2、Anti-inflammatory Effects Of Interleukin-35 In Acquired Aplastic Anemia

Background:Acquired aplastic anemia (AA), characterized by persistent pancytopenia and bone marrow hypoplasia, is an autoimmune disease. The main pathogenesis of AA is the immune-mediated suppression of hematopoiesis. 1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D, is a critical modulator of immune response via binding with vitamin D receptor (VDR). It has been indicated that 1,25(OH)2D3 could inhibit the differentiation and maturation of dendritic cells (DCs). Additionally,1,25(OH)2D3 was a potent inhibitor of T cell proliferation. Moreover, 1,25(OH)2D3 suppressed the production of Thl- and Th17-type cytokines, while promoting the secretion of Th2-related cytokines. More importantly,1,25(OH)2D3 induced the expansion of regulatory T cells (Tregs), further contributing to the maintenance of immune-tolerance. Previous studies have established that 1,25(OH)2D3 and VDR were critically involved in the pathogenesis of some autoimmune diseases, but little was known regarding the function of 1,25(OH)2D3 and VDR in AA.Objective:The present study was aimed to determine the expression of 1,25(OH)2D3 and VDR in patients with AA and evaluate the effects of 1,25(OH)2D3 on peripheral blood mononuclear cells (PBMCs) from AA patients in vitro, thus investigating the role of 1,25(OH)2D3 and VDR in the pathogenesis of AA.Methods:(1) Peripheral blood samples from patients with AA were centrifuged, and plasma was obtained and stored at-80℃ until it was assayed. PBMCs were separated by Ficoll-Hypaque density-gradient centrifugation. (2) The concentrations of 25(OH)D3 in the peripheral blood plasma or cytokines in the cell culture supernatants of PBMCs were measured by ELISA. (3) The mRNA expression of VDR before and after 1,25(OH)2D3 treatment, and transcription factors of T cell differentiation were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). (4) The effect of 1,25(OH)2D3on the proliferation of PBMCs was detected by CCK-8 method. (5) The percentages of Th1, Th2, Tc1, Tc2 and Th17 cells in PBMCs after 1,25(OH)2D3 treatment were measured by flow cytometry.Results:(1) Plasma 25(OH)D3 levels were comparable among untreated AA patients, patients in complete remission and healthy controls. (2) Plasma 25(OH)D3 levels showed significantly positive correlations with platelet counts and the proportion of natural killer T (NKT) cells, while plasma 25(OH)D3 levels were inversely correlated with the percentage of CD19+ B cells in AA patients. (3) The mRNA expression of VDR was significantly lower in untreated AA patients than in healthy controls. (4) Subsequent in vitro stimulation experiments revealed that 1,25(OH)2D3 treatment suppressed the proliferation of PBMCs from AA patients. (5) 1,25(OH)2D3 treatment inhibited the secretion of IFN-y, TNF-a and IL-17A but promoted the production of TGF-β1 in AA patients. (6) 1,25(OH)2D3 inhibited the differentiation of type 1 T and Th17 cells but promoted the differentiation of type 2 T cells, further RT-PCR indicated that 1,25(OH)2D3 decreased the expression of T-bet and RORyt mRNA but promoted the expression of GATA3 and Foxp3 mRNA. (7) VDR mRNA was elevated in healthy controls after 1,25(OH)2D3 treatment, but not in AA patients.Conclusion:Decreased expression of VDR might contribute to the hyperimmune status of AA and appropriate vitamin D supplementation could partly correct the immune dysfunction by strengthening signal transduction through VDR in AA patients.Background:Acquired aplastic anemia (AA), characterized by peripheral blood (PB) pancytopenia and bone marrow (BM) hypoplasia, is a rare and life-threatening bone marrow failure syndrome. Until now, much pathological mechanism has been postulated to account for this disorder, including abnormal immunity, aberrant hematopoietic microenvironment, exhaustion of hematopoietic stem cells and genetic susceptibility. Vitamin D is an important immune regulator, and the effect of vitamin D is mediated by vitamin D receptor (VDR). VDR is coded by the VDR gene, which is located on chromosome 12 and contains more than 60 single-nucleotide polymorphisms (SNPs). VDR gene and its polymorphisms are highlighted as candidate components for susceptibility to various autoimmune diseases. However, to the best of our knowledge, no previous studies have performed to determine the potential relationship between VDR polymorphisms and AA.Objective:The aim of the present study was to investigate the correlation of VDR polymorphisms (rs2228570, rs1544410, rs7975232 and rs731236) with the susceptibility of AA, and evaluate the impact of VDR polymorphisms on the response to treatment, clonal evolution and other clinical and laboratory characteristics of AA patients in a Chinese population.Methods:The DNA of 197 AA patients and 135 healthy controls was extracted. The genotyping of VDR rs1544410 (c.1024+283G>A), rs7975232 (c.1025-49G>T) and rs731236 (c.1056T>C) polymorphisms was conducted using polymerase chain reaction (PCR)-ligase detection reaction, while the genotyping of rs2228570 (c.2T>C) was detected by PCR-restriction fragment length polymorphism. The impact of these four polymorphisms on the susceptibility, response to treatment, clonal evolution and other clinical and laboratory