The Effect Of CCL20/CCR6 Axis On MDSCs In Autoimmune Myocarditis

Part 1 The expression and correlation analysis of CCL20 and MDSCs in patients with acute myocarditisObjective:To detect the expression of chemokine CCL20 and the proportion of MDSCs and CCR6⁺MDSCs in patients with acute myocarditis.And to explore the relationship between MDSCs and CCL20?CCR6⁺MDSCs and CCL20.Methods:20 patients with acute myocarditis and 20 healthy controls in the Wuhan Union Hospital were enrolled in the study.We collected their venous blood on the first day of admission.The level of CCL20 in plasma was detected by enzyme-linked immunosorbent assay?ELISA?.We used flow cytometry to analyze the proportion of CD14⁺HLA-DR^-/lowMDSCs and CCR6⁺CD14⁺HLA-DR^-/lowMDSCs in peripheral blood;Pearson correlation analysis was used to analyze the plasma CCL20 and CD14⁺HLA-DR^-/lowMDSCs?CCR6⁺CD14⁺HLA-DR^-/lowMDSCs in patients with acute myocarditis.Results:Compared with the healthy control group,the level of CCL20 of acute myocarditis was significantly increased?P<0.01?.The plasma level of CCL20 was?10.78±2.57?pg/ml in acute myocarditis and the level of CCL20 in healthy control group was?2.90±0.56?pg/ml.Furthermore,compared with healthy control group,the frequency of CD14⁺HLA-DR^-/lowMDSCs and CCR6⁺CD14⁺HLA-DR^-/lowMDSCs in peripheral blood of patients with acute myocarditis were markedly increased?P<0.001?;In addition,the frequency of CD14⁺HLA-DR^-/lowMDSCs was positively correlated with plasma level of CCL20 in patients with acute myocarditis?R=0.6129,P<0.01?,and the frequency of CCR6⁺CD14⁺HLA-DR^-/lowMDSCs was also positively correlated with plasma level of CCL20,?R=0.7447,P<0.001?.Conclusion:The plasma level of CCL20 and the frequency of CD14⁺HLA-DR^-/lowMDSCs and CCR6⁺CD14⁺HLA-DR^-/lowMDSCs in acute myocarditis patients were markedly increased than those in healthy control group.Morever,the frequency of CD14⁺HLA-DR^-/lowMDSCs and CCR6⁺CD14⁺HLA-DR^-/lowMDSCs were positively correlated with plasma level of CCL20 in acute myocarditis patients.Part 2 The Effect of CCL20 on migration process of CCR6⁺MDSCs in experimental autoimmune myocarditis miceObjective: To observe the dynamic changes of the proportion of CCR6⁺MDSCs in bone marrow,spleen,peripheral blood and heart of autoimmune myocarditis mice at different time points.And to explore the relationship between CCL20 and CCR6⁺MDSCs.Methods: We choosed 96 male Balb/c mice and randomly divided them into four groups: control group?n=36?,EAM group?n=36?,EAM+ isotype control group?n=12?,EAM+CCL20 m Ab group?n=12?.1.We selected randomly six mice from the control group and the EAM group on the 7th,10 th,14th and 21 st day of the experiment.Then we used flow cytometry to analyze the proportion of CCR6⁺CD11b+Gr-1+MDSCs in bone marrow,spleen,peripheral blood and heart;The mRNA expression of CCR6 and CCL20 in the heart of control and EAM group were detected by RT-PCR.2.On the 21 st day of the experiment,We killed all the remaining mice of the four groups?control group,EAM group,EAM+ isotype control group,EAM+CCL20 m Ab group?and we weigh the heart and body weight of mice in each group,then we calculate the ratio of HW/BW?heart weight /body weight?;We used HE/Masson staining to observe the pathological changes of myocardial tissue of mice in four groups;3.We detected the level of CCL20 in peripheral blood of mice in four groups by enzymelinked immunosorbent assay?ELISA?;4.Immunofluorescence technique?IF?was used to detect the co-localization expression of CCL20 and fibroblast marker-Vimentin in the heart;5.We used flow cytometry to analyze the proportion of CCR6⁺CD11b+G-1+MDSCs in the heart and spleen of the mice in four groups.6.The mRNA expression levels of cytokines IL-4,IL-6 and IL-10 in the hearts of four groups were detected by RT-PCR.Results:1.Compared with the control group,the proportion of CCR6⁺CD11b+G-1+MDSCs in the bone marrow of EAM mice peaked at day of 10?p<0.001?,and then it began to decrease;The proportion of CCR6⁺CD11b+G-1+MDSCs in the spleen was increased slightly on day of 10 and began to decrease on day of 14;Compared with control group,the percentage of CCR6⁺CD11b+G-1+MDSCs in peripheral blood and heart were increased on day of 14 and 21?p<0.001?and peaked at D21;The mRNA expression of CCL20 and CCR6 in EAM group were significantly higher than those in control group on day of 21?p<0.05?;2.The clinical scores and HW/BW of EAM group and EAM+ isotype control group were significantly higher than control group?p<0.01?,and EAM+CCL20m Ab group was significantly lower than the other two groups?p< 0.01?;3.Compared with the control group,the plasma level of CCL20 in the EAM group and the EAM+isotype control group were significantly increased?p<0.05?,while that in the EAM+CCL20m Ab group was significantly decreased than the other two groups?p<0.01?.4.We discovered that CCL20 was widely expressed in cardiac fibroblasts of EAM group and EAM+ isotype control group by immunofluorescence,while the expression of CCL20 in EAM+CCL20m Ab group was significantly decreased.5.Compared with the control group,the percentage of CCR6⁺CD11b+G-1+MDSCs in the spleen of EAM group and EAM+ isotype control group were significantly decreased?p<0.01?,while the proportion of EAM+CCL20m Ab group was significantly increased than the two groups?p<0.05?;The proportions of CCR6⁺CD11b+G-1+MDSCs in the heart of EAM group and EAM+isotype control group were significantly higher than that in control group?p<0.05?,while compared with the EAM and EAM+isotype control group,the proportion of EAM+CCL20m Ab group was reduced than that in the other two groups?p<0.05?;6.The results of RT-PCR showed that the mRNA expressions of IL-4,IL-6 and IL-10 in EAM group and EAM+ isotype control group were significantly up-regulated compared with control group?p<0.05?;However,the mRNA expression of IL-6 in EAM+CCL20m Ab was significantly reduced than EAM group and EAM+ isotype control group?p<0.01?.And the mRNA expression levels of IL-10 and IL-4 of CCL20 m Ab group were significantly increased?p<0.05?.Conclusion: 1.In the mice of EAM,CCR6⁺MDSCs were producede in bone marrow,CCR6⁺MDSCs can gradually migrate to the site of cardiac inflammation under the effect of CCL20;2.The cardiac fibroblasts of autoimmune myocarditis mouse can secrete CCL20 and blocking CCL20 can significantly reduce the accumulation of CCR6⁺MDSCs in the heart of autoimmune myocarditis mice.Finally,blocking CCL20 can alleviate myocardial inflammation in EAM mice.