TRIF mediates Toll-like receptor 5-induced signaling in intestinal epithelial cells

Abstract

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.

FIGURE 1.

TLR5 interacts with TRIF and MyD88, but not with TRAM upon flagellin stimulation in human colonic epithelial (NCM460) cells. A and B, NCM460 cells transfected with TLR5-HA were stimulated with flagellin (100 ng/ml). Lysates were immunoprecipitated (IP) using HA antibody and immunoblotted (IB) using antibodies to endogenous TRIF (A) and MyD88 (B). C, NCM460 cells were stably co-transfected with TLR5-HA and TRAM-FLAG constructs. HEK293 cells were co-transfected with TLR5-HA and MyD88-FLAG constructs. After flagellin stimulation, cell extracts were immunoprecipitated using HA antibody and immunoblotted using FLAG antibody. D, THP-1 cells and NCM460 cells were stimulated with LPS (1.0 μg/ml) and flagellin, respectively. Total cell extract was used for immunoblotting to evaluate IRF3 phosphorylation (P-IRF3) and NFκB activation (P-p105). Akt blotting was measured as a loading control. All data are representative of at least three independent experiments.

FIGURE 2.

Silencing TRIF expression reduces TLR5-dependent signaling in NCM460 cells. A, MyD88-KD NCM460 cells (14) and the control cells transfected with the scrambled shRNA construct were stimulated with flagellin (100 ng/ml), followed by evaluating NFκB (P-p105 and P-p65) and MAPK (p38, JNK1/2, and ERK1/2) activation. Silencing MyD88 expression was also confirmed. B, TRIF-KD NCM460 cells were generated by stably transfecting human TRIF shRNA expression construct into NCM460 cells. TLR5-induced signaling was examined in TRIF-KD cells. Silenced TRIF expression was confirmed by immunoblotting. C, MyD88/TRIF-2KD cells were generated by stably transfecting TRIF shRNA encoding construct into MyD88-KD cells. Flagellin-induced NFκB and MAPK activation was examined by immunoblot analysis. Proper loading controls for the immunoblotting were included as indicated. Data presented are representative experiments of at least three independent experiments.

FIGURE 3.

TRIF deficiency inhibits TLR5-induced signaling in primary mouse intestinal epithelial cells. A, epithelial organoids (red arrows) were harvested from small intestine of C57BL/6 mice and attached to fibronectin-coated culture surfaces. Proliferative and viable cells were spread out from these organoids in 4–7 days. A representative image was obtained in 2 weeks after plating. Scale bar, 200 μm. B, the cells from small intestine exhibited evident responses to flagellin (100 ng/ml) as shown by NFκB (P-p65) and MAPK (P-ERK1/2) activation. C and D, the cells from mouse small intestine were immunostained with monoclonal antibodies against epithelial cell markers, β-catenin or cytokeratin (21) (green). DAPI staining (blue) represents nuclei. Scale bars, 50 μm. E and F, primary intestinal epithelial cells from MyD88-KO (E), TRIF-KO (F), and WT (C57BL/6) mice were stimulated with flagellin (100 ng/ml) for the indicated time, followed by immunoblot assays. All data are representative of at least three independent experiments. β-Actin served as a loading control.

FIGURE 4.

Activated intracellular signaling by the flagellin preparation is not affected by the potential LPS contaminant, but rather is specifically mediated by TLR5. A, primary mouse intestinal epithelial cells from C3H/HeJ (TLR4-null) and its control C3H/HeOuJ mice were stimulated with the flagellin preparation (100 ng/ml), followed by evaluating NFκB and MAPK activation. B, TLR5-KD NCM460 and its control cells (14) were stimulated with the flagellin preparation. NFκB activation was abolished in TLR5-KD cells, whereas strong NFκB activation was observed in control cells. C, NCM460 cells are not responsive to TLR2 (Pam3Cys, 2.0 μg/ml) and TLR4-specific ligand (LPS, 2.0 μg/ml), but strongly responsive to TLR5 specific ligand (flagellin, 100 ng/ml). IL-1 receptor ligand (IL-1β, 50 ng/ml) was used as a positive control of NFκB-luciferase reporter assay in NCM460 cells. Error bars indicate ±S.D. All data are representative of at least three independent experiments.

FIGURE 5.

TRIF deficiency inhibits flagellin-induced cytokine expression. A–C, primary mouse intestinal epithelial cells from WT, MyD88-KO, and TRIF-KO mice were stimulated with flagellin (100 ng/ml, 8 h). KC (A), macrophage inflammatory protein 3α (MIP-3α) (B), and IL-6 (C) expression was measured by ELISA. D and E, WT, MyD88-KO, and TRIF-KO cells were stimulated with IL-1β (100 ng/ml, 8 h) (D) and TNFα (100 ng/ml, 8 h) (E), respectively. The expression of KC was measured by ELISA. F, when primary intestinal epithelial cells were stimulated with flagellin, in parallel, mouse monocytes/macrophages (RAW264.7) were stimulated with LPS (1.0 μg/ml, 8 h) to elicit IFN-β expression which was used as a positive control for IFN-β ELISA (D). Error bars indicate ±S.D. *, p < 0.01 (versus WT) as determined by one-way ANOVA followed by Bonferroni's t test.

FIGURE 6.

TRIF deficiency confers protective effects against DSS-induced colitis and flagellin-mediated exacerbation of DSS-induced colitis. A–C, flagellin-exacerbated DSS-induced colitis was relieved in TRIF-KO mice compared with WT mice. TRIF-KO (n = 14) and WT (n = 21) mice were fed with DSS (3%) for total 9 days, and mice were treated with flagellin enema (0.8–1.0 μg/mouse/day) during the last 5 days. A, log-rank test was used to compare significant survival difference, followed by the multiple-comparison Bonferroni method (p < 0.01) B, representative H&E-stained mid- and proximal colon tissues were prepared from flagellin-exacerbated DSS-induced colitis of TRIF-KO and WT mice presented in A. Scale bars, 100 μm. C, body weight change for these mice was evaluated. D, TRIF-KO (n = 16) and WT (n = 16) mice were fed with a sublethal dose (2%) of DSS over 26 days to induce mild colitis, and weight change was measured. Data for body weight change were compared by ANOVA, followed by the multiple-comparison Bonferroni t test to assess differences between groups. E and F, MyD88-KO mice are more susceptible to DSS-induced colitis than TRIF-KO and WT mice. TRIF-KO (n = 27), MyD88-KO (n = 24), and WT (C57BL/6) (n = 12) mice were provided with DSS (4%) to induce colitis. Difference in the survival is shown by a Kaplan-Meier plot. The log-rank test was used to compare significant survival difference between MyD88-KO and TRIF-KO mice group (E) and MyD88-KO and WT mice group (F), and followed by the multiple-comparison Bonferroni method (p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05.