Role And Underlying Mechanism Of Skelemin IQGAP1 During Infection And Granuloma Formation Mediated By Mycobactertial PDIM And Drug Intervention Effects

Background As we all know;tuberculosis is a serious infectious disease.Mycobacterium tuberculosis is an intracellular pathogen that causes tuberculosis.Mycobacterium tuberculosis neither produces endotoxin,nor secretes exotoxin and invasive enzymes.Its pathogenicity is mainly related to the high content of lipid components on the bacterial cell wall.Objective To investigate the role of IQGAP1(IQ motif-containing GTPase-activating protein 1)in the infection and granuloma formation induced by mycobacterial cell wall PDIM lipid,and to clarify the corresponding molecular mechanism during this process.Moreover,this study will explore the internal mechanism of IQGAP1 manipulating p38 MAPK capacity and VEGF expression during mycobacterium infection and subsequent inflammation.Furthermore,small molecule inhibitor of IQGAP1 was administered and the subsequent impact on PDIM-mediated infection was probed.Our research will not only illustrate the pivotal role of IQGAP1 for mycobacteria infection and related diseases,but also provide detailed experimental data and insightful perspective for seeking and identifying new therapeutic targets for anti-tuberculosis.Methods This study will be carried out and accomplished by combining in in vitro Raw264.7 macrophages and in vivo murine infection models.1.To explore the effect of IQGAP1 on the expression profiles of inflammatory cytokines at the early stage of mycobacteria infection and the role of p38 MAPK signal in this process.(1)Raw264.7 macrophages were cultured and randomly divided into three groups: wide-type(WT)Mycobacterium marinum(WT Mm)-infected group,mycobacteria cell wall virulence factor PDIM-deficient mutant(ΔPDIM)-infected group,sterile PBS-treated cells as a control(Mock).At 0.5h,1h,2h and 4h post-infection(hpi),macrophages were harvested and kinetic expression of IQGAP1 protein was detected by western blot analysis.(2)At 4 hpi,q RT-PCR was used to assay the expression levels of pro-inflammatory mediators(TNF-α,IL-6)and anti-inflammatory factors(TGF-β,IL-10).Moreover,cells from different groups were harvested and western blot to detect the expression of TNF-α and TGF-β as well as the totaland/or phosphorylated-p38 MAPK and NF-κBp65.(3)The si RNA and/or overexpression plasmid(GV219＿Iqgap1)for targeting mouse IQGAP1 were designed and constructed and transfected into Raw264.7 macrophages,respectively.Then the Raw264.7 cells were infected with WT Mm and/or ?PDIM strains,respectively.The expression profiles of pro-inflammatory mediators(TNF-α,IL-6)and anti-inflammatory factors(TGF-β,IL-10)were analyzedby q RT-PCR.Moreover,expression of IQGAP1,TNF-α,TGF-β,and the phosphorylated proteins of p38 MAPK and NF-κBp65 were detected by western blot.Furthermore,the effects of IQGAP1 inhibition and/or overexpression on nuclear NF-κBp65dynamics were observed by using Confocal microscope.(4)Raw264.7 cells were infected with WT Mm and/or ΔPDIM and then harvested.q RT-PCR was used to detect the expression of critical activation kinases(e.g.,MKK3,and MKK6)and inactivated kinases(e.g.,MKP1 and MKP5)in the phosphorylation cascade of p38 MAPK signaling pathway.Continuously,those molecules were assay in Raw264.7 cells after IQGAP1 silencing with si RNA and/or transfected with GV219＿Iqgap1 plasmid.Based on the above experiments,the total-and phosphorylated-MKK3 in Raw264.7 cells that infected with WT Mm and/or ΔPDIM was detected by western blot,then the effects of IQGAP1 on the total-and phosphorylatedMKK3 were examined in macrophages after IQGAP1 silencing or overexpression.