Ravi S. Keshari

Inter-α inhibitor protein and its associated glycosaminoglycans protect against histone-induced injury

Extracellular histones are mediators of tissue injury and organ dysfunction; therefore they constitute potential therapeutic targets in sepsis, inflammation, and thrombosis. Histone cytotoxicity in vitro decreases in the presence of plasma. Here, we demonstrate that plasma inter-α inhibitor protein (IAIP) neutralizes the cytotoxic effects of histones and decreases histone-induced platelet aggregation. These effects are mediated through the negatively charged glycosaminoglycans (GAGs) chondroitin sulfate and high-molecular-weight hyaluronan (HMW-HA) associated with IAIP. Cell surface anionic glycosaminoglycans heparan sulfate and HA protect the cells against histone-mediated damage in vitro. Surface plasmon resonance showed that both IAIP and HMW-HA directly bind to recombinant histone H4. In vivo neutralization of histones with IAIP and HMW-HA prevented histone-induced thrombocytopenia, bleeding, and lung microvascular thrombosis, decreased neutrophil activation, and averted histone-induced production of inflammatory cytokines and chemokines. IAIP and HMW-HA colocalized with histones in necrotic tissues and areas that displayed neutrophil extracellular traps. Increasing amounts of IAIP-histone complexes detected in the plasma of septic baboons correlated with increase in histones and/or nucleosomes and consumption of plasma IAIP. Our data suggest that IAIP, chondroitin sulfate, and HMW-HA are potential therapeutic agents to protect against histone-induced cytotoxicity, coagulopathy, systemic inflammation, and organ damage during inflammatory conditions such as sepsis and trauma.

Crosstalk between the coagulation and complement systems in sepsis

Sepsis is a potent activator of the hemostatic and complement systems. While local activation of these proteolytic cascades contributes to the host defense, their uncontrolled systemic activation has major tissue damaging effects that lead to multiple organ failure and death. We have extensively studied the activation of complement and coagulation cascades in experimental sepsis using baboons challenged with live bacteria, such as Gram-negative Escherichia coli or Gram-positive Staphylococcus aureus and Bacillus anthracis, or with the bacterial product peptidoglycan. We observed that these challenges rapidly induce disseminated intravascular coagulation and robust complement activation. We applied a potent C3 convertase inhibitor, compstatin, which prevented sepsis-induced complement activation, reduced thrombocytopenia, decreased the coagulopathic responses, and preserving the endothelial anticoagulant properties. Overall, our work demonstrates that live bacteria and bacterial products activate the complement and coagulation cascades, and that blocking formation of complement activation products, especially during the organ failure stage of severe sepsis could be a potentially important therapeutic strategy.

Mortality among recipients of the Merck V710 Staphylococcus aureus vaccine after postoperative S. aureus infections: An analysis of possible contributing host factors

In a blinded randomized trial, preoperative receipt of the Merck V710 Staphylococcus aureus vaccine was associated with a higher mortality rate than placebo in patients who later developed postoperative S. aureus infections. Of the tested patients, all 12 V710 recipients (but only 1 of 13 placebo recipients) with undetectable serum IL2 levels prior to vaccination and surgery died after postoperative S. aureus infection. The coincidence of 3 factors (low prevaccination IL-2 levels, receipt of V710, and postoperative S. aureus infection) appeared to substantially increase mortality in our study population after major cardiothoracic surgery. Furthermore, 9 of the 10 V710 recipients with undetectable preoperative IL17a levels and postoperative S. aureus infections died. Although the current study is hypothesis-generating and the exact pathophysiology remains speculative, these findings raise concern that immune predispositions may adversely impact the safety and efficacy of staphylococcal vaccines actively under development. The potential benefits of an effective vaccine against S. aureus justify continued but cautious pursuit of this elusive goal.

