Multilevel defects in the hematopoietic niche in essential thrombocythemia

Abstract

The role of the bone marrow niche in essential thrombocythemia (ET) remains unclear. Here, we observed multilevel defects in the hematopoietic niche of patients with JAK2V617F-positive ET, including functional deficiency in mesenchymal stromal cells (MSC), immune imbalance, and sympathetic-nerve damage. Mesenchymal stromal cells from patients with JAK2V617F-positive essential thrombocythemia had a transformed transcriptome. In parallel, they showed enhanced proliferation, decreased apoptosis and senescence, attenuated ability to differentiate into adipocytes and osteocytes, and insufficient support for normal hematopoiesis. Additionally, they were inefficient in suppressing immune responses. For instance, they poorly inhibited proliferation and activation of CD4-positive T cells and the secretion of the inflammatory factor soluble CD40-ligand. They also poorly induced formation of mostly immunosuppressive T-helper 2 cells (Th2) and the secretion of the anti-inflammatory factor interleukin-4 (IL-4). Furthermore, we identified WDR4 as a potent protein with low expression and which was correlated with increased proliferation, reduced senescence and differentiation, and insufficient support for normal hematopoiesis in MSC from patients with JAK2V617F-positive ET. We also observed that loss of WDR4 in MSC cells downregulated the interleukin-6 (IL-6) level through the ERK-GSK3β-CREB signaling based on our in vitro studies. Altogether, our results show that multilevel changes occur in the bone marrow niche of patients with JAK2V617F-positive ET, and low expression of WDR4 in MSC may be critical for inducing hematopoietic related changes.

Figure 1

Transcriptomic analysis revealed multiple abnormalities in bone marrow derived mesenchymal stromal cells (BM-MSC) from patients with JAK2V617F-positive essential thrombocythemia (ET). A. Heatmap of transcriptomic analysis from eight MSC samples (control, n=5; untreated patients with JAK2V617F-positive ET, n=3) demonstrated that MSC from patients with JAK2V617F-positive ET differed from those isolated from HD. Briefly, 1,195 genes were identified with a cut-off of greater than 2.0-fold for gene expression change and P<0.05. B. GO analysis of genes enriched in terms of different function showed changes in gene sets related to the cell cycle, cell differentiation, proliferation, death, and aging. C. KEGG analysis was carried out to identify differential pathway enrichment between ET and control. Rich factor refers to the ratio of the number of genes differentially expressed in the pathway entry to the total number of genes in the pathway entry. A larger rich factor indicates a higher degree of enrichment. The q-value is the P-value after multiple-hypothesis test corrections, ranging from 0 to 1 (a value closer to zero indicates a more significant enrichment). The figure is plotted with the top 20 paths sorted according to the q value from small to large and shows enrichment of genes of inflammation pathways. D. GSEA using MSigDB identified differential gene enrichment between ET and the control. NES, Normal p and FDR q-values for each gene set are shown. The results revealed differential expression of genes involved in cell cycle, inflammatory responses, and hematopoietic support. E. qPCR validation of relevant genes (control, n=16; untreated patients with JAK2V617F-positive ET, n=16). MSC used for gene analysis were isolated and expanded in vitro and identified according to the minimal criteria for defining multipotent mesenchymal stromal cells stated by the International Society for Cellular Therapy position at passage four. *P< 0.05; **P<0.01, ***P<0.001; ****P<0.0001. Data are presented as the mean or mean ± SEM. ET: essential thrombocythemia; HD: healthy donors; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA: gene set enrichment analysis; MsigDB: Molecular Signatures Database; NES: normalized enrichment score; qPCR: quantitative real-time PCR; n: number of unique donors in each group; ns: not significant; SEM: standard error of mean.

