Interleukin 25 regulates type 2 cytokine-dependent immunity and limits chronic inflammation in the gastrointestinal tract

Abstract

The cytokine interleukin (IL) 25 has been implicated in the initiation of type 2 immunity by driving the expression of type 2 cytokines such as IL-5 and IL-13, although its role in the regulation of immunity and infection-induced inflammation is unknown. Here, we identify a dual function for IL-25: first, in promoting type 2 cytokine-dependent immunity to gastrointestinal helminth infection and, second, in limiting proinflammatory cytokine production and chronic intestinal inflammation. Treatment of genetically susceptible mice with exogenous IL-25 promoted type 2 cytokine responses and immunity to Trichuris. IL-25 was constitutively expressed by CD4+ and CD8+ T cells in the gut of mouse strains that are resistant to Trichuris, and IL-25-deficient mice on a genetically resistant background failed to develop a type 2 immune response or eradicate infection. Furthermore, chronically infected IL-25(-/-) mice developed severe infection-induced intestinal inflammation associated with heightened expression of interferon-gamma and IL-17, identifying a role for IL-25 in limiting pathologic inflammation at mucosal sites. Therefore, IL-25 is not only a critical mediator of type 2 immunity, but is also required for the regulation of inflammation in the gastrointestinal tract.

Figure 1.

Exogenous IL-25 promotes immunity to Trichuris in genetically susceptible AKR mice. AKR mice were infected with Trichuris and treated with PBS or rIL-25 intraperitoneally every other day from days 8–18. (A) IL-25 induces a protective T_H2 cytokine response. Antigen-specific IFN-γ and IL-4 production from restimulated mLN cells was determined by ELISA. White bars, medium; black bars, Trichuris antigen. (B) IL-25 promotes lymphocyte-dependent goblet cell responses. Cecal sections from day 20 infected AKR or SCID mice with or without rIL-25 treatment were stained for goblet cells. Bar, 50 μm. (C) IL-25 induces RELMβ production after infection with Trichuris. RELMβ levels were determined by immunoblotting of protein isolated from fecal pellets isolated from infected mice at days 14 and 17 after infection. (D) IL-25 promotes resistance to infection with Trichuris. Worm burdens from infected AKR or SCID mice with or without rIL-25 treatment were determined at day 20 after infection. Results are presented as mean ± SEM and are representative of three independent experiments with three to four mice per group. ND, not detected. *, P < 0.01.

Figure 2.

IL-25 is expressed constitutively in the cecal patch of mice resistant to Trichuris. (A) Increased expression of IL-25 and IL-25R mRNA in the cecum of resistant BALB/c (B/c) mice. IL-25 and IL-25R mRNA levels were determined by real-time PCR from the cecum or mLN of naive BALB/c or AKR mice. (B) CD4⁺ and CD8⁺ T cells in the cecal patch express lacZ/IL-25. Cells from the mLN or cecal patch of IL-25^−/− mice were stained with FDG and CD4 or CD8. Numbers represent frequency of FDG⁺ CD4⁺ or CD8⁺ T cells. Data are representative of three to four independent experiments with three to four mice per group.

Figure 3.

Endogenous IL-25 is required for resistance to Trichuris. (A) IL-25 is required for optimal production of T_H2 cytokines after infection. IL-4, IL-13, and IFN-γ expression in the mLN and cecum in WT and IL-25^−/− mice at day 18 after infection as measured by real-time PCR. (B) Defective goblet cell responses in IL-25^−/− mice after infection with Trichuris. Cecal sections from day 18 infected WT or IL-25^−/− mice were stained for goblet cells. Bar, 50 μm. (C) IL-25^−/− mice fail to up-regulate RELMβ after infection. Protein isolated from fecal pellets on various days after infection was analyzed by SDS-PAGE and immunoblotted for RELMβ. (D) IL-25 is required for resistance to infection with Trichuris. Worm burdens in WT, IL-25^−/− and RAG^−/− mice at day 30 after infection. Data are presented as mean ± SEM and are representative of four independent experiments with three to four mice per group. N, naive; I, infected.

Figure 4.

Type 2 inflammation is IL-25 independent when endogenous type 1 responses are inhibited. Infected IL-25^−/− mice were treated with anti–IL-12 and anti–IFN-γ every 4 d between days 4 and 20. (A) Blockade of type 1 cytokines in infected IL-25^−/− mice recovers T_H2 cytokine responses. Antigen-specific IL-4, IL-5, IL-13, and IFN-γ responses by restimulated mLN cells from control (Ig) or αIL-12/αIFN-γ–treated (α12/γ) IL-25^−/− mice determined by cytometric bead array (CBA; IL-4, IL-5, and IFNγ) or ELISA (IL-13). (B) Anti–IL-12/anti–IFN-γ treatment recovers goblet cell responses in infected IL-25^−/− mice. Goblet cell responses in the cecum of infected WT and control or αIL-12/αIFN-γ–treated IL-25^−/− mice on day 20 after infection. Bar, 50 μm. (C) IL-25 is dispensable for resistance to infection with Trichuris when type 1 responses are neutralized. Worm burdens in infected WT and control or αIL-12/αIFN-γ–treated IL-25^−/− mice were determined at day 20 after infection. Results are presented as mean ± SEM and represent two independent experiments with three to four mice per group. *, P < 0.01.

Figure 5.

IL-25 is required to limit infection-induced intestinal inflammation. WT and IL-25^−/− mice were infected with Trichuris and allowed to progress to the chronic phase of infection (day 30 after infection). (A) IL-25 is required to inhibit infection-induced pathology. Cecal sections from naive or infected (day 30) AKR, B6 WT, or IL-25^−/− mice were stained with hematoxylin and eosin. Bar, 50 μm. (B and C) IL-25 limits expression of proinflammatory cytokines in the draining mLN and gut after infection with Trichuris. (B) Increased Trichuris-specific IFN-γ and IL-17 produced by mLN cells from WT or IL-25^−/− mice at day 18 after infection as measured by CBA (IFN-γ) or ELISA (IL-17). White bars, medium; black bars, Trichuris antigen. (C) Expression of mRNA for IFN-γ and IL-17 in the mLN and cecum of WT and IL-25^−/− mice at day 30 after infection. (D) IL-10 expression in the mLN is intact in infected IL-25^−/− mice. mLN cells from WT or IL-25^−/− mice at day 18 after infection were restimulated with (black bars) or without (white bars) Trichuris antigen and IL-10 levels were determined by CBA. IL-10 mRNA levels in the mLN at day 30 after infection were determined by real-time PCR. Results are presented as mean ± SEM and represent three independent experiments with three to four mice per group. ND, not detected.