Page 148 - Haematologica March 2020

T. Sun et al.
cally transplanted, they help to reconstitute hematopoietic activity in the host region.4 Although malignant hematopoiesis is mainly caused by genetic abnormalities of the mutant stem cells themselves, increasing evidence has shown that abnormal regulation of the hematopoietic niche in the BM has also significant effects.5,6 Recently, several studies have shown that genetic and physiological changes in MSC may accompany hematopoietic disor- ders, such as myelodysplastic syndrome, and leukemia.7-9 However, little is known about BM-derived MSC (BM- MSC) in patients with ET.
Perivascular NES-positive MSC are extensively inner- vated by sympathetic nerve fibers that play vital roles in hematopoiesis and cancer progression by targeting HSPC, MSC, and osteoblasts via the b3 and b2 adrenergic recep- tor (B3AR and B2AR) signaling pathways.4,10,11 The nervous system also regulates immunity and inflammation in the BM, which have long been known to extensively partici- pate in regulation of hematopoiesis.12 Both T-helper 1 cells (Th1) and Th2 cells produce granulocyte-macrophage colony-stimulating factor (GM-CSF), which promotes the differentiation of macrophages from hematopoietic stem cells.13 Th2 cells also produce lineage-specific cytokines, such as IL-4, which can increase production of granulo- cytes and monocytes from mature unipotential hematopoietic progenitor cells, while contributing to thrombocytopenia via inhibitory effects throughout the process of megakaryopoiesis.14,15 Emerging reports have shown that T-cell imbalance in the BM and abnormal secretion of inflammatory factors can impair normal hematopoiesis while accelerating the proliferation of malignant clones that carry mutations.16,17 Moreover, the inflammatory microenvironment can impair sympathetic nerve maintenance and regeneration.18 Recently, neuropa- thy and inflammation have been reported in a mouse model of JAK2V617F-positive myeloproliferative neo- plasms (MPN), which tends to exhibit abnormal hematopoiesis.19 Nonetheless, the sympathetic and inflammatory environment in the BM of patients with ET has not been widely examined.
IL-6 is a multifunctional and pleiotropic cytokine that plays critical roles in the immune system and in a variety of biological processes including hematopoiesis. It is secreted by numerous cell types, including monocytes, dendritic cells, macrophages, T cells, B cells, fibroblasts, osteoblasts, endothelial cells, and particularly MSC.20-23 IL-6 has both pro- and anti-inflammatory properties and is involved in the pathogenesis of nearly all inflammatory diseases. It has been reported that mice homozygous for a mutation in the IL-6 receptor signaling subunit glycopro- tein 130 (gp130Y757F/Y757F) develop a wide range of hematopoietic abnormalities, including splenomegaly, lymphadenopathy, neutrophilia, and thrombocytopenia in addition to elevated myelopoiesis and megakary- opoiesis in the BM.24,25 These mice show glycoprotein 130- dependent signal transduction and hyperactivation of the transcriptional activator STAT3. IL-6 also participates in the pathogenesis of various blood disorders by increasing the number of early pluripotent precursor cells and com- mitted myeloid precursors in the BM.22,26,27 The ERK– GSK3β–CREB signaling pathway has been demonstrated to be involved in regulating IL-6; however, to date, its role in BM-MSC remains unclear.28-30
WDR4, located in human chromosomal region 21q22.3, codes for a member of the WD repeat protein family,
which has been shown to participate in various cellular processes, such as differentiation, apoptosis, cell cycle pro- gression, and stem cell self-renewal by regulating a wide range of signaling pathways via epigenetic regulation of gene expression, or ubiquitin-mediated degradation of proteins.31,32 WDR4 is involved in the regulation of an prometastatic and immunosuppressive microenvironment in lung cancer.33 Downregulation of WDR4 has been detected in the megakaryocytes and platelets of patients with ET, as shown in the GEO Profiles (GEO numbers: GSE2006 and GSE567). However, the role of WDR4 in ET has not been evaluated.
In the present study, we compared the normal BM niche with that of JAK2V617F-positive ET patients to have a comprehensive insight into the changes that occur in the BM microenvironment, and to identify the factors corre- lated with them.
Methods
Patients and samples
Ninety-one untreated ET patients and fifty healthy donors (HD) were included in this study. The characteristics of the subjects are detailed in the Online Supplementary Table S1. This study was approved by the hospital-based ethics committee.
Isolation, expansion, and characterization of MSC
BM-MSC were isolated and expanded in vitro, and their cell morphology, immunophenotype, proliferation, cell cycle, apopto- sis, differentiation, and senescence were evaluated. Additional information on the experimental design is provided in the Online Supplementary Material and Methods.
Transcriptomics and quantitative real-time PCR (qPCR) analyses
Total RNA was isolated from MSC at passage four and used for transcriptomic and qPCR analyses. Detailed information is provid- ed in the Online Supplementary Material and Methods.
Measurement of cytokine levels
Luminex assay (R&D Systems, Minneapolis, MN, USA) was performed on the supernatants obtained from the BM extracts to assess for inflammatory factors according to the manufacturer’s instructions. Enzyme-linked immunosorbent assay (ELISA) was performed on the supernatants obtained from the BM extracts or from the MSC cultures. A list of the ELISA kits is provided in the Online Supplementary Table S3.
Colony-forming unit (CFU) assay
To evaluate the capacity of MSC to sustain normal hematopoiesis, a CFU assay was performed according to the man- ufacturer’s instructions. Detailed information is provided in the Online Supplementary Material and Methods.
Results
Gene expression profiles of BM-MSC derived from HD and patients with JAK2V617F-positive ET
First, we performed RNA sequencing on MSC of HD or patients with JAK2V617F-positive ET. A total of 766 upregulated and 429 downregulated genes were detected in the patient samples (Figure 1A). Gene Ontology analy- sis revealed changes in the gene sets related to cell cycle, differentiation, proliferation, cell death, and aging (Figure
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