Complement C5 inhibition protects against hemolytic anemia and acute kidney injury in anthrax peptidoglycan-induced sepsis in baboons.

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis, leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.

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

Atorvastatin protects against ischemia-reperfusion injury in fructose-induced insulin resistant rats.

PURPOSE: High fructose (HFr) intake is known to cause insulin resistance syndrome (IRS), however its effect against acute coronary events remains elusive. The present study was undertaken to evaluate the effect of HFr (60%) diet on myocardial ischemia-reperfusion (MI-RP) injury and its modulation by atorvastatin treatment. METHODS: Wistar rats kept on HFr/chow feeding for 10 weeks, received atorvastatin (30 mg/kg, per oral) or vehicle for two additional weeks followed by MI-RP injury. RESULTS: MI-RP injury was significantly augmented in HFr fed rats, as evident by the increase in infarct size (IS, 65 ± 5% vs. 43 ± 7%) and activities of cardiac injury biomarkers [serum lactate dehydrogenase (LDH, 698 ± 57 vs. 444 ± 26 U/L), creatinine kinase (CK-MB, 584 ± 58 vs. 435 ± 28 U/L) and tissue myeloperoxidase (MPO, 235 ± 15 vs. 101 ± 11 µM/min/100 mg tissue)]. Insulin resistance (plasma glucose, 64 ± 5 vs. 100 ± 5 mg/dl; AUC (0-120 min), p < 0.05), MI-RP injury (IS 20 ± 5%, LDH 292 ± 28 U/L, CK-MB 257 ± 13 U/L, MPO 95 ± 5 µM/min/100 mg tissue) and triglyceride (TG) level were significantly reduced, while myocardial Akt, p-Akt, eNOS, p-eNOS and iNOS protein expression were significantly enhanced following atorvastatin treatment in comparison to HFr fed rats. Oxidative stress marker, malondialdehyde and circulating levels of inflammatory cytokines (CRP, IL-6, IFN-γ and TNF) were significantly reduced, while total nitrite content in the tissue and plasma was significantly augmented in atorvastatin treated rats. Atorvastatin also ameliorated endothelial dysfunction and significantly enhanced aortic Akt and eNOS protein expression. CONCLUSION: Atorvastatin conferred significant protection against MI-RP injury and alleviated HFr induced IRS possibly by increasing NOS expression through Akt dependent pathway.

Functional and molecular characterization of NOS isoforms in rat neutrophil precursor cells.

Previous studies from this laboratory have demonstrated importance of neutrophil-derived nitric oxide (NO) in free radical generation, characterized nitric oxide synthase (NOS) isoforms, and have reported subcellular distribution of NOS in rat peripheral neutrophils. Maximum number of neutrophils are added per day to the circulation from bone marrow, thus neutrophils might add substantial amount of NO in the bone marrow. NO generating ability and NOS isoforms characteristics in bone marrow neutrophil precursor cells is, however, still unexplored. This study was, therefore, undertaken to investigate NO generation ability and the molecular/biochemical characteristics of NOS isoforms in neutrophil precursor cells. The neutrophil precursors were separated on Percoll density gradient and characterized by Giemsa staining, CD markers, and by their size and granularity at various stages of maturation as Bands 1, 2, and 3. Mature neutrophils were efficient in free radical generation and phagocytosis, whereas immature cells had more mitochondria and myeloperoxidase. Amount of NO augmented from immature to mature neutrophils as assessed by fluorescent probe DAF-2DA and Griess reagent. Measurement of NOS enzyme activity further confirmed the functional status of NOS in these cells. NOS isoforms were differentially expressed during neutrophil maturation as confirmed by enzyme activity, Western blotting, flowcytometry, and RT-PCR. Expression of nNOS was predominantly stable in all the stages of neutrophil maturation. iNOS expression was, however, consistently augmented during maturation, whereas eNOS expression was downregulated with neutrophil maturation. Furthermore, all NOS isoforms proteins were distributed in cytosol as well as nucleus as assessed by confocal microscopy. This study for the first time report biochemical and molecular characteristics of NOS isoforms in rat neutrophil precursor cells.

Nitric oxide-mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: Critical use of probes.

