No abstract

Ischemic stroke prompts a strong inflammatory response, which is associated with exacerbated outcomes. In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and whether they contribute to stroke outcomes. NET-forming neutrophils were found throughout brain tissue of ischemic stroke patients, and elevated plasma NET biomarkers correlated with worse stroke outcomes. Additionally, we observed increased plasma and platelet surface–expressed high-mobility group box 1 (HMGB1) in stroke patients. Mechanistically, platelets were identified as the critical source of HMGB1 that caused NETs in the acute phase of stroke. Depletion of platelets or platelet-specific knockout of HMGB1 significantly reduced plasma HMGB1 and NET levels after stroke, and greatly improved stroke outcomes. We subsequently investigated the therapeutic potential of neonatal NET-inhibitory factor (nNIF) in stroke. Mice treated with nNIF had smaller brain infarcts, improved long-term neurological and motor function, and enhanced survival after stroke. nNIF specifically blocked NET formation without affecting neutrophil recruitment after stroke. Importantly, nNIF also improved stroke outcomes in diabetic and aged mice and was still effective when given 1 hour after stroke onset. These results support a pathological role for NETs in ischemic stroke and warrant further investigation of nNIF for stroke therapy.

Sepsis is a systemic inflammatory response to infection and a leading cause of death. Mucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in mucosal tissues that recognize bacterial ligands. We investigated MAIT cells during clinical and experimental sepsis, and their contribution to host responses. In experimental sepsis, MAIT-deficient mice had significantly increased mortality and bacterial load, and reduced tissue-specific cytokine responses. MAIT cells of WT mice expressed lower levels of IFN-γ and IL-17a during sepsis compared to sham surgery, changes not seen in non-MAIT T cells. MAIT cells of patients at sepsis presentation were significantly reduced in frequency compared to healthy donors, and were more activated, with decreased IFN-γ production, compared to both healthy donors and paired 90-day samples. Our data suggest that MAIT cells are highly activated and become dysfunctional during clinical sepsis, and contribute to tissue-specific cytokine responses that are protective against mortality during experimental sepsis.

Sepsis is a severe disorder characterized by systemic inflammatory responses in the presence of an infection and may progress to multiple organ dysfunction and death. Alterations in cerebral microcirculation fulfill a crucial role in the pathogenesis of severe sepsis, and include a decrease in capillary density and disturbances in leukocyte movement along capillaries. Nevertheless, the mechanisms involved in sepsis-associated cerebral microcirculatory alterations have so far not been defined. We investigated the effect of the peroxisome proliferator-activated receptor gamma (PPARγ) selective agonist rosiglitazone on leukocyte/endothelial cell interaction and functional capillary density in the brain in the cecal ligation and puncture (CLP) model of sepsis. Anti-inflammatory effects of rosiglitazone on the cerebral microcirculation were marked. Functional capillary density increased and leukocyte rolling and adhesion were decreased in animals submitted to CLP and treated with rosiglitazone. Our data provide evidence for involvement of PPARγ activation in leukocyte-endothelium interactions and alterations in capillary density. Improved cerebral perfusion in animals treated with rosiglitazone, suggests that PPARγ activation is protective against cerebral microvascular dysfunction in sepsis.

Dysregulation of the inflammatory response against infection contributes to mortality in sepsis. Inflammation provides critical host defense, but it can cause tissue damage, multiple organ failure and death. Because the nuclear transcription factor peroxisome proliferator-activated receptor γ (PPARγ) exhibits therapeutic potential, we characterized the role of PPARγ in sepsis. We analyzed severity of clinical signs, survival rates, cytokine production, leukocyte influx, and bacterial clearance in a cecal ligation and puncture (CLP) model of sepsis in Swiss mice. The PPARγ agonist rosiglitazone treatment improved clinical status and mortality, while increasing IL-10 production and decreasing TNF-α and IL-6 levels, and peritoneal neutrophil accumulation 24 h after CLP. We noted increased bacterial killing in rosiglitazone treated mice, correlated with increased generation of reactive oxygen species. Polymorphonuclear leukocytes (PMN) incubated with LPS or E. coli and rosiglitazone increased peritoneal neutrophil extracellular trap (NET)-mediated bacterial killing, an effect reversed by the PPARγ antagonist (GW 9662) treatment. Rosiglitazone also enhanced the release of histones by PMN, a surrogate marker of NET formation, effect abolished by GW 9662. Rosiglitazone modulated the inflammatory response and increased bacterial clearance through PPARγ activation and NET formation, combining immunomodulatory and host-dependent anti-bacterial effects and, therefore, warrants further study as a potential therapeutic agent in sepsis.