TRAF3 Regulates Inflammasome Activation And Pyroptosis In Macrophages

Objective Tumor necrosis factor receptor associated factors 3?TRAF3?is a member of the TRAF family with E3 ligase activity,which regulates a variety of tumor necrosis factor receptors,multiple pattern recognition receptors?PRRs?,and a variety of other receptors.TRAF3 is widely involved in inflammatory responses,innate immunity,and tumorigenesis processes.In this study,by analyzing the relationship between TRAF3 and inflammasomes formation and pyroptosis in macrophages,we explored the molecular mechanism of TRAF3 regulating the activation of inflammmasome and pyroptosis in macrophages,and found a new signaling pathway in TRAF3 regulating the inflammatory response of macrophages.Methods 1.The myeloid cell conditional TRAF3 knockouts(TRAF3^MKO)mice were constructed,the genotype was determined by PCR and the protein levels of TRAF3 in macrophages were detected by Western bolt.The inflammasome activation was induced by LPS/Ng?nigericin?or LPS/ATP in macrophages,the landmarks of inflammasomes were detected by Western bolt.Y-VAD,a specific caspase 1 inhibitor,was used to inhibit inflammasome activation in peritoneal derived macrophages?PEMs?and the landmarks of inflammasome were measured using Western blot analysis.TRAF3-knockdown human monocytes THP1 were established,TRAF3 deficient THP1 cells or PEMs were stimulated by LPS/Ng and the pyroptotic cells were detected by flow cytometry.2.The protein level of ULK1 in macrophages was detected by Western blot.The protein and mRNA expression levels of ULK1 in macrophages were detected by Western blot and qRT-PCR.The ubiquitination of ULK1 was detected by Western blot.ULK1-knockdown THP1 cells were established,ULK1-deficient THP1 cells were stimulated by LPS/Ng and the landmarks of inflammasomes was detected by Western blot and pyroptosis was detected by flow cytometry.3.Monosodium urate?MSU?was used to induce peritonitis in mice.The production of IL-1?in peritoneal lavage fluid was detected by ELISA.The proportion and number of neutrophils in peritoneal lavage fluid were detected by flow cytometry.Results 1.TRAF3 was abolished in TRAF3^MKO macrophages,but the expression of TRAF3 in B cells and T cells were not affected.LPS/Ng or LPS/ATP stimulation could significantly induce production of inflammasome landmarks in macrophages.Meanwhile,inflammasome landmarks in TRAF3^MKO macrophages were obviously lower than that in WT.Y-VAD,an inhibitor of capase-1,suppressed activation of inflammasomes both in WT and TRAF3^MKO PEMs.In LPS/Ng-induced PEMs and THP1 cells,knockdown or deletion of TRAF3 significantly suppressed pyroptosis.2.Expression levels of ULK1 in TRAF3^MKO bone marrow derived macrophages?BMDMs?were up-regulated.In inflammasome activated macrophages,expression of ULK1in TRAF3^MKO was obviously higher than that in WT.Although there was no significant difference in mRNA levels of ULK1 between WT and TRAF3^MKO macrophages,the ubiquitination of ULK1 in TRAF3^MKO BMDMs was down-regulated.The levels of inflammasome landmarks and the pyroptosis proportion were significantly increased in LPS/Ng induced ULK1-knockdown THP1 cells.3.In the MSU-induced mouse peritonitis model,the concentration of IL-1?and the proportion and number of neutrophils in the peritoneal lavage fluid of the TRAF3^MKO group was significantly reduced.Conclusion 1.TRAF3 promotes NLRP3 inflammasome activation and pyroptosis in macrophages.2.TRAF3 enhances degradation of ULK1 through facilitating K48 ubiquitination of ULK1 and ULK1 surpresses NLRP3 inflammasomes activation and pyroptosis in macrophages.3.TRAF3 induces NLRP3 inflammasome activation in macrophages in vivo.