The Study On The Mechanism Of Intestinal Flora Regulates Mucosal-associated Invariant T Cells Resistance Salmonella Typhimurium Infection In Mice

Mucosal-associated invariant T cells（MAIT cells）is a group of evolutionarily conserved T lymphocytes.MAIT cells recognize antigens different from traditional lymphocytes.They are activated and play a role through the riboflavin synthesis pathway unique to intestinal bacteria recognized by Major histocompatibility complex class I-like molecule（MR1）.MR1 is an important molecule for MAIT cells to perform normal function.Salmonella Typhimurium is an intracellular parasitic bacterium capable of causing intestinal diseases in humans and livestock,which has attracted much attention because of its great harm.S.typhimurium spreads to the circulatory system after entering the mesenteric lymph nodes through the intestinal epithelial tissue,and then causes the intestinal and the body immune responses.Therefore,in this study,we focused on MR1 null mice and C57BL/6J wild-type（WT）mice to determine the effect of MAIT cell deletion on the ability of the mice to fight S.typhimurium infection and the intestinal flora,and to elaborate on the mechanism by which it regulates S.typhimurium resistance.In this study,we first applied Clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated protein 9（Cas9）based gene editing to obtain injectable mr1guiderna（g RNA）by in vitro transcription,CO-cytoplasmic injection of g RNA and cas9protein into fertilized eggs,and transplantation into recipient mother mice to obtain founder（F0）chimeric mice.The mouse genotype was determined by PCR amplification,agarose gel electrophoresis and sequencing,based on which the animals were bred,and the F1 heterozygote mouse population was obtained by mating the chimeric mice of F0 generation with wild mice,and the heritable MR1 knockout population was established by continuous mating,which eventually succeeded in obtaining MR1^-/-mice.Secondly,to investigate the protective mechanism of MR1^-/-macrophages against S.typhimurium infection,we tried to isolate bone marrow macrophages from wild type and MR1^-/-mice,and the in vitro cell assay proved that during S.typhimurium infection of bone marrow macrophages,macrophages from MR1^-/-mice were inhibited in both phagocytosis and killing,and the expression of costimulatory molecules CD40,CD80 and CD86,NO and ROS were reduced,Impaired macrophage function.In vivo assays confirmed that MAIT cells and macrophages provided good protection to mice infected with S.typhimurium,and depletion of MAIT cells and macrophages significantly increased Salmonella numbers in the spleen and liver and mesenteric lymph nodes.Transcriptome sequencing of isolated bone marrow macrophages cocultured with S.typhimurium identified a total of 170 differentially expressed genes,of which161 m RNAs were significantly downregulated and 9 m RNAs were significantly upregulated,and most of the differentially expressed genes were associated with immune progression.Compared with MR1 overexpression,the CD4 gene in bone marrow macrophages of MR1 null mice was significantly upregulated.MAIT cells,as an important immune cell,under CD4 gene regulation bone marrow macrophages participate in the disease process of S.typhimurium,indicating that MR1 gene has an important role in maintaining the normal immune function of macrophages.Next,the mice were divided into four groups for the test:wild-type mice（WT）,MR1knockout mice(MR1^-/-),wild-type mice infected with S.typhimurium（WT+Sal）,MR1 knockout mice infected with S.typhimurium(MR1^-/-+Sal).Mice in the infection group were gavage with10⁸ CFU/m L S.typhimurium to establish the infection model,the control group was given PBS,the body weight was weighed daily,the death was recorded,the mice were sacrificed on the seventh day,the serum,duodenum and cecum contents were collected,and the tissue bacterial translocation and lesion were observed by the bacterial translocation test and tissue paraffin section.S.typhimurium infection increased mortality and aggravated the extent of lesions in the liver and spleen of MR1^-/-mice.Flow cytometry was used to determine cytokine and nuclear transcription factor expression profiles of CD4⁺T cells from spleen and mesenteric lymph nodes,and depletion of MAIT cells induced mixed Th1/Th2/Th17/Treg cell responses and reduced IFN-γproduction in mice,Expression of IL-4,IL-17,raised expression of IL-10,and raised production of SIg A in the duodenum.Through 16S r DNA high-throughput sequencing,we found that loss of MAIT cells and subsequent infection with S.typhimurium altered the gut microbiota and increased susceptibility and intestinal colonization with Salmonella spp.Taken together,our results indicate that MAIT cells provide conditions for T lymphocyte activation in the mesenteric lymph nodes and spleen,induce specific CD4⁺T cell responses in mice,and that loss of MAIT cells significantly elevates bacterial genera that positively correlate with inflammatory factors,increases the severity of inflammation caused by S.typhimurium infection,and exacerbates dysregulation of gut microbiota homeostasis.Finally,in order to further reveal the role of MAIT cells in Salmonella typhimurium infection,reverse infusion of MAIT cell agonist 5-A-RU activated MAIT cells after supplementation of vitamin B2 precursor 5-A-RU in WT mice,resulting in reduced mortality and bacterial translocation in liver and spleen,but no change in mortality in mice with MR1 gene deletion,indicating that MAIT cells play a protective role in Salmonella typhimurium infection.MAIT agonist induced Th1/Th2/Th17/Treg mixed cell reaction in mice,which increased the expression of IFN-γand IL-4,IL-17,and decreased the expression of IL-10.MAIT cells can regulate the disorder of hydrocortisone and tryptophan metabolites in cecal contents.The main mechanism is to regulate phenylalanine,tyrosine and tryptophan biosynthesis and vitamin B6 metabolic pathway,regulate inflammatory response,and mediate intestinal microbial metabolism.To sum up,this study takes MAIT cell deficient mice and WT mice as research objects,through the construction of Salmonella typhimurium infection model,to determine the mechanism of intestinal flora regulating MAIT cells against Salmonella typhimurium infection,which provides a new idea for the treatment of Salmonella typhimurium.