The Role Of Fbxw7 During The Infection And Granuloma Formation Mediated By Mycobacteria And The Intervening Effect Of Fbxw7-targeted Small Drug

Background: Tuberculosis(TB)is a serious infectious disease caused by the Mycobacterium Tuberculosis(Mtb)infection.China is one of the 30 countries with the highest burden of TB in the world,accounting for 27% of the global TB patients.With the COVID-19(COVID-19)pandemic in 2020,the situation for TB control is even more serious.Objective: To study the role of Fbxw7(F-box and WD repeat domain containing7)during the inflammation and granuloma formation mediated by mycobacteria infection.Moreover,this study will further clarify the significance of Fbxw7-targeted regulation of TNF-α for mycobacteria pathogenesis,and to explore the intervention effect of small molecule drugs targeting Fbxw7 pathway during mycobacteria infection.Therefore,our study will illustrate the critical role of Fbxw7 in mycobacteria infection and provide comprehensive and detailed information of Fbxw7 act as a drug target for anti-tuberculosis research in the near future.Methods: 1.The murine Raw264.7 macrophages infection model was established.Under same conditions,Raw264.7 cells were randomly divided into three groups:sterile PBS-treated Raw264.7 cells as a control(Mock),wide-type(WT)Mycobacterium marinum(Mm)infected group,and mycobacteria cell wall PDIMdificiency mutant infected group(!PDIM)used a virulence-attenuated control.(1)Raw264.7 macrophages were infected with WT Mm-and/or !PDIM-strains according to MOI=5(numbers of bacteria/number of macrophages),respectively.All cells were harvested at 0.5h,1h,2h and 4h post-infection(hpi),and total RNA and protein were extracted,respectively.Then q RT-PCR and western blot were used to detected the profiles of m RNA and protein levels of Fbxw7.(2)Raw264.7 macrophages were infected with WT Mm-and/or !PDIM-strains according to MOI=5 or !PDIM-strains according to MOI=20,respectively.The expression of Fbxw7 protein was detected by Western blot.(3)Raw264.7 macrophages were infected with WT Mm-and/or !PDIM strain,and at 4 hpi all cells were collected,fixed and stained,then the distribution and expression of Fbxw7 were examined by using Confocal microscope.(4)The specific small interfere RNA(si RNA)target Fbxw7 gene was synthesized and transfected into Raw264.7 cells and continuously infected with WT Mm.Then macophages were harvested and the expression profiles of proinflammatory cytokines including TNF-α,i NOS and anti-inflammatory mediators such as IL-10 and TGF-β were analyzed by q RT-PCR and western blot,respectively.(5)After silencing Fbxw7 with si RNA in WT Mm-infected Raw264.7 macrophages,the nuclear NF-κBp65 situation was observed by using Confocal microscope.2.Basedon the above experimental results,we continously explore the effect of Fbxw7 affect TNF-α production during mycobacteria infection.(1)Raw264.7macrophages were transfected with Fbxw7-targeted si RNA and infected with WT Mm,cells were harvested and TNF-α protein level was detected by western blot.(2)A vector specifically overexpressed Fbxw7 was constructed based on pc DNA3.1 backbone and the overexpression efficiency were confirmed by western blot,and finally termed pc DNA-Fbxw7.Then Raw264.7 cells were transefected with pc DNA-Fbxw7 and continuously infected with WT Mm-and/or-!PDIM strains.Cells were harvested and TNF-α was detected by western blot.(3)Raw264.7 cells were transfected with Fbxw7-si RNA and incubated with CHX,then the effects of Fbxw7 on TNF-α half-life upon WT Mm-infection was monitored and analyzed by western blot.(4)Raw264.7macrophages were pretreated with MG132,then the protein’s kinecticsof Fbxw7 and TNF-α in different groups were detected by western blot.