Sepsis is a complex syndrome that is initiated by infection and is characterized by a systemic inflammatory response. Annually, approximately 750,000 people are diagnosed with this condition, resulting in more than 210,000 attributable deaths (1). Death from sepsis is primarily because of the development of sequential organ dysfunction. The preponderance of research in sepsis has focused on dissecting the roles of the immune system cells, innate immune regulation, cytokines, and coagulation factors in response to varying infectious and inflammatory mediators. More recently, the focus has evolved to the identification of genetic variation in crucial genes in the inflammatory response and coagulation pathways. In particular, identification of genetic variation in the Toll-like receptors (TLRs) and proinflammatory cytokines (Fig. 1) has provided valuable insights into the influence of genetic heterogeneity on the response to bacterial infection. Delineating the variation in genes and associated differences in response to infection may contribute to the development of new genetically tailored diagnostic and therapeutic interventions that will improve outcome in this patient population.
A single nucleotide polymorphism (SNP) is the most common type of stable genetic variation in the population (2). A SNP occurs in approximately 1 of 1000 base pairs, with the most frequent being a C to T substitution. There are several different ways that SNPs can lead to an aberrant gene product. First, sequence variation in the 5′ untranslated region (UTR) could disrupt mRNA translation, and mutations in the 3′ UTR could affect mRNA cleavage, stability, and export (3). Second, promoter polymorphisms that alter DNA binding of transcription factors have the potential of decreasing or increasing gene expression. Third, frameshift mutations or variation that results in early termination in transcription may lead to a defective or truncated protein. Fourth, splice site mutations that occur in intron/exon boundaries also have the potential of altering mRNA processing or protein function. Finally, nonsynomous SNPs in exons could alter protein function or activity. It has been estimated that 10% of all SNPs in the genome are functional, thereby having the potential of altering some biological process (3).
In the last 5 years, there has been increasing interest in conducting disease-gene association-based studies aimed at determining the role of genetic variation in the inflammatory response to infection. The majority of these studies have been case-control studies using a candidate gene approach. A second approach has involved the identification and study of multiple SNPs or haplotypes that are in linkage disequilibrium with one another (4). This method increases the statistical power for detecting the association between genotype and outcome (4). Both study designs have generated important information in deciphering the role of genetic variation on clinical outcomes in sepsis.
INNATE IMMUNE RECEPTORS AND RECOGNITION OF BACTERIAL PRODUCTS
Macrophages, dendritic cells, neutrophils, and other cell populations express TLRs that play a central role in the innate immune response to infection through the recognition of distinct bacterial antigens. More specifically, TLR2 and TLR4 play a central role in the recognition of components of gram-positive and gram-negative bacteria, respectively. All TLRs have a conserved cytoplasmic region (∼200 amino acids), known as the Toll-IL-1R (TIR) domain, that is involved in triggering intracellular signals, culminating in the translocation of the transcriptional regulatory factor NF-κB into the nucleus, where it participates in enhancing the expression of proinflammatory cytokines and other immunoregulatory mediators. Because the ability to elicit a prominent inflammatory response to bacteria is so highly dependent on TLRs, there has been interest in understanding if genetic variation in these receptors may be responsible for the disparity seen in the susceptibility, severity, and mortality related to bacterial infections.
The importance of TLR4 in the innate immune response to gram-negative bacteria has been elucidated in mouse models of endotoxemia. Several groups found that C3H/HeJ mice were hyporesponsive to endotoxin (5, 6). A study by Poltorak et al. (7) showed that a mutation in the TLR4 gene at position 712 (a mutation in the TIR domain), in which proline was substituted for histidine, was responsible for this defect. The identification of the TLR4 mutation in these mice, as well as other TLR4-deficient mice (129/SvJ and 10ScCR), has provided valuable insight into the innate immune responses to gram-negative bacteria. For example, Schurr et al. (8) demonstrated in a mouse model of pneumonia that mice deficient in TLR4 signaling (C3H/HeJ and 129/SvJ) had decreased ability to clear Klebsiella pneumoniae from the lung and a profound increase in mortality when compared with mice with a functional TLR4 receptor.
