Study On The Frequency, Subsets And Functional Status Changes Of INKT Cells In Female NOD/LTJ Mice At Different Onset Stages

ObjectiveFemale NOD/LtJ(Non-obese diabetic)mouse model of type 1 diabetes(T1D)was used.Detect the frequency of CD4~+T,CD8~+T,Th cell subsets,iNKT cell and subsets ratios.cytokines and transcription factor levels in different tissues and organs at the non-onset,the early stage of onset,the end onset stage.To explore the immune function of NOD/LtJ mice at different stages of the disease and the role of iNKT cells in the development of T1D,to provide basis for the T1D treatment strategy targeting i NKT cells.Methods1.Establishment of type 1 diabetes mellitus model in NOD/LtJ mice.Female NOD/Lt J mice were tested for urinary glucose twice per week at a fixed time.Fasting blood glucose was measured in urine-positive individuals for a fixed period of two consecutive days.T1D was diagnosed when two consecutive blood glucose levels were?11.1 mmol/L.The animals were divided into 3 groups:non-onset group,early onset group(identified within 1 week after onset),and late onset group(4-5 weeks after onset).Observe and record the mice's blood glucose,body weight,mental status,activity and hairiness,organ index,absolute lymphocyte count.The metabolic cages monitored 24 h food intake,water intake and urine output.Hematoxylin-eosin staining(HE)staining was used to observe the infiltration of pancreatitis cells.The Cytometric Bead Array(CBA)was used to detect serum cytokine levels.The mice were observed to be 30 weeks old.2.Analysis of immune function at different stages of NOD/LtJ miceFlow cytometry(FCM)was used to detect the frequencies of CD4~+T,CD8~+T,Th1/Th2/Th17,and i NKT cells in different tissues at different stages of disease,the ratio of i NKT1/iNKT2 cell subsets,and the absolute number of iNKT cells.CBA was used to detect of spleen,liver,lymphocyte culture supernatant cytokine levels;Western-Blot detection of transcription factors PLZF,T-bet,GATA-3,ROR-?t relative expression.Results1.The mice in the non-onset group and early onset group had good mental state,smooth shiny hair,and sensitive response.At the end of the onset period,the group was apathetic,the hair was dry,and the reaction was unresponsive.Compared with the non-onset group,fasting blood glucose in the onset group showed an upward trend with the progress of the disease.The body weight,organ index,and absolute lymphocyte count at the end of the onset period were significantly lower than those in the non-onset and early onset group(p<0.05).24 h food consumption,drinking water and urine output of mice increased significantly with the progression of the disease(p<0.05).In all three groups,a large number of inflammatory cells infiltrated,and the degree of inflammatory cell infiltration increased with the progress of the disease.The number of islets in the early stage and the late stage of the disease decreased,sparsely distributed,and the number of islet cells decreased(p<0.05).Compared with the non-infected group and the end-stage group,the serum levels of cytokines IFN-?,IL-4,and IL-17A significantly increased,the ratio of IFN-?/IL-4 increased significantly(p<0.05).2.The frequency of CD4~+T cells in the early stage onset groupwas significantly higher in the spleen,liver,thymus and inguinal lymph nodes than in the non-onset group(p<0.05).The frequency of CD4~+T cells in peripheral blood was lower in the late onset group than in the early onset group.In the liver,thymus,and inguinal lymph nodes,there was a significant decrease(p<0.05).Compared with the non-onset group,the frequency of CD8~+T cells in the early stage onset increased significantly in the spleen and thymus(p<0.05);compared with the early stage,The frequency of CD8~+T cells at the end onset group was significantly lower in the liver and thymus(p<0.05).In the spleen,liver,and inguinal lymph nodes,the ratio of Th1 subsets at the late onset group was significantly increased(p<0.05).In the liver and inguinal lymph nodes,the ratio of Th2 and Th17 subsets at the late onset group was significantly higher than that at the early onset group(p<0.05).Absolute counts of i NKT cells in the spleen,liver,and thymus in the early onset group were significantly increased(p<0.05).The ratio of i NKT1 subsets in the early stage and in the late stage group increased significantly,and the ratio of iNKT2 subsets decreased significantly(p<0.05).Compared with the i NKT1 subset,the i NKT2 subset decreased in the three groups of spleen,liver and inguinal lymph nodes.(p<0.05)In the thymus of the non-onset group,the proportion of iNKT2 subset was higher compared with the ratio of i NKT1 subset(p<0.05).In the early stage onset group,the levels of inflammation and anti-inflammatory cytokines in the spleen and inguinal lymph nodes were significantly increased,but the ratio of IFN-?/IL-4 was significantly increased(p<0.05);The levels of inflammatory cytokines(IFN-?,TNF-?,IL-2,IL-6,IL-17A)in the liver and IFN-?/IL-4 gradually increased with the progression of diease(p<0.05);anti-inflammatory cytokines(IL-4,IL-10)levels were highest in the early onset and significantly decreased at the end of the onset group(p<0.05).There was no significant difference in the relative expression of the thymus transcription factor PLZF between the three groups(p>0.05);the relative expression of T-bet in the late stage onset group of the spleen was significantly increased(p<0.05);The relative expression levels of T-bet in liver in the late stage onset group was significantly increased(p<0.05),and the relative expression levels of GATA-3 and ROR-?t were significantly decreased in the early onset group(p<0.05).Conclusion1.The incidence of T1D in female NOD/Lt J mice was concentrated at 22-26 weeks of age.The cumulative incidence of T1D is 70%up to 30 weeks of age.2.The increase frequency of CD4~+T and CD8~+T cells,especially the increase of CD4~+T cells at the early onset group is an important immune basis for insulitis.The increase of the frequency of iNKT cells and the turnover of the iNKT1/iNKT2subsets in the early onset group suggest that iNKT cells may participate in the development of T1D in NOD/Lt J mice.At the late onset group,the Th1/Th2/Th17 subsets in the spleen,liver and inguinallymph nodes all increased,but the Th1 subset increased.The expression levels of related transcription factors T-bet,GATA-3 and ROR-?t were consistent with changes in Th subsets.At the early and late onset groups,the mice exhibited severe imbalance of inflammatory factors in the body,and the inflammatory cytokines were significantly higher than those of the anti-inflammatory cytokines.The iNKT cells,especially the i NKT1 subset,may influence the occurrence of T1Dby inducing the polarization of Th0 to Th1 and Th17 subsets.