Mechanisms Of NLRP3 Inflammasome Activation By Duck Hepatitis A Virus

Duck hepatitis A virus type 1（DHAV-1）is a single-stranded positive RNA virus belongs to the Avihepatovirus genus of the Picornaviridae family,and is one of the major pathogens affecting the duck industry in China.NLRP3 inflammasome can mediate the maturation and secretion of IL-1β,which is a key factor in regulating the inflammatory response and plays an important role in the inflammatory response.However,the role of duck NLRP3 inflammasome in DHAV-1 infection is still unclear.Thus,in this study,we used DHAV-1 to infect duck embryonic fibroblasts（DEF）to clarify the role and activation mechanism of NLRP3 inflammasome during DHAV-1infection.The main results are as follows:1.DHAV-1 induces the maturation and secretion of IL-1βthrough activating NLRP3inflammasome（1）To clarify whether DHAV-1 infection can activate inflammasome,DEFs were infected with different doses of DHAV-1(10² TCID₅₀,10³ TCID₅₀,and 10⁴TCID₅₀).After 48 h post-infection,cells and supernatants were collected and examined by q PCR,Western-blot and ELISA for transcription,protein expression,maturation and secretion of IL-1β,respectively.The results showed that the m RNA level and protein expression of IL-1βincreased after DHAV-1 infection,and mature IL-1β（p17）could be detected in the supernatant.Moreover,the concentration of IL-1β（p17）in the supernatant gradually increased with increasing dose of virus infection.In addition,the cells and supernatants were collected at different times after DHAV-1 infection(10³ TCID₅₀)and examined.The results showed that the transcript,protein expression and secretion of IL-1βwere increased gradually with prolonged infection.These results suggest that DHAV-1 can activate inflammasome and that inflammasome activity is dose-and time-dependent on DHAV-1 infection.（2）Further testing revealed that after DHAV-1 infection,the transcription level of NLRP3 increased.By knocking down NLRP3 gene,the secretion of IL-1βwas significantly decreased.While overexpressing NLRP3,IL-1βsecretion was significantly increased.These results suggest that DHAV-1 mediates IL-1βmaturation and secretion through the activation of NLRP3 inflammasome.（3）In order to detect whether the replication of DHAV-1 would be affected by NLRP3 inflamomsome,we detected viral copies of DHAV-1 after NLRP3 gene overexpression and knockdown,respectively.The results showed that the copies of DHAV-1 were significantly decreased after NLRP3 overexpression.However,when NLRP3 gene was knockdown,there was no significant difference of the copies of DHAV-1 compared to si Control group.This indicates that the activation of NLRP3inflammasome has certain inhibitory effect on the replication and proliferation of DHAV-1.2.Investigation of the mechanism of NLRP3 inflammasome activation by DHAV-1（1）The NLRP3 eukaryotic expression plasmid was transfected into DEF cells following by DHAV-1 infection,the distribution of NLRP3 changed from dispersive in the cytoplasm to perinuclear aggregation after DHAV-1 infection detected by IFA assay,indicating that DHAV-1 could induce NLRP3 oligomerization.Then we used UV-and Hot-inactivated DHAV-1 to infect DEF,there was no secretion of IL-1βin the supernatant detected by ELISA.This suggests that the activation of NLRP3inflammasome by DHAV-1 is dependent on viral replication and/or translation,but not the genomic RNA or capsid protein.（2）To further investigate whether DHAV-1 2B protein is involved in activating NLRP3 inflammasome,2B protein was co-expressed with NLRP3 and it was found that 2B protein could promote the formation of NLRP3 puncta appeared in the perinuclear region and IL-1β（p17）secretion.Then to assess whether the activation of NLRP3 inflammasome by 2B protein depends on its calcium channel activity,DEFs infected with DHAV-1 and transfected with p2B were treated by increasing extracellular Ca²⁺concentration or decreasing cytoplasmic Ca²⁺concentration and detected the secretion of IL-1β.The results showed that the secretion of IL-1βwas gradually increased with the increase of extracellular Ca²⁺concentration.However,the secretion of IL-1βdecreased significantly after chelating cytoplasmic Ca²⁺.It is suggested that2B protein of DHAV-1 activates the NLRP3 inflammasome and is dependent on the increase in cytoplasmic Ca²⁺concentration induced by 2B protein.（3）To explore whether K⁺efflux and mtROS are also involved in the activation of NLRP3 inflammasome after DHAV-1 infection,we increased the extracellular K⁺concentration,and then detected the secretion of IL-1βin the supernatant.The results showed that high extracellular K⁺concentration（>50 mmol/L）inhibited the maturation and secretion of IL-1βinduced by DHAV-1 infection.However,low concentration of K⁺（<25 mmol/L）could promote this process.In addition,the secretion level of IL-1βinduced by DHAV-1 was significantly decreased when DEFs were treated with Mito-TEMPO（10μmol/L）.These results indicate that K⁺efflux and mtROS are also involved in the activation of NLRP3 inflammasome induced by DHAV-1.