No abstract

Sepsis is an infection-induced severe inflammatory disorder that leads to multiple organ failure. Amongst organs affected, myocardial depression is believed to be a major contributor to septic death. While it has been identified that large amounts of circulating pro-inflammatory cytokines are culprit for triggering cardiac dysfunction in sepsis, the underlying mechanisms remain obscure. Additionally, recent studies have shown that exosomes released from bacteria-infected macrophages are pro-inflammatory. Hence, we examined in this study whether blocking the generation of exosomes would be protective against sepsis-induced inflammatory response and cardiac dysfunction. To this end, we pre-treated RAW264.7 macrophages with GW4869, an inhibitor of exosome biogenesis/release, followed by endotoxin (LPS) challenge. In vivo, we injected wild-type (WT) mice with GW4869 for 1 h prior to endotoxin treatment or cecal ligation/puncture (CLP) surgery. We observed that pre-treatment with GW4869 significantly impaired release of both exosomes and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in RAW264.7 macrophages. At 12 h after LPS treatment or CLP surgery, WT mice pretreated with GW4869 displayed lower amounts of exosomes and pro-inflammatory cytokines in the serum than control PBS-injected mice. Accordingly, GW4869 treatment diminished the sepsis-induced cardiac inflammation, attenuated myocardial depression and prolonged survival. Together, our findings indicate that blockade of exosome generation in sepsis dampens the sepsis-triggered inflammatory response and thereby, improves cardiac function and survival.

No abstract

The nuclear enzyme poly (ADP-ribose) synthetase (PARS) has been shown to play an important role in the pathogenesis of ischemia/reperfusion injury and circulatory shock. The aim of this study was to investigate whether PARS activity may modulate endothelial-neutrophil interaction. We present evidence that genetic disruption of PARS provides protection against myocardial ischemia and reperfusion injury by inhibiting the expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) and, consequently, by inhibiting the recruitment of neutrophils into the jeopardized tissue. Furthermore, using in vitro studies, we demonstrate that in fibroblasts lacking a functional gene for PARS, cytokine-stimulated expression of ICAM-1 is significantly reduced compared with fibroblasts from animals with a normal genotype. Similarly, in cultured human endothelial cells, oxidative- or cytokine-dependent expression of P-selectin and ICAM-1 is reduced by pharmacological inhibition of PARS by 3-aminobenzamide. These findings provide the first direct evidence that PARS activation participates in neutrophil-mediated myocardial damage by regulating the expression of P-selectin and ICAM-1 in ischemic and reperfused myocardium, and they also provide the basis for a novel therapeutic approach for the treatment of reperfusion injury.

The role of melatonin as an immunomodulator is well established. Recent reports showed that melatonin exerts protective effects in septic and hemorrhagic shock and in inflammation. The expression of the inducible isoform of nitric oxide synthase (iNOS) makes an important contribution to the pathophysiology of shock and inflammation. We studied, in cultured murine macrophages, the role of melatonin in the regulation of the expression of iNOS and defined the mode of melatonin's action. Our results show that melatonin, at 1 microM-1 mM, decreased the production of nitrite/nitrate (the breakdown products of NO) as well as the production of 6-keto-prostaglandin F1alpha (the major stable breakdown product of prostacyclin) in macrophages stimulated with bacterial lipopolysaccharide (10 microg/ml). We observed that melatonin reduces iNOS steady-state mRNA levels and iNOS protein expression in the same concentration range (1 microM-1 mM). Melatonin, up to 10 mM, exerted only a slight direct inhibitory effect on iNOS activity. Using iNOS promoter-luciferase constructs, we found that melatonin inhibits iNOS promoter activation. Inhibition of iNOS expression was associated with inhibition of activation of the transcription factor nuclear factor kappa B (NFkappaB). We conclude that melatonin inhibits NO production in immunostimulated macrophages mainly by inhibiting the expression of iNOS. This is due to inhibition of iNOS transcription, in part through inhibition of NFkappaB activation. Inhibition of iNOS-derived NO production by melatonin may contribute to the anti-inflammatory effects of this pineal secretory product.