartment, appears to have proinflammatory properties that may play a role in the pathophysiology of sepsis, and may represent a potential therapeutic target for sepsis.52 The attraction of HMGB-1 as a therapeutic target in sepsis stems from the observation that it may be a “late mediator” of sepsis in as much as it appears in the extracellular compartment within a time frame that is considerably later than that seen with the canonical sepsis cytokines such as TNF-a and IL-1b Thus, the kinetics of HMGB-1 expression provide a potential therapeutic window that may be clinically feasible to exploit This temporal observation is evident in both experimental models of sepsis and in humans with established septic shock.53 The biological properties of HMGB-1 appear to involve activation of TLRs and the receptor for advanced glycation end products (RAGE) More recently, it has been suggested that HMGB-1 intrinsically possesses very little proinflammatory biological activity but forms highly proinflammatory complexes with cytokines (e.g., IL-1b) and PAMPs (e.g., bacterial DNA and lipopolysaccharide).54 HMGB-1 is also a prime example of a class of molecules known as alarmins or danger/damage-associated molecular patterns (DAMPs).55 Broadly speaking, DAMPs represent a class of molecules normally existing in the intracellular compartment at baseline but are released from damaged cells into the extracellular compartment during conditions such as trauma or sepsis DAMPs appear to signal through many of the same PRRs recognizing pathogens; therefore, they have the ability to activate the immune/inflammatory system Because DAMPs are released from damaged cells, they can serve to alert the inflammatory system of systemic damage or danger Therefore, they can induce appropriate and adaptive activation of defense mechanisms Alternatively, excessive DAMP-mediated activation of PRRs can lead to unnecessary and maladaptive amplification of inflammation damaging to host tissues Other examples of DAMPs include calgranulins, hepatoma-derived growth factor, heat shock proteins, and uric acid In this regard, heat shock proteins have been reported to be substantially elevated in the serum of children with septic shock.56,57 More recently, formyl peptides released from mitochondria and mitochondrial DNA were reported as novel DAMPs.58 Adhesion Molecules An important breakthrough in the molecular understanding of sepsis-induced organ dysfunction came with the identification of the processes responsible for the infiltration of leukocytes into tissues.59 The leukocyte-endothelial cell adhesion cascade (Fig 110.2) is characterized by early cytokine-mediated activation of the selectin family of endothelial cell adhesion molecules that can mediate a process of neutrophil “rolling” whereby sialylated moieties constitutively present on neutrophils interact with selectins on the endothelial cell membrane (e.g., E-selectin) In the second phase, activation of the rolling neutrophil causes increased expression and activation of the integrin family of adhesion molecules that interact with the similarly upregulated intercellular adhesion molecule (ICAM)-1 on the endothelial cell surface This ligand interaction facilitates firm adhesion of the neutrophil to the endothelium Subsequently, in response to a variety of chemotactic molecules, neutrophils transmigrate through the endothelial junctions to the site of inflammation Release of a variety of radical species, both oxygen and nitrogen based, and proteases by the activated neutrophils can contribute to pathogen eradication but paradoxically can also cause endothelial and tissue injury Nitric Oxide NO was discovered in the 1980s as the molecule responsible for endothelial-derived relaxation of blood vessels.60,61 Since then, NO has received tremendous attention as a potential mediator of septic shock.62 NO is produced by the enzyme nitric oxide synthase (NOS), which converts arginine and oxygen to NO and citrulline Human NOS exists as three different isoforms (NOS1, NOS2, and NOS3) Each isoform has relatively unique tissue localizations, requirements for NO production, and kinetics of NO production Several features of NO-related biology support an important role in the pathophysiology of sepsis First, NOS2 (also known as inducible NOS) is expressed in response to proinflammatory signals (e.g., lipopolysaccharide, TNF-a, and IL-1b) and produces large amounts of NO for prolonged periods of time Second, NO can induce pathologic vasodilation and can function as a myocardial depressant Third, NO can function as an oxidant either alone or by contributing to formation of other highly oxidizing, reactive molecules such as peroxynitrite Fourth, NO has the potential to negatively affect mitochondrial function Finally, elevated levels of NO metabolites have been well documented in children with septic shock and the levels correlate with the degree of cardiovascular dysfunction.63,64 Despite these intriguing biological properties and a wealth of preclinical data testing the efficacy of NOS inhibition, clinical trials targeted at NOS inhibition have failed to