Volume 141, Issue 6, Pages 2200-2209 (December 2011) Accelerated and Progressive and Lethal Liver Fibrosis in Mice That Lack Interleukin (IL)- 10, IL-12p40, and IL-13Rα2  Margaret M. Mentink–Kane, Allen W. Cheever, Mark S. Wilson, Satish K. Madala, Lara Megan Beers, Thirumalai R. Ramalingam, Thomas A. Wynn  Gastroenterology  Volume 141, Issue 6, Pages 2200-2209 (December 2011) DOI: 10.1053/j.gastro.2011.08.008 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Increased fibrosis, inflammation, and mortality in TKO mice. (A) Survival of S mansoni–infected BALB/c (n = 11) and TKO (n = 13) mice, representative of 5 independent experiments. (B) Liver hydroxyproline content as μmol/g of liver tissue, μmol/total liver, and μmol/10,000 S mansoni eggs and (C) granuloma size at 8 weeks postinfection.13 Each circle represents an individual mouse, and bars represent the mean ± SEM. Data are representative of 4 independent experiments. (D) Picrosirius red–stained paraffin-embedded liver sections at 8 weeks postinfection. (E) Cytokine production by liver granuloma-associated CD4+ lymphocytes at 7.5 weeks postinfection. A representative analysis of 2 independent studies is shown. Scatter plot graphs depict CD4+ cytokine analysis (as in E) of 2 combined experiments, with the horizontal bar showing the mean. Asterisks indicate significance in 2-tailed Student t test; **P < .005; ***P < .001. Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Compensatory expression of endogenous negative regulators in the liver. (A–F) Gene expression in the liver of BALB/c (n = 9), IL-13Rα2−/− (n = 7), IL-10−/− (n = 8), IL-12−/− (n = 7), IL-10/IL-12(p40)−/− (n = 8), IL-12(p40)/IL-13Rα2−/− (n = 6), IL-10/IL-13Rα2−/− (n = 7), and IL-10/IL-12(p40)/IL-13Rα2−/− (n = 5) mice 8 weeks after infection with S mansoni. Gene expression is normalized to HPRT and presented as the fold increase relative to expression in livers from naïve BALB/c mice. The analysis is representative of 2 independent experiments. Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Exacerbated liver fibrosis in mice lacking 1, 2, or 3 endogenous regulators of IL-13. (A) The mice shown were infected with 35 cercariae of S mansoni and then killed 8 weeks postinfection, and fibrosis was measured as liver hydroxyproline content. Because of variations in infection intensity between individual mice, the final hydroxyproline (hypro) values were adjusted to take into account the number of S mansoni eggs deposited in the liver. The reported hypro values are hypro/gram adjusted to 10,000 tissue eggs. Data are combined from 2 independent experiments and expressed as box and whisker plots, with the horizontal line representing the median and whiskers extended to the highest and lowest values and outliers shown as circles. (Outliers were defined as lower than the first percentile and greater than the 99th percentile.) Liver hydroxyproline values were significantly lower in all BALB/c WT, single KO, and double KO strains relative to TKO mice (**P ≤ .003 for all groups). (B) Liver procollagen 6a1 mRNA levels at 8 weeks postinfection presented as the fold increase relative to expression in livers from naïve mice. *P ≤ .03 for TKO mice compared with all 7 control strains. (C) Survival following infection with 35 cercariae of S mansoni (≥9 mice per group). TKO survival was significantly accelerated relative to all 7 controls. Data are representative of 3 independent survival experiments (data for single KO mice not shown for figure clarity). Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Anemia, thrombocytopenia, and splenomegaly are exacerbated in infected TKO mice. (A) Red blood cells (RBC), platelets, white blood cells (WBC), hemoglobin, and hematocrit in the blood of 8-week S mansoni–infected mice (n = 5–8 mice per group). (B) Tortuous varix detected along the serosal surface of 8-week infected TKO mice and engorged mesenteric venules are more pronounced in infected TKO mice. (C) Splenomegaly depicted as percent body weight (BW) (n = 8 mice per group) and an image of 3 representative spleens shown from BALB/c and TKO mice. (D) Liver enzyme levels in serum at 8 weeks postinfection, including aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALK) (n ≥ 4 mice per assay). (E) Detection of fecal occult blood at 6 and 8 weeks postinfection. *P ≤ .02, **P ≤ .002, ***P ≤ .0002. Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Blocking IL-13 inhibits liver fibrosis and reverses anemia following S mansoni infection. (A) Mice infected with 35 cercariae of S mansoni and between weeks 5 and 8 treated with either rat anti-mouse IL-13 (IgG2a) or control (GL113) antibody at 0.5 mg per mouse per week intraperitoneally. Mice were killed at week 8 postinfection and fibrosis was measured as liver hydroxyproline (n = 6–9 mice per group). (B) Whole liver procollagen 6a1 messenger RNA expression determined by real-time polymerase chain reaction. (C) Survival of S mansoni–infected TKO mice treated with anti–IL-13 or control IgG (cIgG). (D) Picrosirius red–stained liver sections under polarized light on 5× magnification. (E) BALB/c and TKO mice infected and then treated with cIgG or anti–IL-13 and heparinized blood was analyzed for red blood cells, platelets, hemoglobin, and hematocrit. *P ≤ .03; **P ≤ .005; ***P ≤ .001. Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Anti–IL-13 treatment rescues TKO mice from death following S mansoni infection. (A) Thirty TKO mice were exposed to 25 to 35 cercariae of S mansoni. From week 5 to week 12 postinfection, 10 of the mice were left untreated (closed circles); 10 mice received control IgG (open squares), and 10 mice received rat anti–IL-13 antibody (closed squares). Antibody treatment was administered intraperitoneally at 0.5 mg/mouse twice per week and survival monitored weekly. (B) Representative postmortem images of TKO mice under naïve, control IgG–treated, or anti–IL-13—treated conditions at week 8 postinfection. Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 Gastroenterology 2011 141, 2200-2209DOI: (10.1053/j.gastro.2011.08.008) Copyright © 2011 AGA Institute Terms and Conditions