Aspergillus Fumigatus Induced CD4~+T Cells Downregulate MiR-223-3p And Its Mechanism On Th17 Cell Differentiation

Aspergillus fumigatus is widely present in the environment and there is no disadvantages for healthy individuals inhaling small amounts of Aspergillus fumigatus spores,but it can cause serious infection in immunocompromised individuals.The morbidity and mortality of invasive aspergillosis(IA)is very high and it may be the major cause of death for immunocompromised individuals.Currently the diagnosis of IA mainly depends on pathological and microbiological method,but they are injured and of low success.Because antifungal therapy in early stage of infection can greatly improve the prognosis of patients.Thus,though antifungal therapy has made great progress today,it is still difficult to make patients cured,leading to great threat of life in immunocompromised individuals.Aspergillus fumigatus invasion mainly cause cellular immune response,which include innate and adaptive immnity.Innate immunocyte such as macrophages,neutrophils,monocytes and natural killer cells or inflammation mediated by the cytokines and chemokines can directly kill the spores and hyphaes.When the infection further develop,antigen presenting cells(APC)will present antigens to T cells and trigger the adaptive immune response.Then CD4~+T cells are activated by APC and further differentiate into T helper(Th)subsets and mediate anti-fungal immune response.Th17 involved in anti-fungal infection have been confirmed by numerous studies.There is a close link between inflammation mediated by Th17 or interleukin-17(IL-17)and the outcome after Aspergillus fungal infection.However,as the latest discovery of Th cell subsets,the mechanism of Th17 response to Aspergillus fumigatus has been still unclear.microRNA(miRNA),whose function have been researched in a variety of diseases since they were found.miRNA participate in the regulation of T cell development and differentiation,and regulate the immune response through influencing the differentiation balance among different Th cell subsets.miRNA has been confirmed to involved in the regulation of differentiation and cytokine-mediated inflammation of Th17.Such as miR-17~92 cluster,miR-155 and miR-326 promote the anti-infection immunity or inhibit the immunity by influencing T cell development and differentiation or by influencing cytokines production.Some miRNA can also participate in autoimmune diseases or even cancer progression by regulating other immune cells development and inflammation.However,it is poorly studied by now that the function of miRNA in pathogenesis and immune response mechanism of Aspergillus fumigatus infection.Therefore,the aim of this study was to investigate the differentially expressed miRNA in Aspergillus fumigatus-specific CD4~+T cells and its function in Th17 cell differentiation induced by Aspergillus fumigatus.PartⅠ.Aspergillus fumigatus induce downregulation of mi R-223-3p in CD4~+T cellsand Th17 differentiationFirst,We used monocyte-derived dendritic cells(DC)cultured with heat-inactivated Aspergillus fumigatus in vitro,and then we put CD4~+T cells co-culture with them together,to harvest Aspergillus fungal-specific CD4~+T cells.According to previous results of mi RNA microarray,we selected miR-223-3p which is downregulated in Aspergillus fumigatus-specific CD4~+T cells.Then we used RT-PCR to detect its expression in Aspergillus fumigatus-specific CD4~+T cells.The results were consistent with the miRNA microarray.The expression level of mi R-223-3p in Aspergillus fumigatus-specific CD4~+T cells was downregulated to 35% compared to control group.Then the differentiation and cytokine production in Aspergillus fumigatus-specific CD4~+T cells have been detected by flow cytometry and RT-PCR respectively.The results showed that the differentiation of CD4~+T cells were significantly increased after co-culture with Aspergillus fumigatus,and the expression of cytokines IL-17 has also increased to about 3.4 times of the control group(P <0.05).Part Ⅱ.miR-223-3p have impact on Th17 cell differentiation in CD4~+T cellsTo further observe whether there was some links between the down-regulated mi R-223-3p and Th17 differentiation in Aspergillus fumigatus-specific CD4~+T cells.mi R-223-3p mimic/inhibitor were transfected to Aspergillus fumigatus-specific CD4~+T cells.We found that there was a negative correlation between miR-223-3p and Th17 or IL-17 in Aspergillus fumigatus-specific CD4~+T cells.Aspergillus fumigatus-specific CD4~+T cells transfected with miR-223-3p mimic had decreased differentiation of Th17 and production of IL-17.On the contrary,Aspergillus fumigatus-specific CD4~+T cells transfected with miR-223-3p inhibitor promoted Th17 differentiation and IL-17 production.This results were consistent with the first part results,indicating that miR-223-3p had the ability to regulate Th17 differentiation in Aspergillus fumigatus-specific CD4~+T cells.Part Ⅲ.miR-223-3p promote Th17 differentiation by targetted to PRDM1In this part of study,PRDM1,the target of miR-223-3p was predicted by bioinformatics analysis.Then we verified the interaction between miR-223-3p and 3’UTR of PRDM1 mRNA by RT-PCR,Western Blot and luciferase reporter.To explore whether there was a correlation between miR-223-3p and PRDM1 in regulation of Th17 differentiation,we transfected Aspergillus fumigatus-specific CD4~+T cells with PRDM1 siRNA to silence the expression of PRDM1.The results showed that Th17 differentiation and IL-17 production were statistically reduced(P<0.05)after Aspergillus fumigatus-specific CD4~+T cells transfected with PRDM1 siRNA,which were consistent with the results of miR-223-3p mimic.These results suggest that miR-223-3p promote Th17 differentiation by targetted to PRDM1 in Aspergillus fumigatus-specific CD4~+T cells.In summary,our study show that Aspergillus fumigatus have the ability to induce CD4~+T cells to downregulate the expression of mi R-223-3p,and miR-223-3p can further regulate higher Th17 differentiation and IL-17 production by targetted to transcription factor PRDM1.This mechanism may owe to the relevance of PRDM1 invovled in multiple pathways,and miR-223-3p could regulate anti-fungal immunity just through this mechanism.