Combined inhibition of C5 and CD14 efficiently attenuated the inflammatory response in a porcine model of meningococcal sepsis

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Phagocytosis of live and dead Escherichia coli and Staphylococcus aureus in human whole blood is markedly reduced by combined inhibition of C5aR1 and CD14

Background: Sepsis is a dysregulated host response to infection. The aim of this study was to investigate the effects of complement- and CD14 inhibition on phagocytosis of live and dead Gram-negative and Gram-positive bacteria in human whole blood. Methods: Lepirudin-anticoagulated blood was incubated with live or dead E. coli or S. aureus at 37 °C for 120 min with or without the C5aR1 antagonist PMX53 and/or anti-CD14. Granulocyte and monocyte phagocytosis were measured by flow cytometry, and five plasma cytokines by multiplex, yielding a total of 28 mediators of inflammation tested for. Results: 16/28 conditions were reduced by PMX53, 7/28 by anti-CD14, and 24/28 by combined PMX53 and CD14 inhibition. The effect of complement inhibition was quantitatively more pronounced, in particular for the responses to S. aureus. The effect of anti-CD14 was modest, except for a marked reduction in INF-β. The responses to live and dead S. aureus were equally inhibited, whereas the responses to live E. coli were inhibited less than those to dead E. coli. Conclusion: C5aR1 inhibited phagocytosis-induced inflammation by live and dead E. coli and S. aureus. CD14 blockade potentiated the effect of C5aR1 blockade, thus attenuating inflammation

Staphylococcus aureus-induced complement activation promotes tissue factor-mediated coagulation

There is extensive cross-talk between the complement system, the Toll-like receptors (TLR) and hemostasis. Consumptive coagulopathy is a hallmark of sepsis and often mediated through increased tissue factor (TF) expression.To study the relative roles of complement, TLRs and TF in Staphylococcus aureus (S. aureus)-induced coagulation.Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a-receptor-1 and factor XIIa with peptide inhibitors, CD14, TLR2, and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured by prothrombin fragment 1+2 (PTF1.2) using ELISA, and TF mRNA, monocyte surface expression and functional activity using qPCR, flow cytometry and ELISA, respectively.All three strains generated substantial and statistically significant amounts of C5a, TCC, PTF1.2, TF mRNA, TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently inhibited (P < 0.05) all six markers in strains Cowan and Wood and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly (P < 0.05) reduced PTF1.2 levels to baseline levels.S. aureus-induced coagulation in human whole blood was mainly due to C5a-induced mRNA upregulation, monocyte TF expression and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation. This article is protected by copyright. All rights reserved

Combined inhibition of complement and CD14 improved outcome in porcine polymicrobial sepsis

Introduction Sepsis is an exaggerated and dysfunctional immune response to infection. Activation of innate immunity recognition systems including complement and the Toll-like receptor family initiate this disproportionate inflammatory response. The aim of this study was to explore the effect of combined inhibition of the complement component C5 and the Toll-like receptor co-factor CD14 on survival, hemodynamic parameters and systemic inflammation including complement activation in a clinically relevant porcine model of polymicrobial sepsis. Methods Norwegian landrace piglets (4 ± 0.5 kg) were blindly randomized to a treatment group (n = 12) receiving the C5 inhibitor coversin (OmCI) and anti-CD14 or to a positive control group (n = 12) receiving saline. Under anesthesia, sepsis was induced by a 2 cm cecal incision and the piglets were monitored in standard intensive care for 8 hours. Three sham piglets had a laparotomy without cecal incision or treatment. Complement activation was measured as sC5b-9 using enzyme immunoassay. Cytokines were measured with multiplex technology. Results Combined C5 and CD14 inhibition significantly improved survival (p = 0.03). Nine piglets survived in the treatment group and four in the control group. The treatment group had significantly lower pulmonary artery pressure (p = 0.04) and ratio of pulmonary artery pressure to systemic artery pressure (p < 0.001). Plasma sC5b-9 levels were significantly lower in the treatment group (p < 0.001) and correlated significantly with mortality (p = 0.006). IL-8 and IL-10 were significantly (p < 0.05) lower in the treatment group. Conclusions Combined inhibition of C5 and CD14 significantly improved survival, hemodynamic parameters and inflammation in a blinded, randomized trial of porcine polymicrobial sepsis