There have been two specific SNPs identified in the TLR4 gene in humans. These consist of a substitution of the conserved aspartic acid to glycine at amino acid 299 and the replacement of threonine to isoleucine at amino acid 399 (9). Arbour et al. (9) investigated the relevance of these two polymorphisms in relation to TLR4 function. They recruited a total of 83 subjects to study the effects of inhaled endotoxin on lung function. Genotyping of these individuals revealed a polymorphism at position 299 Asp→Gly that cosegregated with the 399 Thr→Ile polymorphism in 10 subjects. Individuals with the 299/399 polymorphisms were shown to be hyporesponsive to inhaled endotoxin as determined by an attenuated drop in FEV1. Furthermore, in comparison with TLR4 wild type, changes in 299 Asp→Gly, but not 399 Thr→Ile, significantly reduced nuclear levels of NF-κB in lipopolysaccharide (LPS)-stimulated THP-1 cells. In response to LPS, airway epithelial cells, with the 299/399 polymorphisms, had reduced levels of IL-1α; the addition of adenovirus expressing wild-type TLR4 in these cells repaired the 299/399 TLR4 deficiency, resulting in increased levels of IL-1α.
Following observations in mice and human volunteers that variation in TLR4 could alter the response to gram-negative bacteria or endotoxin, studies have been conducted to evaluate the 299 Asp→Gly and 399 Thr→Ile polymorphisms in patients with gram-negative sepsis (10-16). Agnese et al. (10) investigated whether the TLR4 alleles 299/399 were associated with increased risk of gram-negative bacterial infections. In comparison with TLR4 wild type, patients heterozygous for TLR4 299/399 polymorphisms had a significant increase (P = 0.004) in gram-negative bacterial infections and a trend (P = 0.077) toward an increase in 28-day all-cause mortality. Consistent with these findings, a separate study showed that the 299/399 alleles were more prevalent in patients with gram-negative bacterial infections (11). Furthermore, the TLR4 299 polymorphism has been reported to be associated with septic shock (P = 0.05) (11) and a trend (P = 0.076) in mortality in septic patients with systemic inflammatory response syndrome (12). Barber et al. (13) showed that patients with burn injury with the 299 Asp→Gly polymorphism were 1.8 times more likely to develop severe sepsis.
In contrast, there have been several studies in which no association was shown between the TLR4 299/399 alleles and sepsis. Specifically, the TLR4 299/399 polymorphisms did not influence the incidence of sepsis after surgery (14). In addition, a large study involving 1047 patients diagnosed with meningococcal meningitis failed to show an association between patients with meningitis and the 299 polymorphism (15). In that study, the 299 Asp→Gly allele frequencies were 5.9% in volunteers, 6.5% in patients, and 4.1% among 86 nonsurvivors, thus demonstrating that the 299 allele does not influence the susceptibility to or severity of meningococcal meningitis. Another study involving patients with meningococcal disease found similar results; there was no association between alterations in the 299 allele and risk for meningococcal meningitis (16). Together, these studies on TLR4 polymorphisms suggest that individuals with the 299/399 polymorphisms may have an aberrant response to certain, but not all, gram-negative bacterial infections, resulting in an increased susceptibility to infection and severity of disease.
TLR2 plays an essential role in the recognition of gram-positive bacterial components such as peptidoglycan, lipoteichoic acid, and lipoproteins (17-21). There have been two main polymorphisms identified in the TLR2 gene, consisting of the substitution of an arginine with tryptophan at amino acid 677 and the replacement of an arginine with glutamine at amino acid 753 (22, 23). Both of these polymorphisms are situated within the cytoplasmic tail (TIR domain) of the TLR2 receptor, and potentially could affect intracellular signaling.
Two studies were conducted to determine if polymorphisms at amino acid 753 or 677 led to aberrant TLR2 function (23, 24). In the first, 293T cells were transfected with TLR2 wild type or TLR2 753 Arg→Gln and stimulated with gram-positive Borrelia burgdorferi (causative spirochete agent of Lyme disease)-derived lipidated OspA or Treponemia pallidum-derived lipidated 47L (23). Whereas TLR2 wild type-transfected cells had a substantial activation of NF-κB-dependent luciferase expression, cells transfected with TLR2 753 Arg→Gln were unresponsive to OspA or 47L. Second, in comparison with TLR2 wild type cells, TLR2 677 Arg→Trp-transfected cells were shown to have decreased levels of NF-κB-dependent luciferase expression in response to Mycobacterium leprae or Mycobacterium tuberculosis (24). Both of these studies revealed that polymorphisms involving 753 or 677 of the TLR2 gene result in reduced NF-κB-dependent transcriptional activity in response to a variety of gram-positive-derived products.
