| In pathogen-host interactions,some pathogens escape host immune recognition through different mechanisms and successfully colonize,establish replicative ecological niches and latent in the host,eventually causing persistent infection,others are effectively recognized by the host’s immune system and rapidly activate the immune response,so that they are early cleared by the host.The level of immune response determines the fate of pathogen-host interactions.It is important to clear the infecting pathogenic bacteria as early in the infection as possible.Therefore,exploring the regulation of the immune response has great theoretical significance and potential application in the prevention and treatment of infectious diseases.Francisella is a facultative intracellular pathogen that contains four subspecies,the most virulent of which is Francisella tularensis,causing severe human disease called tularemia.Francisella novicida is the least virulent subspecies causes severe disease in mice but not in immunocompetent humans and served as a mouse strain to study Francisella pathogenesis.AIM2(absent in melanoma 2)is a pattern recognition receptor for double-stranded DNA in the cytoplasm,and its mediated inflammasome activation and pyroptosis play crucial roles in host defense against Francisella infection.Lipopolysaccharide(LPS)makes up the outer leaflet of the outer membrane of Francisella,and the LPS modification plays an important role in Francisella escaping host immune defense.However,the effect of LPS structural alterations on the immune response,especially pyroptosis,induced by F.novicida infection,and the mechanisms by which the host regulates AIM2 inflammasome activation are unclear.We focus on the effect of altered Francisella LPS structure on inflammasome activation and the regulation of AIM2 inflammasome to investigate the role of inflammatory response in host resistance to pathogenic bacterial infection.The results mainly contain the followings:(1)AIM2 inflammasome activation via the type I interferon pathway plays an important role in host immune defense to wild-type F.novicida U112.We identified that Francisella novicida mutant XWK4 triggers more inflammatory cytokine production and inflammasome activation than U112 strain.Moreover,the inflammasome activation induced by XWK4 was partially reduced in Ifnar-/-Ifn γ-/double knockout(AG6)BMDMs but not in lfnar-/-BMDMs.Further studies indicate that XWK4 engages with both NLRP3 and AI M2 for inflammasome activation and NLRP3 and AIM2 play independent roles in the inflammasome activation during XWK4 infection.(2)To investigate the regulation of AIM2 inflammasome activation upon F.novicida infection,we performed AIM2 IP-mass spectrometry(IP-MS)analysis of F.novicida-infected WT and Aim2-/-BMDMs.By analyzing the protein composition of IP products from WT and Aim2-/-BMDMs,we identified an E3 ligase,HUWE1,present in the IP products of WT BMDMs.Further studies revealed that HUWE1 interacts with three inflammasome sensor proteins,AIM2,NLRP3 and NLRC4.The interaction was dependent on the 1695-2028 amino acids of HUWE1 containing the BH3 domain and the HIN domain of AIM2,the NACHT domain of NLRP3,and the NACHT domain of NLRC4,respectively.In Huwe1 gene-deleted BMDMs,we noted a substantial reduction of caspase-1 activation of NLRP3,AIM2 and NLRC4 inflammasomes.And BI8622,an inhibitor of HUWE1,also significantly inhibited AIM2,NLRP3 and NLRC4 inflammasomes activation in mouse BMDM,human THP1 and PBMC.Mechanistic studies revealed that HUWE1 enhanced the level of K27linked polyubiquitination of AIM2,NLRP3 and NLRC4,and which was dependent on K23 and K26 sites of AIM2,K21,K22 and K24 sites of NLRP3 and K61 and K71 sites of NLRC4,respectively.AIM2,NLRP3 and NLRC4 wild type and mutant receptors were constructed in HEK293T cells,and it was found that the inflammasome activation was significantly attenuated by mutation of K27-linked polyubiquitination site.These above results demonstrate that HUWE1 promotes activation of inflammasomes by mediating K27-chain ubiquitination modification of receptor molecules.(3)To investigate the role of HUWE1 in host defense against bacterial infection,we treated age-and sex-matched Huwe1+/+-CreER and Huwe1fl/fl-CreER mice with tamoxifen for 5 consecutive days to induce Huwe1 gene deletion.Five days after the last injection of tamoxifen,the mice were infected with bacterial pathogens to analyze host defense and immune responses.Both caspase-1 activation and IL-1β secretion were significantly lower in Huwe1fl/fl CreER mice,with a significant increase in bacterial burdens,demonstrating that HU WE1-mediated inflammasome activation promotes host resistance to F.novicida,A.baumannii and Salmonella infections.However,in F.novicida mutant XWK4-infected WT and Asc-/-mice,WT mice showed higher levels of inflammasome activation than Asc-/-mice,but were more susceptible to XWK4 infection,and TUNEL staining showed significantly increased cell death in lung tissue of XWK4-infected WT mice.This demonstrated that tissue damage caused by XWK4-induced inflammasome activation suppressed host immune defence against XWK4 infection.In summary,the F.novicida mutant XWK4 induced stronger inflammasome activation,and we also found that the E3 ubiquitin ligase HUWE1 promoted the activation of AIM2,NLRP3 and NLRC4 inflammasomes.In different bacterial infections,inflammasome activation may have contrasting effects on host immune defence.HUWE1 promoted AIM2,NLRP3 and NLRC4 inflammasome activation enhancing host immune defence against F.novicida,A.baumannii and Salmonella.However,in XWK4 infection,tissue damage caused by continuously high inflammasome activation hampered clearance of XWK4.Our study provides new insights into the mechanisms of inflammasome activation in host defence and has important potential applications in the treatment of bacterial infections and inflammatory diseases. |
