Screening Of MicroRNAs Expression Profile In CD4+ T Cells Activated By Aspergillus Fumigatus And Study On Its Function

Aspergillus fumigatus(A, fumigatus) is an opportunistic pathogen classified as aspergillus, which was widely exist in nature and easy to be inhaled into the human body because the diameter of the spore are only of 2 ~ 3 microns. The spore of aspergillus fumigatus could be removed by pulmonary immune system if healthy people inhaled it except music bacterium disease such as asthma, allergic bronchial pulmonary aspergillosis, allergic aspergillus sinusitis, and the hypersensitivity pneumonitis etc. In immunocompromised individuals, inhaled conidia are able to develop invasive aspergillosis. In recent years, the prevalence gradually rise, with mortality rates up 50% to 95% in certain situations, but the effect of clinical cure is not very well. The disease prevalence in recent years gradually rising, the fatality rate can be as high as 50% 95%. Because of the drug side effect of antifungal, and the not very ideal therapeutic effect, it is very important for us to discuss smoke aspergillus infection of pathogenic mechanism, search for new therapeutic targets and further develop more effective treatment methods.As smoke aspergillus is opportunistic pathogens, and the main objects of infection is the patients with immunosuppression, the body’s immune response is particularly important in the smoke aspergillosis. Innate immunity is the first line of defense against smoke aspergillums, such as alveolar macrophage phagocytosis clearing away spores, combination of specific fungal peptide and dendritic cells with pattern recognition receptors, activation of dendritic cell and release of cytokines and chemokines chemotaxis for migration of neutrophils and inflammatory cells, and neutrophils mediated NADPH oxidase induced damage, etc. Dendritic cells(dendritic cells, DCs) is one of the key cells of the immune regulation, and is a bridge between innate immunity and adaptive immunity. It not only can identify the spores and hyphae of smoke aspergillus, produce a variety of cytokines and chemokines, raise phagocytes, but also can induce T cells to differentiate into the Th1 and Th2 cells. So it plays an indispensable role in resistance to infection of smoke aspergillus fungal infection.Antigen presented cells could present smoke aspergillus antigen presented to CD4 + T cells, causing adaptive immune response. So Th cell plays a key role in smoke aspergillus infection. Th1 cells secrete proinflammatory factor IFN-γ, inducing protective antifungal defense reaction, while Th2 cells release IL-4 and IL-10 which could activate humoral immunity, and Th17 cells could mainly secrete IL – 17 which could promote the raise of neutrophil, and clearing of smoke aspergillus hypha.Micro RNAs(mi RNA) is the nucleotide of l8 ~ 25 oligomeric bases length inside cell which act in gene transcription level, with target genes m RNA 3 ’Untranslated Regions, suppressing its translation as well as the degradation of m RNA. It plays an important role in the process in regulating the differentiation and development of immune cells, immune response and development of tumor. In recent years, some studys have showed that infection in HIV-1, EB virus, tuberculosis(TB) as well as helicobacter pylori, there are abnormal profiles of mirnas expression in them indeed. The mi R-17 and mi R-19 b can promote the production of Th1, and inhibition of regulatory T cell differentiation in CD4 + T cells. Mi R-146 is not only involved in the toll-like receptor signal transduction feedback negative phase control, but also could reduce type I interferon thouth the TRAF6 / IRAK1 / IRAK2 pathway. Mi R-21 could reduce the level of IL-4 though inhibiting the generation of T lymphocytes. So micro RNA might play an important role in the formation of T helper cells and the secretion of cytokines. Micro RNA has been researched in many fields, but it is still very rarely in the smoke aspergillus infectious diseases. our previous research has shown that in many chronic inflammatory diseases, cells like DC, macrophages and APC are recruited and subgroup Th1, Th2 and Th17 response are triggered. Thus, we speculate that micro RNA could directly or indirectly acts on the CD4+ T cells associated protein so as to affect its function and finally regulate the body’s adaptive immune response against smoke aspergillus in patients with infection of smoke aspergillus.Our aim was to identify the differentially expressed micro RNAs in human CD4+ T cells stimulated by aspergillus fumigatus extract, then make the analysis of the target gene, and finally point out the function and the regulatory mechanism of those micro RNAs. Our study might provide a new route for the aspergillus fumigatus infectious diseases for the clinical in future.Part One: The role of DCs loaded with Aspergillus fumigatus extract on activation of CD4+T cells and effect on its function.Objective: To analyze the role of DCs loaded with Aspergillus fumigatus extract on activation of CD4+T cells.Methods: The Aspergillus fumigatus extract were prepared from Aspergillus fumigatus and loaded onto dendritic cells. DCs were then co-cultured with CD4+ T cells with the ratio of 1:5 in for 24 hours. The activated state was been verified through analyzing the expression of CD69 and secretion of Inflammatory factors IFN-γ, IL-4, IL-17 and IL-22 etc. CCK8 kit was used to analyze its effect on the proliferation of T cells at last.Results: We found that T cell proliferation is significant high and the surface of mature markers CD69 expression were significantly up regulated along with the increase of inflammatory cytokines IFN-γ and IL-22 after