Interleukin-5 (IL-5) augments the progression of liver fibrosis by regulating IL-13 activity

Abstract

Eosinophils are frequently found in increased numbers in a variety of chronic fibrotic diseases; however, their role in the development of hepatic fibrosis has not been dissected in vivo. Here, we used interleukin-5 (IL-5) knockout (KO) mice to determine whether eosinophils contribute to the progressive liver fibrosis that develops in response to chronic Schistosoma mansoni infection. Although infection intensities were similar in C57BL/6 and IL-5 KO mice, the average size of granulomas was significantly smaller in both acutely and chronically infected IL-5 KO mice. Their granulomas were also completely devoid of eosinophils. In addition, the knockout mice displayed over a 40% reduction in hepatic fibrosis by week 16 postinfection. The reduced fibrosis was associated with increased production of the antifibrotic cytokine gamma interferon. Moreover, although IL-13 production did not decrease consistently in the absence of IL-5, IL-13-triggered responses were substantially reduced in the granulomatous tissues. This was confirmed by analyzing the expression of several genes associated with alternative macrophage activation, including arginase 1, Fizz-1, and YM-1. Importantly, all of these IL-13-regulated genes have been linked with the mechanisms of wound healing and fibrosis. In addition to IL-5 polarizing the antigen-specific CD4+ Th2 cell response, we found that granuloma eosinophils were themselves a significant source of IL-13. Thus, by producing profibrotic mediators and polarizing the Th2 response, these findings illustrate both direct and indirect roles for eosinophils and IL-5 in the pathogenesis of schistosomiasis-induced liver fibrosis. Thus, inhibiting the activity of IL-5 or eosinophils may prove effective for a variety of chronic fibrotic diseases.

FIG. 1.

Granuloma formation is reduced in the lungs of IL-5 KO mice. WT C57BL/6 and IL-5 KO mice were sensitized with S. mansoni eggs and then challenged intravenously 2 weeks later. (A) The animals were sacrificed on day 8, and the average percentage of eosinophils in pulmonary granulomas was assessed in individual WT (n = 25) and IL-5 KO (n = 36) mice. (B) The average granuloma size was also determined. The data shown are the individual averages of 30 granulomas/mouse, and the bar denotes the average of all mice. (C) Real-time PCR was used to quantify the expression of procollagen 1 mRNA in the granulomatous tissues (n = 14 mice per group). The data shown are severalfold increases over naïve mice. Asterisks indicate a significant difference (**, P < 0.01; ***, P < 0.001) between WT and IL-5 KO animals.

FIG. 2.

IL-5 KO mice develop unpolarized type 1/type 2 cytokine responses. WT C57BL/6 and IL-5 KO mice were sensitized with S. mansoni eggs and then challenged intravenously 2 weeks later. The animals were sacrificed on day 8, and the LALN and spleens were extracted, processed for in vitro culture, and restimulated with medium alone, ConA, or SEA. Seventy-two-hour culture supernatants were assayed by ELISA for IFN-γ, IL-4, and IL-13. The data shown are averages ± standard deviations of three groups with three mice per group and are representative of several experiments performed. Asterisks denote significant differences (*, P < 0.05) between WT and IL-5 KO mice.

FIG. 3.

IFN-γ mRNA increases in the lungs of egg-challenged IL-5 KO mice. WT C57BL/6 and IL-5 KO mice were sensitized with S. mansoni eggs and then challenged intravenously 2 weeks later. The animals were sacrificed on day 8, and real-time PCR was used to analyze the cytokine response in the lungs. mRNA expression is displayed for individual mice as a severalfold increase compared to the respective naïve control group.

FIG. 4.

Markers of “alternative macrophage activation” are reduced in the lungs of egg-challenged IL-5 KO mice. WT C57BL/6 and IL-5 KO mice were sensitized with S. mansoni eggs and then challenged intravenously 2 weeks later. The animals were sacrificed on day 8, and real-time PCR was used to quantify the levels of Fizz-1 (Relmα), arginase 1, and YM-1 in the lung. The data shown are severalfold increases over the respective naïve control groups for individual mice. The asterisks indicate a significant difference (**, P < 0.01; ***, P < 0.001) between WT and IL-5 KO animals.

FIG. 5.

Inflammation and fibrosis are reduced in infected IL-5 KO mice. WT C57BL/6 and IL-5 KO mice were infected with 25 to 30 S. mansoni cercariae. (A) Groups of animals were sacrificed 9 [acute] and 16 (chronic) weeks (wk) later, and the percentage of eosinophils in liver granulomas was assessed in individual WT and IL-5 KO mice (at least 20 mice per group). (B) The average size of the granulomas was also determined. The data shown are for individual mice and represent the averages of 30 granulomas measured/mouse. The bar denotes the average of all mice in that group. (C) Hepatic fibrosis was quantified with the hydroxyproline assay. Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01; ***, P < 0.001) between WT and IL-5 KO animals, and the values shown in C represent the average decrease in hydroxyproline levels in IL-5 KO versus WT mice shown as a percentage.

FIG. 6.

Eosinophils are replaced with macrophages and fibroblasts in infected IL-5 KO mice. The cellular composition of hepatic granulomas was compared in WT and IL-5 KO mice at 9 weeks postinfection. The data shown in the pie graph represent the averages of 20 IL-5 KO mice and 19 C57BL/6 mice pooled from two separate experiments. PMNs, polymorphonuclear leukocytes; lg, large; Sm, small.

FIG. 7.

Infected IL-5 KO mice fail to polarize the type 2 cytokine response. WT C57BL/6 and IL-5 KO mice were infected with 25 to 30 S. mansoni cercariae. (A) Groups of animals were sacrificed on week 9, and the mesenteric lymph nodes (MLN) were extracted, processed for in vitro culture, and restimulated with ConA or SEA. Seventy-two-hour culture supernatants were assayed by ELISA for IFN-γ and IL-13. The data shown are the averages ± standard deviations of three separate groups with three mice included in each group (9 to 10 mice total). The data are representative of several separate experiments. (B) Real-time PCR was used to analyze the cytokine response in the liver. mRNA expression is displayed for individual mice as severalfold increases over the respective naïve control group.

FIG. 8.

Eosinophils are highly purified from the livers of infected WT mice. WT C57BL/6 mice were infected with 35 S. mansoni cercariae and sacrificed on week 9. Eosinophils were purified from perfused livers by positive selection using the Siglec-F antibody. The cells were separated into nonfractionated, eosinophil-negative, and eosinophil-positive fractions, respectively (left panels). The numbers in the boxes indicate the percentage of Siglec-F-positive eosinophils. Cytospin cells were stained with fast green to document the presence of cytoplasmic green eosinophilic granules (right panel). Magnification, ×400. Gr-1, granulocyte cell surface marker.

FIG. 9.

Granuloma eosinophils produce large quantities of IL-13. WT C57BL/6 mice were infected with 35 S. mansoni cercariae and sacrificed on week 9. Eosinophils were purified from perfused livers by positive selection using the Siglec-F antibody. The cells were separated into nonfractionated (Unsep) (open circles), eosinophil-negative (open squares), and eosinophil-positive (filled triangles) fractions. Real-time PCR was used to quantify the amounts of IL-4, IL-5, IL-13, and IFN-γ (inset) mRNA in each fraction. The data shown in the top panels represent the severalfold increases over naïve, uninfected control liver tissue. Each fraction was also placed in culture so that type 2 cytokine production could be determined by ELISA. Some cells were stimulated for 72 h with the parasite antigen preparations SEA and soluble worm antigen preparation. All of these experiments were repeated, with similar results.