T cell-derived TNF-α mediates myelofibrosis via JAK2/STAT3-ADAM17 axis in JAK2V617F⁺ myeloproliferative neoplasms Free

Background: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by excessive myeloid proliferation, chronic inflammation, and progressive bone marrow fibrosis. Elevated levels of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, have been observed in patients with MPNs and are closely associated with fibrosis grade and disease progression. However, the cellular source of TNF-α and the upstream mechanisms driving its production remain poorly understood. This study explores the role of A Disintegrin and Metalloprotease 17 (ADAM17) in mediating T cell-derived TNF-α release and its transcriptional regulation through the Janus kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) pathway.

Methods: A combination of single-cell RNA sequencing and flow cytometry was used to investigate the association between serum TNF-α levels and fibrosis severity (assessed via reticulin staining, graded MF-0 to MF-3), and to quantify transmembrane TNF-α (tmTNF-α) and ADAM17 expression in peripheral blood immune subsets from MPN patients and healthy donors. Co-culture assays were performed using JAK2V617F-mutant cells (MEG-01 VF and HEL cell lines) and healthy donor T cells to assess their effect on TNF-α production, as measured by enzyme-linked immunosorbent assay (ELISA). To elucidate molecular mechanisms, ADAM17 knockdown models were established using small interfering RNA. Western blotting, co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) were employed to evaluate the interaction between STAT3/phosphorylated STAT3 (p-STAT3) and the ADAM17 promoter.

Results: Single-cell RNA sequencing revealed increased TNF-α expression in T cells of MPN patients compared to healthy controls, with primary myelofibrosis (PMF) patients exhibiting the highest expression levels. Flow cytometry confirmed that T cells are the predominant tmTNF-α-expressing population in peripheral blood, and showed significantly elevated tmTNF-α levels in MPN patients, particularly those with PMF (p < 0.001). ADAM17 expression was markedly upregulated in monocytes and myeloid dendritic cells from MPN patients.

In vitro, JAK2V617F-mutant cells induced both tmTNF-α expression and sTNF-α release in co-cultured healthy T cells, with sTNF-α levels reaching 142.4 ± 15.7 pg/mL in MEG-01 VF co-cultures versus 40.3 ± 5.8 pg/mL in wild-type controls (p < 0.0001). Knockdown of ADAM17 significantly impaired sTNF-α shedding, indicating its essential role in the process. Mechanistically, Co-IP confirmed the interaction between ADAM17 and STAT3/p-STAT3, while ChIP-qPCR demonstrated direct binding of STAT3 to the ADAM17 promoter, supporting transcriptional regulation via the JAK2/STAT3 axis.

Conclusion: Our findings demonstrate that the JAK2V617F mutation promotes ADAM17 overexpression through STAT3-mediated transcriptional activation, leading to cleavage of tmTNF-α on T cells and increased release of soluble TNF-α (sTNF-α). This JAK2/STAT3-ADAM17-TNF-α axis contributes to chronic inflammation and fibrotic progression in MPN. Targeting ADAM17 may offer a novel therapeutic strategy to reduce fibrosis and inflammatory burden in MPNs.