Bacillus anthracis peptidoglycan activates human platelets through FcγRII and complement

Platelet activation frequently accompanies sepsis and contributes to the sepsis-associated vascular leakage and coagulation dysfunction. Our previous work has implicated peptidoglycan (PGN) as an agent causing systemic inflammation in gram-positive sepsis. We used flow cytometry and fluorescent microscopy to define the effects of PGN on the activation of human platelets. PGN induced platelet aggregation, expression of the activated form of integrin αIIbβ3, and exposure of phosphatidylserine (PS). These changes were dependent on immunoglobulin G and were attenuated by the Fcγ receptor IIa-blocking antibody IV.3, suggesting they are mediated by PGN-anti-PGN immune complexes signaling through Fcγ receptor IIa. PS exposure was not blocked by IV.3 but was sensitive to inhibitors of complement activation. PGN was a potent activator of the complement cascade in human plasma and caused deposition of C5b-9 on the platelet surface. Platelets with exposed PS had greatly accelerated prothrombinase activity. We conclude that PGN derived from gram-positive bacteria is a potent platelet agonist when complexed with anti-PGN antibody and could contribute to the coagulation dysfunction accompanying gram-positive infections.

Acute Lung Injury and Fibrosis in a Baboon Model of Escherichia coli Sepsis

Sepsis-induced inflammation of the lung leads to acute respiratory distress syndrome (ARDS), which may trigger persistent fibrosis. The pathology of ARDS is complex and poorly understood, and the therapeutic approaches are limited. We used a baboon model of Escherichia coli sepsis that mimics the complexity of human disease to study the pathophysiology of ARDS. We performed extensive biochemical, histological, and functional analyses to characterize the disease progression and the long-term effects of sepsis on the lung structure and function. Similar to humans, sepsis-induced ARDS in baboons displays an early inflammatory exudative phase, with extensive necrosis. This is followed by a regenerative phase dominated by proliferation of type 2 epithelial cells, expression of epithelial-to-mesenchymal transition markers, myofibroblast migration and proliferation, and collagen synthesis. Baboons that survived sepsis showed persistent inflammation and collagen deposition 6-27 months after the acute episodes. Long-term survivors had almost double the amount of collagen in the lung as compared with age-matched control animals. Immunostaining for procollagens showed persistent active collagen synthesis within the fibroblastic foci and interalveolar septa. Fibroblasts expressed markers of transforming growth factor-β and platelet-derived growth factor signaling, suggesting their potential role as mediators of myofibroblast migration and proliferation, and collagen deposition. In parallel, up-regulation of the inhibitors of extracellular proteases supports a deregulated matrix remodeling that may contribute to fibrosis. The primate model of sepsis-induced ARDS mimics the disease progression in humans, including chronic inflammation and long-lasting fibrosis. This model helps our understanding of the pathophysiology of fibrosis and the testing of new therapies.

Inhibition of complement C5 protects against organ failure and reduces mortality in a baboon model of Escherichia coli sepsis

Significance Complement activation occurs when bacteria invade the circulating blood, leading not only to removal of the pathogen but also to inflammation, organ damage, and poor prognosis for septic patients. We used a baboon model of Escherichia coli bacteremia to determine the effects of a C5 inhibitor on bacteriolysis, bacteria clearance, and sepsis progression. We observed that complement-mediated bacteriolysis has a detrimental effect by inducing release of LPS and fulminant inflammation. Inhibition of C5 cleavage and subsequent formation of the lytic terminal complex C5b-9 diminished LPS release, blocked sepsis-induced inflammation, decreased the associated consumptive coagulopathy, and protected organ function. Overall, treatment with C5 inhibitor significantly improved the survival of septic baboons, suggesting a potentially important strategy to treat bacteremic sepsis. Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli-induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coli-induced “oxidative burst,” as well as leukocyte activation, without affecting host phagocytosis of E. coli. RA101295 treatment reduced plasma LPS content in E. coli-challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death.

Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

Complement inhibition decreases early fibrogenic events in the lung of septic baboons.

