CD4+CD25+ T(R) cells suppress innate immune pathology through cytokine-dependent mechanisms

Abstract

CD4(+)CD25(+) regulatory T (T(R)) cells can inhibit a variety of autoimmune and inflammatory diseases, but the precise mechanisms by which they suppress immune responses in vivo remain unresolved. Here, we have used Helicobacter hepaticus infection of T cell-reconstituted recombination-activating gene (RAG)(-/-) mice as a model to study the ability of CD4(+)CD25(+) T(R) cells to inhibit bacterially triggered intestinal inflammation. H. hepaticus infection elicited both T cell-mediated and T cell-independent intestinal inflammation, both of which were inhibited by adoptively transferred CD4(+)CD25(+) T(R) cells. T cell-independent pathology was accompanied by activation of the innate immune system that was also inhibited by CD4(+)CD25(+) T(R) cells. Suppression of innate immune pathology was dependent on T cell-derived interleukin 10 and also on the production of transforming growth factor beta. Thus, CD4(+)CD25(+) T(R) cells do not only suppress adaptive T cell responses, but are also able to control pathology mediated by innate immune mechanisms.

Figure 1.

CD4⁺CD25⁺ T cells prevent both T cell–dependent and T cell–independent intestinal inflammation. 129SvEvRAG2^−/− mice were reconstituted with purified CD4⁺ T cell subsets as indicated (4 × 10⁵ CD45RB^high T cells and/or 10⁵ CD4⁺CD45RB^lowCD25⁺ T cells or CD4⁺CD45RB^lowCD25⁻ T cells) and infected 24 h later by oral gavage with 2 × 10⁸ cfu H. hepaticus. Mice were killed 8–12 wk later and pathology in the colon (A) and cecum (B) was assessed histologically. Each symbol represents a single animal and results are representative of two similar experiments. **P < 0.01 vs. RB^hi + Hh; *P < 0.05 vs. Hh.

Figure 2.

Representative photomicrographs of hematoxylin and eosin stained intestinal tissues from 129SvEvRAG2^−/− mice that were reconstituted with purified CD4⁺ T cell subsets and/or infected with H. hepaticus as outlined in Fig. 1. (A–G) Colon sections from a control RAG2^−/− mouse (A) and from mice that received; H. hepaticus only (B), H. hepaticus and CD4⁺CD45RB^lowCD25⁺ T cells (C), CD4⁺CD45RB^high T cells only (D), CD4⁺CD45RB^high T cells and H. hepaticus (E), CD45RB^highCD4⁺ T cells plus CD4⁺CD45RB^lowCD25⁺ T cells and H. hepaticus (F), or CD45RB^highCD4⁺ T cells plus CD4⁺CD45RB^lowCD25⁻ T cells and H. hepaticus (G). (H–J) Cecum sections from a control RAG2^−/− mouse (H) and from mice that received; H. hepaticus only (I), or H. hepaticus and CD4⁺CD45RB^lowCD25⁺ T cells (J). Original magnification: ×50.

Figure 3.

H. hepaticus–triggered T cell–independent intestinal inflammation is driven by proinflammatory cytokines. 129SvEvRAG2^−/− mice were infected by oral gavage with 2 × 10⁸ cfu H. hepaticus. Where indicated, mice received 1 mg/wk anti–IL-12p40 for the first 2 wk after infection or 2 mg/wk anti-TNF-α throughout the course of the infection. Mice were killed 8–12 wk later and pathology in the cecum was assessed histologically. Each symbol represents a single animal and results are representative of two similar experiments. **P < 0.01 vs. anti–IL-12p40 or anti-TNF-α.

Figure 4.

H. hepaticus infection of RAG2^−/− mice induces systemic activation of innate immunity that is inhibited by CD4⁺CD25⁺ T cells. 129SvEvRAG2^−/− mice were reconstituted with 10⁵ CD4⁺ CD45RB^lowCD25⁺ T cells and/or infected by oral gavage with 2 × 10⁸ cfu H. hepaticus. Mice were killed 8–12 wk later and spleen cell populations were enumerated using FACS^® analyses. Graphs shown represent numbers of total spleen cells (A), neutrophils (B), monocytes/macrophages (C), dendritic cells (D), NK cells (E), and IFN-γ–producing NK cells (F). Each symbol represents a single animal and results are representative of two similar experiments. **P < 0.01 vs. Hh; *P < 0.05 vs. Hh.

Figure 5.

CD4⁺CD25⁺ T cell reconstitution does not affect H. hepaticus colonization levels in RAG2^−/− mice. 129SvEvRAG2^−/− mice were reconstituted with 10⁵ CD4⁺ CD45RB^lowCD25⁺ T cells and/or infected by oral gavage with 2 × 10⁸ cfu H. hepaticus. Mice were killed 10 wk later, DNA was isolated from cecum and colon samples and H. hepaticus DNA was quantified using a real-time PCR assay. Each symbol represents a single animal and results are representative of two similar experiments.

Figure 6.

CD4⁺CD25⁺ T cell–mediated protection against T cell–independent intestinal inflammation is dependent on IL-10 and TGF-β. 129SvEvRAG2^−/− mice were reconstituted with 10⁵ CD4⁺CD45RB^lowCD25⁺ T cells from wild-type or IL-10^−/− mice and/or infected by oral gavage with 2 × 10⁸ cfu H. hepaticus. Where indicated mice received 0.5 mg/wk anti–IL-10R or 2 mg/wk anti-TGF-β. Mice were killed 8–12 wk later, ceca samples were taken for histological examination. Each symbol represents a single animal and results are representative of two similar experiments. **P < 0.01 vs. Hh.