Fig 2: C. albicans-induced GSDMD cleavage and C. albicans escape from macrophages relies on inflammasome activation.a Casp1/11-mediated C. albicans-induced BMDM death. BMDMs from WT and Casp-1/11-/- mice were infected with C. albicans at MOI 10. BMDM death at the indicated time points was measured as relative LDH release as described in Fig. 3f. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). p = 0.0016 (4 h), p = 0.0282 (8 h), p = 0.0172 (16 h) vs. WT. b Casp1/11-mediated C. albicans escape from macrophages. BMDMs from WT and Casp-1/11-/- mice were infected with C. albicans at MOI 10. Dying cells were stained with PI (red). Experiments were repeated three times. The related time-lapse video is included in the Supplemental Materials. c The percentage of PI-positive BMDMs calculated at each time point. At least 100 cells were assessed for each sample. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). p = 0.0484 (3 h), p = 0.0257 (6 h) vs. WT. d Potassium efflux was essential for C. albicans-triggered GSDMD cleavage in BMDMs. BMDMs from WT or Gsdmd-/- mice were cultured in the indicated concentrations of KCl for 1 h and then infected with WT C. albicans for 6 h at MOI 50. Full-length (p53) and cleaved (p30) GSDMD in the cell lysates were detected by western blotting. Results are representative of data from three biological replicates. Source data are provided as a Source Data file. e C. albicans-induced BMDM death assessed by LDH assay. BMDMs were cultured in the indicated concentrations of KCl for 1 h and then infected (uninfected BMDMs as controls) with WT, candidalysin-deficient (ece1Δ/Δ), or hyphae-deficient (cph1Δ/Δ, efg1Δ/Δ) C. albicans at MOI 10 for 12 h. Relative LDH release was assessed as described in Fig. 3f. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). For cph1Δ/Δ, efg1Δ/Δ-infected BMMCs, p = 0.0016 (KCl 50 mM) vs. KCl untreated macrophages. For KCl -untreated BMMCs, p < 0.0001 (ece1Δ/Δ, cph1Δ/Δ, efg1Δ/Δ, w/o C. albicans) vs. WT C. albicans. For KCl 30 mM -treated BMMCs, p = 0.0001 (cph1Δ/Δ, efg1Δ/Δ), p < 0.0001 (ece1Δ/Δ, w/o C. albicans) vs. WT C. albicans. For KCl 50 mM-treated BMMCs, p = 0.0001 (ece1Δ/Δ), p < 0.0001 (cph1Δ/Δ, efg1Δ/Δ, w/o C. albicans) vs. WT C. albicans. f Candidalysin-dependent LDH release calculated by subtracting the level of LDH release induced by candidalysin-deficient (ece1Δ/Δ) C. albicans from that induced by WT C. albicans. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). p = 0.0052 (KCl 30 mM), p = 0.0036 (KCl 50 mM) vs. KCl untreated macrophages. g Potassium efflux was critical for C. albicans escape from macrophages. BMDMs were cultured in the indicated concentrations of KCl for 1 h and then infected with WT C. albicans at MOI 10. Dying cells were stained with PI (red). Images were acquired as described in Fig. 3c. Shown are representative images at the indicated time points. The related time-lapse video is included in the Supplemental Materials. h The percentage of PI-positive BMDMs calculated at each time point. At least 100 cells were assessed for each sample. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). p = 0.016 (KCl 50 mM) vs. KCl untreated macrophages.

Fig 3: The thioredoxin-interacting protein (TXNIP)-inflammasome-caspase-1 axis regulates the clearance of bacteria from macrophages. (A) The levels of interleukin (IL)-1β in conditioned media from wild-type (WT) and Txnip knockout (KO) mouse peritoneal macrophages after treatment with E. coli for the indicated times. Data are expressed as the mean ± SD (***P < 0.001 compared with WT). (B) The protein expression levels of caspase-1 P45 and caspase-1 P10 in mouse WT and Txnip KO mouse peritoneal macrophages after treatment with E. coli for the indicated times. (C) Caspase-1 activity, with or without ZVAD treatment, in mouse WT and Txnip KO mouse peritoneal macrophages 6 h after the treatment with E. coli. Data are expressed as the mean ± SD (***P < 0.001 compared with WT). (D) Representative images of bacteria-laden WT and Txnip KO mouse macrophages 6 h after a 1-h treatment with GFP-expressing E. coli with or without ZVAD treatment and removal of extracellular bacteria. Scale bar, 10 μm.

Fig 4: AGP downregulated the gene expression of NLRP3 signaling pathway in lung tissue and IL-1β protein levels in BALF of the AECOPD mice. (A–E) The mRNA expression of NLRP3, PYCARD, CASP1, IL1B and IL18, respectively. (F) The protein levels of IL-1β in BALF for each group. The results were presented as the mean ± SEM, n = 6, *P <0.05; **P <0.01; ***P <0.001; ns, P >0.05.

Fig 5: Transcriptional profiling of the liver in NASH mice and verification of NASH associated signaling pathways. A Transcriptional profiling of the liver tissue of C5aR1−/− and WT mice. B KEGG pathway analysis. C Toll-like receptor signaling pathway associated genes were showed in heatmap. D The mRNA level of Tlr4 was measured by qRT-PCR. The data is representative of three independent experiments. E The protein levels of TLR4, p-NFκB P65 and NFκB P65. F The mRNA level of Nlrp3 was determined by qRT-PCR. G The protein levels of NLRP3 and CASP1. H RAW264.7 cells were treated with the recombinant protein C5a (50 ng/mL) for 24 h. The protein levels of TLR4, NLRP3, and CASP1 were determined by western blot. I The protein levels of iNOS, CD86, CD163 and CD206 were measured by Western blot. J The mRNA levels of iNOS and CD163. K. Protein levels of p-AKT and AKT. Experiments were repeated three times. Results are presented as mean ± SD. ***P < 0.001. n = 6 per group