MiR-15b Regulate The Anti-inflammatory Effect Of IL-10,by Enhancing The Induction Of Regulatory T Cells,in Cryptococcus Pneumonia

Objective:Cryptococcus is a very important pathogenic fungus which is difficult to treat after infection.It mainly infects human body through respiratory tract.When Cryptococcus invades the respiratory system,it is usually engulfed by epithelial cells,alveolar macrophages and dendritic cells,leading to transient or asymptomatic cryptococcal pneumonia.But if Cryptococcus evades the host's autoimmune response,it can also invade other organs,such as the nervous system,which will become more serious and lead to a high mortality rate.Cryptococcus neoformans and Cryptococcus getti are the main pathogens causing human diseases.Cryptococcus neoformans infection occurs mostly in people with low immunity.With the increase of HIV infection rate and organ transplantation patients,cryptococcus infection rate is also increasing.All these indicate that the host's immune system regulation plays an important role in Cryptococcus infection.After Cryptococcus invades human body,it activates human immune system,including natural immunity and adaptive immunity.The main effective factors of natural immunity are complement system and phagocytosis effect factor.Adaptive immunity includes humoral immunity and cellular immunity.Complement pathway or phagocytosis is regulated by adaptive immunity.MicroRNA(microRNA)is a kind of endogenous small RNA with a length of about 20-24 nucleotides,which plays a variety of important regulatory roles in cells.We have shown that the expression of microRNA-15 b is high in cryptococcal infection patients,and foreign studies have found that microRNA-15 b can up-regulate the expression of regulatory T cells.Regulatory cells(Tregs)are a subset of T cells that control autoimmune response in vivo and play an important role in natural and adaptive immune systems.Regulatory T cells can be divided into natural regulatory T cells(nTreg)and induced adaptive regulatory T cells(aT-reg or iTreg),such as Th3,Tr1,CD8 T REG and NKT cells,which are closely related to the occurrence of autoimmune diseases,and their abnormal expression can lead to autoimmune diseases.Regulatory T cells exert immunosuppressive effects mainly through IL-10 and TGF-beta.The biological effects of IL-10 are surprisingly multifaceted and have been deeply studied in recent years.The roles of different cell families have been elucidated.From the results,almost all monocyte macrophages are the target cells of IL-10 inhibition.Therefore,based on the previous experiments,this study intends to detect the expression of microRNA-15 b,Treg cells and IL-10 in Cryptococcus pulmonale mice model,preliminarily explore the host's immune response mechanism after Cryptococcus infection,and find out possible therapeutic targets.Research methods:In the first part,It has been reported that the abnormal expression of miR-15 b in THP-1 cells induced by Cryptococcus neoformans.Then Cryptococcus and THP1 cells were co-cultured through cell experiments.The time points of 0h,3h,6h,9h and 12 h were set up respectively.The expression of microRNA-15 b was detected again by polymerase chain reaction.Then the cryptococcal pneumonia mice model was established.The expression of microRNA-15 b in peripheral blood of cryptococcal pneumonia mice was measured at 1d,4d,7d,11 d,14d and 17 D after infection.The proportion of Treg cells in peripheral blood of mice was detected by flow cytometry.The expression of IL-10,TNF-a in peripheral blood of infected mice was measured by ELISA.Meanwhile,the lungs of mice were sliced and stained with argyrophilic staining to observe cryptococcus infection in the lungs of mice.In the second part,microRNA-15 b mimics were injected into tail vein.The expression of microRNA-15 b was detected by polymerase chain reaction.The proportion of Treg cells in peripheral blood was detected by flow cytometry.The expression of IL-10 in peripheral blood of infected mice was measured by ELISA.In the third part,mice were injected with mir-15 inhibits via tail vein,and then the cryptococcal pneumonia model was established.The expression of mir-15 b in peripheral blood of Cryptococcus pulmonale mice was measured at 1,4,7,11,14 and 17 days after infection.The proportion of Treg cells in peripheral blood of mice was detected by flow cytometry.The expression of IL-10,TNF-?in peripheral blood of infected mice was measured by ELISA.Law.Meanwhile,the lungs of mice were sliced and stained with argyrophilic staining to observe cryptococcus infection in the lungs of mice.Result:Part one: The expression of microRNA-15 b in blood of patients was significantly higher than that of normal people.When Cryptococcus was co-cultured with THP1 cells,the expression of microRNA-15 b increased slowly at first and then decreased.The expression of microRNA-15 b in peripheral blood of Cryptococcus pneumonia model mice reached its peak on the 7th day and then decreased.Flow cytometry was used to detect the expression of Treg cells and ELISA was used to detect the expression of IL-10,which was the same as that of microRNA-15 b.The argyrophilic staining of lung slices in mice indicated that Cryptococcus proliferated in the lungs of mice.Part two: Mi-15 b mimics was injected into tail vein.The expression of Mi-15 b in peripheral blood of mice was significantly increased.Treg ratio was increased by flow cytometry and IL-10 expression was increased by ELISA.Part three: Mice were infected with Cryptococcus after injecting Mi-15 inhibits into tail vein of mice.After 7 days,the expression of Mi-15 b in peripheral blood of mice was significantly lower than that of mice without Mi-15 inhibits injection(control group).Treg ratio was measured by flow cytometry.The expression of IL-10 by ELISA was lower than that of control group,while the expression of TNF-?was higher than that of control group.The argyrophilic staining of lung slices in mice showed that the number of Cryptococcus was significantly reduced compared with the control group.CONCLUSION: Cryptococcus infection can stimulate the overexpression of microRNA-15 b in mice,and microRNA-15 b can up-regulate the generation of Treg cells and further promote the expression of IL-10,thus inhibiting the protective effects of cytokines such as TNF-a,i.e.,the clearance of Cryptococcus is reduced,leading to the reproduction of Cryptococcus in the host.