demonstrate efficacy.65 Because NO has a myriad of biological properties important for homeostasis (particularly when NO is produced by the NOS1 and NOS3 isoforms), this lack of efficacy may represent the timing and specificity of NOS isoform inhibition Coagulation Cascade It is now well established that the inflammatory cascade is directly linked to the coagulation cascade, and the coagulation cascade can CHAPTER 110 Pediatric Sepsis 1299 PMN sialyl-Lewisx β2-integrin E-selectin PCAM-1 ICAM-1 Endothelial cell CXCL8 IL-8 Rolling Adhesion Transmigration/ chemotaxis • Fig 110.2 Schematic and corresponding electron micrographs highlighting the process of leukocyte- endothelial cell adhesion and leukocyte transmigration from the intravascular compartment to the extravascular compartment Cytokine-mediated activation of the selectin family of endothelial cell adhesion molecules mediate neutrophil “rolling” followed by ICAM-1–mediated adhesion After adhesion, neutrophils transmigrate across “openings” between endothelial cell junctions to enter the extravascular space The transmigration process is directed by chemokines serving as homing signals for neutrophils and other leukocytes ICAM-1, Intercellular adhesion molecule; IL-8, interleukin-8; PMN, polymorphonuclear (Courtesy Thomas P Shanley, MD, University of Michigan.) be pathologically activated in the context of sepsis.66,67 This pathologic activation leads to disseminated intravascular coagulation, which subsequently leads to endothelial cell dysfunction and microvascular thrombosis If endothelial dysfunction and microvascular thrombosis progress to a critical threshold, end organ failure ensues A complex network of multiple mediators takes part in this pathologic process, including proinflammatory cytokines, tissue factor, antithrombin III, protein C, protein S, tissue factor pathway inhibitor, and plasminogen activator inhibitor type (PAI-1) Increased PAI-1 levels are a particularly strong feature of severe cases of meningococcemia and may be causally linked to a polymorphism in the promoter region of the PAI-1 gene.68,69 Decreased levels of the endogenous anticoagulants antithrombin III, protein S, and protein C are consistently documented in the context of septic shock These observations have led to multiple clinical trials in which recombinant forms of these endogenous anticoagulants have been administered to patients with septic shock The majority of these trials have not demonstrated efficacy Studies in the early 2000s suggested that recombinant activated protein C (APC) reduced mortality in adult patients with septic shock This compound subsequently received approval by the US Food and Drug Administration (FDA).61 The beneficial effects of APC in septic shock are thought to be secondary to both prevention of microvascular thrombosis and an antiinflammatory effect.70 Unfortunately, a phase III trial of APC therapy in children with septic shock, the RESOLVE trial, failed to demonstrate efficacy.71 The RESOLVE trial was terminated after the second interim analysis owing to little chance of reaching the primary efficacy end point In addition, there was an increased risk of serious bleeding in patients younger than months of age Consistent with this finding in children, a subsequent trial in adults with septic shock, PROWESS-SHOCK, also failed to confirm earlier findings of improved mortality, and APC was removed from the market in 2011.72 Nonetheless, the RESOLVE trial represents the largest and most well organized pediatric septic shock trial to date It provides an important context and reference point for all future interventional trials in the field of pediatric critical care medicine Related to the paradigm of altered coagulation playing an important role in the pathophysiology of sepsis is the concept of thrombocytopenia-associated multiple-organ failure (TAMOF).73 New-onset thrombocytopenia in critically ill patients correlates with the evolution of persistent organ failure and poor outcome, including patients with sepsis The mechanistic link between thrombocytopenia and organ failure is thought to involve a form of microangiopathy analogous to thrombotic thrombocytopenic purpura, including substantial decreases of ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 13 motifs) ADAMTS-13 regulates microvascular thrombosis by cleaving the large thrombogenic von Willebrand factor multimers into smaller, less thrombogenic forms Preliminary experience indicates that plasma exchange restores ADAMTS-13 levels and restores organ function in children with TAMOF.74 Fortenberry and colleagues conducted a longitudinal observational study of 81 pediatric patients with sepsis-induced TAMOF and found that therapeutic plasma exchange was associated with improved organ dysfunction and decreased 28-day mortality.75 Ultimately, the efficacy of plasma exchange for TAMOF will require more definitive evidence by way of a formal randomized trial Peroxisome Proliferator-Activated Receptor-g Pathway Peroxisome proliferator-activated receptor-g (PPARg) is a member of the PPAR nuclear receptor superfamily and is a