Keith Walley

Genetic variation contributes to outcome from sepsis. A large number of associations have been observed between genetic variants and sepsis outcome, however, identification of causal single nucleotide polymorphisms (SNPs), or their mechanisms of action, have not been successfully elucidated. The aims of this project are to identify causal variants in two candidate genes and determine whether these variants are involved in the mechanisms leading to altered outcomes in sepsis. The known pathophysiology of sepsis is complex and involves dysregulation of several systemic processes, including the coagulation and inflammatory systems. Based on this knowledge, and known literature on genetic variation in coagulation genes, PROC was chosen as a candidate gene in which to search for causal SNPs. In addition, based on the known role of lipids in sepsis, as well as the already identified causal SNPs in the PCSK9 gene, PCSK9 was selected as a second candidate gene to test the hypothesis that genetic variation in lipid mediators alters outcome in sepsis. Two intronic SNPs were found in the PROC gene (rs2069915 and rs2069916) that are in high linkage disequilibrium and appear to modify untranslated mRNA, leading to lower concentrations of circulating protein C in individuals homozygous for the major alleles of these SNPs. Furthermore, in the PCSK9 gene, an intronic SNP (rs644000) was found that appears to mark known Loss-of-Function and Gain-of-Function coding SNPs, and was associated with outcome in two cohorts of patients with septic shock, and with a reduction of cytokine levels in a subset of these patients. Additionally, using murine genetic Pcsk9 knock-out and pharmacologic inhibition strategies in a murine model of systemic bacteremia, a markedly attenuated global, cardiovascular and inflammatory cytokine response to lipopolysaccharide administration was observed. Furthermore, increased endotoxin clearance was measured after PCSK9 knock-out. Together these results indicate that reduction of PCSK9 activity in both mice and humans reduces the inflammatory response and improves outcome in septic shock.The work presented here furthers the understanding of the role played by non-coding SNPs in protein expression and has implications for a new, potentially personal, drug strategy for sepsis patients in intensive care units.

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Septic shock (sepsis accompanied by cardiovascular failure) is an extreme manifestation ofthe host inflammatory response to severe infection. Tumor necrosis factor alpha (TNFα)super family induced nuclear factor kappa B (NF-κB) signaling pathways play a critical rolein the pathophysiology of the disease. A better understanding of how TNFα induced NF-κBsignaling influences the pathogenesis of septic shock is imperative.NF-κB signaling is activated by a canonical pathway or a non-canonical pathway. Thecanonical NF-κB pathway requires the IκB kinase (IKK) complex comprised of IKKα/β/γ.Activation of the IKK complex in response to inflammatory stimuli, such as TNFα, results inubiquitin-dependent degradation of IκBα or IκBβ, releasing p50 related dimers to thenucleus. In response to TNFα superfamily induced inflammation in the non-canonicalpathway, NF-κB inducing kinase (NIK), a docking molecule, recruits IΚΚα to p100 thusactivating IΚΚα. This phosphorylates p100, which is then degraded, releasing p52containing RelB heterodimers to the nucleus.It has been shown that genetic variation in key inflammatory genes contributes to outcome insepsis. We hypothesized that genetic variation in genes of TNFα super family induced NF-κB signaling would be associated with mortality in septic shock. Specifically, we first testedthe hypothesis that genetic variation within the cytosolic members of the canonical and noncanonicalpathway may be associated with mortality in septic shock. We found that the CCgenotype of NIK rs7222094 is associated with increased mortality and organ dysfunction inseptic shock patients. This is perhaps due to altered regulation of NF-κB pathway genes,including CXCL10. We then tested the hypothesis that genetic variation in genesupregulated by these pathways may be associated with mortality in septic shock. We showedthat the G allele of TNFAIP2 rs8126 is associated with increased mortality and organdysfunction in septic shock patients. We then elucidated a novel biological mechanismwhereby TNFAIP2 is a novel inhibitor of Ras, CREB and NF-κB; TNFAIP2 levels arecontrolled by rs8126; and these by allele differences are reflected in the inhibiton of Ras,CREB and NF-κB.

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