Page 152 - Haematologica March 2020

T. Sun et al.
expression was confirmed by qPCR and Western blotting in MSC derived from patients with JAK2V617F-positive ET (Figure 5 A-B). To further evaluate the function of WDR4 in BM-MSC, we used lentiviruses carrying the WDR4 cDNA (LV-WDR4) or a specific shRNA targeting WDR4 (LV-shWDR4). LV-shWDR4–infected MSC had sig- nificantly lower WDR4 mRNA and protein levels than those in the control groups, and LV-WDR4 effectively increased the WDR4 level in BM-MSC (Figure 5 C-D). We next established coculture systems between normal mononuclear or CD4-positive T cells from the BM, and WDR4 knockdown or overexpressing MSC. Among the subgroups that had different levels of WDR4 expression, no changes were observed in terms of CD4-positive T-cell proliferation and activation, inflam- matory cytokine secretion, or Th2-cell subtype counts (Online Supplementary Figure S2). We next examined the role of WDR4 in BM-MSC regarding functions besides
immunoregulation. In HD MSC infected with LV- shWDR4, we observed biological characteristics similar to those in ET MSC, including enhanced proliferation (Figure 5E), decreased senescence (Figure 5F), and an impaired ability to differentiate into adipocytes, osteocytes, and chondrocytes (Figure 5G). Conversely, when ET MSC were infected with LV-WDR4, the previously detected abnormal biological properties were partially reversed (Figure 5 E–G) except for apoptosis (Online Supplementary FigureS3).
Insufficient action of the WDR4–IL-6 axis decreases hematopoiesis-supporting activities of BM-MSC from JAK2V617F-positive ET patients
We next investigated whether WDR4 affected the hematopoiesis-supporting function of BM-MSC. Hematopoiesis was studied by analyzing CFU. After 14 days of coculture of normal CD34-positive cells with HD
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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.
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haematologica | 2020; 105(3)