Part One Interleukin-35 May Contribute To The Loss Of Immunologic Self-tolerance In Patients With Primary Immune Thrombocytopenia Part Two Systmetic Study Of Biological Properties And Functions Of Bon

Background:Essential thrombocythemia (ET) is one of the classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) characterized by thrombotic and hemorrhagic complications and a long-term risk of evolution to myelofibrosis and acute leukemia. JAK2V617F mutation originated at the stem/progenitor cell level is present in about 55% patients with ET. However, the pathogenetic mechanisms of ET are still unknown. Recent studies suggested that bone marrow microenvironment may involve in the pathogenesis of MPN. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are key components of the hematopoietic microenvironment. Due to its anti-tumor effects, there has been a longstanding interest in using interferon (IFN) to control myeloproliferation in patients with ET. Until now, little is known about whether IFN had effects on the BM-MSCs from patients with ET.Objective:This study aims to systematically and thoroughly assess the biological and functional characteristics of BM-MSCs in patients with ET and explored the effects of IFNa-2b exerted on BM-MSCs.Methods:(1) Bone marrow mononuclear cells were isolated from bone marrow and BM-MSCs were purified by adherent culture and passaging in DMEM/DF12 supplemented with 10% fetal bovine serum. (2) The morphology of BM-MSCs was observed under an inverted microscope. (3) The immunophenotype markers of BM-MSCs were examined by flow cytometry. (4) In the specific-induction mediums, BM-MSCs were induced to differentiate into adipocytes and osteoblasts, which were identified with corresponding staining. (5) The proliferation rate of BM-MSCs was detected. (6) The apoptosis of BM-MSCs were determined by using flow cytometry. (7) Umbilical cord derived CD34+ cells were purified by miniMACS immunomagenetic method and co-cultured with BM-MSCs to assess the function of BM-MSCs to support hematopoiesis including the amplification of hematopoietic cells and clonogenic potential of CFUs (CFU-G, CFU-M, CFU-GM, CFU-Mix, BFU-E and CFU-MK). The expression of hematopoietic growth factors such as SCF, IL-6 and VEGF were detected by ELISA method. (8) Peripheral blood mononuclear cells (PBMCs) were isolated from normal controls and co-cultured with BM-MSCs to assess the suppression capacity of BM-MSCs in the proliferation of PBMCs. PBMCs were also co-cultured with BM-MSCs to assess the contribution of BM-MSCs in promoting the proliferation of Tregs. (9) Compared with ET derived BM-MSCs in the absence of IFNa-2b, the ET derived BM-MSCs were examined the changes in above biological and functional characteristics with the presence of IFNa-2b.Results:(1) BM-MSCs were successfuly isolated and purified from patients with ET and normal controls. (2) No significant differences were found in morphology between BM-MSCs from patients with ET and normal controls. Both BM-MSCs exhibited bipolar spindle-like cells with a whirlpool-like array. (3) BM-MSCs from patients with ET and normal controls also had no discrepancies in immunophenotype characteristics. (4) BM-MSCs from ET showed increased proliferation and lower apoptosis capacity. (5) BM-MSCs from ET patients were more difficult to induce to osteoblasts than healthy controls. (6) Both BM-MSCs from ET patients and healthy controls had the potential to support hematopoiesis. However, BM-MSCs from ET patients had reduced capacity to amplify CD34+ cells and maintain long-term hematopoiesis. BM-MSCs from ET patients expressed lower levels SCF, IL-6 and VEGF than healthy controls. (7) BM-MSCs from ET were defective to suppress the proliferation of PBMCs and could promote PBMCs to differentiate into regulatory T cells. (8) IFNa-2b had no effect on the morphology, immunophenotype and JAK2 mutant allele of BM-MSCs derived from ET patients. IFNa-2b was capable of suppressing the proliferation of BM-MSCs, but increasing its apoptosis and its function of supporting hematopoiesis. IFNa-2b also ameliorated its defective in inhibiting the proliferation of PBMCs and promotes PBMCs to differentiate into regulatory T cells.Conclusion:(1) BM-MSCs from ET patients were multiply defective both in biological properties and functions. (2) BM-MSCs from ET were abnormal in proliferation, apoptosis and differentiation. (3) BM-MSCs from ET had reduced capacity to