Fig 2: IPA suppressed TREM2-dependent pyroptosis following stroke. a KEGG pathway analysis of the 598 common genes identified from the Venn diagram (dMCAO vs. sham and dMCAO+IPA vs. dMCAO) revealed significant enrichment in inflammatory pathways, indicating that IPA treatment modulates the inflammatory response following ischemic stroke. n = 4. b Heatmap depicting the expression of representative genes involved in the pyroptosis pathway, derived from transcriptomic analysis. n = 4. c mRNA expression of NLRP3 at day 3 post-stroke. ***P < 0.001, analyzed by one-way ANOVA followed by LSD multiple comparison tests. n = 6. d mRNA expression of Caspase-1 at day 3 post-stroke. ***P < 0.001, analyzed by one-way ANOVA followed by LSD multiple comparison tests. n = 6. e-j Protein levels of NLRP3, pro-caspase-1, p20 caspase-1, GSDMD and GSDMD-N in each group at day 3 post-stroke. *P < 0.05, **P < 0.01, ***P < 0.001, analyzed by one-way ANOVA followed by LSD multiple comparison tests (NLRP3, pro-caspase-1, GSDMD and GSDMD-N) or Dunnett’s multiple comparison tests (p20 caspase-1). n = 5. k Protein levels of NLRP3, pro-caspase-1, p20 caspase-1, GSDMD and GSDMD-N in each group. l-q Protein levels of NLRP3, pro-caspase-1, p20 caspase-1, GSDMD and GSDMD-N in each group. *P < 0.05, **P < 0.01, ***P < 0.001, analyzed by one-way ANOVA followed by LSD multiple comparison tests (NLRP3, p20 caspase-1, GSDMD and GSDMD-N) or Dunnett’s multiple comparison tests (pro-caspase-1). n = 5

Fig 3: Microbiota-Derived IPA Mitigates Post-Stroke Neuroinflammation by Inhibiting TREM2-Dependent Pyroptosis. (Created in https://BioRender.com)

Fig 4: IPA modulated microglia towards an anti-inflammatory phenotype by activating TREM2 following stroke. a Protein levels of TREM2 in each group at day 3 post-stroke. ***P < 0.001, analyzed by Student’s t-test. n = 5. b Protein levels of TREM2 in each group at day 3 post-stroke. *P < 0.05, **P < 0.01, analyzed by Dunnett’s multiple comparison tests. n = 5. c Iba1 (green) and TREM2 (red) double-immunostaining in the peri-infarct region at day 3 post-stroke. Bar = 50 μm. d Quantification of TREM2+Iba1+/Iba1+ cells. ***P < 0.001, by one-way ANOVA followed by LSD multiple comparison tests. n = 6. e The mRNA expression of TREM2 at day 3 post-stroke. **P < 0.01, ***P < 0.001, analyzed by one-way ANOVA followed by LSD multiple comparison tests. n = 6. f mRNA expression of pro-inflammatory markers (CD16, IL-1β, IL-18, IL-6, and TNF-α) in BV2 microglial cells in vitro. *P < 0.05, **P < 0.01, ***P < 0.001, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison tests (CD16, IL-1β, IL-18, and TNF-α) or LSD multiple comparison tests (IL-6). n = 5. g The mRNA expression of anti-inflammatory markers (CD206, TGF-β, IL-10, Arg-1 and YM-1) in BV2 microglial cells in vitro. *P < 0.05, **P < 0.01, ***P < 0.001, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison tests (CD206) or LSD multiple comparison tests (TGF-β, IL-10, Arg-1 and YM-1). n = 5

Fig 5: Cross-species identification and validation of monocyte/macrophage-associated genes in ACLF. (A) Venn diagram showing the overlap of DEGs across three comparisons in human (LC vs. HC, ACLF vs. HC, ACLF vs. LC) and mouse (Fibrosis vs. HC, ACLF vs. HC, ACLF vs. Fibrosis) liver samples. The top 10 intersecting DEGs are shown, with red-labeled genes associated with monocyte/macrophage functions. (B) Protein-protein interaction (PPI) network of the top 10 conserved DEGs as predicted by STRING analysis. (C) Validation of the mRNA expression levels of six monocyte/macrophage-associated genes (MMP12, TREM2, CAPG, F13A1, LGALS3, and CCR2) in human liver samples using RT-qPCR. (D) Validation of the same six monocyte/macrophage-associated genes in mouse liver samples using RT-qPCR. (E) Western blot of TREM2 and CCR2 protein expression in human and mouse liver tissues. (F) Correlation analysis between mRNA levels of the six genes and MELD scores in human ACLF patients. Spearman correlation coefficients and p-values are indicated. Bars represent mean ± SD; ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0. 05, n.s., no significance.