| Aims:It is known that type 2 diabetes mellitus(T2DM)is a chronic inflammatory disease,inflammatory response can be detected in these patients’ peripheral blood and also can be detected in liver,adipose tissue,pancreas tissues of animal models,the development of T2DM and the progression of its complications are aggravated by the secretion of inflammatory cytokines.Abnormal function of immune system plays an important role in the process of chronic inflammation in T2DM patients.Signaling lymphocyte activated molecular family(SLAMF)was reported to have abnormal expression in a variety of immune disorders,and affects the immunocytes differentiation and function,however,it has not been reported about the expression of SLAMF molecules on the immunocytes in patients with T2DM,and it is unclear about the mechanism of inflammation appearance in T2DM.To investigate this,we used T2DM patients and humanized mice model as the research objects,by analyzing immunocytes differentiation and the SLAMF molecules expression in PBMCs of T2DM patients,treating T cells with palmitic acid in vitro and establishing humanized high-fat mice model,in order to explore the role and mechanism of immunocytes abnormal differentiation and SLAMF molecules abnormal expression in condition of chronic inflammation in T2DM.Methods:(1)We measured the frequencies of T cell,NK cells and their subsets in peripheral blood mononuclear cells of T2DM patients and healthy controls by flow cytometry,SLAMF molecules expression on the surface of T cells and NK cells was detected by flow cytometry.(2)We detected the expression of SLAMF3 molecular on T cells in peripheral blood in patients with T2DM and the secretion of proinflammatory cytokines IFN-y and IL-17 by flow cytometry,analyzed the correlation between the expression of SLAMF3 molecules and cytokine secretion by T cells.(3)We sorted SLAMF3high CD4+ T cells and SLAMF3low CD4+T cells respectively using flow cytometry sorting,then cells were cultured in culture medium with different concentration of anti-CD3/CD28 monoclonal antibody,then cells proliferation was observed respectively.(4)CD4+T cells in cord blood were sorted by magnetic activated cell sorting,and then cultured in different concentration of palmitic acid in vitro under stimulation of anti-CD3/CD28 monoclonal antibody,we detected SLAMF3 molecular expression on CD4+T cells after cultured for 72h;mRNA sequencing of the CD4+T cells mentioned above was in progress and we analyzed the distribution and clinical significance of differential expressed genes;(5)In vitro,Jurkat cells were given different concentrations of palmitic acid and were cultured for 72h,then we detected the expression of SLAMF3 molecular on Jurkat cells.We gave cells with different concentration of PI3K inhibitors(LY294002)or different concentrations of STAT5 inhibitor(sc-355979)when Jurkat cells were induced by palmitic acid in vitro,then the expression of SLAMF3 molecular on Jurkat cells was detected after cultured for 72h.(6)Humanized mice were established by intravenous injection of CD34+hematopoietic stem cells sorted from human embryonic liver and transplanted thymus tissue under renal capsule after 1.75Gy irradiation.After 5 weeks,successful humanized mice were then fed with high fat diet to establish the humanized high-fat mice model.Humanized mice were divided into two groups which were fed with high-fat diet(HFD)or normal diet respectively.Body weight and random blood glucose were measured once a week,humanized immune construction were detected every three weeks,mice were sacrificed after fed with HFD for 12 weeks.We detected serum lipid level with lipid test kit,reconstruction of immunocytes in peripheral blood,liver,spleen bone marrow,thymus,and visceral adipose tissue and the expression of SLAMF3 on T cells were detected by flow cytometry.Liver,spleen,pancreas and visceral adipose tissue of mice were collected to process the hematoxylin-eosi(HE)staining,it is aim to observe the cell morphology,adipocyte infiltration and inflammatory cells infiltration.Results:(1)The frequency of PBMC immunocytes differentiation of T2DM patients was abnormal compared with HCs,it shows that in patients with T2DM the frequency of CD4 T cell was increased,the frequency of CD8 T cells,NKT cells was reduced,the frequency of total NK cells,especially CD56dimNK cells,was decreased obviously.(2)Compared with HCs,patients with T2DM have significant higher MFI level of SLAMF3 expression on total T cells,CD4+T cells,CD4-T cells and NKT cells in PBMC;SLAMF5+CD4 T cells and SLAMF5+NKT cells ratio were increased;Frequency of SLAMF7+cells in NKT cells was significantly reduced.SLAMF3 expression was elevated on NK cells in patients with T2DM compared to HCs;there is significant increased expression of SLAMF5 and SLAMF7 molecular on total NK cells or CD56dimNK cells surface;SLAMF6 expression was decreased on total NK cells or CD56dimNK cells surface.(3)There is a relationship between elevated expression of SLAMF3 on T cells and increased secretion of proinflammatory factor IFN-y and IL-17 in T2DM patients,moreover,SLAMF3high CD4 T cells were more sensitive and had more ability to proliferation against anti-CD3/CD28 monoclonal antibody stimulation.(4)Palmitic acid could induce an agonist in expression of SLAMF3 molecular on T cells through STAT5-PI3K/Akt pathway,enrichment of differentially expressed genes between CD4 T cells that were stimulated in the absence or presence of PA was tend to associated with T2DM and its complications.(5)We established humanized high-fat mice by transferring human embryonic liver CD34+hematopoietic stem cells and thymus tissue transplantation under renal capsule on NCG mice combine with a HFD feeding.We can detect a marked increase in serum total cholesterol and triglyceride,and the weight of visceral adipose tissue was increased significantly after fed with HFD for 12 weeks,but there is no obvious adipose degeneration or inflammatory cells infiltration in vital organs such as liver and spleen.It is shown that there is elevated expression of SLAMF3 on T cells in PBMC and VAT in HFD group.Conclusions:(1)The differentiation of immunocytes was abnormal in PBMC of T2DM patients compared to HCs,expression of variety of SLAMF molecules was abnormal on surface of T cells and NK cells.(2)There is a relationship between elevated expression of SLAMF3 on T cells and the increased secretion of proinflammatory factor IFN-y and IL-17 and then abnormal activation in T cells in T2DM patients.(3)Palmitic acid could slow down the proliferation of human T cells,and induce an agonist in expression of SLAMF3 molecular on T cells through STAT5-PI3K/Akt pathway,enrichment of differentially expressed genes between CD4 T cells that were stimulated in the absence or presence of PA was tend to associated with T2DM and its complications.(4)humanized high-fat mice model was established by transferring human embryonic liver CD34+hematopoietic stem cells and thymus tissue transplantation under renal capsule on NCG mice combine with a HFD feeding.HFD may be one of the causes of elevated expression of SLAMF3 molecular on T cells in PBMC and VAT in humanized mice.This investigation provides strong evidence for mechanism of chronic inflammation in T2DM,and it proves that the saturated fatty acid such as palmitic acid may promote on expression of SLAMF3 molecular on T cells which leads to the appearance of inflammation it may be a new target for T2DM immunotherapy by changing expression of SLAMF3 molecular on T cells in T2DM patients. |
PDF Full Text Request