RELL1 Regulates Inflammation And Autophagy Pathwaysand Improves Mycobacterium Tuberculosis Survival In Macrophages

Tuberculosis(TB)has existed for long time and remains a major global health problem.It causes approximately 10 millions new infections each year and is one of the top ten causes of death worldwide.Mycobacterium Tuberculosis(MTB),the pathogen of TB,is an intracellular bacteria spreading through respiratory route.Macrophages are the main host of MTB as well as important immune cells fighting against MTB in innate immunity.After invading the human body,MTB can express virulence factors to modulate the immune system and inhibit the bactericidal effects of host.The functions of virulence factors include but not limited to inhibition of phagososome acidification and phagolysosome formation,prevention of autophagy,suppression of ROS/RNI production,and inhibition of apoptosis.RELL1 belongs to the RELT family,which is a member of the TNF receptor superfamily.This receptor is especially abundant in hematologic tissues.It has been shown to activate the inflammatory pathway and selectively bind TNF receptor-associated factor-1(TRAF1).In a high-throughput screening,RELL1 was identified as one of the host proteins that may affect the survival of MTB in macrophages,without any known function or mechanism.In this thesis,we aimed to study the function and mechanism of RELL1 in MTB infected macrophages.In this study,we constructed viral vectors up-regulating or down-reguating RELL1,packaged virus and infected RAW264.7 cells,to get the stable cell lines with up-regulated or downregulated expression of RELL1(RAW264.7-RELL1,RAW264.7-sh RELL1).In order to investigate the effects of RELL1 on the survival of MTB inmacrophages,we infected the stable cell lines with H37 Rv,and detected the intracellular bacterial loads at different time points.We found that up-regulation of RELL1 could improve H37 Rv survival in macrophages,while down-regulation of RELL1 inhibited the survival of H37 Rv.In order to study the mechanism of RELL1 supporting the survival of H37 Rv,we stimulated the stable cell lines with H37 Rv or TLR2 agonist,and detected the expression levels of inflammatory cytokines and activation status of signal molecules on NF-?B or MAPK pathway by Luciferase assays,ELISA and Western blot.The results revealed that RELL1 can enhance the phosphorylation of NF-?B and MAPK signal pathway molecules,including p65,IKK,I?b,Erk1/2,SAPK/JNK and p38,and promote the expression of TNF-? and IL-6 in macrophages.Meanwhile,we found that RELL1 can inhibit both H37 Rv or Rapamycin induced autophagy in macrophages by Western blot and Confocal.To further explore the mechanisms of these functions,we identified interacting proteins of RELL1 by co-IP and Mass spectrometry,and found that RELL1 interacted with m TOR and SPAK.Analysis of the phosphorylation levels of m TOR substrate p70S6 K showed that RELL1 increased the activity of m TOR,and thus inhibited cell autophagy.Taken together,our results indicated that RELL1 can activate inflammatory cytokine expressions in macrophages,and at the same time inhibit cell autophagy by interacting with m TOR and activating its activity.As autophagy is one of the most important strategies for macrophages to eliminate MTB,RELL1 improves H37 Rv survival in macrophages by inhibiting autophagy.