Regulation And Molecular Mechanism Of RIPK3 On Host Cell Autophagy Induced By BCG Infection

Background:Tuberculosis is the second most infectious disease and a serious threat to human health in today’s world and is caused by the human pathogen Mycobacterium tuberculosis(Mtb).As the main host cells for invasive Mtb,macrophages interact with Mtb,playing a crucial role in the occurrence and development of TB.Importantly,diverse cell death modes may be induced during Mtb infection,and of these types,some propose limiting bacterial intracellular growth and facilitating anti-Mtb immune responses,others benefit Mtb replication and transmission.In fact,autophagy promotes fusion of Mtb phagosomes with autophagosomes and facilitates subsequent clearance of bacilli in autophagolysosomes,while Mtb escapes host defense by inhibiting phagosome maturation.Therefore,it is of great scientific significance to study the mechanism of Mtb-induced autophagy of macrophages to clarify the interaction between Mtb and macrophages.However,the autophagy process is regulated by a variety of complex signals and the mechanisms of its regulation have not been fully elucidated.Recently,receptor-interacting protein kinase 3(RIPK3)has been found to be involved in the regulation of autophagy,but whether it is involved in the regulation of Mtb-induced macrophage autophagy has not yet been reported.A previous study by our research group found that BCG infection promoted up-regulation of RIPK3 expression in macrophages.Studies have found that RIPK3 promotes macrophage autophagy after BCG infection,but the mechanism is still unclear.Based on the above analysis,our research questions were:(1)What key steps does RIPK3 play in regulating autophagy of macrophages induced by BCG infection?(2)What molecular mechanism does RIPK3 regulate macrophage autophagy?(3)How does RIPK3 regulate autophagy in lung tissue and the body’s ability to fight infection after BCG infection in mice?To answer the above scientific questions,the following research is proposed:Purpose and methods:1.In this study,we explore the regulatory effect of RIPK3 on autophagy of BMDMs induced by BCG infection in vitro.First,we constructed and isolated the bone marrow-derived macrophages from C57BL/6J mice and RIPK3-/-mice.In addition,we detected expression of related proteins at different autophagy stages in WT BMDMs and RIPK3-/-BMDMs with BCG infection,observed the number of autophagosomes in cells,and autophagy flow by Western blot,immunofluorescence,and flow cytometry.These studies will clarify that RIPK3 is involved in regulating the key stage of BCG-induced macrophage autophagy.2.We further explored the molecular mechanism of RIPK3 regulating autophagy in BCG-induced BMDMs in vitro.We screened proteins interacting with RIPK3 in macrophages after BCG infection by immunoprecipitation mass spectrometry(IP-MS).Autophagy proteins were further screened,and the interaction between RIPK3 and protein at endogenous and exogenous levels was verified by co-precipitation(CO-IP)and immunofluorescence.Next,after confirming the interaction between RIPK3 and protein X,the RIPK3 mutant plasmid was further constructed to explore the RIPK3-protein interaction domain.Finally,direct correlation factors of RIPK3 interacting protein regulating autophagy were elucidated through literature.The relationship between RIPK3,RIPK3 interaction protein and autophagy-related factors was verified from endogenous and exogenous sources.3.To verify the regulatory effect of RIPK3 on lung autophagy and anti-infection ability of mice after BCG infection in vivo,the following studies were performed.The level of autophagy in lung tissue of WT and RIPK3-/-mice after BCG infection was further detected by Western blot and immunohistochemistry,and the regulatory role of RIPK3 on autophagy in lung tissue of mice after BCG infection was clarified.Furthermore,the effects of RIPK3 on lung tissue injury,bacterial load in lung tissue,expression of inflammatory factors in mouse serum,and immune cell recruitment in mouse lung tissue were detected by HE staining,CFU colony count,ELISA,and flow cytometry.Results:1.Our findings reveal that RIPK3,p-RIPK3,and LC3II expression are up-regulated with BCG infection in BMDMs,and promote p62 degradation.Further research confirmed that p-PI3K,p-AKT,and p-mTOR protein expression were down-regulated in BCG-infected WT BMDMs that were reversed in RIPK3-/-BMDMs.After BCG infection,compared to WT BMDMs,multiple autophagy levels in RIPK3-/-BMDMs changed.The expression of the autophages formation proteins Beclin1,Atg5,Atg7,Atg12 and LC3Ⅱ was down regulated,the number of autophages was reduced,utophages and lysosomal fusion proteins Rab7 and LAMP2 were down regulated,the expression of p62 was up regulated,co-localization of LAMP2 and LC3 was reduced,and autophagic flow was blocked.Meanwhile,the intracellular bacterial load of RIPK3-/-BMDMs increased and cell survival decreased compared to WT BMDMs with BCG infection.2.We screened 204 proteins interacting with RIPK3 in BCG-infected macrophages by immunoprecipitation(IP)and liquid chromatography tandem mass spectrometry(LC-MS/MS).Three proteins directly involved in autophagy were further screened,including Hspa8,ANXA2 and Etf1.We further verified that ANXA2 interacts with RIPK3 in BMDMs with BCG infection by CO-IP and immunofluorescence.We found that RHIM domain is associated with RIPK3 binding ANXA2,by constructing expression vectors with different RIPK3 domains.Importantly,the results show that RIPK3 knockout can inhibit the expression of ANXA2 in the cell membrane and cytoplasm,increase its expression in the nucleus,enhance the interaction between ANXA2 and TFEB,and reduce the expression of TFEB in the nucleus of BMDMs with BCG infection.The above results were verified in 293T cells,and RIPK3-HA,ANXA2-Flag and TFEB-Myc plasmids were simultaneously expressed in cells.The results showed that RIPK3 promoted the translocation of ANXA2 membrane,inhibited the interaction between ANXA2 and TFEB,and promoted the expression of TFEB in the nucleus.3.The in vivo study further confirmed the above findings.Our results showed that RIPK3 and LC3II expression in the lungs of mice was upregulated and p62 expression was inhibited with BCG infection.Compared with WT mice,the expression of LC3II,Beclin1,Atg5 and Atg12 in the lung tissue of RIPK3-/-mice decreased,and the expression of p62 increased,and the number of autophagic lysosomes in the lung tissue decreased with BCG infection.In addition,pathological score results showed that RIPK3-/-mice had relatively severe lung tissue damage.After BCG infection,compared with WT mice,the bacterial load in the lung tissue of RIPK3-/-mice increased,the body weight of mice decreased in the first 10 days,and TNF-α,IL-6 and IL-1β were increased in the serum of mice,the expression of IL-10 decreased,and the number of macrophages increased in lung tissue.And CD8+ T cells were reduced in mice’s lungs.Conclusion:1.RIPK3 promotes autophagosome induction,autophagosome formation and fusion of autophagosomes and lysosomes in BMDMs after BCG infection,and promotes intracellular autophagy flow.Meanwhile,RIPK3 inhibits bacterial load in BMDMs after BCG infection and promotes cell survival.2.RIPK3 interacts with ANXA2 via RHIM domain,promotes ANXA2 membrane translocation,inhibits ANXA2-TFEB interaction,releases TFEB from cytoplasm into nucleus,and promotes autophagy of BMDMs after BCG infection.3.RIPK3 promotes autophagy levels in lung tissue of BCG-infected mice,and reduces bacterial load in lung tissue of mice,inhibits pathological lung tissue damage in mice after BCG infection,which has a protective effect on anti-infection in mice.