Phenotypic Analysis And Preliminary Exploration Mechanism Of CD169~+ Macrophages In Aging Mice

Research background:By 2050,China will have 400 million people over the age of 65.China is facing great medical challenges caused by aging.The mechanism research,diagnosis,treatment and intervention of age-related diseases have become hot topics in basic and clinical medicine research.Studies have shown that chronic inflammation is a common feature of mammalian body and organ aging,but how inflammation regulates aging is still unclear.Age-related changes in gut structure and function can lead to the entry of bacteria and their products into the circulation,activation of macrophages,and initiation of inflammatory response.Our previous study has shown that the loss of CD169+macrophages distributed in the lamina propria of the colon can improve the symptoms of intestinal inflammation and reduce the production of cytokines.However,how the number and function of CD169+ macrophages change in the colon of aging mice,and whether CD169+ macrophages can be used as a target for regulating aging are still not reported.CD169+ macrophages are a unique macrophage subset that is different from M1 and M2 macrophages.CD169+macrophages play an important role in antigen presentation,immune tolerance induction,autoimmune inflammatory response and tumor development.The differentiation mechanism of CD169+macrophages in the colon is not dependent on lymphotoxin a signaling,but closely related to vitamin A.Analyzing the phenotype and mechanism of intestinal CD169+macrophages in aging is crucial for delaying intestinal aging and resisting diseases and aging..Research Purposes:In this study,the colon was used as the entry point to clarify the changes in the number,gene expression and efferocytosis of CD169+macrophages in aged mice and the related mechanism of regulating inflammation,and to explore the intervention measures to improve aging by targeting CD169+macrophages,so as to provide new data and new ideas for intervening the aging process and finding methods for healthy agingResearch methods:First,in this study,HE staining,RT-qPCR,and Western blot were used to detect the degree of intestinal barrier damage and the levels of inflammatory factors under physiological and pathological conditions in aging colon,and to clarify the effect of CD169+ macrophages on inflammation in aging mice.Secondly,the number and efferocytosis function of CD169+macrophages in aging mice were detected by flow cytometry,RT-qPCR and immunofluorescence.RNA sequencing technology and 16S rDNA sequencing technology were used to detect the gene expression changes of CD169+macrophages and the changes of fecal microbiota and metabolites in aging mice,respectively.Finally,the efferocytosis of CD169+macrophages regulated by bacterial metabolite sodium butyrate was analyzed by in vitro and in vivo apoptotic cell phagocytosis experiments.Results:Under physiological conditions,there were different degrees of inflammatory infiltration of barrier damage in the colon of aged mice.The cytokine IL-6 and TNF-α were significantly higher than those of young mice,and the accumulation of apoptotic cells in the colon was obvious.The number of CD169+macrophages in the colon of aging mice was significantly increased.After DSS-induced acute colitis,the degree of inflammation was more severe in aged mice.The number Uf CD169+ macrophages increased even more in the wolon.Moreover,depletion of CD169+macrophages alleviated symptoms of colitis in aging mice.RNA-seq results and RT-qPCR analysis showed that the expression of pathways related to apoptotic cell clearance and efferocytosis receptors was significantly down-regulated in the aging group.Moreover,immune tolerance and immune homeostasis were impaired in aging mice.16S rDNA sequencing results showed that the diversity of gut microbiota in aging mice decreased significantly,and the composition of gut microbiota also changed significantly.The proportion of bacteria related to acetate and butyrate production decreased significantly.Further studies showed that sodium butyrate reversed the aging-induced decline in efferocytosis of macrophages and inhibited the differentiation of CD169+macrophages in vitro..Conclusion:This study identified the changes in the number,efferocytosis,immune tolerance and gene expression level of CD169+macrophages in aging mice,and sodium butyrate could improve the efferocytosis and differentiation level of aging CD169+macrophages.These results suggest that CD169+ macrophages are the key cells in the regulation of aging in mice..1.There are physiological changes and immune microenvironment changes in the colon of aging mice,and CD169+ macrophages are significantly increased in the colon of aging nice.2.The symptoms of DSS-induced colitis were more severe in aging mice,and the increase in the number of CD169+ macrophages was more significant.Depletion of CD169+macrophages leads to reduced symptoms of colitis in aged mice.3.The expression of Tim-4 on CD169+macrophages decreased,and the ability to efferocytosis decreased in aging mice.4.Butyrate,a metabolite of gut microbiota,significantly enhanced the ability of CD169+macrophages to efferocytosis and inhibited the differentiation of CD169+ macrophages.