Accelerated and progressive and lethal liver fibrosis in mice that lack interleukin (IL)-10, IL-12p40, and IL-13Rα2

Abstract

Background & aims: Progressive fibrosis contributes to the morbidity of several chronic diseases; it typically develops slowly, so the mechanisms that control its progression and resolution have been difficult to model. The proteins interleukin (IL)-10, IL-12p40, and IL-13Rα2 regulate hepatic fibrosis following infection with the helminth parasite Schistosoma mansoni. We examined whether these mediators interact to slow the progression of hepatic fibrosis in mice with schistosomiasis.

Methods: IL-10(-/-), IL-12/23(p40)(-/-), and IL-13Rα2(-/-) mice were crossed to generate triple knockout (TKO) mice. We studied these mice to determine whether the simultaneous deletion of these 3 negative regulators of the immune response accelerated mortality from liver fibrosis following infection with S mansoni.

Results: Induction of inflammation by S mansoni, liver fibrosis, and mortality increased greatly in TKO mice compared with wild-type mice; 100% of the TKO mice died by 10 weeks after infection. Morbidity and mortality were associated with the development of portal hypertension, hepatosplenomegaly, gastrointestinal bleeding, ascites, thrombocytopenia, esophageal and gastric varices, anemia, and increased levels of liver enzymes, all features of advanced liver disease. IL-10, IL-12p40, and IL-13Rα2 reduced the production and activity of the profibrotic cytokine IL-13. A neutralizing antibody against IL-13 reduced the morbidity and mortality of the TKO mice following S mansoni infection.

Conclusions: IL-10, IL-12p40, and IL-13Rα2 act cooperatively to suppress liver fibrosis in mice following infection with S mansoni. This model rapidly reproduces many of the complications observed in patients with advanced cirrhosis, so it might be used to evaluate the efficacy of antifibrotic reagents being developed for schistosomiasis or other fibrotic diseases associated with a T-helper 2 cell-mediated immune response.

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 μmoles/gram of liver tissue, μmoles/total liver and μmoles/10,000 S. mansoni eggs (C) granuloma size at 8 wk post-infection. Each circle represents an individual mouse and bars the mean ± SEM. Data representative of 4 independent experiments. (D) Picrosirius red-stained paraffin-embedded liver sections at 8wk pi (E) Cytokine production by liver granuloma-associated CD4+ lymphocytesat 7.5wk pi.A representative analysis of two independent studies is shown. Scatter plot graphs depict CD4+ cytokine analysis (as in E) of two combined experiments with the horizontal bar showing the mean. Stars indicate significance in two-tailed Student's t-test; **P<0.005.

Figure 2. Compensatory expression of endogenous negative regulators in the liver

(A) Gene expression in the liver of BALB/c (N=9), IL-13α^−/− (N=7), IL-10^−/− (N=8), IL-12^−/− (N=7), IL-10/IL-12(p40)^−/− (N=8), IL-12(p40)/IL-13α2^−/− (N=6), IL-10/IL-13Rα2^−/− (N=7) and IL-10/IL-12(p40)/IL-13α2^−/− (N=5) mice after 8wk 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 two independent experiments.

Figure 3. Exacerbated liver fibrosis in mice lacking one, two, or three endogenous regulators of IL-13

(A) The mice shown were infected with 35 cercariae of S.mansoni then sacrificed wk8 pi and fibrosis measured as liver hydroxyproline content. Because of variations in infection intensity between individual mice, the final 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 combined from two independent experiments and expressed as box-and whisker plots with the horizontal line representing the median and whiskers extended to highest and lowest values with outliers shown at circels. (Outliers defined as lower than the 1st percentile and greater than the 99th percentile). Liver hydroxyproline values were significantly lower in all BALB/c WT, single, and double KO strains relative to TKO mice (p≤0.003 for all groups) (B) Liver procollagen 6a1 mRNA levels at 8wk pi presented as the `fold increase' relative to expression in livers from naïve mice. (*, p ≤ 0.03 for TKO mice compared to all seven control strains. (C) Survival following infection with 35 cercariae of S.mansoni (≥9 mice per group). TKO survival 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).

Figure 4. Anemia, thrombocytopenia and splenomegaly are exacerbated in infected TKO mice

(A) RBCs, platelets, WBCs, hemoglobin and hematocrit in the blood of 8wk S.mansoni-infected mice (n=5–8 mice per group). (B) Tortuous varix detected along the serosal surface of 8 wk infected TKO and engorged mesenteric venules are more prounounced in infected TKO mice (C) Splenomegaly depicted as % body weight (n=8 mice per group) and an image of three representative spleens shown from BALB/c and TKO mice. (D) Liver enzymes in serum at 8wk pi including: aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALK) (n ≥ 4 mice per assay) (E) Fecal occult blood detection at 6 and 8WK pi. *, P ≤ 0.02, **, P ≤ 0.002, ***, P ≤ 0.0002.

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 wk 5 and 8 treated with either rat-anti-mouse IL-13(IgG2a) or control (GL113) Ab at 0.5mg per mouse/week intraperitoneally (IP). Mice were sacrificed on wk8 pi and fibrosis measured as liver hydroxyproline (n= 6–9 mice per group). (B) Whole liver procollagen 6a1 mRNA expression determined by real time PCR. (C) Survival of S.mansoni infected TKO treated with anti-IL-13 or cIgG. (D) Picrosirius red stained liver sections under polarized light on 5× magnification. (E) BALB/c and TKO mice infected then treated with control IgG or anti-IL-13 and heparinized blood was analyzed for RBCs, platelets, hemoglobin and hematocrit.

Figure 6. Anti-IL-13 treatment rescues TKO mice from death following S.mansoni infection

Thirty TKO mice were exposed to 25–35 cercariae of S.mansoni. From WK5 through WK12 pi, 10 of the mice were left untreated (closed circle); 10 mice received cntrl IgG (open square) and 10 mice received rat anti-IL-13 Ab (closed square). Ab treatment was administered I.P. at 0.5mg/mouse at 2×/week and survival monitored weekly. (B) Representative post-mortem images of TKO under naïve, control IgG- or anti-IL-13- treated conditions at 8wk pi.