The Commensal Microbiota Regulates The Long Non-coding RNA Expression Of Alveolar Macrophages From Mice

Objectives There are a large number of commensal microbiota in normal mucosal tissues,and their components and metabolites can not only maintain immune homeostasis,but also participate in the occurrence and development of a variety of immune diseases.The respiratory tract is a complex mucosal tissue,and the regulation of lung immune response by commensal microbiota is precise and specific.Alveolar macrophages are the most abundant innate immune cells distributed on the surface of alveolar cavities.They play an important role in immune response and immune regulation,and are involved in the occurrence and development of various pulmonary diseases.Commensal microbiota signals in the lungs can regulate the function and polarization of alveolar macrophage.Our existing studies have revealed that respiratory commensal microbiota promote the anti-tumor immune response by maintaining alveolar macrophages in M1 state and low expression of CCL24.LncRNA is an important regulator of immune homeostasis and function of macrophages.Previous studies have confirmed that a variety of lncRNAs play a key role in maintaining the homeostasis and function of innate and adaptive immune cells,and are closely related to the occurrence and development of various diseases.Studies have shown that lncRNAs can regulate the inflammatory response and the polarization of macrophages.Therefore,it is of great significance to explore the effect of commensal microbiota on the expression of lncRNAs in alveolar macrophages.The purpose of this study was to detect the expression level of lncRNAs in alveolar macrophages of mice with commensal microbiota deficiencies,as well as its regulatory role on the expression of CCL24 and Arg1,so as to provide strong evidence for further understanding of the molecular mechanism of commensal microbiota regulating lung immune homeostasis.Methods Commensal microbiota-deficient mouse models were established by feeding mice with drinking water containing a combination of antibiotics,Flow cytometry was used to isolate and purify alveolar macrophages from antibiotic-treated mice and normal mice,lncRNA microarray technology was performed to screen out differentially expressed lncRNAs,GO and KEGG bioinformatics analysis was used to predict their target genes,and the results were verified by RT-PCR.The subcellular localization of lncRNA-30162 in RAW264.7 macrophages was performed by using FISH technique,lncRNA-30162 was knocked out by RNA interference method and the effect of lncRNA-30162 on gene expression of RAW264.7 cell was also detected,finally,the results were verified by ELISA.Results 1.The purity of alveolar macrophages was greater than 95% after sorting,compared with normal mice,there were a total of 634 differentially expressed lncRNAs of alveolar macrophages from commensal microbiota-deficient mice(variation multiple≥2,p<0.05),including 363 up-regulated lncRNAs and 271 down-regulated lncRNAs.2.Target genes of differentially expressed lncRNAs of alveolar macrophages from commensal microbiota-deficient are related to a variety of bioinformatics pathways,including immune system regulation,cell differentiation,chemotaxis,etc.3.Knockout of lncRNA-30162 expression in RAW264.7 cells can significantly reduce the expression levels of CCL24 and Arg1,while the expression levels of TIMP1,IGF1,and MMP12 were not significantly changed.Conclusion 1.Commensal microbiota can regulate the expression of multiple lncRNAs of alveolar macrophages from mice,and differentially expressed lncRNAs are closely related to immune system regulation,cell differentiation,chemotaxis,etc.2.LncRNA-30162 can regulate the expression of CCL24 and Arg1 in RAW264.7 cell.