The TLR2 753 Arg→Gln and 677 Arg→Trp polymorphisms have been investigated in patients with gram-positive infections. Specifically, a case-control study of 45 controls and 45 patients showed that the 677 Arg→Trp polymorphism was associated with an increased susceptibility to lepromatous leprosy (22). In addition, the TLR2 677 and 753 polymorphisms have been shown to be a risk factor for susceptibility to M. tuberculosis (25, 26). A study by Ben-Ali et al. (25) reported that the allele frequency of the 677 Arg→Trp polymorphism was significantly (P < 0.0001) elevated in patients with tuberculosis when compared with healthy volunteers. A second study showed similar results, in that individuals with the 753 polymorphism also had an elevated risk of developing tuberculosis (26). Recently, it has been described that TLR1 and TLR2 can form heterodimers, further potentiating responses to certain gram-positive components. A study by Takeuchi et al. (27) showed that mice deficient in TLR1 had decreased TNFα production when stimulated with a 19-kDa lipoprotein from M. tuberculosis. This result suggests that TLR1 may also be involved in the pathogenesis of M. tuberculosis.
The relationship between TLR2 753 polymorphism and Staphylococcus aureus infection has been examined in several studies. Lorenz et al. (23) showed that two out of 91 patients heterozygous for the 753 polymorphism had S. aureus infections. In contrast, a study by Moore et al. (28) failed to show an association between the 753 polymorphism and morbidity or mortality caused by S. aureus. These studies illustrate that the TLR2 753 Arg→Gln and 677 Arg→Trp polymorphisms may predispose individuals to certain gram-positive infections such as tuberculosis or leprosy. However, more studies are warranted to confirm the association between these alleles and other gram-positive infections.
TLR5, which recognizes bacterial flagellin from gram-positive and gram-negative bacteria, plays an important role in mediating responses to this bacterial antigen through activating NF-κB and the release of proinflammatory cytokines (29). A stop codon polymorphism at amino acid 392 Arg→TER (termination) has been identified in the TLR5 gene and is associated with an increased susceptibility to Legionnaires' disease (30). To determine if this polymorphism alters TLR5 function, CHO and HEK293T cells were transfected with TLR5 wild type or TLR5 392 Arg→TER polymorphism and stimulated with flagellin. In comparison with TLR5 wild type, TLR5 392 Arg→TER CHO and HEK293T cells stimulated with flagellin were unable to elicit an NF-κB response, demonstrating that this polymorphism may be biologically functional (30). Interestingly, in the same study, A-549 and Calu-3 cells (lung cell lines) stimulated with Legionellapneumophila had significant release of IL-8. However, these same cell lines were unable to produce any IL-8 when stimulated with Salmonellaminnesota derived LPS at 1 μg/mL. This suggests that TLR5 plays an important role in pulmonary epithelial responses to L. pneumophila and that the TLR5 392 Arg→TER polymorphism may increase susceptibility to pneumonia associated with flagellated organisms.
CD14 functions as an anchor protein and enhances TLR4 or TLR2 responses (31). The soluble form of CD14 is also essential for TLR2 or TLR4 responses in cells that do not possess membrane-bound CD14 (32). Increased serum CD14 levels have been shown to correlate with shock and greater mortality in patients with gram-positive and gram-negative bacterial infections (33, 34). A genetic polymorphism within the promoter region at position −159 C→T of the CD14 gene has been identified and correlates with increased CD14 serum levels (35). A study by Baldini et al. (35) in subjects with asthma or allergies demonstrated that serum levels in subjects with the CC allele were 4.1 μg/mL compared with subjects with the TT allele at 4.5 μg/mL (P = 0.01).
There have been two case-control studies associating the CD14 −159 promoter polymorphism with sepsis. Gibot et al. (36) conducted a multicenter study that included 90 patients with sepsis and 122 age- and gender-matched controls. They reported that the frequency of the TT genotype was higher in patients with sepsis than in controls and that the mortality rate in patients with the TT genotype was significantly increased when compared with patients with the CC or CT genotypes (P = 0.008). In addition, a study by Sutherland et al. (37) confirmed that TT and CT genotypes correlated with increased prevalence of gram-negative bacterial infections (P < 0.03). Therefore, it is plausible that the −159 C→T polymorphism of the promoter region of CD14 is associated with increased serum levels of CD14, the prevalence of gram-negative bacterial infections, and mortality.
Mannose-binding lectin (MBL)
MBL is an acute-phase protein that is involved in the innate immune responses. This acute-phase protein has the ability to bind carbohydrate structures on microbial pathogens, enhancing phagocytosis, and promotes the activation of the complement system (38, 39). Within exon 1 of the MBL gene, three different polymorphisms have been described: 52 Arg→Cys (D allele), 54 Gly→Asp (B allele), and 57 Gly→Glu (C allele) (40-42). Several studies have shown that all the variant alleles are associated with low serum MBL levels and increased susceptibility to a wide range of bacterial infections (41, 43, 44). In addition to variants in exon 1, promoter region polymorphisms have been described at position −221 G→C (allele Y/X) and −550 G→C (allele H/L) (44, 45).
