MiR-31 Regulates The Differentiation Of CD4~+ Treg By Targeting IL-34 In Chronic Obstructive Pulmonary Disease

Part I: IL-34 regulates the differentiation of CD4~+ Treg in chronic obstructive pulmonary diseaseObjectives: Previous studies have reported that lower expression and insufficiency of CD4~+CD25~+Foxp3~+ T cells(CD4~+ Treg)were associated with abnormal immune response in patients with chronic obstructive pulmonary disease(COPD).However,the mechanism has not been fully elucidated.The purpose of this study was to clarify the role of interleukin-34(IL-34)on the differentiation of CD4~+ Treg and the pathway involved.Methods: 1.Firstly,the clinical characteristics of healthy non-smoking group(HC group),smoke with normal pulmonary function group(HS group)and COPD group were analyzed,we then detected the expression of IL-34 in peripheral blood mononuclear cells(PBMC)and serum from the three groups by flow cytometry and enzyme-linked immunosorbent assay(ELISA).2.The effects of exogenous IL-34 on the differentiation and function of na(?)ve CD4~+ T cells was analysed by flow cytometry.3.The expression of IL-34 in cytokines(such as IL-1?,IL-4,IL-6,IL-10,IL-12,TNF-?,TGF-?1,IFN-?,CSE)treated na(?)ve CD4~+ T cells was detected by flow cytometry.4.ELISA was used to detect the expression of IL-4,IL-6,IL-10,IL-12,TNF-?,TGF-?1,IFN-? in IL-34 treated na(?)ve CD4~+ T cells.5.Finally,the mechanism of IL-34 regulating Treg differentiation was studied by Western Blot experiment.Results: 1.The expression of IL-34 in serum and PBMC in HS group and COPD group was higher than that in HC group,and the level of IL-34 was positively correlated with smoking index(p<0.05).The expression of IL-34 in serum from severe and very severe COPD patients was significantly higher than that in mild to moderate COPD patients(p<0.05),suggesting that the expression of IL-34 can reflect the severity of COPD.2.Exogenous IL-34 could decrease the proportion of CD4~+ Treg,while the addition of neutralizing antibody of IL-34 could inhibit the process.However,IL-34 had no effect on the expression of other CD4~+ Th cell subsets,nor the function of CD4~+ Treg.3.Inflammatory factors IL-17,TNF-?,IFN-? and CSE can induce the expression of IL-34 significantly.4.Exogenous IL-34 can also promote the levels of IL-6 and IL-12 in na(?)ve CD4~+ T cells and decrease the secretion of IL-10.5.Exogenous IL-34 could up-regulate the expression of p-PI3 K,p-Akt and p-m TOR in na(?)ve CD4~+ T cells,while the expression of p-PI3 K,p-Akt and p-m TOR decreased after the addition of LY294002,an inhibitor of PI3 K pathway.The data suggests that PI3K/Akt/m TOR pathway was involved in the process of IL-34 inhibiting Treg expression.Conclusion: IL-34 is highly expressed in patients with COPD than healthy controls,and the expression of IL-34 can reflect the severity of COPD.IL-34 could reduce the expression of Treg through p-PI3K/p-Akt/p-m TOR signalling pathway.Part II: miR-31 regulates the differentiation of CD4~+ Treg via inhibiting IL-34 in chronic obstructive pulmonary diseaseObjectives: micro RNA can regulate the expression of target genes and widely participates in the proliferation and differentiation of immune cells.The purpose of this study is to screen the micro RNAs that regulate the expression of IL-34 and verify the mechanism involved.Furthermore,clarify whether CSE can regulate the levels of IL-34 through targeting miR-31.Methods: 1.Bioinformatics analysis was used to screen the micro RNAs that specifically regulate the expression of IL-34.Double luciferase reporter assay was used to verify whether miR-31 could bind to the 3'UTR of IL-34.2.The expression of miR-31 in PBMC from HC,HS and COPD groups was detected by q RT-PCR.We further analyse the effect of CSE on the expression of miR-31 in na(?)ve CD4~+ T cells.3.miR-31 inhibitors or mimics was transfected into na(?)ve CD4~+ T cells by lentivirus,and the expression levels of IL-34 were tested by flow cytometry,and then neutralizing antibody of IL-34 or exogenous IL-34 was added to detect the expression changes of IL-34.4.The expression Foxp3 and CD25 were tested by flow cytometry by transfecting miR-31 inhibitors or mimics into na(?)ve CD4~+ T cells to see whether miR-31 could affect the function of IL-34.Results: 1.We screened out that IL-34 might be a target gene of miR-31 through bioinformatics analysis.The double luciferase report assays further proved that miR-31 could directly bind to the 3'UTR region of IL-34 and affect the expression of IL-34.2.miR-31 expression in PBMC from COPD groups was lower than that of healthy controls, and CSE could down-regulate the expression of miR-31 in na(?)ve CD4~+ T cells.3.Increased IL-34 expression was observed in na(?)ve CD4~+ T cells transfected with miR-31 inhibitors,while the expression of IL-34 decreased after miR-31 mimics transfection.4.After miR-31 inhibitors was transfected into na(?)ve CD4~+ T cells,decreased Foxp3 and CD25 expression was detected,and the proportion of Foxp3 and CD25 increased after the addition of IL-34 neutralizing antibody.Similarly,na(?)ve CD4~+ T cells transfected with miR-31 mimics showed a higher expression of Foxp3 and CD25,and the addition of exogenous IL-34 could reverse the effect.Finally,up-regulating of miR-31 in na(?)ve CD4~+ T cells can influence the ability of CSE to increase IL-34 expression.Conclusion: miR-31 can directly bind to the 3'UTR region of IL-34 to inhibit its transcriptional activity,which affecting the expression of IL-34.CSE can down-regulate the expression of miR-31 in na(?)ve CD4~+ T cells.