Small RNA-omics Of MiRNAs Mediated Immune Response In Host-related Organs During Schistosoma Japonicum Infection

Schistosomiasis caused by schistosome is a serious zoonotic disease in humans and animals.It’s mainly distributed in Asia,Africa and Latin America,endemic in 78 countries and regions.Extracellular Vesicles(EVs)play an essential role in various biological processes such as antigen presentation,immune response.T cell immune response plays an important role in Schistosomiasis;whereas miRNAs play an essential role in the regulation of T cells activation,differentiation and proliferation.Therefore,the miRNA profiles associated with plasma extracellular vesicles and T cells during the immune responses are critical for understanding the immune regulation of the host during schistosomiasis infection.This paper including the following research:Ⅰ.Isolation,purification and characterization of EVs.Murine EL-4 cells and mouse plasma extracellular vesicles were isolated and purified by size exclusion chromatography combined with differential ultra-centrifugation.The structure of EVs was observed by transmission electron microscopy,the size of EVs was detected by nanoparticle tracking analysis,and the marker molecules of EVs were analyzed by western blot.This study provided a fundamental for subsequent experiments.Ⅱ.miRNAome profile of EVs cargo isolated from the plasma at different stages of Schistosoma japonicum-infected mice.We investigated the micro RNA(miRNA)profiles of EVs isolated from host plasma at different stages of S.japonicum infection(lung stage and liver stages).Mi RNAs expression profiles and differential characteristics were obtained at lung stage(3dpi)and liver stages(14 and21dpi).Compared with the uninfected group,the expression of miR-1a-3p and miR-122-5p showed higher abundance,whereas miR-150-3p and miR-126 a showed lower abundance in the plasma EVs of infected mice at 3,14,and 21 dpi.In addition,several worm-specific miRNAs were detected in 21 dpi plasma EVs.Our results revealed that the abundance of miRNA cargo of the host plasma EVs was related to S.japonicum infection.Moreover,the differentially abundant miRNA cargo in host EVs associated with S.japonicum infection and parasite-specific miRNAs may also provide valuable clues for identifying novel biomarkers for schistosomiasis diagnosis.Ⅲ.Characterization of T cell miRNA profile isolated from the blood and liver at hepatic stages of S.japonicum infection.In this study,T cells from the blood and liver of S.japonicum-infected mice at different stages(14dpi and 21dpi)were isolated by flow cytometry and subjected to small RNA sequencing,respectively.To explore the immune mechanism of host T cells and the key miRNAs involved in T cell immune regulation during S.japonicum-infected.Further analysis indicated that a total 508 and 504 miRNAs were identified in peripheral blood and liver T cells,respectively.Compared with the uninfected group,comparative analysis of miRNAs in peripheral blood T cells showed that miR-486b-5p /3p expression was significantly down-regulated and miR-375 expression was highly up-regulated.The miRNAs in liver T cells showed that miR-466b-3p,miR-486b-3p,miR-1969,and miR-375 were differentially expressed.Meanwhile,miR-181c-5p and miR-30d-5p were significantly down-regulated both in peripheral blood and liver T cells.These results reveal the dynamic changes of T cells in host peripheral blood and liver during S.japonicum infection,and the different miRNAs composition may be related to the regulation of host T cell immune response,providing a piece of essential information to better understand the regulation of T cell immune response during S.japonicum infection.Ⅳ.EVs artificially modified by loading miRNAs in vitro.We standardized electroporation conditions for loading miRNA into EVs and evaluated the electroporated efficiency.The results showed that EVs can successfully uptake by different cells,and more labeled EVs were uptake by monocytes than lymphocytes.The maximum efficiency was achieved when the concentration of miRNAs mimics was 1μg and the voltage was 100 V.Compared with the control group,flow cytometry showed that miRNA modified EVs(PE and FITC)could be uptake by more cells.These results provide a foundation for subsequent studies which using miRNA-modified EVs as vaccine and therapy.In summary,this study conducted a comparative analysis of small RNAs at different stages of plasma extracellular vesicles and T cells isolated from peripheral blood and liver of S.japonicum infected mice.The results help to understand the key miRNAs involved in host plasma EVs and T cell immune response during S.japonicum infection and providing a new method for modifying EVs content in vitro that can be useful for further studies to prevent and control of schistosomiasis.