| Aspergillus fumigatus(Af)is an opportunistic pathogen that widely exists in the natural environment such as water,soil,air,plants and survives and grows on organic debris.At present,Aspergillus fumigatus has become the most common airborne fungal pathogen with abundant conidia,each of conidial head produces thousands of conidia and the conidia released into the atmosphere have a diameter small enough to reach the lung alveoli.Environmental surveys show that all humans inhale at least hundreds of Aspergillus fumigatus conidia per day.Inhalation of Aspergillus fumigatus conidia by immunocompetent people hardly caused disease,because the lung innate immune mechanism can effectively remove conidia.Inhalation of Aspergillus fumigatus conidia triggers IgE-mediated allergic reactions in asthmatic subjects causing aspergillosis such as asthma,allergic bronchopulmonary aspergillosis,and allergic pneumonia.Immunosuppressed patients inhaled Aspergillus fumigatus conidia are likely to develop invasive aspergillosis(IA).Invasive aspergillosis is one of the most serious infectious diseases in immunocompromised patients,especially in bone marrow transplants recipients and solid-organ recipients.Due to the increased number of transplants,the development of new intensive chemotherapy for hematological disease and solid tumors,AIDS and the use of immunosuppressant greatly increased in recent years,the incidence of invasive aspergillosis increased dramatically,while the diagnosis of the disease is more difficult,the detection rate of fungal pathogens is not high,in addition to the side effects of antifungal drug,the treatment effect is not obvious,so it is difficult to completely remove the Aspergillus fumigatus in the patient’s body,which leads to an extremely high mortality,that can be as high as 50%~95%.Therefore,researching the pathogenic mechanism of Aspergillus fumigatus infection,looking for new related therapeutic targets,and further exploring and formulating effective treatment methods are urgent issues that are needed to be solved for Aspergillus fumigatus infection.The body’s anti-fungal immune response is crucial in Aspergillus fumigatus infection.Aspergillus fumigatus invades the body and primarily elicits a cellular immune response,including an innate immune response and an adaptive immune response.The innate immune response is the body’s first line of defense against Aspergillus fumigatus infection through the direct phagocytosis of macrophages,neutrophils,monocytes and natural killer cells that colonize the lungs and the secretion and release of a series of cytokines and chemokines,causing the relevant inflammatory response,which can directly kill the conidia and hyphaes.When Aspergillus fumigatus infection further develops,Antigen presenting cells(APC)present Aspergillus fumigatus antigen to CD4~+T cells,thereby triggering an adaptive immune response.CD4~+T cells activated by APC further differentiate into T helper cells to mediate the anti-fungal immune response in the body,so Th cells play a particularly crucial role in Aspergillus fumigatus infectious diseases.Th1 cells mainly secrete cytokine IFN-γ(Interferon-γ),which mediates protective anti-fungal immune response.Th2 cells mainly secrete cytokines IL-4(Interleukin-4)and IL-10(Interleukin-10)to activate humoral immunity.Th17 cells mainly secrete cytokines IL-17(Interleukin-17),promoting the recruitment of neutrophils for the effective removal of Aspergillus fumigatus.Th17 cells have been demonstrated to be able to mediate anti-fungal immune responses in vivo and the immune response caused by Th17 cells and cytokine IL-17 is closely related to the body response after Aspergillus fumigatus infection.However,as a newly discovered member of the Th cell subsets,the regulatory mechanism of Th17 cell mediated anti-fungal immune response against infection by Aspergillus fumigatus is not yet clear and remains to be further studied.In recent years,a series of studies have emphasized the importance of microRNAs(miRNAs),and their related functions and mechanisms of action continue to be studied and explored in various diseases.miRNAs are a group of highly conserved endogenous non-coding small RNA,about 18-25 nucleotides in length,can negatively regulate the expression of the target gene at the post-transcriptional level by targeting the binding and cleaving of the target gene or inhibiting the protein translation of the target gene.Numerous studies have confirmed that miRNA plays a crucial role in the differentiation and development of immune cells and in the regulation of immune responses.miRNAs are involved in the regulation of CD4~+T cell development and differentiation,for example,miR-155 inhibits the differentiation of Th1 cells by inhibiting the expression of IFNγRα;miR-17-92a cluster is required for effective Th1 cell differentiation,miR-17 and miR-19b are important specific miRNAs;miR-21 promotes Th2 cell differentiation by inhibiting Spry1 expression;miR-29a