(5)Based on the above experimental results,Raw264.7 cells were infected with ΔPDIM strain and simultaneously treated with SB203580,a specific inhibitor of p38 MAPK signaling pathway.Samples were collected and the phosphorylation of NF-κBp65 at different time-points was detected.Finally,Raw264.7 cells were infected with ΔPDIM and co-incubated with Gossypetin,a novel inhibitor of MKK3/MKK6,then the phosphorylation level of MKK3 was detected by western blot.(6)A murine caudal tail vein infection model with Mycobacterium marinum was established according to the published protocol.At day 7 post-infection,Nocodazole,a skeleton protein inhibitor,was administered intraperitoneally once every 2days.After seven consecutive injections with Nocodazole,all mice were sacrificed and tail tissues were collected to grind the coating plate to calculate the bacterial loading.The gross tail lesions and histopathology were examined accordingly.Meanwhile,tails were homogenated and total protein were extracted for IQGAP1 detection.2.To investigate the role and underlying mechanism of IQGAP1 regulating VEGF expression during mycobacterium infection.(1)Raw264.7 infection model was established as previously described and VEGF contents in cellular and supernatant was detected by western blot and ELISA,respectively.(2)Raw264.7 macrophages was transfected with IQGAP1 specific si RNA and/or GV219＿Iqgap1 plasmid to silence or overexpressing IQGAP1,then infected with WT Mm and/or ?PDIM strains,and VEGF expression was detected by western blot.(3)Axitinib,a selective inhibitor of VEGF-receptor,was used to treat Raw264.7 cells and the expression of VEGF and IQGAP1 were detected by western blot.(4)The mouse infection model and Nocodazole intervention was performed as abovely described.And mice tail tissue was collected and the expression of VEGF was detected by western blot.The expression and distribution of IQGAP1 and VEGF were assay by immunohistochemistry staining.(5)Expression and distribution of IQGAP1 and VEGF were measured in lung tissues from H37Rv-infected mice,rabbit and monkey as well as clinical tuberculosis patients.Results 1.IQGAP1 affects the expression profiles of inflammatory cytokines in the early stage of mycobacterium infection by regulating p38 MAPK signaling pathway.(1)Compared with ΔPDIM-infected group,IQGAP1 in the WT Mm-infected Raw264.7 macrophage was significantly increased(**P<0.01).(2)q RT-PCR detection showed that the expression of anti-inflammatory factors(TGF-β,IL-10)in WT Mm-infected cells was highly expressed than that in ΔPDIM group(**P<0.01),while the expression of pro-inflammatory factors(TNF-α,IL-6)was significantly lower than that in PDIM group(**P<0.01).Western blot analysis showed that highly expressed TNF-α,phosphorylated p38 MAPK and NF-κBp65(**P<0.01),and lower TGF-β in ΔPDIM-infected group(*P<0.05),while a opposite expression profile of these same factors in WT Mm-infected macrophages.Confocal observation showed that a large amount of NF-κBp65 into the nucleus in ΔPDIM group,but less in WT Mm-infected cells(**P<0.01).(3)Raw264.7 cells was transfected with a si RNA to silencing mouse IQGAP1 and then infected with WT Mm and ΔPDIM,respectively.q RT-PCR and western blot analysis showed that the expression of TNF-α in WT Mm-infected group was significantly increased(*P<0.05),while TGF-β was dramatically decreased(**P<0.01).Western blot detection also found that phosphorylated p38 MAPK and NF-κBp65 were increased in WT Mm-infected cells(*P<0.05).Confocal observation revealed that inhibiting IQGAP1 significantly enhanced the ratio of nuclear NF-κBp65 in WT Mm-infected group(**P<0.01).However,Raw264.7 cells transfected with GV219＿Iqgap1 to overexpress IQGAP1,TNF-α was dramatically decreased as well as increased TGF-β in ΔPDIM-infected cells(*P<0.05),meanwhile the phosphorylated p38 MAPK and NF-κBp65 were also reduced significantly(**P<0.01).