The Significance And Molecular Mechanism Of Mycobacterial Cell Wall PDIM Lipid Inducing Gal-3 To Regulate Inflammation And Granuloma Formation

Objective To identify the potential Toll-like receptor pathway of Gal-3 induction by mycobacterial cell wall lipid PDIM with established Raw264.7 macrophage cell line.Then to explore the role and mechanism of elevated Gal-3 on the production of inflammatory cytokines mediated by PDIM.Finally,SPF C57BL/6 mice was intravenously infected with mycobacterium marinum and TD139(Gal-3 specific inhibitor)was used to clarify the significance of Gal-3 during PDIM-mediated tissues injury and granuloma formation.Methods Raw264.7 cells were randomly divided into three groups: the control group(Mock),WT and ?PDIM infected groups.(1)Western blot was used to detect the Gal-3 protein level of each group during 0.5-4 h.The Gal-3 mRNA was detected at the same time-point by qRT-PCR.The expression and distribution of Gal-3 protein was also observed by immunofluorescence at 4 h after infection.The average numbers of intracellular bacteria at different time-point of each group was reviewed and counted under confocal microscope.(2)The protein of TLR2 and TLR4 levels during 0.5-4 h of each group were detected respectively.The mRNA of TLR2 and TLR4 was also detected by qRT-PCR at the corresponding time-point.Moreover,C29 and JSH-23,the specific inhibitor for TLR2 receptor and NF-?B,were used respectively,then the levels of Gal-3 protein and mRNA during 0.5-4 h in each group were detected by western blot and qRT-PCR.(3)After mycobacterium marinum infection,qRT-PCR was used to detect the expression of TNF-?,IL-6,iNOS,IL-10 and TGF-? at 4 h of each group.Western blot was used to detect the total protein of ERK,NF-?Bp65,IKK and its phosphorylated status during 0.5-4 h in each group was detected by western blot.The colocalization of Gal-3 and NF-?Bp65 at 4 h in each group was observed under confocal microscopy.Co-immunoprecipitation(Co-IP)was used to testify the interaction between Gal-3 and NF-?Bp65 molecules.(4)Gal-3 specific siRNA plasmid was successfully transfected into Raw264.7 cells,then WT and ?PDIM strains were infected for 4 h.qRT-PCR was used to detect the production of pro-inflammatory cytokines including TNF-?,IL-6,iNOS and anti-flammatory mediators including IL-10,TGF-? in each group.Confocal microscope was used to detect the intracellular expression and localization of Gal-3 and NF-?Bp65 in each igroup.(5)The Gal-3 over-expression plasmid was successfully constructed and transfected into Raw264.7 cells,and then ?PDIM strain was infected for 4 h.Since then,the inflammatory mediator including TNF-?,iNOS and TGF-? was detected by qRT-PCR.And the Gal-3 expression and NF-?Bp65 nuclear importing was observed by confocal microscope.(6)Firstly,a mouse model of tail vein infection with M.marinum was established,and then TD139(Gal-3 specific inhibitor)was injected into each animal.The gross histopathology lesions were recored and bacterial numbers was calculated to evaluate the efficacy of TD139 on mice.H.E and acid-fast staining were used to evaluate the pathological changes and bacterial distribution in mouse tissue.The expression and distribution of Gal-3,TNF-? and MMP-9 in mouse tissues were detected combinedly by immunohistochemical staining and image analysis.Results(1)In M.marinum infected Raw264.7 cells,the Gal-3 protein and mRNA in the WT-infected group showed an upward trend.Moreover,the average number of intracellular bacteria between WT and ?PDIM-infected group was calculated,which demonstrated that the elevated Gal-3 expression was independent of the intracellular bacteria.(2)Compared with the ?PDIM-infection group,the expression levels of TLR2 protein and mRNA in the WT-infected group increased dramatically,while the expression of TLR4 protein and mRNA decreased in both groups.Interestingly,either C29(TLR2 specific inhibitor)or JSH-23(NF-?B specific inhibitor)was used to pretreat Raw264.7 cell,the levels of Gal-3 protein and mRNA in the WT-infected group decreased obviously,which suggesting that PDIM maybe induce Gal-3 expression via TLR2/NF-?B signaling pathway.(3)Compared with the WT-infected group,the expression of pro-inflammatory cytokines including TNF-?,IL-6 and iNOS was significantly increased in the ?PDIM-infected group,while the anti-inflammatory mediators such as IL-10 and TGF-? was decreased,western blot detection found that the phosphorylation levels of ERK,NF-?Bp65 and IKK proteins were lower in the WT-infected group,while they developed a sustaining phosphorylation status in the ?PDIM-infected group,which implicating that the PDIM can inhibit the early immune response of host cells,Confocal observation found that there was a visible colocalization of Gal-3 and NF-?Bp65.Co-IP also showed there was an obvious interaction between the Gal-3 and NF-?Bp65 molecules,which indicated that Gal-3 can inhibit the NF-?B activity and its downstream inflammation-related factors production.(4)Transfection of Gal-3 siRNA plasmid can greatly reverse the production of pro-inflammatory and anti-inflammatory factors in the WT-infected group and significantly enhancing the nuclear activation of NF-?Bp65.(5)Transfection of Gal-3 over-express plasmid can also reverse the expression of inflammatory mediators in ?PDIM-infected group and significantly reduce the nuclear activation of NF-?Bp65.(6)The pathological examination showed that there was obvious swelling and ulceration in the tails of WT-infected mice,especially the typical granulomatous lesions were formed.However,the ?PDIM-infected mice just developed mild swelling in the tail,in which a few bacteria was detected by acid-fast staining.Immunohistochemistry staining revealed that Gal-3,TNF-? and MMP-9 were strongly expressed in the WT-infected mice.Amazingly,the WT-infected mice treated with TD139 significantly improved the tail injury,the expression of TNF-? and MMP-9 decreased accordingly.Conclusion Mycobacterial cell wall PDIM lipid maybe induce Gal-3 expression via TLR2/NF-?B signal pathway.Gal-3 can inhibit NF-?B activity to restrain the production of pro-inflammatory cytokines such as TNF-? and IL-6 in the early infection,which would benefit bacteria to escape early host antimicrobial activity.Gal-3 promotes TNF-? and MMP-9 secretion and mediates tissue damage and granuloma formation in animal infection experiments.