Association Of Vitamin D-mediated Immunoregulation And VDR Gene Polymorphism With Autoimmune Diabetes

Part one Association between the VDR gene Fok I polymorphism and autoimmune diabetesObject:To study the association between the VDR gene Fok I polymorphism and autoimmune diabetes.Subject and Methods:595 autoimmune diabetic patients including 241 typical type 1 diabetic patients and 354 latent autoimmune diabetes in adults(LADA),473 type 2 diabetic patients and 380 unrelated healthy subjects were recruited for this study from China.Genotyping for VDR gene Fok I polymorphism were performed by RFLP-PCR technique. Multiple linear regression was used to study for associations between autoimmune antibodies levels of GADA and IA-2A and VDR gene Fok I genotype in T1DM and LADA.Results:1) The distribution of VDR gene Fok I genotypes of all subjects was in compliance with the Hardy-Weinberg equilibrium (P=0.776).2) The distribution of VDR gene Fok I genotype and allele frequencies between diabetic patients and controls differed significantly (P＜0.05) with the ff genotype occurring more frequently in diabetic patients.3) Moreover,there was a significant difference in frequencies of VDR gene Fok I genotype and allele between T1DM and LADA (P=0.041) whereas this difference was not observed between T1DM and controls.4) VDR gene Fok I ff genotype(odds ratio[OR]=1.707,95% CI 1.201～2.427) was the main susceptibility genotype in LADA.5) Multiple linear regression showed no association of the autoimmune antibodies,IA-2A and GADA with VDR Fok I genotype in T1DM. However,in LADA ff genotype was associated with higher GADA titer compared with FF genotype(P＜0.05).6) LADA patients were divided into LADA-1 and LADA-2 according to the titers higher than 0.8 or not. The distribution of VDR gene Fok I genotype between LADA-1 and LADA-2 differed significantly(P=0.009).The distribution of VDR gene Fok I genotype in LADA-2 was no difference compared with that in controls.7) In T1DM,Ff and ff genotypes showed lower levels of FCP and PCP than FF genotype.Moreover,in LADA,Ff and ff genotypes showed lower level of FCP than FF genotype.Although low HOMA-IS and high HOMA-IR in LADA ff genotype,the difference was not significant compared with that in FF genotype(P=0.242,P=0.790; respectively).Conclusions:1) VDR gene Fok I genotype showed no association with T1DM.2) VDR gene Fok I ff genotype may be a genetic mark for predicting risk of LADA and was associated with higher GADA titer. Part two Monocyte surface CD14,TLR2 and TLR4 expression in autoimmune diabetesObject:To investigate the CD14,TLR2 and TLR4 expression of monocyte from T1DM,LADA and T2DM.Methods:Peripheral blood monocytes were collected from 23 healthy controls,16 type 1 diabetes mellitus(T1DM),18 latent autoimmune diabetes in adults(LADA),and 22 type 2 diabetes mellitus (T2DM),respectively.CD14,TLR2 and TLR4 expression were analyzed by flow cytometer.Serum IL-1βand TNF-αlevel were measured by enzyme linked immunosorbent assay(ELISA).Results:Monocytes showed significantly higher surface CD14 expression from LADA compared with that from T1DM,T2DM and controls(P＜0.001).However,surface CD14 expression on monocytes from T1DM was lower than controls(P=0.002).Monocyte surface CD14 expression from T2DM showed no difference compared with controls (P＞0.05).Higher TLR4 expression on freshly isolated monocytes from LADA and T2DM compared that from T1DM and controls(P＜0.05).And no difference of monocyte TLR4 expressin between LADA and T2DM was found(P＞0.05).The levels of TLR2 expression were no significant differences between patients and controls(P＞0.05).Levels of IL-1βand TNF-α,as inflammation biomarkers,was high in patients,concentration of IL-1βin controls was under-detected.Conclusions:Changes of monocyte surface CD14,TLR2 and TLR4 expressions in T1DM and LADA suggests that the change of the innate immune may be involved in autoimmune diabetes. Part three Modulation of 1,25-dihydroxy-vitamin D3 on monocyte hyperresponsiveness from autoimmune diabetesObject:To investigate modulation of 1,25-dihydroxy-vitamin D3 on monocyte activity from autoimmune diabetes.Methods:Peripheral blood monocytes were collected from 23 healthy controls,16 type 1 diabetes mellitus(T1DM),18 latent autoimmune diabetes in adults(LADA),and 22 type 2 diabetes mellitus (T2DM),respectively.The effect of 1,25-dihydroxy-vitamin D3 (1,25(OH)₂D3) on monocyte response to TLR2 ligand(lipoteichoic acid, LTA) and TLR4 ligand(lipopolysaccharide,LPS) was evaluated in vitro by measuring phosphorylation level of NF-κB-p65 and associated cytokine production(IL-1βand TNF-α).In additional,effects of preincubation with 1,25(OH)₂D3 on moncyte CD14,TLR2 and TLR4 expression stimulated by LTA and LPS were observed.CD14,TLR2 and TLR4 expression and phosphorylation level of NF-κB-p65 were determined by flow cytometer(FCM).IL-1βand TNF-αconcentrations were measured by enzyme-linked immunosorbent assay(ELISA).Results:LPS and LTA decreased surface expressions of CD14 were observed on monocytes from T1DM,T2DM and controls,in contrast to increased on monocytes from LADA(P＜0.05).After incubation with LPS,the levels of monocyte TLR4 expression remarkably decreased in controls,as compared with T1DM and LADA(P＜0.05).There was no significant difference in the expression of TLR2 on monocyte between patients and controls after stimulation with LTA(P＞0.05).Additionally, expressions of CD14,TLR2 and TLR4 on monocytes surface did not show significant changes between patients and controls when monocytes were pretreated with 1,25(OH)₂D3,and afterwards incubated with LPS or LTA(P＞0.05).Activation of NF-κB and amounts of IL-1βand TNF-αproduction by stimulation with LTA and LPS significantly increased in T1DM and LADA,which was modulated by 1,25(OH)₂D3 to similar level,as compared to controls.Conclusions:Monocytes from T1DM and LADA showed similar high cellular reactivity towards ligands and 1,25(OH)₂D3 was observed to restore this defect to a certain extent in vitro.The modulation of 1,25(OH)₂D3 on monocytes makes us to consider more potency of vitamin D3 as therapy in autoimmune diabetes.