Cisatracurium Besylate Inhibits Necroptosis And Regulates Macrophage Anti-tuberculosis Immunity

Background:Tuberculosis(TB)is the world’s leading infectious disease,caused by infection with the bacterium M.Tuberculosis(MTB).Innate immunity is the first line of defense against tuberculosis infection,in which macrophages(Mφ)play an important role.MTB can cause a variety of death pathways in Mφ to execute immune evasion and accelerate the spread of the bacteria and expand the damage of host lung tissue,including apoptosis,necroptosis,ferroptosis,pyroptosis and etc.Therefore,the development of drugs to combat cell death induced by MTB infection would be of great benefit to TB patients.Objectives and methods:1.RNA sequencing combined with Western blot were used to verify the cell death mode caused by MTB infection.2.Using CCK-8 assay to preliminarily screen the small molecule drugs which may play a protective role against Mφ infection in the FDA drug library.3.Employing scanning electron microscope(SEM)and transmission electron microscope(TEM)to observe the number and morphology of necrotic cells following H37Rv infection.4.Morphological evaluations using HE staining and IHC detection of p-MLKL within spleen and lung tissues between tuberculosis(TB)compared with patients harboring chronic inflammation(CI).5.Western blot and CCK-8 assay were used to detect changes in molecular phosphorylation levels of(BMDMs)infected with MTB using Nec-1 or Resibufogenin(RES)along or simultaneously with CIS.6.Detection and quantification of p-MLKL translocation in H37Rv-infected BMDM using immunofluorescence.7.TNF-α and zVAD stimulated L929 cells were used to establish the classical necroptotic cell model in vitro.The oligomerization of MLKL on the plasma membrane of L929 cells treated with TNF-α and zVAD or simultaneously with CIS was detected following the nuclear and cytoplasmic fraction.8.DARTS and Co-IP were used to verify the target of CIS and comparing the interaction between necroptosis pathway molecular with or without CIS treatment.9.Constructing plasmids of MLKL and RIPK3 protein domains.Using DARTS to detect the specific domains binding with CIS.Co-IP was used to explore the change of interaction between RIPK3 domains and MLKL domains with or without CIS.10.Detecting RIPK3 and MLKL phosphorylation when ZBP1 silencing,RIPK1 silencing and TRIF silencing to determine the upstream molecules that initiate necroptosis in Mφ infected with MTB.11.The cell models and animal model of CIS for MTB infection were constructed in vitro and in vivo,respectively,to detect the bactericidal effect of CIS alone and/or isoniazid combined with macrophage.Results:1.Mtb infection induces differential modes of cell death in macrophages.2.CIS is a potent protective drug in rescuing macrophages from Mtb infection-induced cell death.3.CIS rescue macrophages from H37Rv infection-induced necroptosis.4.CIS suppress MLKL phosphorylation by inhibiting the association between RIPK3 and MLKL.5.CIS suppress both the RIPK1/RIPK3 and the ZBP1/RIPK3 branches of necroptosis in Mtb-infected Mφ but not through interfering the association between RIPK1 or ZBP1 and RIPK3.6.CIS exert protective effects against Mtb infection in vitro and in vivo.Conclusions:In this study,cisatracurium besylate(CIS)was screened out from the FDA approved drug library and showed to be highly protective for the viability of macrophages during Mtb infection.Transcriptomic sequencing combined with death modes discrimination and cytomorphological observation confirmed CIS functions in inhibiting Mtb-induced necroptosis but no other death modes.CIS inhibited RIPK3 to interact with the executive molecule MLKL to form intact necroptotic complex for preventing MLKL phosphorylation and pore formation on cell membrane,while did not interfere the auto-phosphorylation of RIPK3 induced by binding with it upstream RIPK1 or ZBP1 during Mtb infection.Moreover,CIS treatment significantly inhibited the expansion of intracellular Mtb both in vitro and in vivo and displayed a strong auxiliary bacteriostatic effect to support isoniazid and rifampicin,the first-line anti-tubercular drugs.The anti-necroptotic and anti-Mtb roles of marketed CIS potentiates the rapid development of CIS to be an adjuvant drug for antituberculosis therapy,assisting the battle against drug-resistant tuberculosis.