maintain hematopoiesis. (4) BM-MSCs from ET were defective in maintaining immune homeostasis. (5) IFNα-2b affects BM-MSCs from ET patients in multiple differentiation capacities, proliferation, apoptosis and the capacity to support long-term hematopoiesis and immune regulation.Background:Primary immune thrombocytopenia (ITP) is a relatively common blood disorder characterized by autoimmune-mediated platelet destruction and suboptimal platelet production. The mechanism is involved with impaired platelet production and effects mediated by antigen-presenting cells, T cells, and B cells. IL-35 is a cytokine expressed in stimulated human Tregs during inflammatory responses, which can suppress T cell proliferation and elicit the development of inducible regulatory T cells (Tregs). Previous studies have shown decreased plasma IL-35 levels and dysfunctional T cells in patients with ITP. These results suggested that a decrease in IL-35 might contribute to the pathogenesis of ITP; however, the mechanisms of this contribution are still unknown.Objective:The present study was aimed to investigate the associations between IL-35 levels and immunologic abnormalities in ITP patients and evaluate the effects of IL-35 on the proliferation of T cells and the production of representative cytokines in vitro.Methods:(1) Peripheral blood samples from patients with ITP were centrifuged, and plasma was obtained and stored at -80℃ until it was assayed. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation using Ficoll-Hypaque gradients centrifugation. (2) Isolated PBMCs were suspended in complete culture medium (RPMI-1640 with 10% fetal bovine serum,2 mmol/L glutamine, and 100 U/ml penicillin/streptomycin) with or without stimulation with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), 1μg/mL ionomycin, and 10μg/mL brefeldin A (BFA) for 4 hours at 37℃ under 5% CO2. Then, the cells were harvested and stained for flow cytometry to detect the proportion of nTreg, Th1 and Th17 respectively. (3) Isolated PBMCs were cultured in the above complete culture medium containing 5 mg/ml phytohemagglutinin (PHA) and 5 ng/ml rhIL-2 with or without stimulation with 100 ng/mL recombinant human IL-35 (rhIL-35) for 72 hours at 37℃ under 5% CO2. In the last 6 hours of the 72-hour incubation, BFA was added at a final concentration of 10μg/mL into the cultures, and after incubation, cells were collected for flow cytometry to detect the proportion of CD4+, CD8+ and Treg respectively. (4) Aliquots of PBMCs were cultured as described in step (3). Cultures were pulsed with BrdU at a final concentration of 10μM for the last 18 hours of the 72-hour assay, and the cell culture supernatants were stored for detection of IFN-y, IL-10, IL-17 and TGF-β1 by ELISA. The concentrations of IL-35 in the plasma from patients with ITP were also measured by ELISA.Results:(1) The mean percentage of Tregs was significantly lower in active ITP patients than in patients in remission and in healthy controls. The proportion of Thl cells in ITP patients was higher than that in healthy controls. ITP patients and healthy controls did not significantly differ in proportions of Th17 cells. (2) Patients with active ITP had significantly lower plasma IL-35 levels than did patients in remission and healthy control. Patients in remission also had lower plasma IL-35 levels than healthy controls did. Plasma IL-35 levels did not significantly differ between untreated and treated patients with active ITP. (3) The Spearman rank correlation coefficient assay showed a statistically significant positive correlation between plasma IL-35 levels and platelet counts and the mean percentage of nTregs in ITP patients. However, plasma IL-35 levels were inversely related to the proportion of Thl cells and did not correlate with the proportion of Th17 cells, age, sex, or disease duration. (4) IL-35 stimulation decreased the proliferative capacity of PBMCs from ITP patients and from normal controls. IL-35 also significantly decreased the level of CD4+ T cells and CD8+ T cells but increased the proportion of nTregs. (5) IL-35 promoted the production of TGF-β1 and IL-10 but suppressed the expression of IFN-γ and IL-17 in culture supernatants of PBMCs from patients with ITP and from healthy controls.Conclusion:In summary, our data suggest that IL-35 appears to contribute to the loss of immunologic self-tolerance in ITP patients by modulating T cells and immunoregulatory cytokines.