Certain haplotypes are associated with varying serum levels of MBL. For example, individuals with a YA haplotype (Y allele at −221, and A wild-type allele at codons 52, 54, and 57) have high serum MBL levels and those with an O haplotype (Y allele at −221, D allele at codon 52, B allele at codon 54, and A allele 57) have low serum MBL concentrations (37). There is an increase in the incidence of infection and sepsis when individuals have two copies of low MBL haplotypes (37). A study by Sutherland et al. (37) examined the association of specific haplotypes with prevalence of infection, septic shock, and 28-day survival. In this study, patients were divided into a high-MBL haplotype group (YAAA) or low-MBL haplotype group (O), which included the Y allele in the promoter and a combination of at least two allelic variants in D, B, or C. Patients with low-MBL haplotypes were shown to have an increased prevalence of positive cultures (gram-negative or gram-positive; P < 0.02). However, no association between high- or low-MBL haplotypes with regard to survival was demonstrated.
Interleukin-1 receptor-associated kinase 4 (IRAK4)
IRAK4 is an important intracellular kinase in the TLR and IL-1 receptor pathways and is responsible for transducing TIR-related intracellular signals that ultimately lead to nuclear translocation of NF-κB and NF-κB-dependent expression of critical proinflammatory mediators (46, 47). There have been two allelic variants described in the IRAK4 gene that are associated with recurrent gram-positive bacterial infections in children (48, 49). These consist of a deletion at nucleotide position 821 that resulted in a premature stop codon at amino acid 287, and a stop codon mutation at amino acid position 293 Gln→TER. Patients homozygous for either of these mutations were shown to have no IRAK4 mRNA or protein. Furthermore, HEK293T cells transfected with the 287 or 293 mutant alleles had an impaired activation of NF-κB and cytokine production (48, 49).
HEAT SHOCK PROTEINS (HSP)
HSP are a class of proteins that are induced in response to stress, including infection, inflammation, and trauma. HSP play an important role in protein transport, protein folding, and assisting in antigen presentation (50, 51). The 70-kD HSP (HSP70) is one of these family members and is encoded by three different genes: HSPA1A, HSPA1B, and HSPA1L. HSPA1A and HSPA1B encode the same protein, whereas HSPA1L shares 90% homology (52). Several genetic polymorphisms have been identified in the coding region (2075 HSPA1B G→A and 1209 HSPA1L C→T) and the promoter region (HSPA1B −179 C→T, HSPA1B −1538 G→A, and HSPA1L −2437 C→T) of the HSPA1B and HSPA1L genes (53-55). Temple et al. (53) examined the functional effects of HSPA1B −179 C→T promoter polymorphism on HSPA1A and HSPA1B mRNA expression in mononuclear cells stimulated with 10 μg/mL LPS. Individuals heterozygous (CT) for HSPA1B −179 C→T had significantly elevated HSPA1A and HSPA1B mRNA levels when compared with CC homozygotes. A second study evaluated the relationship between HSPA1B −1538 G→A and HSPA1L −2437 C→T promoter polymorphisms and IL-6 and TNF-α plasma levels in patients after traumatic injury (54). Patients heterozygous (GA) at position −1538 were shown to have significantly elevated levels of plasma TNF-α on days 2 and 5 post-trauma when compared with GG homozygotes. In contrast, there was not a significant difference between the −1538 promoter polymorphisms and IL-6 plasma concentrations. In addition, whereas there was no difference between the CT-2437 C→T promoter polymorphism with respect to TNF-α plasma levels, IL-6 plasma levels were significantly elevated at 6 h, and 2 and 5 days in injured patients with the CT genotype compared with the TT genotype.
Several studies have evaluated HSP70 polymorphisms and outcomes in patients with sepsis or trauma. A study by Schroeder et al. (55) investigated the HSPA1B G→A substitution at nucleotide position 2075 (synonymous mutation) and HSPA1L C→T transition at nucleotide position 1209 Met→Thr in patients with severe sepsis. This study failed to show an association between HSPA1B G→A and HSPA1L C→T polymorphisms with susceptibility to infection or survival in patients with severe sepsis. Shroder et al. (54) evaluated the association between HSPA1B −1538 G→A and HSPA1L −2437 C→T promoter polymorphisms and outcomes in patients with traumatic injury. Whereas the HSPA1B −1538 G→A polymorphism was not associated with outcome in this patient population, HSPA1L −2437 CT genotype was associated with an increase in liver failure (odds ratio 4.6, 95% confidence interval 1.5-14.1). There have not been any studies evaluating the HSPA1B −179 C→T promoter polymorphism and outcome in patients with sepsis (54).