Figure 2

Bone marrow derived mesenchymal stromal cells (BM-MSC) from patients with JAK2V617F-positive essential thrombocythemia (ET) show enhanced proliferation and attenuated apoptosis, senescence, and differentiation. A. Growth curves of BM-MSC isolated from HD (n=12) and patients with JAK2V617F-positive ET (n=12). The ET MSC grew progressively faster than controls. B. Decreased apoptosis of BM-MSC derived from patients with JAK2V617F-positive ET as determined by flow cytometry (control, n=16; ET, n=16). C. The number of β-galactosidase–positive cells were lower in BM-MSC derived from patients with JAK2V617F-positive ET compared to those from control patients (control, n=16; ET, n=16). D. Differentiation potentials of MSC toward adipocytes, osteocytes, and chondrocytes were assessed by Oil Red O, Alizarin Red, and Alcian Blue staining, respectively, after induction for 14–21 days. Representative micrographs of BM-MSC derived from HD, and patients with JAK2V617F-positive ET are shown. Variations in the differentiation between HD (n=12) and ET samples (n=16) were quantified by the staining index described in the Methods section. E. Cell cycle status was determined by flow cytometry. Patients with JAK2V617F-positive ET had less MSC in the G1 phase and more in the S and G2 phases relative to those in the HD controls (control, n=12; ET, n=12). F. NES mRNA expression in BM cells of the controls (n=17) and patients with JAK2V617F-positive ET (n=16). G. NES-positive cells in the bone marrow of an HD control (n =1) and patient with JAK2V617F-positive ET (n=1) shown by immunofluorescence. H. BM sections of the controls and patients with JAK2V617F-positive ET immunostained with NES (brown). (control, n=8; ET, n=37; upper panels); BM sections of the controls and patients with JAK2V617F-positive ET immunostained with NES (red) and CD34 (brown). (control, n=8; ET, n=37; lower panels). MSC used in each assay were at passage four (except for those immunostained with NES or NES and CD34). *P<0.05; **P<0.01, ***P<0.001; Data are presented as the mean ± SEM. ET: essential thrombocythemia; HD: healthy donors; n: number of unique donors in each group; IF: immunofluorescence; ns: not significant; SEM: standard error of mean.

Figure 3

Bone marrow derived mesenchymal stromal cells (BM-MSC) from patients with JAK2V617F-positive essential thrombocythemia (ET) show insufficient ability to support normal hematopoiesis. A. Representative micrographs of CFU formed by purified normal CD34-positive cells in the presence of BM-MSC from HD (n=12) and patients with JAK2V617F-positive ET (n=16). B. Numbers of BFU-E, CFU-E, CFU-GM, CFU-Mix, CFU-Total, and CFU-MK formed by purified normal CD34-positive cells after coculture with BM-MSC from HD (n=12) or from patients with JAK2V617F-positive ET (n=16) for 7 or 14 days. After 7 or 14 days of coculture of purified normal CD34-positive cells and MSC from patients with JAK2V617F-positive ET, the numbers of CFU-GM and CFU-Total were significantly lower, with no sig-nificant changes in the numbers of BFU-E, CFU-E, CFU-Mix, and CFU-MK. C. Cytokines secreted by BM-MSC into the culture medium analyzed by ELISA (control, n=16; JAK2V617F-positive ET, n=16). MSC used in each assay were at passage four. *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001. Data are presented as the mean ± SEM. ET: essential thrombocythemia; HD: healthy donors; ELISA: enzyme-linked immunosorbent assay; n: number of unique donors in each group; ns: not significant; BFU-E: burst-forming unit-erythroid; CFU-E: colony-forming unit-erythroid; CFU-GM: colony-forming unit-granulocyte and macrophage; CFU-Mix: colony-forming units mixed; CFU-Total: total colony-forming units; CFU-MK: colony-forming unit-megakaryocyte; SEM: standard error of mean.

