TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Eunok Im,Charalabos Pothoulakis,Yoon Jeong Choi,Sang Hoon Rhee,Hyo Kyun Chung

doi:10.1074/jbc.m110.158394

Eunok Im, Charalabos Pothoulakis + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.158394

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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.

Highlights

Toll-like receptors (TLRs)3 are the most intensively characterized member of pattern-recognition receptors and detect microbe-associated molecular patterns present in a wide range of microorganisms [1]
TLR5 interacts with TRIF in response to flagellin, we examined whether flagellin stimulation induces the interaction between
We found that TLR5 did not recruit TRAM upon flagellin stimulation, whereas flagellin stimulation resulted in an evident interaction between TLR5 and MyD88 (Fig. 1C)

Summary

EXPERIMENTAL PROCEDURES

Materials and Mice—Human colonic epithelial cells (NCM460) were cultivated as described previously [9, 14]. This finding suggests that TRIF physically erate stably transfected cells, cells were plated in the proper interacts with TLR5 and thereby participates in mediating medium containing selecting antibiotics. Measuring KC, IL-6, Macrophage Inflammatory Protein 3␣, the phosphorylation of IRF3 in flagellin-treated NCM460 cells, and IFN-␤ Production—An enzyme-linked immunosorbent whereas LPS stimulation clearly induces IRF-3 phosphorylaassay (ELISA) was performed to measure the level of the cyto- tion in human monocytes (THP-1) (Fig. 1D). This observation kine expression using the appropriate kits from BIOSOURCE implies that TRIF adaptor in the TLR5-dependent signaling. Luciferase Reporter Assays and Immunoblot Assay—These Cells—The interaction between TLR5 and TRIF upon flagellin assays were performed as described in our previous publica- prompted us to investigate whether TRIF is involved in meditions [9, 14]

RESULTS

Our data show that the inhibitory

Findings

DISCUSSION