co-cultured of CD4 + T cells with dendritic cells which have been loading of Aspergillus fumigatus extract antigen for 24 hours.Summary: Our study showed that DC cells with antigen of Aspergillus fumigatus extract not only could activate CD4+T cells but also could induce the release of inflammatory factors associated with the defense responses Aspergillus fumigatus which laid the foundation for our following study.Part Two: Detection and verification of abnormal expression of micro RNA in CD4+T cells activated by Aspergillus fumigatus extractObjective: To select differentially expressed micro RNAs in CD4+ T cells active by Aspergillus fumigatus extract.Methods: The Aspergillus fumigatus extract were prepared and were loaded onto dendritic cells. DCs were then co-cultured with CD4+ T cells for 24 hours. CD4+T cells were sorted using a magnetic column and total RNA was extracted. Cyanine 3(Cy3)-PCP markers were used to scan the micro RNA sequence.The six random micro RNAs were selected to verify those abnormal micro RNAs using Real-time PCR.Results: The results of Chip scan showed that there are 54 different expressed micro RNAs of which 26 up-regulated expression and 28 down-regulated expression in CD4+T cells activated by DC cells with Aspergillus fumigatus extract. The six micro RNAs, micro RNAs:mi RNA-142-3P,micro RNA-630, micro RNA-130b-3P, micro RNA-652-3P, micro RNA-494,micro RNA-574-5P, were chose to verified through PCR.Summary: We found that different micro RNA profile was expressed in CD4+T cells activated by DC cells with Aspergillus fumigatus extract after analyzing with Chip scan and PCR.Part Three: Selection of objective micro RNA, prediction of target gene and analysis of its functionObjective: To screen the objective micro RNA of differentially expressed micro RNA and analyze its biological functions and signaling pathways in Aspergillus fumigatus infection.Methods: The comprehensive fluorescence value and difference of multiples of differentially expressed micro RNA were ranked in descending order. The interested micro RNA was selected then. Target genes were predicted though the databases of TARGETMINER, mi RDB, micro RNA, Tar Base, RNA22. GO and Pathway analysis were conducted on the target genes predicted.Results: The target genes micro RNA we selected is mi RNA-142-3P. GO analysis shows that these target genes involved in regulating the significant function of mainly protein, cell adhesion, signal transduction, regulation of cellular processes, and regulating metabolism etc. Pathway analysis shows that the signaling pathway is mainly gobble up for cells and MAPK pathway, etc.Summary: The results above showed that mi RNA-142-3P target genes screened before was involved in many biological functions and metabolic pathway of regulation, which have been confirmed involving in T cell activation, proliferation and differentiation process.Part Four Effect of mi RNA-142-3P on the secretion of cytokines in CD4+T cells activated by Aspergillus niger and its mechanismObjective: To investigate the mechanism of cytokine secretion regulated by mi RNA-142-3P in CD4+T cellsMethods:Mi RNA-142-3P was selected from the chip to study the effect of micro RNA on the function of CD4+T cells by respectively transferred to the precursors and the inhibition. The expression of RICTOR, phosphorylation level of AKT, IFN-γ and IL-22 were all detected. The phosphorylation level of AKT and secretion level of IFN-γwere both detected when RNA interference technique was used to interfere with the expression of RICTOR.Results: When transfected into CD4+ T cells with mi RNA-142-3P precursor, the expression of RICTOR, phosphorylation level of AKT and IFN-γ were significantly lower. Instead, when transfected with mi RNA-142-3P inhibitor, the expression of RICTOR phosphorylation level of AKT and IFN-γ were significantly increased, while IL-22 stable. What`s more, the phosphorylation level of AKT and secretion level of IFN-γwere both decreased when RNA interference technique was used to interfere with the expression of RICTOR.Summary: The results above showed that mi RNA-142-3P in CD4+ T cells could be down regulated when activated by Aspergillus fumigatus extract mentioned above and could up regulated the expression of RICTOR and the protective cytokine IFN-γ to enhancing the defensive reaction of the organism against Aspergillus fumigatus.Conclusion: Firstly, DC cells with antigen of Aspergillus fumigatus extract not only could activate CD4+T cells but also could induce the release of inflammatory factors associated with the defense responses Aspergillus fumigatus. Secondly, as a result, different micro RNA profile was expressed in CD4+T cells activated by DC cells with antigen of aspergillus fumigatus. It suggests that the change in expression of some micro RNA may be one of the mechanisms though which to regulate the adaptive immune response once infected by Aspergillus fumigatus. Finally, down regulated of mi RNA-142-3P in CD4+T cells activated by DC cells with antigen of Aspergillus fumigatus could not only up regulated the expression of RICTOR but also could promote the secretion of protective cytokine IFN-γ to anti the infective of Aspergillus fumigatus.Above all, in this study, we found that different micro RNA profile was expressed in CD4+T cells activated by DC cells with Aspergillus fumigatus extract and its function in the adaptive immunity was studied then. As a result, mi RNA-142-3P could be down regulated when activated by Aspergillus fumigatus extract mentioned above and could up regulated the expression of RICTOR and the protective cytokine IFN-γ. Micro RNA-142-3P could be new therapeutic targets on infection of Aspergillus fumigatus in future.