The control and maintenance of the immune response to infection is highly regulated. This process involves the rapid release of proinflammatory cytokines, followed by the secretion of anti-inflammatory proteins. Defects in immune regulation occur when this balance is shifted to an excessive proinflammatory response, potentiating injury to various tissues and organs, or to a greater anti-inflammatory response, which may result in the inability to clear invading pathogens. Genetic polymorphisms in proinflammatory cytokines (TNF-α, IL-1, IL-6, IL-8, and macrophage migration inhibitory factor [MIF]) and anti-inflammatory cytokines (IL-10 and IL1RA) have been identified.
Interindividual variation in TNF-α production has been described (56), and family-based studies have shown that the variation in TNF production is largely genetic (57). Thus, SNPs in the promoter region of the TNF-α gene may explain the large degree of variation seen in the human population. There have been three SNPs identified within the promoter region of the TNF-α gene, consisting of −308 G→A, −376 G→A, and −238 G→A (58-60). A number of studies have evaluated the significance of the -308 A allele in relation to TNF-α production (61, 62). A study by Louis et al. (61) reported elevated levels of TNF-α in the -308 A group when whole blood was stimulated with LPS. In addition, reporter constructs that harbor the -308 SNP had a significant increase in transcription of TNF-α (62).
A number of studies investigated the role of the TNF-α −308 promoter polymorphism in patients with sepsis. A multicenter study by Mira et al. (63) evaluated a cohort of 89 patients with septic shock and reported a significant increase in the -308A allele frequency in patients that died (P = 0.008) and concluded that patients with the -308A allele had a 3.7-fold increase risk of death. Interestingly, circulating TNF-α levels were not found to be different in the control and −308 TNF-α polymorphism groups. Consistent with this data, other studies have shown a significant increase (P = 0.03) in the -308A allele frequency among nonsurvivors in children with meningococcal disease (64) and a significant increase (P < 0.05) in mortality in surgical patients with the −308 polymorphism once septic shock developed (65).
Additional studies have failed to demonstrate a functional effect in the transcriptional rate of TNF-α with the -308A polymorphism (66, 67) and associations between the -308A allele and sepsis (66). More specifically, Stuber et al. (66) reported no association in allele frequency of -308A and incidence of sepsis. They also showed no significant increase in the transcription of TNF in reporter constructs that contained the -308A allele. Consistent with this result, a second study found no functional difference between the -308A and -308G allele with respect to transcription of TNF (67). These mixed results make it difficult to determine the significance of this polymorphism in the pathogenesis of sepsis.
A second polymorphism in the TNFα promoter has been identified at position -376. An increased frequency of G→A transition at this site was shown to be present in nonsurvivors of septic shock (63). A third promoter polymorphism was found at position −238 and was reported to be associated with community-acquired pneumonia (60).
There are two forms of IL-1, IL-1α and IL-1β. Both have similar proinflammatory roles by engaging the same receptor, IL-1R (68). Genetic polymorphisms in the promoter region (position −511) and gene (exon 5) have been described for IL-1β (69). A study by Pociot et al. (70) showed that homozygosity for the polymorphism at position -511 resulted in significantly increased production of IL-1β in monocytes stimulated with LPS. In addition, a polymorphic region in the IL-1α gene (intron 6: VNTR, 46 base pairs) has been identified (71). However, functional studies involving intron 6 of the IL-1α gene have not been described.
There have been several association studies that have examined IL-1 polymorphisms in relation to sepsis (72, 73). A study by Ma et al. (72), compared 60 patients with sepsis with 60 healthy volunteers and failed to show an association between IL-1α polymorphisms (intron 6: VNTR, 46 base pairs) or IL-1β (−511 promoter) and susceptibility to sepsis. A second study of 93 patients with sepsis and 263 healthy volunteers also found no association between IL-1β polymorphism and susceptibility to sepsis (73).
IL-6, which has multiple biological functions, has also been demonstrated to be a marker of severity in sepsis (74-77). This pleiotropic cytokine plays an important role in bacterial clearance and the initiation of the adaptive immune responses, including the production of IL-4 from antigen-presenting cells, the enhancement of CD4+ activity, and the maturation of plasma cells. Allelic variation has been described in the promoter region (-174 G→C) and in the IL-6 gene (1753 C→G and 2954 G→C) (4, 78). A study by Fishman et al. (78) showed an increase in IL-6 plasma levels in healthy volunteers with the −174 CC allele and lower levels in volunteers with the −174 GG allele. Moreover, HeLa cells transfected with IL-6 −174 GG allele had a significant decrease in IL-6 expression (78).
