Effects Of Mannan-binding Lectin On The Differentiation Of Th17 Cells

BackgroundMannan-binding lectin（MBL）is a key PRR（Pattern recognition receptor,PRR）in the natural immune system and where a vital role plays.Th17 cells（T help cells17,Th17）are CD4⁺T cell subsets that secrete IL-17 cytokines.It is reported that dectin-1,which is a C-type lectin family with MBL,can promote the differentiation of Th17 cells.SO what is the relationship between MBL and Th17 cells?Does MBL also regulate the differentiation of Th17 cells?It is still unclear.Therefore,it is important to study the effect of MBL on the differentiation of Th17 cells,and to clarify the new role of MBL in acquired immunity.ObjectiveTo investigate the effects of MBL on the differentiation of Th17 cells,and to provide a new experimental basis for the treatment of Th17 cell-related inflammatory diseases and autoimmune diseases.Methods1.Ficoll density centrifugation:Human Cord blood mononuclear cells（Cord blood mononuclear cells,CBMCs）were isolated by Ficoll density centrifugation.2.Immunomagnetic beads sorting:CD4⁺T cells were purified in vitro from CBMCs by immunomagnetic beads sorting.3.Flow cytometry（FACS）detect:Anti-CD3 monoclonal antibody（McAb）and anti-CD28 McAb were used to stimulate CD4⁺T cells with IL-6 and TGF-β1 as inducers.Then,these cells were treated with（MBL group）or without（control group）different concentrations（1μg/mL,5μg/mL and 10μg/mL）of MBL.Percentages of Th17 cells in different groups were detected by fluorescence-activated cell sorting（FACS）after 72 hours of culturing.4.Quantitative real-time PCR（qRT-PCR）analysis:qRT-PCR was used to analyze the gene expression of RORγt（Retinoid-related orphan receptor-γ-t）at mRNA level in both control and MBL groups.5.Enzyme-linked immunosorbent assay（ELISA）detect:ELISA was performed to detect the levels of IL-17A in supernatants of cell culture from different groups.6.Identification of MBL knockout mice:Genotyping and Sequencing to identify the homozygous mice of MBL-A knockout(MBL-A^-/-),MBL-C knockout(MBL-C^-/-)and MBL-A and MBL-C double knockout(MBL^-/-).7.FACS analysis:FACS was used to detect the percentages of Th17 cells in MBL^-/-mice and wild type（WT）mice.8.Statistical analysis:All the experimental data was analyzed with the SPSS22.0software,the experimental results were expressed by mean±standard deviation（xˉ±s）,and the data was statistically analyzed using one-way ANOVA（ANOVA）and Independent samples t test,P<0.05 indicated statistical difference（*P<0.05,**P<0.01）.Results1.MBL could significantly reduce the proportion of CD4⁺RORγt⁺Th17 cells:The FACS showed that the proportion of CD4⁺RORγt⁺Th17 cells in the 5μg/mL MBL group and 10μg/mL MBL group was significantly reduced after 72 hours of cell culture as compared with the control group.2.MBL could significantly reduce the expression of RORγt mRNA:qRT-PCR results showed that MBL could significantly down-regulated the expression of RORγt at mRNA level in the 5μg/mL MBL group and 10μg/mL MBL group as compared with the control group.3.MBL could significantly decrease the expression level of IL-17A:ELISA analysis showed that MBL could significantly decrease the expression of IL-17A in the 5μg/mL MBL group and 10μg/mL MBL group as compared with the control group.4.Obtained MBL knockout mice:Genotyping and Sequencing showed that successfully obtained that the homozygous mice of MBL-A knockout(MBL-A^-/-),MBL-C knockout(MBL-C^-/-)and MBL-A and MBL-C gene double knockout(MBL^-/-).5.The proportions of Th17 cells after MBL knockout was significantly increased:The FACS showed that the percentages of CD4⁺RORγt⁺Th17 cells in MBL^-/-mice was significantly higher than those in WT mice.ConclusionMBL can inhibit the differentiation of CD4⁺T cells to Th17 cells.