MiR155-deficient Alleviated Intestinal Inflam-Tion By Regulating The Development And Functionof DC Subsets In The Lamina Propria Of Intestine

ObjectiveIn this study,miR155-deficient mice were used as a model to detect the effect of changes in miR155 expression on the development and function of intestinal DC subsets.Methods1.We take miR155 deficient mouse tissue,RNA extraction,genetic identification,determine the use of mice in the experimental group lack of microRNA155.2.In the steady state,the small intestine was taken to prepare single cells of miR155-deficient mice and control group(C57BL/6)small intestine mucosal lamina propria.The total DC and subpopulations in the small intestine mucosal lamina propria were detected by flow cytometry.3.At steady state,single cells of the intestinal mucosal lamina propria of miR155-deficient mice and control group(C57BL/6)were prepared,and the proliferation and apoptosis of DC subpop?lations of the intestinal lamina propria was detected by flow cytometry using staining methods of Ki67 and Annexin V.4.In steady state,the mesenteric lymph nodes(mLN)were taken from mice with miR155-deficient mice and control mice.The number and proportion of DC subpopul-ations in mLN were detected by flow cytometry.5.In the steady state,femurs were taken to prepare bone marrow mononuclear cells of miR155-deficient mice and control group(C57BL/6).Flow cytometry was used to detect the effect of miR155 deletion on DC precursors in bone marrow.6.The bone marrow-derived CD103~+CD11b~+DC of miR155-deficient mice were induced with GM-CSF and FLT3-L.The changes of CD103~+CD11b~+DC subsets were de-tected on day 8,day 12 and day 16 after culture.7.The bone marrow-derived CD103~+CD11b~+DC s were induced in vitro.On day 15,the culture supernatants were taken and stimulated overnight with LPS.The supernatants were taken on day 16.ELISA was used to detect inflammatory cytokines TNF-?and IL-6secreted by bone marrow-derived DCs in miR155-deficient mice and controls.8.The spleen DCs of miR155-deficient mice and control mice were sorted by flow cytometry.The Na?ve T cells of the spleen of CD45.1~+OT-II mice were sorted,and DC and Na?ve T cells were co-cultured at ratio of 1:10 in vitro.The proliferation of Na?ve T cells was measured after 3 days.9.IBD model was established to compare the differences in intestinal inflammation between miR155-deficient mice and control mice.(1)Establishment of mouse IBD modelDSS was dissolved in sterile distilled water so that the final concentration of DSS was2.5%,and the mice were given continuous 2.5%DSS for 5 days and then replaced with normal sterile distilled water for 3 days.(2)Severity assessment of enteritisDuring the modeling process,mouse body weight was recorded at the same time every day.We observed the degree of faeces and the presence of blood in the stool and its severity.The disease activity index(DAI)was used to evaluate whether the mouse colitis model was successfully constructed and evaluate the severity of intestinal inflammation.(3)Acute enteritis was induced in control mice(C57BL/6)and miR155-deficient mice,respectively,and the severity of both acute enteritis was compared.Result1.We took miR155-deficient mouse tissues,extracted DNA,and then amplified the gene.After electrophoresis,we found that the DNA band was distributed at 600 bp,which is consistent with the mouse identification standard provided by the jackson laboratory.Therefore,we determined that the mice used were miR155-deficient mice.2.Under steady-state conditions,the number and proportion of total DCs in the intestinal lamina propria and the CD103~+CD11b~-DC and CD103~-CD11b~+DC groups did not change significantly after miR155 deletion,but the number of CD103~+CD11b~+DC subpopulations decreases significantly.3.In the steady state,Ki67 and Annexin V were used to detect the proliferation and apoptosis of CD103~+CD11b~+DC in miR155-deficient mice LP.It was found that loss of miR155 did not affect the proliferation and apoptosis of this group of cells.4.At steady state,there was no significant change in DC subsets in the mesenteric lymph nodes of miR155-deficient mice.5.In steady state,the mouse femur was taken to prepare bone marrow single cells of miR155-deficient mice and control group.We used flow cytometry to detect and found the loss of miR155 did not affect the development of precursor cells of other blood cells such as MEP,CMP,GMP,and total pre-DC.But the development of cDC1(CD103~+DC)and its precursor cells(pre-cDC1)was down-regulated,which in turn may have affected the development of CD103~+DC,especially CD103~+CD11b~+DC in the small intestine lamina propria.6.The bone marrow-derived CD103~+CD11b~+DC were induced by GM-CSF and FLT3-L in miR155-deficient mice and control groups.The ratio of total bone marrow-derived DCs was detected by flow cytometry on day 8,12 and 16 after culture.we find no significant difference in the ratio of total bone marrow-derived DCs on day 8,day12 and day 16 after culture.However,the proportion of bone marrow-derived CD103~+CD11b~+DC was significantly reduced in miR155-deficient mice.7.The bone marrow-derived CD103~+CD11b~+DC was induced in vitro.On day 15,the culture supernatant was taken to stimulate with LPS for one night,and the supernatant was taken on day 16.The inflammatory factors secreted by the two groups of bone marrow-derived DC were detected by ELISA.It was found that the inflammatory factors TNF-?and IL-6,especially the latter,had a downward trend in secretion after stimulation with LPS compared with the control group of bone marrow-derived DCs.8.The spleens of miR155-deficient mice and control mice were taken to prepare single cells.After fluorescent labeling,the total spleen DCs were sorted by flow cytometry.Single cells of spleen of CD45.1~+OT-II mice were prepared,Na?ve T cells were sorted by flow cytometry.DC and Na?ve T cells were co-cultured at a ratio of 1:10,and the prolife-ration of Na?ve T cells was detected after 3 days.After loss of miR155,the ability of DCs to stimulate the proliferation of Na?ve T cells was reduced.9.In the IBD model,miR155-deficient mice have significantly reduced colitis.(1)DSS-induced IBD enteritis model was successfully constructed.On day 1 to 4,there was no significant change in body weight in mice.On day 5,day6,day 7,and day 8,the body weight of mice continuously decreased,reaching the lowest on day 8.(2)Acute enteritis was induced in the control group(C57BL/6)and miR155-deficient mice,respectively.The results showed that compared with control,the miR155-deficient mice had a lower disease activity index score,a lower weight loss.(3)Acute enteritis was induced by DSS.Intestinal lamina propria lamina propria(LP)single cells were prepared.Th17 in intestinal mucosal lamina propria in control and miR155-deficient mice were determined by flow cytometry and we found that the number of Th17 in the intestinal lamina propria of miR155-deficient mice was reduced compared to the control group.ConclusionLoss of miR155 at steady state can lead to a decrease in the number of intestinal lamina propria CD103~+DC,especially CD103~+CD11b~+DC,but this does not affect the pro-liferation,apoptosis and migration of the cell.Moreover,experiments have found that loss of miR155 leads to a decrease in cDC1 precursor cells in the bone marrow,suggesting that miR155 regulates the development of CD103~+DC.The decrease of somatic cells indicates that miR155 regulates the development of CD103~+DC.A further study in the IBD model revealed that the miR155 deficiency has protective effects on DSS-induced enteritis,which may be related to a decrease in the Th17 cell response caused by a decrease in the number of intestinal lamina propria CD103~+DC,especially CD103~+CD11b~+DC.