characteristics of AA patients was determined using SPSS 17.0 statistical software.Results:(1) Distribution frequencies of VDR rsl544410, rs7975232, rs731236 and rs2228570 genotypes were in agreement with Hardy-Weinberg equilibrium for both AA patients and healthy controls. (2) rs1544410, rs7975232 and rs731236 polymorphic loci were in a strong linkage disequilibrium in both AA patients and healthy controls. (3) The frequencies of GG genotype and G allele of rs1544410 were significantly higher in AA patients than in controls, while no significant differences were revealed regarding the genotype and allele distributions of rs7975232, rs731236 and rs2228570. (4) The GG genotype and G allele of rs 1544410 was significantly increased in NSAA patients than in controls. For rs7975232, the genotype distribution was significantly different between NSAA and controls:CC genotype of rs7975232 showed a positive while AC genotype a negative association with NSAA. For rs2228570, the TT genotype and T allele were over-represented in SAA patients when compared with controls. Altogether, rs 1544410 and rs7975232 polymorphisms were correlated with the risk to non-severe AA while rs2228570 was relevant to severe AA. (5) The response to treatment of AA patients was related to rs2228570 polymorphism:the presence of C allele (CC and CT genotypes) tended to be associated with a favorable response. (6) The clonal evolution of AA patients was also correlated with rs2228570 polymorphism:TT carriers were associated with a higher rate of transformation to myelodysplastic syndrome or acute myeloid leukemia, while CT carriers had a greater risk to evolve to overt paroxysmal nocturnal hemoglobinuria.Conclusion:VDR polymorphisms may contribute to susceptibility to AA and influence the severity and prognosis of AA in a Chinese population.Background:Acuqired aplastic anemia (AA) is an immune-mediated bone marrow (BM) failure syndrome attacked by auto-reactive effector T cells and BM is the main target organ. Interleukin (IL)-35, the newest member of the IL-12 family, is a novel regulatory cytokine primarily produced by regulatory T cells (Tregs). IL-35 is a heterodimeric cytokine composed of the IL-12 p35 subunit and the IL-27 Epstein-Barr virus-induced protein 3 (EBI3) subunit. Recently, IL-35 is considered to be the essential effector molecule for Treg-mediated suppression. Additionally, IL-35 has been shown to induce a potent regulatory population, named inducible IL-35-producing Tregs (iTR35 cells). Accumulating evidence has established that IL-35 plays an important role in the regulation of immune homeostasis, but little is known regarding the function of IL-35 in acquired aplastic anemia (AA).Objective:The aim of the present study was to evaluate the expression of IL-35 and its effects on T cell response in patients with AA, thus investigating the potential roles of IL-35 in the pathogenesis of AA.Methods:(1) The concentrations of cytokines in the peripheral blood plasma or in the cell culture supernatants of peripheral blood mononuclear cells (PBMCs) were measured by ELISA. (2) The mRNA expression of the IL-35 subunits (p35 and EBI3) in PBMCs, and the transcription factors of T cell differentiation were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). (3) The effect of IL-35 on the proliferation of PBMCs was detected by the incorporation of BrdU. (4) The absolute number and percentages of Thl (CD3+CD8-IFNγ+), Th2 (CD3+CD8-IL4+), Tel (CD3+CD8+IFNγ+), Tc2 (CD3+CD8+IL4+) and Th17 (CD3+CD8-IL17+) cells in PBMCs after IL-35 treatment were measured by cell counting combined with flow cytometry.Results:(1) Plasma IL-35 levels were significantly lower in untreated AA patients than in normal controls and patients in CR, while patients in CR had IL-35 levels comparable with normal controls. Further analysis indicated that IL-35 levels correlated inversely with disease severity:patients with VSAA and SAA expressed considerably lower levels of IL-35 compared with Non-SAA patients. (2) Plasma IL-35 levels showed a significant positive correlation with the platelet counts, absolute neutrophil counts and absolute reticulocyte counts, while plasma IL-35 levels were negatively correlated with the percentage of lymphocytes in untreated AA patients. (3) The mRNA expression of p35 and EBI3 in untreated S AA patients was significantly lower than that of normal controls, while their expression in Non-SAA patients was not significantly different from normal controls. (4) Subsequent in vitro stimulation experiments revealed that IL-35 treatment suppressed the proliferation of PBMCs from AA patients. IL-35 also significantly decreased both the absolute number and percentage of CD4+ and CD8+T cells. (5) IL-35 treatment inhibited the secretion of IFN-y, TNF-α and IL-17A but promoted the production of TGF-β1 in AA patients. (6) IL-35 inhibited the differentiation of type 1 T and Th17 cells but promoted the differentiation of type 2 T cells, further RT-PCR indicated that the expression of T-bet and RORyt mRNA was inhibited while the expression of GATA3 mRNA was induced after IL-35 treatment in AA patients.Conclusion:Our findings suggested that decreased expression of IL-35 might contribute to the loss of immune-tolerance and be critically involved in the pathogenesis of AA.