Fig. 3: Lung MKs are activated for inflammatory responses.

a–c RNA-seq analysis of CD41⁺ MKs from lung single-cell suspension obtained from mice treated with 2 mg/kg body weight CB particles for 24 h. The volcano plots show the Log2 fold change of gene expression in CB group relative to control group (Ctrl). a Volcano plot showing upregulated and downregulated genes in lung MKs, two-tailed Wilcoxon rank-sum test. b KEGG pathway enrichment analysis of annotated upregulated genes in CB particle-treated lung MKs were associated with thrombocytosis, immunity, and cell regulation signals, one-side Fisher’s exact test. c Heatmap showing differentially expressed genes in lung MKs after CB particle treatment, categorized by biological functions. Columns represent samples (n = 3). d, e RT-qPCR analyses of Caspase-1 (d) and IL-1β (e) mRNA expression in lung MKs sorted from mice treated with CB particles for 24 h (n = 6 biological replicates per group). f H&E-stained images of lung sections show that c-mpl^-/- mice treated with CB particles for 24 h had fewer inflammatory foci (red arrows) than treated WT animals. g Flow cytometer analysis of lung single-cell suspension from animals in f (n = 6 per group). h ELISA assay of IL-1β content in sera and BALF from WT and c-mpl^-/- mice upon CB particles for 24 h (n = 6 per group). i H&E (top) and Masson (bottom) stained images of lung sections from WT and c-mpl^-/- mice at 72 h post-CB administration show accumulated inflammatory cells (red arrows) and collagen deposition (blue arrows). j Immunofluorescent images of vascular junction with CD31⁺ cells in lung sections from WT and c-mpl^-/- mice at 72 h post-CB particle exposure. White arrowheads indicate damaged junctions. White dash-dots outline the vessel. Inset: magnified view of vascular junction. k, l Immunofluorescent images (k) and counts per field (l) of neutrophils (MPO⁺Ly6G⁺ cells, white arrowheads) in lung sections from WT and c-mpl^-/- mice at 72 h after CB exposure (n = 6 mice per group). m Immunofluorescence-stained images of lung sections show that mice receiving MK transplant promoted inflammatory responses and recruited significantly more neutrophils than control mice without transplants. n ELISA assay of IL-1β content in sera of mice with (+) and without (-) MK transplant at 24 h post 2 mg/kg body weight CB exposure (n = 6 mice per group). o Schematic shows c-mpl^-/- mice displayed greater lung damage and 1–2 days slower recovery from particle-triggered inflammation than WT mice. Box and whisker plots indicate median, interquartile range and 10th to 90th percentiles of the distribution (h, n). Data are presented as the mean values of 3 independent experiments ± SEM (d–e, g, h, l and n), two-side student’s t-test (d, e and g) and one-way ANOVA (h, l and n). N.S, not significant.