ligandactivated transcription factor having well-known effects on lipid 1300 S E C T I O N X I Pediatric Critical Care: Immunity and Infection metabolism and cell proliferation.76 The thiazolidinedione class of insulin-sensitizing drugs are well-known PPARg ligands (activators) that are currently widely used in the management of type II diabetes Recently, it has become evident that pharmacologic activation of PPARg has important antiinflammatory effects of significant benefit in experimental models of critical illness, including sepsis.77–82 The recent demonstration that PPARg expression and activation is altered in children with septic shock, coupled with the availability of FDA-approved PPARg ligands, provides an opportunity to test the efficacy of PPARg ligands in sepsis.83 Kaplan et al recently reported a phase I safety and pharmacokinetic study of pioglitazone, a PPARg ligand, in critically ill children with sepsis.84 Myeloid-Derived Suppressor Cells The neutrophil is often considered the first line in the innate immune response to infection Patients with sepsis can present with elevated or extremely low neutrophil counts In either case, the physiologic response to infectious stimuli is “emergency granulopoiesis” to generate a large number of myeloid cells to deal with the onslaught of infectious particles This can lead to a much needed increase in cells to both contain and eliminate pathogens However, in patients with sepsis, this can also give rise to a recently described myeloid-derived suppressor cell (MDSC) population.85 These cells produce antiinflammatory cytokines and suppress T-cell activation; thus, they may contribute in part to the failure of the adaptive immune system.86 These cells have been primarily characterized in the mouse model of sepsis, in which up to 30% of cells in the spleen 10 days after sepsis are MDSCs.87 Whether these cells are helpful to the immune response or lead to further immunosuppression is not entirely clear.87,88 MDSCs can also be found in patients with other inflammatory conditions, such as cancer and autoimmunity.85 Further research is needed to determine if MDSCs represent a physiologically orchestrated part of the immune response or a pathologic arrest of developing myeloid cells leading to immunosuppression Paradigm of Sepsis as an Adaptive Immune Problem Our conceptual framework of the pathophysiology of sepsis has evolved to include the concept of immune paralysis Whereas sepsis has been traditionally viewed as being a reflection of uncontrolled hyperinflammation (i.e., an innate immunity problem), it is now thought that sepsis also has a strong, perhaps predominant, “antiinflammatory” component manifested as immunosuppression and the relative inability to effectively clear an infectious challenge (an adaptive immunity problem).22,23,89,90 For example, monocyte deactivation related to decreased MHC gene mRNA expression and decreased surface expression of MHC molecules have been previously demonstrated in patients with septic shock, including children.50,51,91,92 With regard to lymphocyte dysfunction, Heidecke and colleagues demonstrated that adult patients with intraabdominal infections and septic shock have defective T-cell proliferation and defective T cell-dependent cytokine secretion, all of which is consistent with anergy/immunosuppression.93 Felmet and colleagues identified prolonged lymphopenia and apoptosis-associated depletion of lymphoid organs as independent risk factors for the development of nosocomial infections and multiple organ failure in critically ill children.94 Muszynski and colleagues recently provided evidence for early adaptive immune dysfunction in children with septic shock, as measured by ex vivo production of interferon-g by CD4 cells.95 Animal studies have well documented the requirement of an intact T-cell system to adequately combat a septic challenge.90,96 Interestingly, however, neonatal mice (4–6 days of age) not appear to require an intact adaptive immune system to clear infection.97 More recently, animal-based experiments have demonstrated that experimental septic shock is characterized by widespread apoptosis of T cells and that preventing T-cell apoptosis positively impacts the outcome of experimental sepsis.98–105 Importantly, the concept of T-cell apoptosis in human sepsis has been indirectly corroborated by autopsy studies, including children,94,106 and lymphocyte-based immunophenotyping was recently demonstrated to effectively stratify septic shock outcome in adults.107 Finally, it has been recently demonstrated in experimental models that alterations of the adaptive immune system in sepsis can persist well beyond the acute period (up to at least weeks) via epigenetic mechanisms involving dendritic cells.108,109 Despite these data, formal studies of T-cell function and adaptive immunity in pediatric septic shock have never been conducted in a systematic and comprehensive manner Such studies hold the promise of radically changing our conceptual approach to the long sought, but not yet realized, goal of rational immunomodulation in septic shock Recently, Hotchkiss et al