Complement C5 Inhibition Blocks the Cytokine Storm and Consumptive Coagulopathy By Decreasing Lipopolysaccharide (LPS) Release in E. coli Sepsis

Abstract

Complement is a "dual-edged sword" that is critical for the innate immunity against pathogens, but its uncontrolled activation contributes to disseminated intravascular coagulation (DIC), inflammation, cell death, immune paralysis and cardiac dysfunction, leading to multiple organ failure and death. E. coli bacteremia is accompanied by robust complement activation triggered by the pathogen. The end products of the complement cascade are the cytolytic terminal complement complex C5b-9 and the C5a anaphylatoxin, both known mediators of sepsis pathology. We hypothesized that complement activation during bacteremia is the culprit of sepsis-induced inflammation and DIC by inducing rapid bacteria lysis and massive LPS release. Thus, C5 complement inhibition could block LPS release and downstream signaling through toll-like receptor 4 (TLR4) leading to nuclear factor (NF)-kappa B activation and production of inflammatory cytokines and procoagulant proteins.We used in vitro whole blood (WB) assays and a baboon model of E. coli sepsis to test the effects of a novel C5 inhibitor, the macrocyclic peptide RA101295, on sepsis-induced complement activation, LPS release, inflammation, oxidative stress and phagocytosis of the bacteria.WB was incubated with 5x107 cfu/ml live E. coli in presence/absence of RA101295. The treatment abolished complement activation measured as C5b-9 and C5a generation. RA101295 decreased the "oxidative burst" induced by E. coli in neutrophils and monocytes, a C5a dependent process that leads to rapid generation of highly toxic peroxide. C3b opsonization and bacteria phagocytosis were not affected. Concordantly, plasma samples treated with E. coli and RA101295 showed significantly lower TLR4 signaling in HEK-TLR4 reporter cells (Invivogen). These data indicate that RA101295 blocks the lysis of bacteria and release of LPS in plasma, without affecting E. coli phagocytosis. To test the in vivo effects of C5 inhibition we used a baboon model of sepsis. Animals challenged with E. coli (1-2x1010 cfu/kg; LD100) by iv infusion show robust generation of soluble C5b-9. Treatment with RA101295 fully inhibited complement activation and improved the clinical parameters and provides significant survival benefit. Notably, RA101295 treatment during the bacteremic sepsis did not impede bacteria clearance; on the contrary, the colony counts in the blood at 8 hours were slightly lower in the treated than in non-treated septic baboons. Plasma LPS levels were also significantly lower in treated animals suggesting that complement inhibition can block bacterial lysis without impairing clearing by phagocytosis. Remarkably, RA101295 abolished six sepsis-induced proinflammatory cytokines (TNF-α, IL6, IL8, IL1RA, MCP1 and G-CSF), reduced hypotension/shock and decreased the febrile response. Distinctly, LPS-driven expression of TNF-α is >57 fold decreased in RA101295 treated group, suggesting that the treatment blocks E. coli induced cytokine storm by inhibiting bacteria lysis and LPS release. The expression of IL6 and IL8, two cytokines induced via C5a signaling was almost fully inhibited.Moreover, RA101295 treatment significantly lowered consumptive coagulopathy, reflected by APTT, PT, and fibrin/fibrinogen degradation products tests. Furthermore, plasma levels of plasminogen activator inhibitor 1, a LPS and TNF regulated fibrinolysis inhibitor, were decreased by >2.4 fold in the treated versus non-treated septic animals.Altogether, our data show that RA101295 has protective effects when administered during the bacteremic stage by inhibiting C5b-9 induced lysis of the bacteria and explosive release of LPS. Equally important from a therapeutic point of view, C5 inhibition does not interfere with the phagocytosis and intracellular digestion of the pathogen, which ensures that efficient removal of the bacteria is unaffected. These data suggest that targeting complement activation can be an effective therapy to prevent damaging inflammation and DIC in sepsis patients. DisclosuresDeMarco:Ra Pharmaceuticals: Employment. Lupu:Ra Pharmaceuticals: Research Funding; National Institutes of Health: Research Funding.