Cellular And Molecular Mechanisms Of Lung Injury In MicroRNA-21 Transgenic Mice

Background and ObjectiveAutoimmune disease is characterized by the immune response of T cells or B cells against the autoantigens,the normal level of autoimmunity is very important to clear the damaged,mutated and cancerous cells and maintain homeostasis.However,when the autoreactive T cells or B cells are abnormally activated due to some factors,it will lead to damage and dysfunction of tissues and organs.In addition to T cells and B cells,there are many kinds of cells and molecules participate in this process,because of the the different tissues and organs involved,the pathogenesis of different diseases various considerably.MiR-21 is one of the most widely studied microRNAs.It participates in the development of a variety of autoimmune diseases by directly or indirectly acting on target genes,regulating cell proliferation,apoptosis,metabolism,et al.MiR-21 plays different roles in different tissues and cells,the pathological mechanisms involved differs in autoimmune diseases.In this study,we will investigate the following aspects:（1）the immunophenotype of miR-21 transgenic mice;（2）the pathological changes of miR-21 transgenic mice;（3）the relationship between dysregulated immune cells and tissue damage;（4）the cellular and molecular mechanisms of tissue damage.The results of this study will provide a theoretical reference for further exploring the role of miR-21 in autoimmune diseases.Methods1.The development of T cells and B cells in thymus and bone marrows,as well as their activation and differentiation levels in lymph nodes and spleens of miR-21 transgenic mice were detected using flow cytometry.2.The pathological changes of miR-21 transgenic mice were measured by HE and immunohistochemical staining.3.The numbers,proportions and locations of dysregulated immune cells in lymph nodes and spleens were detected by multilabeled immunofluorescence staining.4.The levels of immunoglobulin subtypes,antinuclear antibodies and complement in serum of wild-type and miR-21 transgenic mice were determined by ELISA and flow cytometry.5.Mice were immunized with NP-CGG,the NP-specific antibodies:anti-NP4-Ig G1and anti-NP20-Ig G1 were detected by ELISA at different time points after immunization to analyze the immune response ability of miR-21 transgenic mice.4.Na（?）ve CD4+T cells and B220+B cells from WT and miR-21 transgenic mice were sorted by immunomagnetic beads enrichment and flow cytometry.The cells were mixed according to different groups and then transferred to Rag1^-/-mice.The related pathological changes were analyzed.5.RNA-seq was preformed to detected the difference of gene expression and the related pathways.Results1.MiR-21 transgenic mice developed lung lesions,lymphocyte infiltration and heterotopic germinal center（GC）appeared in the lung tissue;plasma cells（PCs）increased reactively in lymph nodes and spleens（all p<0.05）.2.Immunofluorescence staining showed that in lymph nodes and spleens of miR-21transgenic mice,the increase of PCs in extrafollicular（EF）region was higher than that in GC（all p<0.05）;the ratios of PCs:Tfh cells in EF regions was significantly higher than that of wild type（all p<0.05）.3.The number of PCs in lymph nodes and spleens of miR-21 transgenic mice that had direct contact with a Tfh cell（distance≤15μm）was significantly higher than that of WT mice（all p<0.05）;the distances between each PCs and the nearest TFH cell in miR-21 transgenic mice were greater than that of WT mice in lymph nodes.4.The contents of immunoglobulin,antinuclear antibodies and complement in serum of miR-21 transgenic mice were increased（all p<0.05）,and they were deposited on the surface of alveolar wall,glomerulus and renal tubule;their abilities of responding to external antigens was enhanced.5.No significant pathological changes were found in the lungs of Rag1^-/-mice after adoptive transfer of WT-T cells+WT-B cells or WT-B cells+miR-21-T cells;obvious inflammatory cells infiltration were found in the lungs of Rag1^-/-mice after adoptive transfer of miR-21-T cells+miR-21-B cells;Rag1^-/-mice received WT-T cells+miR-21-B cells displayed milder lung lesions.6.RNA-seq showed that the expression of differentiation,survival and function related molecules in PCs and Tfh cells of miR-21 transgenic mice were up-regulated;enrichment results showed that the differential genes mainly focus on immune system,complement system and autoimmune disease related functions and pathways.ConclusionIn miR-21 transgenic mice,the overexpressed miR-21 promotes the differentiation,development,function and survival of PCs and TFH cells through direct and indirect ways;the GC and EF responses were enhanced and PCs increased reactively;the booming autoantibodies and the activation of complement system caused the lung injury.In this process,B cells played central roles as driver,T cells also function as important helper.In conclusion,this study provides new clues for understanding the interaction and mechanism between Tfh and PCs in EF and GC regions,expands the knowledge of T-B cell interaction,and provides theoretical foundation and experimental system for further study of the role of miR-21 in autoimmune diseases.