conducted a phase I trial of immune checkpoint inhibition in adults with sepsis using an antiprogrammed cell death–ligand antibody.110 Genomic Medicine and Sepsis The initial completion and publication of the human genome, the development of molecular biology tools for efficient highthroughput data generation, and the evolution of the field of biomedical informatics have combined to generate a new field termed genomic medicine and the related field of systems biology All aspects of medicine are potentially amenable to the concepts of genomic medicine and systems biology, including pediatric sepsis One skeptical perspective of this concept is that clinical pediatric sepsis is too heterogeneous and multifactorial to be credibly interrogated by the current genomic and systems biology approaches An alternative and more optimistic perspective is that the concepts of genomic medicine and systems biology are ideally suited to more effectively address the complex syndromes that we encounter in pediatric critical care medicine, such as septic shock Herein, we will address the two areas of genomic medicine most well developed in the field of pediatric septic shock: candidate gene association studies and genome-wide expression profiling Genetic Influence and Septic Shock Susceptibility to sepsis and the clinical course of patients with sepsis are both highly heterogeneous, raising the strong possibility that the host response to infection is, at least in part, influenced by heritable factors (i.e., genetics).111 A landmark study by Sorensen et al., published more than 25 years ago, provides strong evidence linking genetics and susceptibility to infection.112 This study involved a longitudinal cohort of more than 900 adopted children born between 1924 and 1926 The adopted children and both their biological and adoptive parents were followed through 1982 If a biological parent died of infection before the age of 50 years, the relative risk of death from infectious causes in the child was 5.8 (95% CI, 2.5–13.7), which was higher than for all other causes studied, including cancer and cardiovascular/cerebrovascular CHAPTER 110 Pediatric Sepsis disease In contrast, the death of an adoptive parent from infectious causes did not confer a greater relative risk of death in the adopted child More recently, investigations attempting to link genetics with sepsis focused mainly on candidate gene association studies and gene polymorphisms A gene polymorphism is defined as the regular occurrence (.1%) in a population of two or more alleles at a particular chromosome location The most frequent type of polymorphism is called a single-nucleotide polymorphism (SNP): a substitution, deletion, or insertion of a single nucleotide occurring in approximately per every 1000 base pairs of human DNA SNPs can result in an absolute deficiency in protein, an altered protein, a change in the level of normal protein expression, or no discernible change in protein function or expression There is a growing body of literature linking SNPs within several genes regulating inflammation, coagulation, and the immune response with critical illness and several excellent reviews exist on the topic.113–116 The signaling mechanisms involved in pathogen recognition, immune response, and inflammation were described in previous sections In this section, we will provide an overview of relevant SNPs described in many of the genes involved in these signaling mechanisms TLR-4 (the primary receptor for recognition of lipopolysaccharide) mutations have been described in humans, all of which increase susceptibility to infections secondary to gramnegative organisms.117 While several SNPs in the TLR-4 receptor gene have been described, few have been found to be associated with an increased risk of septic shock or septic shock–related mortality in children For example, an adenine for guanine substitution 896 base pairs downstream of the transcription start site for TLR-4 (1896) results in replacement of aspartic acid with glycine at amino acid 299 (Asp299Gly) The Asp299Gly polymorphism has been associated with reduced expression and function of the TLR-4 receptor in vitro.117,118 Furthermore, adults who carry the Asp299Gly polymorphism appear to be at increased risk for septic shock and poor outcome in several cohort studies.119–121 While children who carry the Asp299Gly polymorphism appear to be at increased risk of urinary tract infection, this SNP does not appear to influence either the susceptibility or severity of meningococcal septic shock in children.122,123 These results were further corroborated in a cohort study involving over 500 Gambian children.124 SNPs related to other members of the LPS-receptor complex (e.g., CD14, MD-2, and MyD88) have been studied in adult populations, but no such studies have yet to be performed in children.120,125–128 SNPs in other classes of TLRs have also been studied For example, gene polymorphisms of TLR-1 and TLR-2, the primary PRRs for gram-positive bacteria, have been associated with increased length of hospitalization in pediatric sepsis and risk of infection in children and