Study On Maturation Of DCs Regulated By TLR2 Expression In Corneal Epithelial Cells

Research BackgroundCorneal epithelial cells(CECs)are located in the outermost layer of the cornea,not only as the first physical barrier against exogenous pathogens,but also an important immune barrier that identifies the receptors by expressing multiple patterns Recognition receptors,PRRs)participate in corneal resistance to bacteria,viruses and other microbial invasion of the natural immune and acquired immune process.Toll-like receptors(TLRs)are a wide range of widely distributed PRRs,which rely mainly on pathogen-associated molecular patterns(PAMPs),activation of myeloiddifferentiationfactor88(Myd88)dependent/non-dependent Sexual signal pathway,start the body congenital immune response,and to communicate the natural immune response and access to the immune response of the bridge.TLRs can be involved in the pathogenesis of microorganisms and regulate the development of inflammatory responses by identifying exogenous ligands such as peptidoglycan(PGN),LPS,lipoteichoic acid and HSP70,HMGB1 and other endogenous ligands.Among them,Toll-like receptor 2(TLR2)is the most widely expressed receptor in TLRs,which has become a hotspot.Corneal epithelium specific immune cells such as dendritic cells(DCs)are also involved in corneal-acquired immune response,DCs is the most powerful antigen presenting cells(Antigen presenting cells,APC)The DCs in normal corneal epithelium are mainly in the form of immature(imDCs)in the corneal surrounding area,when the cornea occurs inflammatory reaction,the corneal peripheral area of the imDCs to the central cornea gathered and mature,with activated T lymphocytes and other functions.DCs are regulated by a variety of endogenous and exogenous factors.The study found that TLRs signaling can trigger DCs by up-regulating MHCII and co-stimulatory molecules,inflammatory cytokines such as TNF-a,IL-6 and IL-12.However,there are few studies on the corneal immune response of corneal epithelial cells,and the expression of TLR2 in corneal epithelial cells has not been reported in the study of corneal DCs maturation.Therefore,the experimental study is carried out.Part.l Effects of Peptidoglycan up-regulated TLR2 and TLR4 in corneal epithelial cells of miceObjectiveTo investigate the effects of Peptidoglycan(PGN)with different concentrations and times on Toll-like receptor 2(TLR2)?MyD88expression in corneal epithelial cells of mice.MethodsCorneal epithelial cells of mice were cultured in vitro.Cells were divided into blank control group and PGN-treated group,the cells in PGN-treated group were divided into 10ug/ml gruop.30ug/ml gruop and 80 ug/ml group(treated by different concentration of PGN for 12h).In the meantime,the cells in 30 ug/ml gruop were cultured for different times(named 12h group?24h group?36h group).Expressions of TLR2 and myd88 mRNA and protein in different group were measured by RT-PCR and Flow cytometry.ResultsCompared with control group(1.00±0.14?1.00±0.01)?the expression of TLR2?myd88mRNA in 10 ug/ml gruop(4.35±0.46?3.53±0.5)?30ug/ml gruop(8.06±0.72?5.31 ±0.34)?80ug/ml group(2.93±0.46?2.23±0.04)were increased,the difference was statistically significant(P<0.05).Compared with control group(1.00±0.14?1.00±0.01),the expression of TLR2,myd88mRNA in 12h group(2.93±0.46?2.23±0.5)?24h group(2.08±0.72?1.71±0.34)?36h group(1.66±0.46?1.49±0.04)were increased,the difference was statistically significant(P<0.05).The results of flow cytometry were consistented with RT-PCR.ConclusionPeptidoglycan can stimulate the expression of TLR2 and Myd88 in mouse corneal epithelial cells,suggesting that corneal epithelial cells participate in corneal immune response through TLR2-Myd88 signaling pathway.Part.2 Study on stimulatory effect of TLR2 in mouse corneal epithelial cells on the maturation of dendritic cellsObjectiveTo study the expression of Toll like receptor 2(TLR2)in mice corneal epithelial cells(CECs)on Maturation of Dendritic Cells(DCs),to explore the regulation of natural immunity to acquired immunity.MethodUsing enzyme digestion method to obtain CECs,divided into Control group(CECs from wild mice);Stimulation group(wild mice CECs stimulate by peptidoglycan);Knockout group(CECs from TLR2-/-mice).CECs of each group were adjusted to 2×106,and cultured with Bone Marrow-derived Immature dendritic cell(imDCs)(2:1)for 24h,then collect suspended DCs.DCs were detected by three methods:(1)RT-PCR detecte the expression of CD86?CD80?MHCII mRNA;(2)Flow cytometry detecte the expression CD80?CD86 and MHCII;(3)co-cultured with T cells from lymph nodes of mice for 72h,CCK-8 detecte the proliferative index of T lymphocyte.ResultsRT-PCR showed the expression of CD80(4.23+0.21)?CD86(4.40±0.12)?MHCII(11.52±0.54)mRNA in stimulation group were higher than the control group(P=0.02?0.01?0.03);Flow cytometry showed CD80 0.50±0.03)?CD86(0.25±0.04)?MHCII(4.52±0.34)in stimulation group were higher than the control group(P=0.04?0.01?0.02);CCK-8 showed that T cell proliferation rate of stimulation group(45.4±0.09%)were higher than the control group(25.4±0.09%),(P=0.01).ConclusionThe high expression of TLR2 in CECs can promote the maturation of DCs,suggested that the TLR2 in CECs may facilitate maturation of DCs and accelerate the innate immune response of cornea.