Acute respiratory distress syndrome (ARDS) induced by severe sepsis can trigger persistent inflammation and fibrosis. We have shown that experimental sepsis in baboons recapitulates ARDS progression in humans, including chronic inflammation and long‐lasting fibrosis in the lung. Complement activation products may contribute to the fibroproliferative response, suggesting that complement inhibitors are potential therapeutic agents. We have been suggested that treatment of septic baboons with compstatin, a C3 convertase inhibitor protects against ARDS‐induced fibroproliferation. Baboons challenged with 109 cfu/kg (LD50) live E. coli by intravenous infusion were treated or not with compstatin at the time of challenge or 5 hrs thereafter. Changes in the fibroproliferative response at 24 hrs post‐challenge were analysed at both transcript and protein levels. Gene expression analysis showed that sepsis induced fibrotic responses in the lung as early as 24 hrs post‐bacterial challenge. Immunochemical and biochemical analysis revealed enhanced collagen synthesis, induction of profibrotic factors and increased cell recruitment and proliferation. Specific inhibition of complement with compstatin down‐regulated sepsis‐induced fibrosis genes, including transforming growth factor‐beta (TGF‐β), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase 1 (TIMP1), various collagens and chemokines responsible for fibrocyte recruitment (e.g. chemokine (C‐C motif) ligand 2 (CCL2) and 12 (CCL12)). Compstatin decreased the accumulation of myofibroblasts and proliferating cells, reduced the production of fibrosis mediators (TGF‐β, phospho‐Smad‐2 and CTGF) and inhibited collagen deposition. Our data demonstrate that complement inhibition effectively attenuates collagen deposition and fibrotic responses in the lung after severe sepsis. Inhibiting complement could prove an attractive strategy for preventing sepsis‐induced fibrosis of the lung.

In vivo–generated thrombin and plasmin do not activate the complement system in baboons

Sepsis concurrently activates both coagulation and complement systems. Although complement activation by bacteria is well documented, work in mice and in vitro suggests that coagulation proteases can directly cleave complement proteins. We aimed to determine whether generation of coagulation proteases in vivo can activate the complement cascade in 2 highly coagulopathic models. We compared temporal changes in activation biomarkers of coagulation (thrombin-antithrombin [TAT]), fibrinolysis (plasmin-antiplasmin [PAP]), and complement (C3b, C5a, C5b-9) in baboons infused with factor Xa (FXa) and phospholipids (FXa/phosphatidylcholine-phosphatidylserine [PCPS]) vs LD100 Escherichia coli We found that, albeit with different timing, both FXa/PCPS and E coli infusion led to robust thrombin and plasmin generation. Conversely, only E coli challenge activated the complement system, reaching a maximum at 2 hours postchallenge during the peaks of lipopolysaccharide and bacteremia but not of TAT and PAP. Despite inducing a strong burst of thrombin and plasmin, FXa/PCPS infusion did not produce measurable levels of complement activation in vivo. Similarly, ex vivo incubation of baboon serum with thrombin, plasmin, or FXa did not show noticeable complement cleavage unless supraphysiologic amounts of enzymes were used. Our results suggest that in vivo-generated thrombin and plasmin do not directly activate the complement in nonhuman primates.

Serum Amyloid P and IgG Exhibit Differential Capabilities in the Activation of the Innate Immune System in Response to Bacillus anthracis Peptidoglycan

ABSTRACT We showed that human IgG supported the response by human innate immune cells to peptidoglycan (PGN) from Bacillus anthracis and PGN-induced complement activation. However, other serum constituents have been shown to interact with peptidoglycan, including the IgG-like soluble pattern recognition receptor serum amyloid P (SAP). Here, we compared the abilities of SAP and of IgG to support monocyte and complement responses to PGN. Utilizing in vitro methods, we demonstrate that SAP is superior to IgG in supporting monocyte production of cytokines in response to PGN. Like IgG, the response supported by SAP was enhanced by phagocytosis and signaling kinases, such as Syk, Src, and phosphatidylinositol 3-kinase, that are involved in various cellular processes, including Fc receptor signaling. Unlike IgG, SAP had no effect on the activation of complement in response to PGN. These data demonstrate an opsonophagocytic role for SAP in response to PGN that propagates a cellular response without propagating the formation of the terminal complement complex.