A German study investigated the -174 IL-6 promoter polymorphism in 50 patients with severe sepsis (79). Patients with the −174 GG genotype had an improved survival rate when compared with patients with the CC genotype (P = 0.008), suggesting that the GG allele may be protective. A very recent study of critically ill patients showed a correlation between IL-6 haplotypes and mortality (4). In this study, a cohort of 228 patients was genotyped at positions -174, 1753, and 2954, and the association between haplotypes and mortality was evaluated. Compared with patients with one copy or no copies of a polymorphism in any of the genotyped regions, patients with two copies of -174C/1753C/2954G (C/C/G), G/G/G, or G/C/C haplotypes had an increase in mortality (P = 0.02) (4).
A study involving IL-8 genetic polymorphisms has revealed a SNP in the promoter region consisting of a T→A at position -251 (80). This T→A transition has been shown to be associated with increased IL-8 production in whole blood stimulated with LPS (P = 0.07) (80). Furthermore, this polymorphism was associated with diseases that include respiratory syncytial virus infection (80), Helicobacter pylori-induced duodenal ulcer disease (81), and multiple system atrophy (82). However, the association of this polymorphism in the sepsis has not yet been reported.
MIF is a proinflammatory cytokine that has a wide variety of functions, such as the activation of innate and adaptive immune responses, by inducing the release of TNF-α, IL-1β, and nitric oxide, as well as suppressing the glucocorticoid release of anti-inflammatory mediators (83-85). Mice treated with anti-MIF antibodies were protected from LPS-induced lethality (86, 87). Furthermore, increased MIF levels have been shown in patients with sepsis and septic shock, and elevated levels are associated with poor outcomes (88, 89). A recent study by Bozza et al. (90) showed that MIF plasma levels were elevated in patients with sepsis versus controls (P < 0.05), and in nonsurvivors versus survivors (P = 0.003).
Polymorphisms in the promoter region of the MIF gene have been identified and include −173G→C and -794 VNTR of five to eight repeats (91-93). Several studies have shown an association with the −173G→C polymorphism and various inflammatory diseases, including juvenile arthritis (91-93) and sarcoidosis (94). There has been one study examining the relationship between the -174 allele and sepsis. Gao et al. (95) showed an increase frequency in the C allele in patients with sepsis or sepsis-induced acute lung injury compared with controls.
IL-10 is a potent anti-inflammatory cytokine that is involved in the suppression of innate and adaptive immune responses. Studies have revealed that higher levels of IL-10 are associated with worse outcomes in patients with sepsis (96). Whole blood from volunteers stimulated with LPS showed interindividual variation in IL-10 production (57). There are three polymorphisms in the promoter region of the IL-10 gene occurring at -1082, −819, and −592 (97, 98). A study by Lowe et al. (97) found that LPS-stimulated whole blood in volunteers with the −592 C/C genotype had significantly elevated levels of IL-10 release when compared with the A/C and A/A genotypes. Furthermore, they demonstrated that the A allele was associated with a decrease in IL-10 production and an increase in mortality in patients with sepsis. However, this same study did not find an association with the -1082 SNP with respect to IL-10 secretion, sepsis, or mortality.
Shu et al. (98) investigated the association between -1082, −819, and −592 IL-10 promoter polymorphisms and sepsis severity and survival. They found a significant increase (P < 0.05) in allele frequency involving -1082 in patients with sepsis when compared with controls. However, they failed to demonstrate an association between the −819 and −592 alleles and the incidence of sepsis or mortality. Schaaf et al. (99) showed a significant increase (P = 0.04) in IL-10 concentration in patients with sepsis that were homozygous for the -1082 G/G genotype when compared with A/A or A/G genotypes. Moreover, they demonstrated that patients homozygous for the G allele had a significantly (P = 0.024) elevated risk of developing septic shock from pneumococcal disease. Gallagher et al. (100) identified an association between the -1082 G/G genotype and severity of illness and elevated mortality in patients with community-acquired pneumonia.
IL1RA is a member of the IL-1 family that binds to the IL-1 receptor, thereby inhibiting the proinflammatory actions of IL-1α and IL-1β. Polymorphisms (VNTR of 86 base pairs) within the IL1RA gene have been reported in intron 2. Five alleles have been identified and include A1 (four repeats), A2 (two repeats), A3 (five repeats), A4 (three repeats), and A5 (six repeats) (101, 102). A study involving IL1RA allele 2 has revealed a relationship between this allele and higher plasma concentrations of IL1RA (103). In addition, it has been shown that IL1RA allele 2 monocytes stimulated with granulocyte-macrophage colony-stimulating factor had increased levels of IL1RA protein (104).
