Essential contribution of IRF3 to intestinal homeostasis and microbiota-mediated Tslp gene induction

Abstract

The large intestinal epithelial cells and immune cells are exposed to a variety of molecules derived from commensal microbiota that can activate innate receptors, such as Toll-like receptors (TLRs) and retinoic acid-inducible gene-I-like receptors (RLRs). Although the activation of these receptors is known to be critical for homeostasis of the large intestine, the underlying gene regulatory mechanisms are not well understood. Here, we show that IFN regulatory factor (IRF)3 is critical for the suppression of dextran sulfate sodium-induced colitis. IRF3-deficient mice exhibited lethal defects in the inflammatory and recovery phases of the colitis, accompanied by marked defects in the gene induction for thymic stromal lymphopoietin (TSLP), a cytokine known to be essential for protection of the large intestine. We further provide evidence that DNA and RNA of the large intestinal contents are critical for Tslp gene induction via IRF3 activation by cytosolic nucleic acid receptors. We also demonstrate that IRF3 indeed activates the gene promoter of Tslp via IRF-binding sequences. This newly identified intestinal gene regulatory mechanism, wherein IRF3 activated by microbiota-derived nucleic acids plays a critical role in intestinal homeostasis, may have clinical implication in colonic inflammatory disorders.

Fig. 1.

DSS-induced colitis in IRF3-deficient mice. A total of 2% DSS is administered for 6 d and replaced with water on day 7. (A) Survival of WT (n = 6), IRF3-deficient (n = 5), or IRF5-deficient mice (n = 5) monitored every 24 h. (B) Colons from WT, IRF3-deficient, and IRF5-deficient mice on day 8. (C) Quantification of the colon length described in B. *P < 0.05 compared with colon from WT mice. (D) Histological analysis of H&E-stained colon sections from WT and IRF3-deficient mice treated with 2% DSS for 8 d. (Original magnification: 200×.) (E) Body weight of WT (n = 6) or IRF3-deficient mice (n = 5) monitored every 24 h. **P < 0.01 and *P < 0.05 compared with WT mice. (F) qRT-PCR analysis of Tslp and Il33 mRNA in colons from WT or IRF3-deficient mice (n = 5) on day 8. **P < 0.01 and *P < 0.05, compared with WT mice. Statistical data are presented as mean ± SD.

Fig. 2.

IRF3-dependent Tslp and Il33 gene induction by treatment with large intestinal contents. (A) qRT-PCR analysis of Tslp and Il33 mRNA in colons from WT mice treated (n = 5) or untreated (n = 3) with antibiotics. **P < 0.01 compared with untreated mice. Data are presented as mean ± SD (B) qRT-PCR analysis of Tslp and Il33 mRNA in MEFs (Left), peritoneal macrophage (PEC; Center), or bone marrow–derived dendritic cells (BMDC; Right) stimulated with large intestinal contents (enteral contents) or feces for 3 h. Data are presented as mean ± SD of triplicate determinations. (C) qRT-PCR analysis of Tslp and Il33 mRNA in WT or IRF3-deficient MEFs stimulated with feces for 3 h. **P < 0.01 compared with WT cells. Data are presented as mean ± SD of triplicate determinations.

Fig. 3.

IRF3-dependent Tslp and Il33 gene inductions by fecal nucleic acids. (A) qRT-PCR analysis of Tslp and Il33 mRNA in Trif, Mavs, or Sting knocked-down MEFs stimulated with feces for 3 h. **P < 0.01 and *P < 0.05 compared with control siRNA-treated MEFs. (B) qRT-PCR analysis of Tslp and Il33 mRNA in WT MEFs stimulated with nontreated or nuclease-treated feces for 3 h. **P < 0.01 and *P < 0.05 compared with MEFs treated with feces. (C) qRT-PCR analysis of Tslp and Il33 mRNA in WT MEFs stimulated with fecal nucleic acids (2.5, 5.0, or 10.0 μg/mL) for 6 h. (D) qRT-PCR analysis of Tslp and Il33 mRNA in WT and IRF3-deficient MEFs stimulated with B-DNA (10 μg/mL) or poly(I:C) (10 μg/mL) for indicated periods. **P < 0.01, compared with WT MEFs. (E) qRT-PCR analysis of Tslp and Il33 mRNAs in WT or IRF3-deficient MEFs transduced with retrovirus expressing WT or mutant IRF3. Cells were stimulated with B-DNA for 6 h. **P < 0.01 compared with WT MEFs. All data are presented as mean ± SD of triplicate determinations.

Fig. 4.

IRF3 activates Tslp promoter in cooperation with p65. (A) Schematic view of murine Tslp promoter and reporter genes (Upper). There are six putative IRF binding sites (ISREs) and four NF-κB sites in the Tslp promoter. The previously reported NF-κB site is κB3 (22); the arrow indicates the transcription start site. For simplicity, binding sites for other transcription factors are not denoted (32). Reporter plasmids containing ISRE sequences (Lower). (B) Reporter assay in HEK293T cells transiently cotransfected with a Tslp reporter plasmid (pTSLP-4k–Luc) and combinations of expression plasmid for IRF3-5D (100 ng) and/or p65 (1 ng); results are presented in relative light units (RLU) relative to Renilla luciferase activity. (C) Reporter assay in HEK293T cells with pTSLP-ISRE reporter plasmids performed as described in B. (D) Schematic view of pTSLP-ISRE1mt reporter genes (Upper). Reporter assay in HEK293T cells with pTSLP-ISRE1-luc and pTSLP-ISRE1mt-luc reporter plasmids and combinations of expression plasmid for IRF3-5D (100 ng) and p65 (1 ng) (Lower); results are presented as in B. **P < 0.01 and *P < 0.05 compared with RLU by pTSLP-ISRE1 reporter gene. All data are presented as mean ± SD of triplicate determinations.