Heme oxygenase-1/CO signaling suppresses NLRP3 inflammasome-mediated stromal fibrosis in primary myelofibrosis

Inflammation and oxidative stress are critical drivers of bone marrow (BM) fibrosis in primary myelofibrosis (MF). Here, we investigated the interplay between the heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway and NLRP3 inflammasome activation in MF microenvironment. Bioinformatic analysis of patient datasets (GSE53482 and GSE41812) revealed an approximately two-fold reduction of HO-1 expression in CD34+ peripheral blood cells compared with healthy controls (p < 0.01). Consistently, JAK2V617F-mutated cell lines (HEL, SET2) displayed respectively lower HO-1 and higher NLRP3 expression than JAK2 wild-type UT7 cells. Ruxolitinib, a potent pharmacological inhibitor of JAK/STAT pathway, restored HO-1 (≈1.7-fold increase) and reduced NLRP3 by ∼50% (p < 0.01). Similarly, in mesenchymal stromal cells (HS-5), tumor-conditioned medium induced a two-fold increase in NLRP3 and Caspase-1 and a ∼40% decrease in HO-1. Pharmacological induction of HO-1 by hemin (10 μM) or CO-releasing molecule CORM-A1 (25 μM) restored HO-1 (∼1.8-fold) and reduced NLRP3 activation (∼50%), limiting αSMA expression and collagen deposition by ∼55–60%. In a TPOhigh zebrafish model, nlrp3, caspase-a, and il1β were upregulated ≈2.5-fold, while hmox1a/b decreased by ∼50%; CORM-A1 co-treatment reversed these effects, increasing hmox1a (∼1.7-fold) and decreasing cd41 and col1a1 (∼45%). NLRP3 inhibition by MCC950 (1 μM) produced similar results. Consistently, BM biopsies from MF patients showed ∼65% lower HO-1 expression (p < 0.001) and nuclear BACH1 localization in megakaryocytes. In conclusion, our results demonstrate that HO-1/CO signaling suppresses NLRP3 inflammasome activation and fibrotic remodeling, suggesting that this pathway may represent a potential therapeutic target in MF.