Previous reports from this laboratory and others demonstrated NO-mediated biphasic modulation of NADPH oxidase and attenuation of neutrophil reactive oxygen species generation, whereas recently we reported augmentation in DCF fluorescence following NO treatment. These discrepancies seem to be due to utilization of different probes/methods to assess effect of NO on reactive oxygen and nitrogen species (ROS/RNS, reactive species) generation. This study aims to look into this and evaluate NO-mediated enzymatic reactive species formation by using multiple probes, human neutrophils/HL60 cells and various interventions. Addition of NO donor, SNP or SNAP (100 nM-1 mM) to PMNs suspension, exhibited a concentration- and time-dependent augmentation in DCF fluorescence, but reduced DHE fluorescence. Collective generation of reactive species was confirmed by enhanced DMPO-nitrone adduct, dityrosine and rhodamine-123 and quenching of scopoletin. NO also enhanced bacterial killing, without altering phagocytosis. Addition of NO to HL-60 cells lacking functional NADPH oxidase enhanced reactive species formation, indicating importance of other enzyme(s) too. NO-dependent ROS/RNS generation was substantially reduced by NADPH oxidase inhibitor (DPI), MPO inhibitor (ABAH), or NOS inhibitor (7-NI). However, 7-NI reduced MPO activity, warranting reappraisal of those reports, which implied NOS in reactive species formation. The results obtained demonstrated NO-mediated reactive species augmentation in human PMNs. Furthermore, superoxide scavenging by NO seems to be the key process in the decrease of DHE fluorescence and suggest usefulness of DCF as the most appropriate probe to measure the NO-mediated modulation of reactive oxygen species in particular in various pathological situations.

Nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation.

High availability of NO, oxidative stress and neutrophil extracellular trap (NETs) contents are often noticed at the site of inflammation/infection. Studies from this lab and others have reported NO mediated free radical generation from neutrophils; role of NO in NETs formation however remains undefined so far. The present study was therefore undertaken to explore the effect of NO donors on NET release from human neutrophils (PMNs), using confocal/scanning microscopy, measuring the extracellular DNA content and NET-bound elastase activity. Addition of NO donors (SNAP and SNP) to adhered PMNs led to a time and concentration dependent NETs release, which was blocked by N-acetyl cysteine, suggesting involvement of free radicals in NETs formation. Free radical formation by NO donors was assessed by using DCF-DA, DMPO-nitrone antibody and by p47 phox migration to the neutrophils membrane. NO mediated formation of free radicals and NETs was significantly reduced by the pretreatment of neutrophils with diphenyleneiodonium (DPI), a NADPH-oxidase inhibitor and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase inhibitor, suggesting role of enzymatic free radical generation by NO donors. We thus demonstrate that NO by augmenting free radical formation in human neutrophils mediates NETs release.

Inhibition of contact-mediated activation of factor XI protects baboons against S aureus-induced organ damage and death.

Staphylococcus aureus infections can produce systemic bacteremia and inflammation in humans, which may progress to severe sepsis or septic shock, even with appropriate antibiotic treatment. Sepsis may be associated with disseminated intravascular coagulation and consumptive coagulopathy. In some types of mouse infection models, the plasma coagulation protein factor XI (FXI) contributes to the pathogenesis of sepsis. We hypothesize that FXI also contributes to the pathogenesis of sepsis in primates, and that pharmacological interference with FXI will alter the outcome of Staphylococcus aureus-induced lethality in a baboon model. Pretreatment of baboons with the anti-FXI antibody 3G3, a humanized variant of the murine monoclonal 14E11 that blocks FXI activation by FXIIa, substantially reduced the activation of coagulation, as reflected by clotting times and plasma complexes of coagulation proteases (FXIIa, FXIa, FIXa, FXa, FVIIa, and thrombin) with serpins (antithrombin or C1 inhibitor) following infusion of heat-inactivated S aureus 3G3 treatment reduced fibrinogen and platelet consumption, fibrin deposition in tissues, neutrophil activation and accumulation in tissues, cytokine production, kininogen cleavage, cell death, and complement activation. Overall, 3G3 infusion protected the structure and function of multiple vital organs, including lung, heart, liver, and kidney. All treated animals reached the end point survival (7 days), whereas all nontreated animals developed terminal organ failure within 28 hours. We conclude that FXI plays a role in the pathogenesis of S aureus-induced disseminated intravascular coagulation and lethality in baboons. The results provide proof of concept for future therapeutic interventions that may prevent sepsis-induced organ failure and save lives in certain forms of sepsis.

