Serp-2, a virus-derived apoptosis and inflammasome inhibitor, attenuates liver ischemia-reperfusion injury in mice.

BACKGROUND: Ischemia-reperfusion injury (IRI) is an antigen-independent, innate immune response to arterial occlusion and ischemia with subsequent paradoxical exacerbation after reperfusion. IRI remains a critical problem after vessel occlusion and infarction or during harvest and surgery in transplants. After transplant, liver IRI (LIRI) contributes to increased acute and chronic rejection and graft loss. Tissue loss during LIRI has been attributed to local macrophage activation and invasion with excessive inflammation together with hepatocyte apoptosis and necrosis. Inflammatory and apoptotic signaling are key targets for reducing post-ischemic liver injury.Myxomavirus is a rabbit-specific leporipoxvirus that encodes a suite of immune suppressing proteins, often with extensive function in other mammalian species. Serp-2 is a cross-class serine protease inhibitor (serpin) which inhibits the inflammasome effector protease caspase-1 as well as the apoptotic proteases granzyme B and caspases 8 and 10. In prior work, Serp-2 reduced inflammatory cell invasion after angioplasty injury and after aortic transplantation in rodents. In this report, we explore the potential for therapeutic treatment with Serp-2 in a mouse model of LIRI. METHODS: Wildtype (C57BL/6 J) mice were subjected to warm, partial (70%) hepatic ischemia for 90 min followed by treatment with saline or Serp-2 or M-T7, 100 ng/g/day given by intraperitoneal injection on alternate days for 5 days. M-T7 is a Myxomavirus-derived inhibitor of chemokine-GAG interactions and was used in this study for comparative analysis of an unrelated viral protein with an alternative immunomodulating mechanism of action. Survival, serum ALT levels and histopathology were assessed 24 h and 10 days post-LIRI. RESULTS: Serp-2 treatment significantly improved survival to 85.7% percent versus saline-treated wildtype mice (p = 0.0135), while M-T7 treatment did not significantly improve survival (p = 0.2584). Liver viability was preserved by Serp-2 treatment with a significant reduction in serum ALT levels (p = 0.0343) and infarct scar thickness (p = 0.0016), but with no significant improvement with M-T7 treatment. Suzuki scoring by pathologists blinded with respect to treatment group indicated that Serp-2 significantly reduced hepatocyte necrosis (p = 0.0057) and improved overall pathology score (p = 0.0046) compared to saline. Immunohistochemistry revealed that Serp-2 treatment reduced macrophage infiltration into the infarcted liver tissue (p = 0.0197). CONCLUSIONS: Treatment with Serp-2, a virus-derived inflammasome and apoptotic pathway inhibitor, improves survival after liver ischemia-reperfusion injury in mouse models. Treatment with a cross-class immune modulator provides a promising new approach designed to reduce ischemia-reperfusion injury, improving survival and reducing chronic transplant damage.

Mouse Gamma Herpesvirus MHV-68 Induces Severe Gastrointestinal (GI) Dilatation in Interferon Gamma Receptor-Deficient Mice (IFNγR-/-) That Is Blocked by Interleukin-10.

Inflammatory bowel disease (IBD) and Clostridium difficile infection cause gastrointestinal (GI) distension and, in severe cases, toxic megacolon with risk of perforation and death. Herpesviruses have been linked to severe GI dilatation. MHV-68 is a model for human gamma herpesvirus infection inducing GI dilatation in interleukin-10 (IL-10)-deficient mice but is benign in wildtype mice. MHV-68 also causes lethal vasculitis and pulmonary hemorrhage in interferon gamma receptor-deficient (IFNγR-/-) mice, but GI dilatation has not been reported. In prior work the Myxomavirus-derived anti-inflammatory serpin, Serp-1, improved survival, reducing vasculitis and pulmonary hemorrhage in MHV-68-infected IFNγR-/- mice with significantly increased IL-10. IL-10 has been investigated as treatment for GI dilatation with variable efficacy. We report here that MHV-68 infection produces severe GI dilatation with inflammation and gut wall degradation in 28% of INFγR-/- mice. Macrophage invasion and smooth muscle degradation were accompanied by decreased concentrations of T helper (Th2), B, monocyte, and dendritic cells. Plasma and spleen IL-10 were significantly reduced in mice with GI dilatation, while interleukin-1 beta (IL-1ß), IL-6, tumor necrosis factor alpha (TNFα) and INFγ increased. Treatment of gamma herpesvirus-infected mice with exogenous IL-10 prevents severe GI inflammation and dilatation, suggesting benefit for herpesvirus-induced dilatation.

Viral serpin therapeutics from concept to clinic.

Over the past 19 years, we have developed a novel myxoma virus-derived anti-inflammatory serine protease inhibitor, termed a serpin, as a new class of immunomodulatory therapeutic. This review will describe the initial identification of viral serpins with anti-inflammatory potential, beginning with preclinical analysis of viral pathogenesis and proceeding to cell and molecular target analyses, and successful clinical trial. The central aim of this review is to describe the development of two serpins, Serp-1 and Serp-2, as a new class of immune modulating drug, from inception to implementation. We begin with an overview of the approaches used for successful mining of the virus for potential serpin immunomodulators in viruses. We then provide a methodological overview of one inflammatory animal model used to test for serpin anti-inflammatory activity followed by methods used to identify cells in the inflammatory response system targeted by these serpins and molecular responses to serpin treatment. Finally, we provide an overview of our findings from a recent, successful clinical trial of the secreted myxomaviral serpin, Serp-1, in patients with unstable inflammatory coronary arterial disease.

Inhibition of chemokine-glycosaminoglycan interactions in donor tissue reduces mouse allograft vasculopathy and transplant rejection.

BACKGROUND: Binding of chemokines to glycosaminoglycans (GAGs) is classically described as initiating inflammatory cell migration and creating tissue chemokine gradients that direct local leukocyte chemotaxis into damaged or transplanted tissues. While chemokine-receptor binding has been extensively studied during allograft transplantation, effects of glycosaminoglycan (GAG) interactions with chemokines on transplant longevity are less well known. Here we examine the impact of interrupting chemokine-GAG interactions and chemokine-receptor interactions, both locally and systemically, on vascular disease in allografts. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of GAG or CC chemokine receptor 2 (CCR2) deficiency were coupled with the infusion of viral chemokine modulating proteins (CMPs) in mouse aortic allograft transplants (n = 239 mice). Inflammatory cell invasion and neointimal hyperplasia were significantly reduced in N-deacetylase-N-sulfotransferase-1 (Ndst1(f/f)TekCre(+)) heparan sulfate (GAG)-deficient (Ndst1(-/-), p<0.044) and CCR2-deficient (Ccr2(-/-), p<0.04) donor transplants. Donor tissue GAG or CCR2 deficiency markedly reduced inflammation and vasculopathy, whereas recipient deficiencies did not. Treatment with three CMPs was also investigated; Poxviral M-T1 blocks CC chemokine receptor binding, M-T7 blocks C, CC, and CXC GAG binding, and herpesviral M3 binds receptor and GAG binding for all classes. M-T7 reduced intimal hyperplasia in wild type (WT) (Ccr2(+/+), p< or =0.003 and Ccr2(-/-), p