Arnalich et al. (105) investigated the role of IL1RA polymorphisms in intron 2 and mortality in patients with sepsis. In this study, a cohort of 78 patients with severe sepsis was genotyped for A1, A2, A3, A4, and A5. Homozygosity for allele 2 of the IL1RA gene strongly correlated with mortality (P = 0.04). Two other studies have shown an increase in frequency of IL1RA allele 2 in patients with severe sepsis when compared with healthy controls (P < 0.01) (73) and a significant (P < 0.05) increase in allele frequency in patients with sepsis compared with controls (72).
Inflammation and coagulation are intimately related phylogenetically, with invertebrates, in fact, possessing a single common pathway for both (106). In higher organisms, crosstalk exists between the two separate cascades. For example, LPS and cytokines (TNF-α, IL-1, and IL-6) activate the extrinsic pathway of coagulation through enhanced tissue factor expression (107), and inhibit fibrinolysis through augmenting plasminogen activator inhibitor-1 (PAI-1) levels (108). Coagulation of human blood in vitro induces release of cytokines from peripheral blood mononuclear cells (PBMCs) and endothelium by a mechanism dependent upon thrombin, factor Xa, and fibrin (109-111). In animal models of septic shock, genetic deficiency of tissue factor reduces mortality (112), whereas deficiencies of the anticoagulants thrombomodulin, antithrombin III, and protein C (PC) all increase mortality (113-115). To date, several SNPs and other genetic polymorphisms have been described in genes of hemostatic factors, including thrombin, fibrinogen, factor V, PAI-1, PC, and endothelial PC receptor. Whereas most have been studied in reference to cardiovascular and thrombotic disease, reports have also confirmed a modulatory role for some of these polymorphisms in sepsis.
Recombinant human activated PC (APC) is the sole pharmacotherapy to date proven to reduce mortality in severe sepsis (116). APC not only inhibits factors Va, Xa, and PAI-1 (117), but also neutrophil adherence (118), chemotaxis (119), and PBMC cytokine release (120). Conceptually, the PC and intersecting fibrinolysis pathways present mechanistically unifying frameworks for understanding the role of SNPs relating to coagulation in sepsis (121).
Physiologic activation of PC involves interaction among thrombin, thrombomodulin (TM), PC, and endothelial PC receptor (EPCR) on the surface of endothelial cells, where factor V is an integral target of APC activity. Factor V, a component of the prothrominase complex, in turn, influences PC activation by promoting thrombin generation, underscoring its central and bidirectional role in the PC pathway. Of these molecules, only a SNP of factor V has, to date, been confirmed to influence morbidity and mortality in severe sepsis in humans, whereas SNPs of the remainder represent targets for future study.
Three independent SNPs of factor V in different populations have been described: factor V Cambridge (Arg306Thr), factor V Hong Kong (Arg306Gly), and factor V Leiden (Arg506Gln), and all make factor Va partially resistant to inactivation by APC, thereby yielding a prothrombotic state (122). The surprisingly high prevalence of these SNPs (e.g., factor V Leiden is present in ∼5% of Caucasians) has raised questions as to whether they might offer a survival advantage. In a large study of children with meningococcal disease, Factor V Leiden heterozygosity was associated with increased incidence of purpura fulminans; nevertheless, it was also associated with a nonstatistical trend to reduced mortality (123). In a substudy of the original clinical trial showing efficiency of rhAPC in sepsis (116), factor V Leiden carrier status was associated with significantly lower 28-day mortality (13.9% versus 27.9%) and with less vasopressor use at baseline, but did not determine responsiveness to recombinant human APC infusion (124). The investigators also reported lower mortality in factor V Leiden mice than wild-type controls in an endotoxin exposure model and, in parallel, increased thrombin generation and PC activation (124). These observations, taken together with previous reports that thrombin infusion improves mortality in endotoxemia (125), led the authors to speculate that an optimum generation of thrombin may be beneficial in sepsis, perhaps through activation of endogenous PC.
A number of genetic polymorphisms of the remaining components of the PC pathway have been demonstrated to be functional in the context of thrombosis, but have yet to be formally studied in sepsis. EPCR, found in membrane-bound and soluble forms, catalyzes thrombin-thrombomodulin-mediated PC activation (126). Two EPCR polymorphisms responsible for reduced PC activation have been associated with venous thrombosis: a rare 23-base pair insertion in exon 3 associated with a truncated protein that is not expressed on the cell surface (127), and the A3 haplotype of EPCR, which is associated with increased soluble EPCR levels (128). Regarding thrombomodulin (TM), a point mutation impairing TM-mediated PC activation is associated with increased mortality to endotoxin in mice (113), whereas rare TM mutations have been described in families with thromboembolic disease (129). Prothrombin, the precursor to thrombin, has an SNP (G20210A) that has been associated with hyperprothrombinemia and thromboembolism (130), and therefore represents a promising target for study. Tissue factor, a membrane protein inducible by endotoxin and thought to be a late factor in sepsis-induced coagulation, has two promoter haplotypes, 1208 D (deletion) and 1208 I (insertion) (131). Of these, 1208 I has been associated with higher plasma levels of tissue factor and higher risk for venous thrombosis (131), and thus may well represent a risk factor for severe sepsis.
