Fig 1: Control of GM-CSF Production in Synovial ILCs by IL-2, IL-33, and TLR-9 Ligands(A) Quantity of the active form IL-33 assessed by ELISA (Biolegend, Mouse IL-33 ELISA kit) in indicated tissue homogenates.(B) Quantitative RT-PCR for Il33 expression and ELISA for IL-33 protein in arthritic or control joints. Symbols represent individual mice.(C) Production of cytokines by synovial ILCs. Synovial ILCs (5 × 103) from arthritic joints were purified and cultured for 24 hr with rhIL-2 (20 U/mL), rmIL-7 (20 ng/mL), and rmIL-33 (20 ng/mL) alone or in combination. The concentration of IL-5, IL-13, and GM-CSF in the supernatant was measured (n = 3).(D) Quantitative RT-PCR analysis of the expression of indicated TLR genes in naive CD4+ T cells and synovial ILCs as shown in Figure 4M (n = 3).(E) GM-CSF production by synovial ILCs. Synovial ILCs (5 × 103) were cultured for 24 hr with poly(I:C) (1 μg/mL), LPS (1 μg/mL), and CpG DNA (1 μM) alone or in combination with rmIL-33 (20 ng/mL). The concentration of GM-CSF in the supernatant was measured (n = 3).∗∗p < 0.01. Data are representative of at least two independent experiments. Horizontal bars indicate the means in (A) and (B). Vertical bars indicate SD in (C)–(E).
Fig 2: Tropisetron suppresses chronic pancreatitis in WT mice. (a) Representative images of hematoxylin and eosin (H&E)-stained pancreases from WT mice treated with tropisetron or PBS, following the caerulein-induced chronic pancreatitis protocol. (b) Representative images of CD45-stained pancreases from WT mice treated with tropisetron or PBS, following the caerulein-induced chronic pancreatitis protocol. (c) CD45+ immune cell counts in the pancreases of WT mice treated with tropisetron or PBS at the conclusion of the caerulein treatment protocol. Each dot represents cell counts from a high power field (HPF) image. Five randomly selected HPF images are included per sample (n = 7 in tropisetron and n = 4 in the PBS group). (d) Representative images of F4/80-stained pancreases from WT mice treated with tropisetron or PBS, following the caerulein-induced chronic pancreatitis protocol. (e) F4/80+ macrophage counts in the pancreases of WT mice treated with tropisetron or PBS at the end of the caerulein treatment protocol. Each dot represents cell counts from an HPF image. Two or three randomly selected HPF images are included per sample (n = 7 in tropisetron and n = 6 in the PBS group). (f) Il33 expression in the pancreases of WT mice treated with tropisetron or PBS at the conclusion of the caerulein treatment protocol (n = 7 in the tropisetron group and n = 7 in the PBS group). (g) IL-33 protein levels in the pancreases of WT mice treated with tropisetron or PBS at the conclusion of the caerulein treatment protocol (n = 7 in the tropisetron group and n = 6 in the PBS group). (h) Representative images of IL-33 and p-IRF3-stained pancreas from WT mice treated with tropisetron or PBS, following the caerulein-induced chronic pancreatitis protocol. (i) IL-33 and p-IRF3 double-positive cell counts in the pancreas of WT mice treated with tropisetron (n = 7 mice) versus PBS (n = 5 mice) at the conclusion of the caerulein treatment protocol. Each dot represents cell counts from an HPF image. Three randomly selected HPF images are included per sample. Scale bars: 100 μm. *: p < 0.05, ***: p < 0.0001, two-sided unpaired t-test. Bar graphs show mean + SD.
Fig 3: Tropisetron blocks IRF3 phosphorylation and IL-33 expression. (a) Il33 expression in poly(I:C)-stimulated Pam212 cells treated with either received tropisetron or DMSO (n = 4 in each group). (b) Immunoblot of p-IRF3, IRF3, and GAPDH proteins in whole cell lysates of poly(I:C)-treated Pam212 cells that received tropisetron versus DMSO. (c) The ratio of p-IRF3/IRF3 and IRF3/GAPDH protein band intensity from Pam212 immunoblots (n = 3 in each group). (d) Il33 expression in poly(I:C)-treated 839WT cells that received tropisetron or DMSO (n = 6 in each group). (e) Immunoblot of p-IRF3, IRF3, and GAPDH proteins in whole cell lysates of poly(I:C)-treated 839WT cells that received tropisetron versus DMSO. (f) The ratio of p-IRF3/IRF3 and IRF3/GAPDH protein band intensity from 839WT immunoblots (n = 3 in each group). The uncropped blots are shown in Figure S4. *: p < 0.05, **: p < 0.01, ***: p < 0.0001, ns: not significant, one-way ANOVA with Tukey’s multiple comparisons test. Bar graphs show mean + SD.