inhibits Th1 cells differentiation and cytokine IFN-γproduction,etc.Multiple miRNAs have been found to promote Th17 cell differentiation,such as miR-155 promotes Th17 cell differentiation by inhibiting the transcription factor Ets1;miR-301 participates in Th17 cell differentiation by inhibiting PIAS3,a negative regulator of STAT3 signaling,etc.Thus,miRNAs can promote or inhibit the body anti-infective immune response by regulating the development and differentiation of helper T cells and the secretion of related cytokines.In addition,miRNAs are rarely reported in the pathogenesis of Aspergillus fumigatus infection and the regulatory effect of the body on the immune response to Aspergillus fumigatus infection.Therefore,the aim of this study was to investigate and explore the regulatory mechanism of differentially expressed miRNAs and their related target genes in Aspergillus fumigatus-specific CD4~+T cells on the differentiation of Aspergillus fumigatus-specific CD4~+T cells into Th17 cells.PartⅠ.Aspergillus fumigatus induce up-regulation of miR-17-5p in CD4~+T cells andTh17 cells differentiationWe first stimulated the PBMC from healthy people with heat-inactivated Aspergillus fumigatus in vitro,then sorting CD4~+T cells magnetic beads those are Aspergillus fumigatus-specific CD4~+T cells after 24 hours.According to our laboratory research group’s previous results of miRNA microarray,we selected miR-17-5p that was up-regulated in Aspergillus fumigatus-specific CD4~+T cells to carry on research.At the same time,we used Real-time fluorescent quantitative PCR to detect the expression of miR-17-5p in Aspergillus fumigatus-specific CD4~+T cells.The results were consistent with the microRNA microarray.The results showed as follows:the expression of miR-17-5p in Aspergillus fumigatus-specific CD4~+T cells increased,or about 1.49 times of the control group(P<0.05).Then the differentiation and cytokine production in Aspergillus fumigatus-specific CD4~+T cells have been detected by flow cytometry and Real-time fluorescent quantitative PCR respectively.The results showed as follows:the differentiation of CD4~+T cells were significantly increased after induced with Aspergillus fumigatus,and the expression of cytokine IL-17 also significantly increased to about 7.19times of the control group(P<0.05).PartⅡ.The expression level of miR-17-5p have impact on Th17 cells differentiationin Aspergillus fumigatus-specific CD4~+T cellsIn order to further understand whether the up-regulation of miR-17-5p in Aspergillus fumigatus-specific CD4~+T cells was related to Th17 cells differentiation,we transfected miR-17-5p mimic or miR-17-5p inhibitor into PBMC,then stimulated with heat-inactivated Aspergillus fumigatus when 24 hours post transfection,and CD4~+T cells were sorted by magnetic beads after 24 hours.The results showed as follows:there was a positive correlation between the expression level of miR-17-5p and Th17 cells differentiation or IL-17 production in Aspergillus fumigatus-specific CD4~+T cells,the transfection of miR-17-5p mimic promoted Th17 cells differentiation and IL-17production in Aspergillus fumigatus-specific CD4~+T cells(P<0.05).On the contrary,the transfection of miR-17-5p inhibitor suppressed Th17 cells differentiation and IL-17production in Aspergillus fumigatus-specific CD4~+T cells(P<0.05).Thus it can be seen that miR-17-5p had the ability to regulate and control Th17 cells differentiation in Aspergillus fumigatus-specific CD4~+T cells.PartⅢ.miR-17-5p promote Th17 cells differentiation by targeting to PTEN inAspergillus fumigatus-specific CD4~+T cellsWe first predicted the target genes of miR-17-5p by multiple microRNA target prediction websites,and selected the target gene-PTEN which was related to Th17 cells differentiation after some analysis.Then we verified the interaction between miR-17-5p and 3’UTR of PTEN mRNA by Real-time fluorescent quantitative PCR,Western Blot and luciferase reporter.To investigate whether there was a correlation that miR-17-5p regulated and controlled Th17 cells differentiation by PTEN,we designed and synthesized PTEN siRNA to silence the expression of PTEN.The results showed as follows:Th17 differentiation and IL-17 production were statistically increased(P<0.05)in Aspergillus fumigatus-specific CD4~+T cells when 24 hours post transfection of PTEN siRNA.Thus it can be seen that miR-17-5p promoted Th17 cells differentiation by targeting to PTEN in Aspergillus fumigatus-specific CD4~+T cells.In conclusion,our study showed that Aspergillus fumigatus have the ability to induce CD4~+T cells to up-regulate the expression of miR-17-5p,and miR-17-5p can further regulate and control Th17 cells differentiation and IL-17 production by targeting to PTEN.This mechanism may be related to the PI3K-AKT signaling pathway that PTEN was involved in,so miR-17-5p may regulate and control anti-fungal immune response just through the mechanism. |
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