Fibrin is generated by thrombin-mediated cleavage of fibrinogen, and is subsequently cross-linked into a polymeric form by factor XIII. The balance between formation and degradation of fibrin is, in turn, regulated by the balance between the profibrinolytic enzymes urokinase and tissue plasminogen activator, and their inhibitor, PAI-1. Sepsis is characterized by impaired fibrinolysis and consequent microvascular thrombus formation. A functional polymorphism of the PAI-1 promoter that governs its expression has been shown in several studies to influence outcome in sepsis. In addition, PAI-1 has been reported to be a potent inhibitor of the PC pathway (132).
PAI-1 is synthesized principally by endothelial cells, platelets, and hepatocytes, and is inducible by cytokines (133). Elevated plasma levels are an independent risk factor for cardiovascular disease and predict worse outcome in meningococcal disease and severe trauma (134-138). A common PAI-1 promoter SNP is characterized by a single base pair insertion/deletion of a G residue, yielding “4G” and “5G” alleles. As only the 5G allele binds a transcriptional repressor, 4G homozygosity is associated with an increase in PAI-1 transcription in vitro and in vivo (139), as well as with an increased risk for visceral thrombosis (140) and myocardial infarction (141). In a cohort of 175 children with meningococcal disease and 226 controls, the 4G/4G genotype compared with 4G/5G or 5G/5G was associated with higher PAI-1 levels and with an increased risk of death (134). However, the 4G genotype was not correlated with susceptibility to meningococcal disease. In a larger follow-up study by the same investigators, 4G was associated with mortality among patients with meningococcal sepsis and with a higher incidence of vascular complications among survivors (136). In another study of meningococcal disease, patients whose relatives were carriers of the 4G/4G genotype had a 6-fold higher risk of developing septic shock when compared with all other genotypes (135). Another group confirmed higher mortality and incidence of sepsis in meningococcal disease patients with the 4G/4G genotype (138). Finally, in a study of severe trauma, 4G/4G was associated with higher PAI-1 and cytokine levels and with worse outcome (137).
Other participants in the fibrin formation and degradation pathways likely also deserve further study. Thrombin-activatable fibrinolysis inhibitor is a thrombin/thrombomodulin-activated carboxypeptidase that directly links the PC and fibrinolysis pathways. Of interest, a recent report indicates that the thrombin-activatable fibrinolysis inhibitor 325 Ile/Ile genotype was more prevalent among nonsurvivors of meningococcal disease than controls, and that this genotype is associated with increased mortality from meningococcal infection (142). Fibrinogen has a number of genetic polymorphisms that determine its level in the plasma, some of which have been associated with thromboembolic disease (143). Factor XIII, the cross-linking transglutaminase of fibrin monomers, has several common polymorphic forms, some of which have also been associated with altered outcomes in cardiovascular disease (144).
Studies using mouse models of infection and investigating genetic polymorphisms in patients with sepsis have provided a starting point for unraveling the complex relationships associated with genetic heterogeneity in the immune response and predisposition to infection, as well as the severity of and mortality from sepsis. Although positive or negative associations between a polymorphism and outcome have been identified in studies involving patients with critical illness, the confidence in such conclusions is often tenuous because of small sample sizes. It will be important for future case-control genetic association studies to have adequate power to identify with appropriate statistical rigor whether an association between a particular genotype and clinical outcome actually does or does not exist.
Because sepsis is a polygenic syndrome, further advancement in the development of new technologies will yield critical information on the impact of allelic variants in multiple genes on clinical outcomes. Thus, it is unlikely that one polymorphism will result in a particular phenotype, but when several or many polymorphisms act together in the presence of an infection, the disease phenotype associated with a defined outcome, such as increased risk for death, may be apparent. Recently, a genome-wide SNP genotyping assay has been developed that can genotype hundreds of thousands of SNPs accurately in a single experiment (146). This technology will facilitate the identification of genotypes associated with specific clinical outcomes. It is likely that such classification will highlight fundamental cellular mechanisms involved in the pathogenesis of sepsis. SNP genotyping also promises to categorize patients more precisely, thereby allowing treatment to be targeted to pathways altered in individual patients. Future clinical therapeutic trials for sepsis are likely to be designed to be directed toward specific genotypes, allowing greater homogeneity of study groups to be achieved.
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