Figure 4

Bone marrow derived mesenchymal stromal cells (BM-MSC) from patients with JAK2V617F-positive essential thrombocythemia (ET) have an impaired immunomodulatory capacity. A–B. Proliferation (A) and activation (B) of CD4-positive T cells from HD (n=12) and patients with JAK2V617F-positive ET (n=12). C. Expression of T-cell subset transcription factors in bone marrow mononuclear cells (BMMC) derived from HD (n=12) and patients with JAK2V617F-positive ET (n=12). T-cell–expressed transcription factors T-bet, GATA-3, RORγt, and FOXP3, representing Th1, Th2, Th17, and Treg cells, respectively. D. Flow-cytometric analysis of the T-cell subset in the bone marrow of HD (n=16) and patients with JAK2V617F-positive ET (n=16). CD4-positive cells were sorted into Th1, Th2, Th17, and Treg subsets according to the expression of IFN-γ, IL-4, IL-17 and FOXP3 with CD25. The number of the Th2 subset in patients with JAK2V617F-positive ET was lower relative to that in the control group. E. A Luminex assay performed using the supernatant of bone marrow extract from HD (n=20) and from patients with JAK2V617F-positive ET (n=24), revealed a decreased level of IL-4 and elevated IL-1β and sCD40L levels in ET. F–G. Proliferation (F) and activation (G) of normal CD4-positive T cells were higher after coculture with BM-MSC from patients with JAK2V617F-positive ET (n=8) relative to those observed with HD MSC (n=8). H. Flow-cytometric analysis of T-cell subsets showed lower number of Th2 cells formed from normal BMMC after coculture with BM-MSC from patients with JAK2V617F-positive ET (n = 12) relative to those with HD MSC (n=12). I. Decreased level of IL-4 and increased level of sCD40L were found in the supernatant of cell coculture medium of normal CD4-positive T cells and BM-MSC isolated from patients with JAK2V617F-positive ET, as determined by ELISA (control, n=12; ET, n=12). MSC used in each assay were at passage four. *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001. Data are presented as the mean ± SEM. ET: essential thrombocythemia; HD: healthy donors; BMMC: bone marrow mononuclear cells; ELISA: enzyme-linked immunosorbent assay; n: number of unique donors in each group; SEM: standard error of mean.

Figure 5

Low expression of WDR4 is correlated with enhanced proliferation, decreased senescence, and impaired differentiation of bone marrow derived mesenchymal stromal cells (BM-MSC) isolated from patients with JAK2V617F-positive essential thrombocythemia (ET). A. WDR4 mRNA expression in BM-MSC isolated from the controls (n=20) and patients with JAK2V617F-positive ET (n=15).B. WDR4 protein expression in BM-MSC isolated from the controls (n=4) and patients with JAK2V617F-positive ET (n= 4). C–D. WDR4 shRNA and cDNA decreased or increased WDR4 expression in BM-MSC efficiently, as determined by qPCR (C) and Western blotting (D). E. WDR4 cDNA treatment decreased the proliferative capacity of BM-MSC, while WDR4 shRNA treatment increased the proliferation of BM-MSC, as measured by the CCK-8 assay. F. WDR4 cDNA increased senescence of BM-MSC as measured by β-galactosidase staining while WDR4 shRNA had the opposite effect. G. WDR4 increased the differentiation potential of BM-MSC into adipocytes, osteocytes, and chondrocytes as indicated by Oil Red O, Alizarin Red, and Alcian Blue staining, respectively. MSC used in each assay were at passage four. All the experiments (except for the quantitation of WDR4 mRNA and WDR4 protein expression in clinical samples) were repeated at least three times. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Data are presented as the mean ± SD (except for mRNA expression of WDR4 in clinical samples, mean ± SEM). ET: essential thrombocythemia; CCK-8: Cell Counting Kit 8; n: number of unique donors in each group; NC: normal control; SD: standard deviation; SEM: standard error of mean.

