Lipopolysaccharide Mediates Macrophage M1/M2 Polarization And Function Of Barrier Regulation Through The Tim-3/Galectin-9 Signaling Pathway

ObjectiveTo determine lipopolysaccharide(LPS)induced bone marrow derived macrophages(BMDMs)M1/M2 polarization and its effect on barrier function of pulmonary microvascular endothelial cells(PMVECs)which were observed from cell monolayer permeability and cell migration ability.Then,investigate the molecular mechanisms of T-cell immunoglobulin mucin-3/Galectin-9(Tim-3/Gal-9)pathway on LPS induced macrophage M1/M2 polarization.MethodsBMDMs and PMVECs were cultured in vitro.BMDMs were stimulated with 0.1 mg/L LPS,M1/M2 phenotypic macrophage markers CD86 and CD206 were measured by flow cytometry,inducible nitric oxide synthase(iNOS)and arginase-1(Arg-1)were measured by western blot at 0,1,3,6,12,and 24 h after stimulation.Tim-3 and Gal-9 interaction was observed by immunofluorescence combined with confocal laser scanning microscopy,physical association between Tim-3 and Gal-9 was detected by co-immunoprecipitation.Co-culture the PMVECs with BMDMs in different groups,cell monolayer permeability was detected using Transwell-Evans blue(EB)monolayer permeability assay,cell migration ability was measured using an in vitro wound healing assay.The other BMDMs were divided into blank control group,LPS treatment group,a-lactose/Tim-3 monoclonal antibody(mAb)pretreatment groups,LPS+a-lactose treatment group,LPS+Tim-3 mAb treatment group(pretreated with serum-free DMEM containing 40 μmol/L Gal-9 signal antagonist or 10mg/L function blocking agent 1 h before LPS 3 h stimulation).Blocking Tim-3 or Gal-9 signal was used to verify the role of Tim-3/Gal-9 in macrophage M1/M2 subtype polarization and function regulation.Results1.The percentage of M1 macrophages increased in the 6,12,and 24 h groups compared to the 0 h group and iNOS protein expression increased with increasing stimulation times(all P<0.01),however,the percentage of M2 macrophages and the expression of Arg-1 were upregulated after stimulation with 0.1 mg/L LPS for 1 h and peaked at 3 h(compared to 0 h group,P<0.01);then the percentage of M2 macrophages gradually decreased to the 0 h level.Moreover,the expressions of Arg-1 were significantly downregulated when stimulated with 0.1 μg/mL LPS for 12 h and 24 h(both P<0.01).2.BMDMs with short-term LPS stimulation(1 h,3 h)upregulated the cell migration rates(compared to 0 h group,1 h group P<0.05;3 h group P<0.01)and downregulated the PMVECs monolayer permeability(both P<0.01).While BMDMs with long-term LPS stimulation(12 h,24h)downregulated the cell migration rates and upregulated the PMVECs monolayer permeability(all P<0.01).3.The level of Tim-3/Gal-9 protein in macrophages were significantly up-regulated with increasing stimulation times,peaked at 3 h,and then gradually decreased.The interaction of tim-3 and Gal-9 reflected similar trends(all P<0.01).4.Compared to the LPS(3 h)group,the percentage of M1 macrophages(both P<0.01)and the expression of iNOS(LPS+a-lactose group P<0.05;LPS+Tim-3 mAb group P<0.01)in LPS(3 h)+a-lactose and LPS(3 h)+Tim-3 mAb treatment groups were significantly increased,while the percentage of M2 macrophages(LPS+a-lactose group P<0.05;LPS+Tim-3 mAb group P<0.01)and the expression of Arg-1(both P<0.01)were decreased.5.Compared to the LPS(3 h)group,the BMDMs in LPS(3 h)+a-lactose and LPS(3 h)+Tim-3 mAb treatment groups downregulated the migration rates and upregulated the monolayer permeability of co-cultured PMVECs(all P<0.01).Meanwhile,the blocking of tim-3 and Gal-9 signaling could also reduce the expressions and binding ability of tim-3 and Gal-9(all P<0.01).ConclusionLPS had a biphasic effect on BMDMs polarization and function regulation through the Tim-3/Gal-9 pathway.Short-term LPS stimulation upregulated Tim-3/Gal-9 signaling,eventually inhibiting M1 polarization,induce a potent recovery capacity in barrier function,however,long-term stimulation downregulated Tim-3/Gal-9 signaling,eventually promoting M1 polarization,induce injury in barrier function.