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T. Sun et al.
ous changes in IL-6 levels in the BM extract. This may be the case because the myelofibrosis grade of the patients enrolled in this study was 0-1. The significance of the hematopoiesis-supporting role of IL-6 has been well-docu- mented.24-27 Most HSPC are in contact with MSC in the BM, forming a highly sophisticated interaction network through direct contact and paracrine effects.2 Thus, one possible explanation is that IL-6 levels in MSC contribute to the regulation of hematopoietic progenitors through local signals. Interestingly, our CFU assays showed that although some types of colonies depended on WDR4-IL6 axis, CFU-MK did not. JAK2V617F leads to increased HSC survival and biased differentiation to the lineages that sig- nal through JAK2, including MK-platelets.41 Therefore, it is reasonable to hypothesize that these JAK2V617F CFU-MK would outgrow other lineages that are more dependent on the WDR4-IL6 axis in the BM of ET patients. Further work is required. In the present study, CD34-positive cells cul- tured with MSC infected with LV-shWDR4 showed a dif- ferent phenotype to those with ET MSC in terms of BFU- E. We observed a trend towards BFU-E reduction in patients with ET, but the difference was not significant. The difference is likely related to the diversity between these groups. Both groups treated with WDR4 shRNA or control shRNA showed similar characteristics in terms of clinical or laboratory features, while an overlap existed between BFU-E levels in patients and control samples, pos- sibly because of the heterogeneity of clinical specimens and variable disease progression of the patients. Together these findings indicate that dysregulation of the WDR4-IL6 axis is involved in most dysfunctions in ET MSC.
Neuropathy was detected in the present study in BM sections of patients with JAK2V617F-positive ET. Although 10 mm sections are not optimal for accurate identification and quantification of BM fibers, no thicker sections were approved due to the scarcity of the clinical specimens. Aberrant expression of NE and B3AR were also detected in the BM of the patients. IL-1β, secreted by hematopoietic progenitors and many types of stromal cells, has been shown to mediate neuropathy in the BM of JAK2V617F-positive MPN mice and patients.19 We also found elevated IL-1β levels in the BM extract of patients with JAK2V617F-positive ET, which may explain why neuropathy was detected. Neuropathy has been linked to the loss of NES-positive cells in MPN mice and patients,19 while denervation of the BM in HD or patients with acute myeloid leukemia can lead to increased numbers of NES- positive cells.9 A higher number of NES-positive cells was estimated in this study when thinner sections (5 μm) were used. Given that combining data derived from sections with different thickness may introduce significant bias,
further studies are required to understand the relevance between neuropathy and NES-positive cells. IL-1β has previously been reported to upregulate NES,48 suggesting that immune cues are involved in altering both the sympa- thetic nervous system and MSC. NE inhibits nuclear fac- tor-κB activity and pro-inflammatory cytokine release by binding to B2AR and B3AR on MSC and other immune cells.12,18 The decreased production of NE, possibly caused by increased neurotoxic inflammatory cytokines, in ET can facilitate the formation of an inflammatory environ- ment. Further studies are required to elucidate the connec- tion between MSC, immunity, and sympathetic nerves.
In summary, this study revealed multilevel defects in the hematopoietic microenvironment of patients with JAK2V617F-positive ET and demonstrated that one of the differentially expressed genes, WDR4, underlies most dys- functions in ET MSC via downregulation of IL-6 expres- sion and secretion through suppression of the ERK– GSK3β–CREB pathway. Activation of the JAK-STAT3 pathway through IL-6 binding and its receptor is essential for maintaining normal hematopoiesis. A study recently revealed that IL-6 stimulated JAK2-STAT3 signaling in PV and PMF, but suppressed this signaling in ET.49 Our results, alongside with those of previous studies, provide a com- pelling rationale for exploring the in vivo effect of the WDR4-IL-6 axis against ET in the future. Due to technical limitations, we also suggested a number of hypotheses and potential explanations for the paradoxes with other groups and future work is required for their validation.
Acknowledgments
The authors would like to thank Liyan Zhang, Chengwen Li, and Qi Sun for providing the cells used in the experiments.
Funding
This work was supported by the National Natural Science Foundation of China [grant numbers 81600099, 81770128, 81970121 and 81900126], The Beijing-Tianjin-Hebei basic research project [grant number 18JCZDJC44600/ H2018206423], Tianjin Municipal Science and Technology Commission Grant [grant numbers 18JCQNJC11900 and 19JCZDJC33000], Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences [grant number 2019PT310022], CAMS Innovation Fund for Medical Sciences [grant numbers 2016-I2M-1-018, 2016-I2M-3-002, 2017- I2M-1-015, and 2019-I2M-1-006], National Key Research and Development Program of China [grant numbers 2017YFA0103102 and 2019YFA0110802], Novo Nordisk Haemophilia Research Fund in China and Novartis Foundation. The authors would like to thank all the funding agencies that supported this research.
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