(5)Raw264.7 cells were pretreated with SB-216763 and its effects on the expression profile of Fbxw7 and TNF-α in different infection groups were detected.(6)Different plasmids including MycFbxw7,Flag-TNF-α,Ha-Ub,Ha-Ub48/63 mutated constructs(Ha-Ub K48R/HaUb K63R)were co-transfected into MG132 pretreated Raw264.7 macrophages,then tag proteins were detected by Western blot.3.A murine caudal vein infection model in C57BL/6 mice infected with WT Mm(Mycobacterium marinum)was established,and then SB-216367 was intraperitoneally administrated to explore the effects of inhibiting Fbxw7 on WT Mm-mediated inflammation and tissue injury.In mice infected with WT Mm and treated with SB-216763,the tail gross lesions and abscess areas and tissue injury were monitored.Histopathology examination and and red-rod bacteria in the tail of mice were observed by HE and acid-fast staining,respectively.Combination of Immunohistocheminstry staining and image-acquisition system to assay the expression and distribution of Fbxw7 positive signal in mice tail.The protein expression and distribution of Fbxw7 were detected by immunohistochemistry in mice rabbits and clinical patients infected with Mycobacterium tuberculosisResults: 1、(1)At MOI=5,the m RNA and protein levels of Fbxw7 were upregulated in WT Mm group,while reduced dramatically in !PDIM group.(2)When increase the MOI of !PDIM group at 20,the Fbxw7 protein level still lower compared with WT Mm MOI 5.(3)After WT Mm-infection,anti-inflammatory cytokines of IL-10 and TGF-β were increased and pro-inflammatory factors(TNF-α and i NOS)were decreased.However,silencing Fbxw7 in WT Mm-infected Raw264.7 cells with si RNA significantly down-regulate the expression of IL-10 and TGF-β(**P<0.01),as well as transparently increased the pro-inflammatory mediators such as TNF-α and i NOS(**P<0.01).(4)Althrough confocal observation don’t find obvious co-colocalization of Fbxw7 and NF-κBp65 after WT Mm-infection,inhibiting Fbxw7 greatly increased NF-κBp65 nucleation during Mm-infection(**P<0.01).2、(1)TNF-α expression was increased in WT Mm-infected group after Fbxw7 silencing with si RNA.(2)TNF-α decreased in Raw264.7 macrophages after Fbxw7 overexpression.(3)Raw264.7 macrophages combinedly treated with Fbxw7 si RNA and cyclohexane(CHX)found that inhibiting Fbxw7 obviously prolong the TNF-αhalf-life.(4)MG132,a protease inhibitor,its administration ib Raw264.7 macrophages greatly enhance the protein accumulation of TNF-α in both WT Mm-and/or !PDIM groups.(5)Moreover,in SB-216763-pretreated macrophages,western blot detection found that the protein level of TNF-α was increased in both WT Mm-and/or !PDIM groups.(6)Raw264.7 macrophages were co-transfected with Myc-Fbxw7,Flag-TNF-α,Ha-Ub,Ha-Ub K48 R and/or Ha-Ub K63 R expression profile of TNF-α demonstrated that it was regulated by Fbxw7 ubiquitination modification by which possible through the K63 pathway.3、In a murine caudal vein infection model,the tail in mice WT Mm-infected formed larger skin abscess and developed progressive tissue injury,surprisingly,the tail injury was greatly relieved by SB-216367 application.Moreover,CFU counting demonstrated that the bacterial loading in WT Mm-infected mice were gradually increased(*P<0.05),in contrary,which was dramatically reduced after SB-216367 treatment.Immunohistochemical detection of Fbxw7 in mice rabbits and clinical patients infected with Mycobacterium tuberculosis showed positive expression near the granuloma tissue.Conclusion:(1)Fbxw7 induction by mycobacteria is mainly related to the virulence component such as PDIM.(2)Fbxw7 may regulate TNF-α expression via K63-linked ubiquitination during mycobacterium infection.(3)SB-216763,a small molecule drug targeting Fbxw7 pathway,could significantly alleviate the tissue damage induced by Mycobacterium marinum in mice.