Fig 2: Protective effects of IL-37 in TLCS- and ARG-induced AP models.(A and B) Taurolithocholic acid 3-sulfate disodium salt (TLCS; 2.5%, 2 mg/kg) was injected in retrograde into the pancreatic bile duct to induce AP in IL37tg and WT mice. The sham group was used as the control in TLCS-induced experimental AP models. (A) H&E staining of pancreatic tissues from the indicated groups at 24 hours, and representative IHC staining of CD68 and MPO in pancreatic tissues. Scale bars: 200 or 50 µm. (B) Percentages of pancreatic cell death area, and serum amylase, lipase, and IL-1ß levels at 24 hours (n = 5–6 per group). (C and D) WT and IL37tg littermates were given i.p. injections of 8% l-arginine (ARG; 3.3 g/kg, hourly, 3 times total) to induce AP, while an equivalent volume of PBS was injected into mice from the control group. (C) H&E staining at 72 hours, and representative IHC staining of CD68 and MPO in pancreatic tissues. Scale bars: 200 or 50 µm. (D) Percentages of pancreatic cell death area, and serum amylase, lipase, and IL-1ß levels (n = 5–6 per group). Statistical comparisons were made using 1-way ANOVA. Data are presented as mean ± SD, and statistical significance is denoted as **P < 0.01, and ***P < 0.001.

Fig 3: Recombinant IL-37 protects mice from experimental AP.(A and B) C57BL/6J WT mice were injected with CAE to induce AP, and after 1 hour, the indicated doses of rIL37 were administered (n = 5–6 per group). Mice injected with PBS were used as normal controls. All mice were sacrificed and harvested after 24 hours. (A) H&E staining of pancreatic tissue from the indicated mice. Scale bars: 200 or 50 µm. (B) Percentages of pancreatic cell death area, and serum amylase, lipase, and IL-1ß levels at 12 hours. (C and D) Pancreatic acinar cell line 266-6 or primary acinar cells were treated with CCK, together with gradient doses of rIL37 for 12 or 6 hours, respectively. The levels of LDH release are shown. (E) 266-6 cells were treated with CCK together with rIL37 for 12 hours; then cells were harvested for propidium iodide (PI) staining. Representative flow cytometry histograms and the proportion of PI-positive cells are shown. Experiments were repeated 3 times. Statistical comparisons were made using 1-way ANOVA. Data are presented as mean ± SD, and statistical significance is denoted as *P < 0.05, **P < 0.01, and ***P < 0.001. NC, normal control.

Fig 4: Reduced severity of experimental AP in IL37tg mice.(A) Schematic diagram of the generation of human IL-37 transgenic mice (IL37tg). (B–F) WT and IL37tg mice were injected with PBS and caerulein (CAE; 200 µg/kg, 1-hour intervals, 10 times total) to generate control and AP groups, respectively. Mice were harvested at 24 hours after the first injection. (B) Quantitative real-time PCR analysis of IL-37 mRNA expression in pancreatic tissues from IL37tg and WT littermates in normal condition. (C) H&E staining of pancreatic tissues from the indicated groups (n = 5–6 per group). Scale bars: 200 or 50 µm. (D) Percentages of pancreatic cell death area, and serum amylase, lipase, and IL-1ß levels at 12 hours. (E) IHC staining of CD68 and MPO in pancreatic tissues, which marked macrophages and neutrophils, respectively (n = 3 per group). Scale bars: 50 µm. (F) Flow cytometry analysis of pancreatic leukocytes from the WT-CAE and IL37tg-CAE groups. Representative flow cytometry gating and the proportion of M1 (CD11b+F4/80+TNF-a+ of CD45.2+) and M2 (CD11b+F4/80+CD206+ of CD45.2+) macrophages (n = 4 per group). Experiments were repeated 3 times. Statistical comparisons were made using 1-way ANOVA or Student’s 2-tailed t test. Data are presented as mean ± SD, and statistical significance is denoted as *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Specific knockdown of GSDMD in pancreatic acinar cells neutralizes the protective effect of IL-37.(A) Primary pancreatic acinar cells (PACs) were isolated from Gsdmdfl/fl and Pdx1cre Gsdmdfl/fl (Gsdmd?Pan) mice. PACs were treated with CCK with or without rIL37 (50 ng/mL). The LDH levels are shown. (B–D) AP was induced in Gsdmdfl/fl and Pdx1cre Gsdmdfl/fl mice with CAE, and rIL37 (5 µg/kg) was administered 1 hour after AP induction. (B) H&E staining of pancreatic tissues. Scale bars: 200 or 50 µm. (C) Percentages of pancreatic cell death area, and serum amylase, lipase, and IL-1ß levels at 12 hours (n = 5–6 per group). (D) Representative images and quantitative analysis for IHC staining of CD68 and MPO (n = 3 per group). Scale bars: 50 µm. (E) Schematic diagram showing that IL-37 protects against AP through suppression of the pyroptosis pathway. Statistical comparisons were made using 1-way ANOVA. Data are presented as mean ± SD, and statistical significance is denoted as *P < 0.05, **P < 0.01, and ***P < 0.001.