Fig 2: Prdx1 activates the NLRP3 inflammasome in macrophages.A–E Primary peritoneal macrophages (PPMs) isolated from wild-type mice were stimulated with rPrdx1 (25 nM) for 12 and 24 hours (h) with or without pretreatment of MCC950 (10 μM for 1 h). Cells were collected and analyzed for the expression levels of pro-caspase-1 (pro-Casp1) (A), NLRP3 (B), and pro-IL-1β (C). Conditioned medium was harvested and analyzed for cleaved Casp1 (D) and mature IL-1β (E) expression levels. Typical images and quantification are included (n = 3 biological repeats). F, G NLRP3 protein expression and quantification in colon tissues from Prdx1–/– mice and their littermates treated with DSS (n = 6 mice per group). H IL-1β levels in colon tissues from the indicated mouse groups were detected by ELISA (n = 6 mice per group). I–K WT mice were depleted of macrophages using clodronate liposomes (CLs) and subsequently administered 3% DSS, accompanied by i.v. injection of either PBS or rPrdx1 (10 μg/kg; n = 5 mice per group). NLRP3 protein expression and quantification (I, J), along with IL-1β levels (K) were detected in colon tissues from the indicated mouse groups. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA. Results presented were repeated at least three independent experiments for each experimental condition.

Fig 3: Prdx1 neutralization protected against intestinal inflammation.Wild-type mice were administered 3% DSS in their drinking water, followed by intraperitoneal (i.p.) injection of either control IgG or Prdx1-neutralizing antibody (150 μg/mouse) on Days 1, 3, and 5. The mice were sacrificed 7 days after DSS administration (n = 5–7 mice per group). A Schematic diagram of administration of Prdx1-neutralizing antibody to DSS-treated mice. B Body weight of the mice was monitored daily and the data was plotted as a percentage of the initial body weight on Day 0. C The disease activity was monitored and scored daily for each mouse. D Typical images of H&E staining and histopathological scores of colon sections from the indicated mouse groups. Scale bar, 50 μm. E–G The mRNA expression levels of IL-1β, IL-6, and TNF-α in the colons of indicated mouse groups were measured by RT-qPCR. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by non-paired Student’s t test (B, C) or one-way ANOVA (D–G). Results presented were repeated at least three independent experiments for each experimental condition.

Fig 4: Reintroduction of recombinant Prdx1 (rPrdx1) facilitated intestinal inflammation.Prdx1–/– mice and their littermates were administered 3% DSS in their drinking water, followed by intravenous (i.v.) injection of either phosphate-buffered saline (PBS) or rPrdx1 (10 μg/kg) on Days 4, 5, and 6. The mice were sacrificed 7 days after DSS administration (n = 5–7 mice per group). A Schematic diagram of administration of rPrdx1 to DSS-treated Prdx1–/– mice. B Body weight of the mice was monitored daily and the data was plotted as a percentage of the initial body weight on Day 0. C The disease activity was monitored and scored daily for each mouse. D Typical images of H&E staining and histopathological scores of colon sections from the indicated mouse groups. Scale bar, 50 μm. E–G The mRNA expression levels of IL-1β, IL-6, and TNF-α in the colons of indicated mouse groups were measured by RT-qPCR. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01 by non-paired Student’s t test (B, C) or one-way ANOVA (D–G). Results presented were repeated at least three independent experiments for each experimental condition.

Fig 5: Serum Prdx1 was increased in mice with experimental colitis.Wild-type mice were treated with 4% DSS for the indicated time (n = 5 mice per group). A Typical images of H&E staining and histopathological scores of colon sections from control mice and those treated with DSS for varying durations. Scale bar, 50 μm. B Immunofluorescence staining for Prdx1 in colon sections from the indicated mouse groups. Typical images are shown. Scale bar, 200 μm. C, D Prdx1 mRNA and protein expression in colon tissues from the indicated mouse groups were measured by RT-qPCR and western blot, respectively. Typical images and quantification are shown. E Serum Prdx1 levels in the indicated mouse groups were measured by ELISA and quantified using a standard curve. F The linear correlation between serum Prdx1 levels and disease activity index (DAI) scores in mice was analyzed by Pearson’s correlation coefficient. G–I The linear correlations between serum Prdx1 levels and levels of serum IL-1β, IL-6, and TNF-α were analyzed by Pearson’s correlation coefficient. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by non-paired Student’s t test. Results presented were repeated at least three independent experiments for each experimental condition.