Page 157 - Haematologica March 2020

Bone marrow niche dysfunction in ET
Discussion
In the present study, we observed transcriptional and functional abnormalities in the hematopoietic niche of patients with JAK2V617F-positive ET, including function- al deficiency in MSC, immune imbalance, and sympathet- ic neuropathy, relative to those in HD controls. BM-MSC from patients with JAK2V617F-positive ET showed an altered transcriptome, faster proliferation, attenuated apoptosis and senescence, decreased potential to differen- tiate into adipocytes and osteocytes, and insufficient sup- port for normal hematopoiesis. These findings partially agree with those found in previous studies of the myelodysplastic syndrome, leukemia, and MPN by other groups, but differ from the conclusions obtained from an MPN mouse model and patients.7-9,19,34 NES-positive cells have been reported to be heterogeneous populations com- prising mesenchymal cells and endothelial cells in the BM.4 We co-stained bone marrow samples with NES and CD34 (endothelial cell marker), and found that patients with JAK2V617F-positive ET showed higher numbers of NES-positive mesenchymal cells in the BM relative to those in the HD controls. This observation contradicts the results obtained from the MPN mouse model and patients mentioned above but is consistent with the results obtained in patients with ET, polycythemia vera (PV), and primary myelofibrosis (PMF) by other group.34 NES−/leptin receptor−/CXCL12+ MSC subpopulations have been reported to be essential for the maintenance of HSC in mouse models.35 Furthermore, in BM sections from patients with myelodysplastic syndrome, HSC are mostly in close contact with CD271+/NES− MSC.36 Hock et al. con- firmed that HSC with elevated proliferation rates were functionally compromised.37 Therefore, given that MSC of patients with JAK2V617F-positive ET showed enhanced proliferation, we hypothesize that functional deficits are also present in these MSC. These studies, as well as our data, may explain the poor ability of the expanded MSC isolated from patients with JAK2V617F-positive ET to support normal hematopoiesis. Regarding the paradox with the mouse models of MPN, it is possible that the het- erogeneity of the patients with MPN contributed to the observed discrepancy since our study involved only patients with JAK2V617F-positive ET as subjects, but not those with PV or PMF. Because of the low number of the primitive non-passaged BM-MSC (0.08% of BM mononu- clear cells), MSC used for functional analysis were expanded ex vivo in the present work. It is possible that this process caused changes that were not completely con- sistent with the most primitive state in vivo.38 Additionally, although mouse models are powerful tools for studying MSC in vivo, animals may not fully recapitulate the med- ical conditions in humans, and inter-species differences in structure, function, and immunophenotype may have contributed to the contradictory results. Additionally, JAK2V617F mutation is detected in approximately 95% of patients with PV and 50% of patients with ET and PMF, and mouse models of JAK2V617F show a tendency to develop a PV phenotype more often than ET.39-40 Furthermore, the hematopoietic niches of different MPN phenotypes may differ to some extent. Therefore, both animal models and clinical specimens help us to better understand the intrinsic state of MSC under medical con- ditions in humans. The ratio of mutant to wild-type JAK2 has been proved to be critical for the phenotypic manifes-
tation.41 Nonetheless, a successful model that accurately recapitulates the human manifestations of JAK2-positive ET is currently not available for us. Collectively, the pres- ent in vitro findings revealed a perturbed transcriptome and aberrant biological characteristics of BM-MSC of patients with JAK2V617F-positive ET.
It has been reported that expansion of BM CD4-positive T cells can lead to exhaustion of hematopoietic cells.42 In this study, we found that MSC of patients with JAK2V617F-positive ET showed reduced inhibition of CD4-positive T-cell proliferation and activation, and secretion of the inflammatory cytokine sCD40L. In addi- tion, they showed decreased induction of mostly immunosuppressive and antineoplastic Th2 cell forma- tion, and secretion of the anti-inflammatory cytokine IL-4. Th2 formation is highly dependent on the activation of signal transducers and activators of transcription 6 by IL- 4.43 Thus, low secretion of IL-4 may be both the cause and consequence of blockage of the Th2 response. These results are mostly compatible with the results of previous studies.14,15,42 Attenuated senescence of MSC from patients with JAK2V617F-positive ET was observed in the present study. Thus, one paradox arises, given that decreased senescence of MSC is typically linked to their anti-inflam- matory status. The concept that MSC are highly plastic and the local inflammatory environment thus can shape the immunomodulatory effects of MSC may help to improve the understanding of the state of MSC in patho- logical processes. Specific inflammatory signals prompt MSC to switch between the proinflammatory and anti- inflammatory phenotypes.44 One possible explanation for the contradiction between aging and their inflammatory phenotype is that aging-related changes may be compen- sated to some extent by the local inflammatory milieu. Additionally, MSC can be polarized by downstream Toll- like receptor (TLR) signaling into two homogenous phe- notypes. TLR4-primed MSC mostly produce pro-inflam- matory cytokines, while TLR3-primed MSC express mostly immunosuppressive cytokines.45 TLR4 has been confirmed to inhibit senescence via epigenetic silencing of senescence-related genes.46 Thus, another possible expla- nation is that upregulation of TLR4 and downregulation of TLR3 polarize the MSC from patients with JAK2V617F-positive ET to a proinflammatory phenotype with an anti-senescence effect. Collectively, these results indicate that MSC contribute at least partially to the immune imbalance in the BM of ET patients.
The changes mentioned above provide a possible link between the alterations in hematopoietic niches to the pathophysiology of JAK2V617F-positive ET in humans. Nonetheless, the underlying mechanisms remain unclear. In this study, we determined a mechanism whereby WDR4 deficiency impairs the ability of BM-MSC to sup- port normal differentiation of hematopoietic progenitors in patients with JAK2V617F-positive ET. This effect occurs due to decreased IL-6 expression and secretion through suppression of the ERK–GSK3β–CREB pathway. Overall patients with MPN have been described to have higher IL-6 levels in the BM and there is published data on the oncogene-dependent mechanisms of fibroblasts expansion and IL-6 upregulation in fibroblasts in patients with JAK2V617F-positive MPN.47 Nonetheless, in the pres- ent study, a prominent reduction in IL-6 levels was found in the supernatants of the culture medium of BM-MSC from patients with JAK2V617F-positive ET, with no obvi-
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