Roles And Mechanisms Of RIP3 In Mouse Models Of Avian Influenza H7N9 Virus Infection And Sterile Inflammatory Diseases

The receptor-interacting protein kinase 3(RIP3/RIPK3)is a member of RIP family,whose gene is located on chromosome 14 in humans.RIP3,a polypeptide of 518 amino acids,has an active serine/threonine kinase domain in its N terminus which is essential for programmed necrosis or termed necroptosis.Necroptosis is a programmed form of non-apoptotic cell death driven by RIP3 and its downstream substrate mixed lineage kinase domain-like protein(MLKL)and it exerts key functions in infectious and sterile inflammations.RIP3 interacts with its upstream kinase RIP1 via the RIP-homotypic interaction motif(RHIM)to form an amyloid-like complex termed the necrosome to signal necroptosis in response to death receptor or toll-like receptor stimulation,pathogen infection,or sterile cell injury.And the necrosome is essential for recruiting and phosphorylating the MLKL,activated MLKL forms oligomers then translocate to the plasma membrane and eventually induce membrane rupture and causes the leakage of cellular contents including danger-associated molecular patterns(DAMPs)to promote inflammatory reactions.More recently,emerging data suggest that RIP3 also has necrotosis-independent function.In this study,we establish several mouse disease models including mouse infected with avian influenza H7N9 virus model,dextran sodium sulfate(DSS)-induced acute colitis and cearulein-induced acute pancreatitis models to identify the specific function of RIP3 and the precise molecular machinery,and which will enhance our comprehension on the role of RIP3 in a certain disease setting and pathogenesis of H7N9 virus infection,colitis and pancreatitis as well as provide experimental basis for prevention measures.Part 1: RIP3 deficiency ameliorates inflammatory response in mice infected with Avian Influenza H7N9 virusHuman infection with avian influenza H7N9 virus causes an acute,highly contagious respiratory illness and sufferers may manifest a progressive pneumonia leading to acute respiratory distress syndrome(ARDS).H7N9 virus infection triggers cell death and airway epithelial destruction.Indeed,there were a great number of studies revealed that influenza H7N9 virus induced a wide variety of proinflammatory cytokines in the lung and serum also called cytokine storm.Inflammation brings about diffuse alveolar damage with disruption of bronchoalveolar network,increased lung tissue permeability,pulmonary edema,leading to blood-air barrier disruption.Necroptosis is a newly discovered pathway of regulated necrosis that requires RIP3 and MLKL,strikingly,RIP3 is a key regulator of necroptosis to a growing number of viral and microbial agents.However,the precise mechanism of RIP3 in the pathogenesis of influenza H7N9 virus infection-associated cytokine storm is still unclear.Here,RIP3 knock out(RIP3-/-)mice and littermate wild type mice were used to be infected intranasally with influenza H7N9 virus(A/Fujian/S03/2015)to determine the contribution of RIP3 to the cytokine storm of influenza H7N9 virus infection.We found that RIP3-/-mice infected with H7N9 virus showed higher survival and less weight loss compared with wild type littermate mice.In addition,RIP3-/-mice had fewer regions of edema,infiltration with inflammatory cells,and alvelolar collapses in the lungs.Consistently,the secretions of IL-1β,IL-6,RANTES and MIP-1α in bronchoalveolar lavage fluid(BALF)were significantly decreased when compared with WT mice.It is noteworthy that caspase-1/IL1β signaling was found to be involved in RIP3 associated inflammation of influenza H7N9 virus,but not RIP3/MLKL dependent necrosis.Our results indicated that RIP3 is involved in the production of cytokine storm following H7N9 virus infection and RIP3 deficiency can protect mice from the infection of influenza H7N9 virus by downregulating caspase-1/IL1β signaling,which provided evidence of the RIP3 involved necroptosis independent manner.Furthermore,this study suggests a novel therapeutic target of influenza H7N9 virus infection.Part 2: Roles and mechanisms of RIP3 in mouse models of sterile inflammatory diseasesRIP3 was reported to be involved in cell death and many inflammatory diseases,and previous studies inferred that RIP3-mediated necroptosis exacerbates tissue injury because massive inflammatory tissue injury could be rescued by additional deletion of RIP3.Moreover,inactived RIP3 also ameliorates tissue injury in mouse disease models of pancreatitis,atherosclerosis,kidney ischemia–reperfusion injury,myocardial infarction and so on.However,the contribution of necroptosis to inflammation in human diseases is still less clear,and emerging results suggest that RIP3 exerts roles in a various manners.In this part,RIP3-/-mice and littermate wild type mice were used to establish the colitis and pancreatitis models to determine the contribution of RIP3 in the pathogenesis of the diseases.We found that in DSS-induced colitis model,RIP3-/-mice showed significantly enhanced colitis symptoms,including increased weight loss,colon shortening and colonic mucosa damage and severity,but decreased production of interleukin-6(IL-6)and IL-1β.Furthermore,it was found that m RNA expressions of several repair-associated cytokines including IL-6,interleukin-22(IL-22),cyclooxygenase-2(Cox2),epithelial growth factor receptor ligand Epiregulin(Ereg)and matrix metalloproteinase 10(MMP10)were siginificant decreased in RIP3-/-mice.The results showed that RIP3 deficiency could not ameliorate but exacerbate the severity of colitis and RIP3-/-mice exhibited an impaired tissue repair in response to DSS.On the other hand,in cearulein-induced pancreatitis model,there is no significant difference in pancreatic tissue injury and serum amylase activity between RIP3-/-and WT mice.Thus,RIP3 play a neither protective nor detrimental role in the cearulein-induced pancreatic damage model.Collectively,these results suggest that the same RIP3 can have both protective and destructive roles which are context-dependent.