Interaction of inducible nitric oxide synthase with rac2 regulates reactive oxygen and nitrogen species generation in the human neutrophil phagosomes: implication in microbial killing.

AIMS: Present study explores importance of inducible nitric oxide synthase (iNOS) and its interaction with Rac2 in reactive oxygen species (ROS)/reactive nitrogen species (RNS) generation, protein-nitration and in microbial killing by neutrophils. RESULTS: The iNOS transcript and protein were constitutively present in human as well as in mice neutrophils. iNOS protein was found in cytosol, granules containing elastase and gelatinase, and in other subcellular organelles in resting human neutrophils. After phagocytosis of bovine serum albumin (BSA) coated beads, both human and mice neutrophils showed significant elevation in superoxide radicals, nitric oxide (NO), ROS/RNS and consequent BSA nitration. These responses were significantly reduced in presence of iNOS, NADPH oxidase (NOX), myeloperoxidase or Rac inhibitors, as well as in iNOS, Nox2 and Rac2 silenced human or iNOS-knockout mice neutrophils. Complex formed on interaction of iNOS with Rac2 coprecipitated with anti-Rac2, predominantly in cytosol in resting human neutrophils, while iNOS-Rac2 complex translocated to phagosomes after phagocytosis. This was accompanied by generation of superoxide radicals, NO, ROS/RNS and consequent BSA-nitration. Importance of Rac2 in iNOS mediated NO formation and microbial killing was confirmed by pretreatment of mice with Rac inhibitor, NSC23766 that significantly abrogated NO release and microbial killing in vivo. INNOVATION: Present study highlights previously undefined role of Rac2-iNOS interaction, in translocation of iNOS to phagosomal compartment and consequent NO, superoxide radicals, ROS/RNS generation, BSA nitration and microbial killing. CONCLUSIONS: Altogether results obtained demonstrate the role of iNOS in NO and ROS/RNS generation, after phagocytosis of coated latex beads by human polymorphonuclear neutrophils. These studies imply functional importance of iNOS and its interaction with Rac2 in pathogen killing by the neutrophils.

A time course study on prothrombotic parameters and their modulation by anti-platelet drugs in hyperlipidemic hamsters.

The present study was undertaken to assess the chronology of major pathological events associated with high cholesterol (HC) diet and their modulation by anti-platelet drugs. Male Golden Syrian hamsters were fed HC diet up to 90 days. Plasma lipid, glucose and coagulation parameters (commercial kits), platelet activation (whole blood aggregation and static adhesion), endothelial dysfunction (aortic ring vasoreactivity), splenocyte TNF-α, IFN-γ and iNOS mRNA transcripts (RT-PCR), and ferric chloride (time to occlusion) induced thrombosis were monitored at 15, 30, 60, and 90 days after HC feeding and compared with normolipidemic hamsters. A significant increase in plasma lipid levels was observed at 15 days of HC feeding, but other parameters remain unaltered. Enhanced ADP, collagen, and thrombin-induced platelet aggregation, splenocyte TNF-α expression along with endothelial dysfunction were observed from 30 to 90 days of HC feeding. Platelet adhesion on collagen-/fibrinogen-coated surface and IFN-γ expression were augmented only after 60 days, while enhanced iNOS expression, reduction in thrombin time, and potentiation of ferric chloride-induced thrombosis was observed only at 90 days of HC feeding. Thus, pathological changes induced by HC diet depend on the duration and extent of hyperlipidemia. Moreover, hamsters treated with anti-platelet drugs aspirin (5 mg/kg) or clopidogrel (10 mg/kg) along with HC feeding exhibited reduction in platelet activation as well as subsequent changes observed in the abovementioned parameters following HC feeding. Since reduction in TNF-α was associated with reversion in endothelial dysfunction and prothrombotic state, the role of platelets is implicated in the pathological changes associated with HC feeding.