Regulation Of RIP3 On Autophagy Of Macrophage Induced By Bacillus Calmette-Guerin

Background:Tuberculosis(TB)is a severe infection disease of human and animals caused by Mycobacterium tuberculosis(Mtb).As the main target cell of Mtb,macrophage could eliminate the intracellular bacteria and suppress Mtb proliferation through autophagy.However,Mtb destroy autophagy which further help Mtb avoid immune effects.The interaction between Mtb and macrophage are complex and has not been fully studied.Receptor-interacting protein 3(RIP3),a serine/threonine kinase,played an essential role in regulating cell death.The previous study showed that RIP3 can not only swiches apoptosis to necrosis,but also is involved in cell autophagy.Our previous study found that Mtb infection can induce autophagy with a increasing RIP3 expression in RAW264.7 cells.However whether RIP3 is invovled in macrophage autophagy infected with BCG(Bacillus Calmette-Guerin)is still unclear.Taken together,to investigate the role of RIP3 in regulating the autophagy induced by BCG in RAW264.7 cells,we carried out the studies as follows:Methods:We used small interfering RNA(siRNA)to knocked down RIP3 expression in RAW264.7 cells infected with BCG.TEM and immunofluorescence q-PCR and Western blotting were used to detected the autophagy-related genes indicators which further revealed the effect of RIP3 on macrophage autophagy induced by BCG.Furthermore,rapamycin was employed to deternmined whether RIP3 could regulate the autophagy induced by BCG through mTOR-dependent pathways.And the interaction and colocalization of RIP3 and P62 were further explored.The results are shown as below:Results:(1)BCG infection could induce macrophage autophagy.Protein expression of LC3? was significantly increased by BCG infection with 10 MOI for 6 hours.Meanwhile,the expression of RIP3 in RAW264.7 cells were dramatically increased by BCG infection and suppressed by siRNA targeting RIP3(si-RIP3).(2)siRNA-RIP3 suppressed the accumulation of autophagosome and autolysome and autophagy flux in BCG-infected RAW264.7 cells;Furthermore,siRNA-RIP3 decreased the expression of autophagy associated genes such as LC3,ULK1,Beclinl ATG5/7/12 and aggravated the accumulation of P62 and inhibited autophagy process.(3)siRNA-RIP3 enhanced the expression of p-PI3K,p-AKT and p-mTOR in macrophages with BCG infection.Furthermore,the promotion of autophagy associated genes LC3?,ATG5 and ATG12 induced by rapamycin could be inhibited by siRNA-RIP3.The results indicated that RIP3 is involved in regulating autophagy of RAW264.7 cell infected with BCG via mTOR-dependent pathway.(4)Immunoprecipitation experiments results showed that RIP3 could interact with P62.BCG infection increased the expression of RIP3 and promoted the colocalization of RIP3 and p62.Furthermore,RIP3 and P62 colocalized with lysosomal membrane protein LAMP2 respectively which were suppressed by siRNA-RIP3 in RAW264.7 cells infected with BCG.The results suggested that RIP3 induced by BCG-infection promoted the formation of autolysosome by regulating colocalization withP62 in RAW264.7 cells which were inhibited by si-RIP3.To sum up,BCG infection induced autophagy with an increasing RIP3 expression in RAW264.7 cells.While siRNA-RIP3 down-regulated the expression of autophagyassociated genes induced by BCG-infection via mTOR-dependent pathway;In addition,siRNA-RIP3 inhibited RAW264.7 cells autophagy by suppressing interation of RIP3 and P62 following with the reduced autolysosome maturation.