Fig. 5. Inhibition of p38 activation is an effective immunotherapy against C. neoformans infection.

a KEGG analysis of differentially expressed genes involved in the signal transduction or the immune system in BM-derived MDSCs from wild-type (WT) or Clec2d^−/− mice, which were generated by GM-CSF (GM, 40 ng/ml) + GXM (10 μg/well) and then treated with GXM (10 μg/well) for 3 h. b Gene set enrichment analysis (GSEA) plots (Right) of the indicated signaling and Heatmap (Left) of differentially expressed MAPK-related genes in BM-derived MDSCs from WT or Clec2d^−/− mice as shown in a. P values were calculated by hypergeometric test and adjusted for multiple comparisons. c Western blot assay of the indicated proteins in BM-derived MDSCs from WT or Clec2d^−/− mice, which were generated by GM (40 ng/ml) + GXM (10 μg/well) and then treated with GXM (10 μg/well) for the indicated time. Representative blots were shown from three independent experiments. d Heatmap of differentially expressed MDSC-related and pro-inflammatory genes in wild-type BM-derived MDSCs, which were generated by GM (40 ng/ml) + GXM (10 μg/well) and then treated with GXM (10 μg/well) combined with or without p38 inhibitor SB202190 (p38i, 10 μM) for 3 h. e mRNA levels of Arg1 and Nos2 in cell line MSC-2, which were treated with PBS (control), IL-4 (10 ng/ml), GXM (10 μg/well), GXM + p38 inhibitor SB202190 (p38i, 10 μM), or GXM + ERK1/2 inhibitor U0126 (ERKi, 10 μM) for 6 h. f Proliferation frequency of CD4⁺ and CD8⁺ T-cells, which were co-cultured for 72 h with MSC-2 cells pretreated as shown in e. g Proliferation frequency of CD4⁺ and CD8⁺ T-cells, which were co-cultured with wild-type (WT) and Clec2d^−/− BM-derived MDSCs with or without treatment of p38 inhibitor SB202190 (p38i, 10 μM) for 72 h. h Survival assay of C. neoformans strain H99-infected mice (n = 8, 1 × 10³ CFU/mouse), which were intraperitoneally treated with or without p38 inhibitor SB202190 (p38i, 25 μg/kg every other day). i–m Fungal burden (i), representative histological images with hematoxylin-eosin (H&E) and Periodic Acid-Schiff (PAS) staining (j), the percentage of MDSCs, M-MDSCs, and PMN-MDSCs (k), mRNA levels of Arg1 and Nos2 (l), and the frequency of IFNγ⁺ T_H1 and IL-17A⁺ T_H17 cells (m) in the lungs of C. neoformans strain H99-infected mice (1 × 10³ CFU/mouse) on Day 14, which were intraperitoneally treated with or without p38 inhibitor SB202190 (p38i, 25 μg/kg every other day). Scale bars = 1000 μm; arrows indicate yeasT-cells in granulomatous lesions. Data were presented as mean ± SEM, n = 3 (e–g, I, k–m), n = 8 (h) biologically independent samples. Data were analyzed by unpaired two-sided Student’s t-test in e, f, i, k–m, one-way ANOVA adjusted for multiple comparisons in g and two-sided log-rank (Mantel–Cox) tests in h. Source data are provided as a Source Data file.