Toll-like receptor 9-induced type I IFN protects mice from experimental colitis

Abstract

Experimental colitis is mediated by inflammatory or dysregulated immune responses to microbial factors of the gastrointestinal tract. In this study we observed that administration of Toll-like receptor 9 (TLR9) agonists suppressed the severity of experimental colitis in RAG1-/- but not in SCID mice. This differential responsiveness between phenotypically similar but genetically distinct animals was related to a partial blockade in TLR9 signaling and defective production of type I IFN (i.e., IFN-alpha/beta) in SCID mice upon TLR9 stimulation. The addition of neutralization antibodies against type I IFN abolished the antiinflammatory effects induced by TLR9 agonists, whereas the administration of recombinant IFN-beta mimicked the antiinflammatory effects induced by TLR9 agonists in this model. Furthermore, mice deficient in the IFN-alpha/beta receptor exhibited more severe colitis than wild-type mice did upon induction of experimental colitis. These results indicate that TLR9-triggered type I IFN has antiinflammatory functions in colitis. They also underscore the important protective role of type I IFN in intestinal homeostasis and suggest that strategies to modulate innate immunity may be of therapeutic value for the treatment of intestinal inflammatory conditions.

Figure 1

Histological evaluation. (A–F) WT mice (A and B), RAG1^–/– mice (C and D), and SCID mice (E and F) were treated with ISS-ODN (DSS + ISS; A, C, and E) or were injected with saline (DSS; B, D, and F) and 2 hours later were given 1.5% DSS in the drinking water. After 7 days, paraffin sections of the colon were prepared and stained with H&E. In WT and RAG1^–/– mice, ISS treatment markedly attenuated colitis, as shown by the absence of mucosal ulceration and inflammatory cell infiltration in the same strain not treated with ISS (A and C). In contrast, SCID mice exhibited extensive mucosal ulceration and inflammatory cell infiltration into mucosa, submucosa, and muscle regardless of ISS treatment (E and F).

Figure 2

Cytokine production after ISS-ODN stimulation. (A) Splenocytes (10⁶/ml) from RAG1^–/– and SCID mice were stimulated with ISS-1 (10 μg/ml) and cytokine levels in the supernatants were measured 48 hours later. (B) Serum levels of cytokines 2 hours after i.v. injection of 50 μg of ISS-1. Results represent 1 of 2 experiments (n = 4 per group per experiment). Similar results were obtained for ISS-2 (data not shown). The cytokine levels in naive mice were below detectable levels (data not shown). *P < 0.05 versus RAG1^–/–.

Figure 3

DNA-PK mediates TLR9-induced type I IFN production via IRF-1 and IRF-8. (A) BM-MDCs (10⁶/ml) from RAG1^–/– and SCID mice (B6) were stimulated with ISS-1 (10 μg/ml). Cytokine levels in the supernatants were measured 24 hours later. *P < 0.05 versus RAG1^–/–. (B) BM-MDCs were treated with ISS-1 (10 μg/ml) and nuclear extracts were prepared. NF-κB was detected by electrophoretic mobility-shift assay (EMSA); activation of DNA-PK, by in vitro kinase assay; and activation of IRFs, pSTAT1, and pERK, by Western blotting. Results represent 1 of 3 experiments. (C) BM-MDCs (10⁶/ml) from RAG1^–/– and SCID mice (B6) were stimulated with 50 ng/ml of LPS. After 24 hours, the cytokine levels in the supernatants were measured. (D) BM-MDCs were treated with LPS (50 ng/ml) and nuclear extracts were prepared. Signaling assays were performed as described for B. Results represent 1 of 3 experiments.

Figure 4

TLR9-activated DNA-PK mediates activation of IRFs and type I IFN via MyD88. (A) BM-PDCs were treated with ISS-1 (10 μg/ml) and nuclear extracts were prepared. NF-κB was detected by EMSA; activation of IRFs, pSTAT1, and pERK, by Western blotting. Results represent 1 of 2 experiments. (B) BM-PDCs (10⁶/ml) from RAG1^–/– and DNA-PK^–/– mice (B6) were stimulated with ISS-1 (10 μg/ml). Cytokine levels in the supernatants were measured 24 hours later. (C) BM-MDCs (10⁶/ml) from RAG1^–/– and DNA-PK^–/– mice (B6) were stimulated with ISS-1 (10 μg/ml). Cytokine levels in the supernatants were measured 24 hours later. (D) BM-MDCs (10⁶/ml) from WT and MyD88^–/– mice were stimulated with 10 μg/ml ISS-1 (ISS) or 50 ng/ml LPS. Activation of DNA-PK was measured by an in vitro kinase assay using GST-p53 as a substrate, as described in Methods. Lysates from BM-MDC from RAG1^–/– and SCID mice were stimulated with ISS-1 (10 μg/ml) and then were subjected to immunoprecipitation with anti-ubiquitin, SDS-PAGE, and immunoblotting with anti–IRF-7. Ub-, ubiquitinated. (E) Proposed localization of DNA-PK in the TLR9 signaling pathway. Ub, ubiquitin.