Fig 4: IL-33/Treg axis is essential for HBV+DEN-induced liver cancer.a Representative macroscopic image of TregST2CKO and WT liver treated with HBV+DEN at 8 months post-infection. The arrow points to a liver tumor. b Tumor burden measured as % liver surface area of TregST2CKO (n = 12) and WT (n = 6) mice that received HBV+DEN at 8 months post-infection. Each dot represents a mouse. Experimental data were verified in two independent experiments. c Representative images of hematoxylin and eosin (H&E) stained TregST2CKO and WT liver treated with HBV+DEN at 8 months post-infection. d Representative images of PCNA stained TregST2CKO and WT liver treated with HBV+DEN at 8 months post-infection. e Quantification of PCNA+ hepatocytes in TregST2CKO (n = 8) and WT (n = 5) mice that received HBV+DEN at 8 months post-infection. PCNA+ cells per 100 hepatocytes were counted in five to eight randomly selected HPF images of each liver. Each dot represents an HPF image. f IL-10+ Treg frequency in TregST2CKO (n = 7) and WT (n = 7) liver treated with HBV+DEN at 4 months post-infection. Each dot represents a mouse. g Representative images of CD3 and CD8 stained TregST2CKO and WT liver treated with HBV+DEN at 4 months post-infection. Insets highlight T cells in the liver. h Quantification of CD3+ T cells in TregST2CKO (n = 4) and WT (n = 3) liver treated with HBV+DEN at 4 months post-infection. CD3+ cells were counted in three to four randomly selected HPF images per liver sample. Each dot represents an HPF image. i Quantification of CD8+ T cells in TregST2CKO (n = 4) and WT (n = 3) liver treated with HBV+DEN at 4 months post-infection. CD8+CD3+ cells were counted in three to four randomly selected HPF images per liver sample. Each dot represents an HPF image. Graphs show mean + SD, two-sided unpaired t-test, scale bars: 1 cm (liver images) and 100 μm (histology). Source data are provided as a Source Data file.
Fig 5: Pitavastatin blocks liver cancer development in chronic inflammation marked by elevated IL-33 expression.a Representative macroscopic images of pitavastatin- and PBS-treated WT liver exposed to HBV+DEN at 8 months post-infection. Arrows point to liver tumors. b Tumor burden measured as % liver surface area of pitavastatin- (n = 10) and PBS-treated (n = 5) WT mice that received HBV+DEN at 8 months post-infection. Each dot represents a mouse. Experimental data were verified in two independent experiments. c Representative images of H&E stained pitavastatin- and PBS-treated WT liver exposed to HBV+DEN at 8 months post-infection. d Representative images of PCNA stained pitavastatin- and PBS-treated WT liver exposed to HBV+DEN at 8 months post-infection. e Quantification of PCNA+ hepatocytes in pitavastatin- (n = 4) and PBS-treated (n = 5) WT mice that received HBV+DEN at 8 months post-infection. PCNA+ cells per 100 hepatocytes were counted in five to eight randomly selected HPF images of each liver. Each dot represents an HPF image. f Serum IL-33 levels in HBV-positive hepatitis patients (n = 40), HBV-negative hepatitis patients (n = 40), and healthy controls (n = 24). Each dot represents a patient serum sample. g Representative images of IL-33 stained liver tissues from HBV-positive hepatitis patients, HBV-negative hepatitis patients, and healthy control. h A retrospective cohort analysis of hepatitis and HCC in hepatitis risk in matched cohorts of patients treated with pitavastatin (test) versus ezetimibe (control) (two-sided two-proportion z-test). i The correlation between IL33 expression levels and CD8+ T cell and Treg cell infiltration in HCC samples (n = 371) from TCGA. j The correlation between IL1RL1 expression levels and CD8+ T cell and Treg cell infiltration in HCC samples (n = 371) from TCGA. k Schematic diagram outlining the mechanism that links HBV infection to increased hepatitis and HCC risk (Created in BioRender. Demehri, S. (2025) https://BioRender.com/taqks8w). Graphs show mean + SD, (b, e) two-sided unpaired t-test, (f) one-way ANOVA with Tukey’s multiple comparison test, (i, j) two-sided t-test for Pearson correlation coefficient, scale bars: 1 cm (liver images) and 100 μm (histology). Source data are provided as a Source Data file.
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