Differentiation Of Regulatory T Cels Induced By Vitamin D And Its Role In The Pathogenesis Of Autoimmune Hepatitis

BackgroundThe auto-immune hepatitis(AIH)is an inflammation of the liver characterized by the presence of peri-portal hepatitis,hypergammaglobulinemia,and serum autoantibodies.The disease is classified into 2 distinct types according to the nature of autoantibodies.Type 1 AIH is characterized by anti-nuclear antibodies and/or smooth muscle antibodies in serum.Type 2 AIH is characterized by antibodies to the liver-kidney microsome type 1(anti-LKM1)and primarily affects children between the ages of 2 and 14 years.The mechanism of AIH is not clear at present,scholars have been considered that it islinked with genetic factors,environmental factors,viral infection and autoimmune disorders.A variety of factors cause innate immune response and adaptive immune response to damage liver tissue.The disordered balance of regulatory T cells/helper T 17 cells has an important role in the development of AIH disease.The proportion of Treg cells and the secretion of IL-10 cytokines in the peripheral blood of AIH patients was decreased.Disturbances of the calcium-parathyroid hormone-vitamin D axis are frequently associated with chronic liver disease.Patients with AIH have a high prevalence of vitamin D deficiency.Genetic studies have provided the opportunity to determine which proteins link vitamin D to AIH pathology,namely,the major histocompatibility complex class ? molecules,vitamin D receptors,toll-like receptors,cytotoxic T lymphocyte antigen-4,cytochrome P450 CYP2D6,regulatory T cells(Tregs)and the forkhead/winged helix transcription factor 3.Vitamin D can affect a variety of immune cell functions,such as B cells,T cells,etc..Whether the Treg cells in peripheral blood of AIH patients is related to the deficiency of vitamin D,and how the mechanism of interaction has not been reported in recent years.This paper intends to use flow cytometry and ELISA technique to analysis of the relationship between the percentage of Treg cells,IL-10 and the content of vitamin D in peripheral blood of AIH patients,and then investigate the effects of vitamin D on the differentiation of Treg cells in vitro.ObjectiveThis paper intends to uncover the proportion of Treg cells and its relation with vitamin D in AIH patients through experiment;To prove vitamin D can promote the differentiation of Treg cells in AIH patients,and then reveal the lack of vitamin D participate in the occurrence and development of AIH disease by inhibiting the differentiation of Treg cells.The project also provides a theoretical basis for the mechanism of vitamin D supplementation in the treatment of AIH.At the same time,AIH is expected to provide new targets for immune therapy.Methods1.Subjects investigated:Referring to the diagnosis criteria of autoimmune hepatitis in the 2010 American Society of liver disease,30 patients with AIH were enrolled in our hospital.The control group was 18 cases of healthy people in the hospital during the same period with the age and gender matched.2.The collection and processing of samples:Peripheral blood mononuclear cells were collected from HC and AIH.The separated plasma were stored in-80?refrigerator for ELISA.3.Detection the level of plasma vitamin D:The content of vitamin D in plasma of AIH and HC was detected by automatic biochemical analyzer.4.Detection the level of plasma IL-10:using human IL-10 ELISA kit to detect the level of plasma IFN-?.5.Detection of CD4+CD25+Foxp3+IL-10+Treg cell proportion:Detection and analysis of CD4+CD25+Foxp3+IL-10+Treg cell proportion in samples by flow cytometry.6.Measurement data with standard deviation of mean and subtract(X�s),compare the difference between the two groups using t test,the data were compared by chi-square test,correlation using Pearson correlation analysis.Inspection level of alpha = 0.05,P<0.05 for the difference was statistically.Results1.The plasma 25-(OIH)-D3 content in patients with AIH is(38.6515.38)nmol/L,that in healthy controls is(61.43�14.90)nmol/L,the former was higher than the latter,and there was a statistical difference(P=0.014).2.The content of IL-10 in peripheral blood of patients with AIH was(33.43�6.50)pg/ml,lower than that of healthy controls(57.51�12.68)pg/ml,and there was a statistical difference(P<0.001 AIH);PBMC from AIH were stimulated with PMA,IL-10 content of supernatant was(57.26�20.20)pg/ml in the present of 1,25-(OIH)2-D3,without 1,25-(OH)2-D3,that was(19.99�6.72)pg/ml,and these two groups were statistically significant.3.25-(OH)-D3 in peripheral blood were correlated both with IL-10 and CD4+CD25+Foxp3+IL-101+T cell proportion(R2=0.379,P=0.0003;R2=0.151,P=0.0341)in AIH.4.CD4+CD25+Foxp3+1L-10+T of AIH patients was(5.09�1.50)%,the proportion decreased significantly compared with that of HC group(10.0814.03)%.PBMC from AIH were stimulated with PMA,the proportion of CD4+CD25+Foxp3+IL-10+T cells was(12.05�4.59)%in the present of 1,25-(OH)2-D3,while that was(5.54�2.17)%in the absence of 1,25-(OH)2-D3,and these two groups were statistically significant.Conclusions1.The decreased proportion of Treg cells and IL-10 content in AIH patients may be caused by the deficiency of vitamin D.2.Vitamin D can promote the differentiation of Treg cells in patients with AIH,and and stimulate Treg cells to secret IL-10,to a certain extent,it is revealed that vitamin D deficiency is involved in the development of AIH disease by inhibiting the differentiation of Treg cells.