Neuropathy in the Altered Bone Marrow Microenvironment of Patients with Essential Thrombocythemia and the Effects of Interferonα-2b on Them

Background: ET is one of the classical Philadelphia chromosome-negative MPNs . BM-MSCs may involve in the pathogenesis of MPN. Bone marrow neural tissue, closely related to hematological diseases , is composed of sympathetic nervous system (SNS) fibres , ensheathing Schwann cells, supporting nestin+ MSCs . 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 and SNS in patients with ET and explored the effects of IFNα-2b exerted on them.

Methods: (1) The morphology of BM-MSCs was observed under an inverted microscope. (2) The immunophenotype markers of BM-MSCs were examined by flow cytometry. (3) BM-MSCs were induced to differentiate into adipocytes and osteoblasts, which were identified with corresponding staining. (4) The proliferation rate, senescence and apoptosis of BM-MSCs was detected.(5)Nestin+cell number ,sympathetic nerve fibres , schwann cells and nestin mRNA expression were detected by IHC and RT-PCR respectively.(6)CXCL12 and norepinephrine was detected by ELISA . (7) Umbilical cord derived CD34+ cells were purified and co-cultured with BM-MSCs to assess the function of BM-MSCs to support hematopoiesis . Hematopoietic growth factors such as SCF, IL-6 and VEGF were detected by ELISA . (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 IFNα-2b, the ET derived BM-MSCs were examined the changes in above biological and functional characteristics with the presence of IFNα-2b.

Results: (1) 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. (2) BM-MSCs from patients with ET and normal controls also had no discrepancies in immunophenotype characteristics. (3) BM-MSCs from ET showed increased proliferation, lower apoptosis capacity and decreased senescence. (4)In MPN patients, nestin+cell number and nestin messenger RNA expression were markedly increased. (5)Nevertheless , BM-MSCs from ET patients expressed lower levels CXCL12 than healthy controls. (6)Sympathetic nerve fibres and ensheathing Schwann cells were markedly reduced in BM of ET patients , with lower levels of norepinephrine secreted. (7) BM-MSCs from ET patients were more difficult to induce to osteoblasts than healthy controls. (8) 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. (9) BM-MSCs from ET were defective to suppress the proliferation of PBMCs and could promote PBMCs to differentiate into regulatory T cells. (10) IFNα-2b had no effect on the morphology, immunophenotype and JAK2 mutant allele of BM-MSCs derived from ET patients. IFNα-2b was capable of suppressing the proliferation of BM-MSCs, but increasing its apoptosis and its function of supporting hematopoiesis. IFNα-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,senescence, 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) Neuropathy is obvious in ET patients. (6) 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.