(4)q RT-PCR assay the expression profiles of key activated kinases(e.g.,MKK3,and MKK6)and inactivated kinases(e.g.,MKP1 and MKP5)in the phosphorylation cascade of p38 MAPK pathway,and just found MKK3 was markedly upregulated in ΔPDIM-infected cells compared with that in WT Mm-infected macrophages(****P<0.0001).Interfering with IQGAP1 by si RNA could promote the expression of MKK3,while overexpression of IQGAP1 inhibits MKK3 expression in Raw264.7 macrophages.Continuously,western blot analysis showed that the inhibiting IQGAP1 by si RNA significantly increased phosphorylated MKK3 in WT Mm-infected group(*P<0.05),while IQGAP1 overexpression dramatically decreased expression of phosphorylated MKK3 inΔPDIM-infection group(*P<0.05).(5)Raw264.7 cells were firstly treated with SB203580,a novel inhibitor of p38 MAPK pathway,then infected with ΔPDIM strain,the phosphorylated NF-κBp65 presented a dynamic change and decreased markedly at 1 hpi(*P<0.05).Moreover,Raw264.7 cells were co-incubated with ΔPDIM and Gossypetin,a selective inhibitor of MKK3/MKK6,its application significantly reduced the phosphorylated MKK3(**P<0.01).(6)A murine caudal vein infection model with WT Mm and/or ΔPDIM strain was established according to the published protocol.WT Mm-infected mice tails formed larger skin abscess and developed progressive tail lesions compared with the scattered lesions in ΔPDIM-infected mice.Histopathology examination found numerous infiltrations of inflammatory cells and granulomatous lesions in WT Mm-infected mice.CFU counting demonstrated that bacteria loading was gradually increased in WT Mm-infected mice.Surprisingly,Nocodazole treatment not only effectively alleviated tail tissue injury in mice infected with WT Mm,but also the CFU counting presented a gradual decrease in tissue bacteria loading(*P<0.05).Immunohistochemistry staining showed that the positive signals of IQGAP1 was strongly upregulated and distributed extensively in WT Mm-infected mice,and Nocodazole intervention significantly reduced the expression of IQGAP1.Western blot analysis demonstrated that the expression of IQGAP1 and TGF-β in the tail tissue of mice infected with WT Mm were increased,while dramatically decreased in Nocodazole treated mice(**P<0.01).2.Mycobacteria induced IQGAP to promote VEGF expression and forms a feedback circle between IQGAP1 and VEGF.(1)VEGF was highly expressed in WT Mm-infected Raw264.7cells compared with the diminished expression in ΔPDIM group(**P<0.01).(2)IQGAP1silencing with si RNA significantly reduced VEGF production in ΔPDIM-infected Raw264.7cells,while IQGAP1 overexpression greatly upregulated the VEGF creation inΔPDIM-infected cells(**P<0.01).(3)Axitinib,a specific inhibitor for VEGF-receptor,its application in Raw264.7 cells not only decreased the VEGF generation(*P<0.05),but also down-regulated the IQGAP1(*P<0.05).(4)Western blot analysis found that IQGAP1 was highly expressed in WT Mm-infected mice,while Nocodazole administration dramatically reduced the production of VEGF(*P<0.05).(5)Immunohistochemistry staining showed that VEGF was strongly expressed and distributed extensively in WT Mm-infected mice.(6)Immunohistochemistry staining found that IQGAP1 and VEGF were strongly expressed in the lung tissue from H37Rv-infected mice,rabbit and monkey as well as samples of clinical tuberculosis patients especially they were distributed in the granulomatous lesions.Conclusion 1.Mycobacterium can up-regulate the expression of skeleton protein IQGAP1,and this effect is mainly associated with bacteria virulence factor such as PDIM in this study.2.IQGAP1 can inhibit the phosphorylation level of p38 MAPK by down-regulating MKK3 production,and subsequently affect the activation of NF-κBp65 and expression profile of pro-inflammatory factors,thus inhibiting the early host immune response during mycobacteria infection.3.Mycobacteria induced IQGAP1 could promote the expression of VEGF.4.Inhibiting IQGAP1 could alleviate histopathology induced by mycobacteria infection and presents a promising therapeutic target for anti-tuberculosis.