Figure 6

Insufficient action of the WDR4–IL-6 axis decreases hematopoiesis-supportive activities of bone marrow derived mesenchymal stromal cells (BM-MSC) from patients with JAK2V617F-positive ET. A–B. Numbers of BFU-E, CFU-E, CFU-GM, CFU-Mix, and CFU-MK formed by purified normal CD34-positive cells after coculture with BM-MSC infected with LV-shWDR4, or LV-WDR4. WDR4 increased the number of BFU-E, CFU-GM, and CFU-Total formed by normal CD34-positive cells (A). No changes were observed in the number of CFU-MK (B). C. WDR4 increased the secretion of IL-6 from BM-MSC as determined by ELISA on the supernatant obtained from the MSC cultures. D–E. WDR4 increased the intracellular expression of IL-6 in BM-MSC as determined by Western blotting (D) and qPCR (E). MSC used in each assay were at passage four. All the experiments were repeated at least three times. *P 0.05; **P<0.01; ***P<0.001; ****P<0.0001. Data are presented as the mean ± SD. qPCR: quantitative real-time polymerase chain reaction; CFU-E: colony-forming unit-erythroid; CFU-GM: colony-forming unit-granulocyte and macrophage; CFU-Total: total colony-forming units; CFU-MK: colony-forming unit-megakaryocyte; n: number of unique donors in each group; ns: not significant; NC: normal control; SD: standard deviation.

Figure 7

WDR4 acts through the ERK–GSK3β–CREB pathway to enhance IL-6 expression and secretion by bone marrow derived mesenchymal stromal cells (BM-MSC). A. Graphic representation of the quantification of 12 proteins with the most significant difference in phosphorylation status between MSC infected with LV-shWDR4 and MSC in the control group, as measured by a phosphokinase array of 43 phosphorylated kinases. B. Western blot analysis of phosphorylation levels of GSK3β (S9), AKT1/2/3 (S472/S473/S474), ERK1/2 (T202/Y204, T185/Y187), and CREB (S133) in MSC infected with LV-shWDR4 or LV-WDR4 and their respective controls. C. CREB-specific siRNA decreased CREB expression in BM-MSC efficiently, as determined by qPCR and Western blotting. D–F. IL-6 induction by WDR4 overexpression was at least partially suppressed by an ERK1/2 inhibitor (SCH772984), GSK3 inhibitor (SB216763), or CREB-specific siRNA as determined by qPCR (D), Western blotting (E), and ELISA (F). MSC used in each assay were at passage four. All the experiments were repeated at least three times. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Data are presented as the mean ± SD. qPCR: quantitative real-time polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; NC: normal control; SD: standard deviation.

Figure 8

Neuropathy and aberrant expression of IL-1β in the bone marrow (BM) of patients with JAK2V617F-positive essential thrombocythemia (ET). A. Sympathetic nerve fibers quantitated with the help of an anti-TH antibody (control, n=9; ET, n=42), and Schwann cells visualized using an anti-GFAP antibody (control, n=9; ET, n=40) decreased in the BM of patients with JAK2V617F-positive ET relative to those in the HD, as determined by immunohistochemistry. B. Increased expression of B3AR in the BM of patients with JAK2V617F-positive ET (n=4) relative to the HD (n=4) as determined by Western blotting. C–D. Lower NE levels (C) (control, n=20; ET, n=20) and higher IL-1β levels (D) (control, n=20; ET, n=20) in the BM of patients with JAK2V617F-positive ET relative to the control, as measured by ELISA on the supernatant of the BM aspirates. E. A model illustrating BM hematopoietic dysfunction in JAK2V617F-positive ET. *P<0.05; **P<0.01, ***P<0.001; ****P<0.0001. Data are presented as the mean ± SEM. ET: essential thrombocythemia; HD: healthy donors; n: number of unique donors in each group; TH: tyrosine hydroxylase; GFAP: glial fibrillary acidic protein; NE: norepinephrine; B3AR: β3 adrenoceptor; SEM: standard error of mean.