Role Of Zebrafish Caspy2-mediated Non-canonical Inflammsome Activation In Anti-infection And Endotoxic Shock

The innate immunity is a host natural defense against pathogens,and it plays critical role in triggering adaptive immunity.The inflammasomes,which compose with the multi-protein assembly complex,are one of the most important cytosolic pattern recognition receptor components of the innate immune system.Recently,the inflammasomes in mammals are identified as caspase-1-mediated canonical inflammasome and caspase-11-(mouse)or caspase-4/5(human)mediated non-canonical inflammasome.The non-canonical inflammasome is activated by caspase-4/5/11 that recognition of intracellular bacterial lipopolysaccharide(LPS,also known as endotoxin)through its CARD domain to trigger the pyroptosis,and engaged the maturation of inflammatory cytokines,such as IL-1(3 and IL-18.This process plays a key role in anti-bacterial infection,and also regulates the effects of endotoxic shock.From our previous results,we found a significant pyroptosis in ZF4 cells infected with the hemolysin-over-expressed E.piscicida 09901,compared with the wild-type E.piscicida EIB202 infection.Moreover,we identified that the zebrafish caspy2 was crucial for recognization of intracellular LPS.However,the critical roles of caspy2-meidiated non-canonical inflammasome activation in zebrafish during bacterial infection remains poorly understood.Based on the results above,this study will aim to analysis the caspy2 non-canonical inflammasome activation-mediated anti-bacteria effects in zebrafish larvae,and will try to analysis the role of caspy2 non-canonical inflammasome activation in regulating endotoxic shock.Firstly,we examined the expression of caspy2 during zebrafish development,and found that the protein was significantly expressed from 48 dpf.Meanwhile,the whole mount in situ hybridization was performed to find that caspy2 was mainly expressed in the mouth.pharyngeal arch and intestine during zebrafish embryonic development,which suggesting that caspy2 might play a role in mucosal immunity in response to infection.Then.we utilized the morpholino antisense oligonucleotides to develop the caspy2 knockdown zebrafish model.Based on the results above,we stepped forward to use the 5 dpf zebrafish larvaes to develop the E.piscicida immersion infection model.Compared with EIB202 infection,we detected a significant activation of caspy2 in zebrafish larvaes infected with 09091.Moreover.we utilized 09091 to infect wild-type or caspy2 knockdown zebrafish,and found that caspy2 knockdown zebrafish larvaes showed a higher mortality and increased bacterial colonization.compared with wild-type larvaes during infection.Interestingly,through in vivo microscopic analysis,we found that during 09091 infection in caspy2 knockdown zebrafish larvaes,the bacterial could break through the intestinal barrier and spread to the dorsal arch and muscle tissue.In addation,through histopathological analysis,we found that caspy2 plays an important role in maintaining the intestinal integrity of zebrafish larvaes during 09901 infection.Collectively,the results demonstrated that the caspy2-mediated non-canonical inflammasome activation plays an important role in restricting bacterial infection in intestine.To further investigate whether the caspy2-mediated non-canonical inflammasome activation plays a role in endotoxic shock,we utilized a lethal dose of LPS to soak the 5 dpf zebrafish larvaes,and established an endotoxic shock model.The wild-type zebrafish larvaes showed a significantly mortality,compared with in caspy2 knockdown zebrafish larvaes.Moreover,through in vivo microscopic analysis,we found that caspy2 knockdown zebrafish larvaes showed a significantly attenuation about the symbol of pericardial edema and tissue erosion caused by the endotoxic shock.In addition,through analysis the transcripts of IL-1?,TNF-a,IL-6,IL-8,IL-10,and IFN-y,we found a significantly reduced 'cytokines storm' in caspy2 knockdown zebrafish larvae.Collectively,the results suggest that inhibiting the caspy2-mediated non-canonical inflammasome activation could effectively reduce the endotoxic shock in zebrafish larvae.Taken together,this study analyzed the role of caspy2-meidated non-canonical inflammasome activation in response to bacterial infection,and it is crucial for restricting bacterial conolization in vivo.Meanwhile,the knockdown of caspy2 could significantly reduce the endotoxic shock in zebrafish larvae.The results offer a new insight for further investigation on non-canonical inflammasome activation in zebrafish,and also provide a platform for anti-endotoxic shock drug screening.