adults.129–132 Several SNPs affecting cytokine expression have been described, but the corresponding gene association studies in critically ill adults with septic shock have been conflicting.115,116,133 For example, two allelic variants of the TNF-a gene have been described: the wild-type allele TNF1 (guanine at 2308A), and TNF2 (adenosine at 2308A) The TNF2 allele has been associated with higher expression of TNF-a and increased susceptibility to septic shock and mortality in at least one study involving critically ill adults.134 Nadel and colleagues found an increased risk of death in critically ill children with meningococcal septic shock who carried the TNF2 allelic variant.135 Several additional SNPs in TNF-b, IL-1, IL-6, IL-8, and IL-10 have also been shown to influence susceptibility to and severity of septic shock in children.136–142 1301 Because dysregulation of the coagulation cascade plays an important role in the pathophysiology of septic shock, several studies have examined polymorphisms of key genes involved in coagulation For example, the 4G allele of a deletion/insertion (4G/5G) SNP in the promoter region of the PAI-1 gene has been associated with higher plasma concentrations of PAI-1 The 4G allele increases susceptibility to and severity of septic shock as well as increasing the risk of mortality in children with meningococcal septic shock.68,143–145 In addition, an SNP in the protein C promoter region has been associated with susceptibility to meningococcemia and illness severity in children.146 SNPs in genes involved in phagocytosis and the complement cascade have also been studied in the context of septic shock For example, SNPs affecting function have been described in virtually all family members of the Fcg receptor (important for phagocytosis) Several of these SNPs have been associated with susceptibility to meningococcal sepsis, severity of meningococcemia, and poor outcome from meningococcal septic shock 147–153 In addition, an association between the FcgRIIa polymorphism and infection with other encapsulated bacteria has also been reported.154,155 Several SNPs in the mannose binding lectin (MBL) gene have been associated with increased susceptibility to infection, as well as increased illness severity.156–161 Finally, an SNP of the bactericidal permeability increasing protein (BPI) gene has also been associated with increased mortality from septic shock in children.162 This polymorphism is particularly interesting because a well-conducted phase III trial of recombinant BPI in children with septic shock failed to demonstrate efficacy.163 It is likely that many more studies are forthcoming that will attempt to link SNPs with the susceptibility and/or outcome of pediatric septic shock; all need to be carefully considered and evaluated With respect to validity and wide clinical acceptance, the ideal candidate gene association study requires several important qualities, including biological plausibility; large sample sizes; a priori hypothesis statements and power calculations, accounting for confounding factors; and independent validation.164 Genome-Wide Expression Profiling in Children With Septic Shock The development of high-throughput technologies to measure gene expression has provided an unprecedented opportunity to efficiently measure genome-wide mRNA expression patterns in clinical samples Over the last decade, this approach has been leveraged to enable more comprehensive understanding of the pathophysiology of pediatric septic shock and as a means of discovery and hypothesis generation Comprehensive reviews of these studies were recently published.165–167 The first studies to characterize the transcriptomic response of pediatric septic shock confirmed that septic shock is characterized by upregulation of gene programs corresponding to innate immunity and the inflammatory response.168,169 These studies also noted concomitant downregulation of gene programs corresponding to adaptive immunity, which is consistent with the immune paralysis paradigm described earlier These gene expression patterns are evident within 24 hours of admission to the ICU and persist at least through ICU day 3.170 Other notable data generated from these transcriptomic studies include (1) the observation that pediatric septic shock is characterized by repression of gene programs that either depend on zinc homeostasis or directly participate in zinc homeostasis168; (2) characterizing distinct gene programs across the spectrum of SIRS, sepsis, and septic shock171; ... onslaught of infectious particles This can lead to a much needed increase in cells to both contain and eliminate pathogens However, in patients with sepsis, this can also give rise to a recently... may be a “late mediator” of sepsis in as much as it appears in the extracellular compartment within a time frame that is considerably later than that seen with the canonical sepsis cytokines... HMGB-1 expression provide a potential therapeutic window that may be clinically feasible to exploit This temporal observation is evident in both experimental models of sepsis and in humans with established