S100A9 initiates pyroptosis in MDS. (A) Enzyme-linked immunosorbent assay (ELISA) assessment of BM plasma concentration of S100A9 in normal donors (n = 12) vs MDS (n = 33 lower risk, n = 27 higher risk). (B) S100A9 BM plasma concentration analyzed according to IPSS risk score. (C) HMGB1 BM plasma concentration assessed by ELISA in normal donors (n = 11) and MDS patients (n = 55). (D) qPCR analysis of S100A9 mRNA levels in normal (n = 2) vs LR-MDS BM-MNCs (n = 8). (E) HMGB1 mRNA levels in normal (n = 6) vs MDS BM-MNCs (n = 10). (F) Representative histograms of intracellular levels of S100A9 by hematopoietic lineage in BM-MNCs isolated from MDS patients (n = 6) and normal donors (n = 5). (G) Mean percentage of S100A9⁺ cells by hematopoietic lineage. (H) qPCR analysis of untreated normal BM-MNCs (n = 3), normal BM-MNCs treated with 1 µg/mL rhS100A9 for 24 hours (n = 2), and MDS patient specimens (n = 5). (I) Representative micrograph (original magnification ×2520, 7.5 µm scale) depicting inflammasome formation in normal, untreated BM-MNC or normal BM-MNC treated with 5 µg/mL rhS100A9 for 24 hours (DAPI, blue; a–caspase-1, green; and NLRP3, red; merged images show inflammasome formation). (J) Quantitative analysis of confocal images of BM-MNCs from normal donors (n = 6), normal BM-MNCs treated with 5 µg/mL rhS100A9 (n = 2), and MDS patients (n = 10). Error bars represent SE. *P < .05, ** P < .01, and ***P < .001.