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. 2005 Sep 20;102(38):13610-5.
doi: 10.1073/pnas.0502174102. Epub 2005 Sep 12.

Pathophysiological role of Toll-like receptor 5 engagement by bacterial flagellin in colonic inflammation

Sang Hoon Rhee  1 , Eunok Im, Martin Riegler, Efi Kokkotou, Michael O'brien, Charalabos Pothoulakis
Affiliations

Pathophysiological role of Toll-like receptor 5 engagement by bacterial flagellin in colonic inflammation

Sang Hoon Rhee et al. Proc Natl Acad Sci U S A. .

Abstract

Commensal and enteroinvasive microbes in the human gut release bacterial flagellin, a specific microbial ligand of Toll-like receptor 5 (TLR5). However, the pathophysiological role of bacterial flagellin in gastrointestinal inflammation has not been determined. Here we evaluated the role of bacterial flagellin using native human colonic mucosa and the mouse colitis model of dextran sulfate sodium (DSS). We demonstrate that, in intact human colonic mucosa, the flagellin/TLR5 response occurs only after exposure to the basolateral, not the apical, surface, implying a basolaterally polarized TLR5 response in human colonic mucosa. In this context, flagellin exposure to injured colonic mucosa due to DSS administration in mice resulted in a TLR5-associated response evaluated by in vivo activation of mitogen-activated protein kinase/extracellular signal-related kinase 1/2 (MEK1/2) and elevated IL-6, TNF-alpha, and keratinocyte-derived chemokine production, whereas intact colonic mucosa did not respond to flagellin. Moreover, flagellin exposure to injured mouse colon in vivo, but not to intact colon, also significantly aggravated colonic inflammation, increased mouse mortality, and enhanced histopathological damage in the colonic mucosa. However, the TLR2-specific agonist, peptidoglycan or lipoteichoic acid, did not cause an inflammatory response in intact or DSS-injured mouse colon. Furthermore, intracolonic flagellin administration in mice causes severe apoptosis in colonic epithelium disrupted by DSS administration. These data suggest that intracolonic flagellin via TLR5 engagement is able to elicit inflammatory responses in disrupted colon, whereas the normal colon is not responsive to bacterial flagellin. These results demonstrate that bacterial flagellin plays an important role in the development and progress of colitis.

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Figures

Fig. 1.
Fig. 1.
The basolateral, but not apical, aspect of human colonic mucosa is responsive to flagellin stimulation. Human colonic mucosa mounted in Ussing chambers was stimulated either apically or basolaterally with flagellin (100 ng/ml) for 2 and 4 h. IL-8 production was evaluated in the respective supernatants by ELISA. Data are expressed as mean ± SEM. Results are representative of six separate experiments, each with triplicate determinations. *, P < 0.05.
Fig. 2.
Fig. 2.
Flagellin exposure to disrupted colon induces in vivo activation of MEK1/2 in colonic mucosa. C3H/HeJ mice received either water alone or water containing 2.5% DSS through the entire experimental period. Four days after initiation of DSS treatment, mice were colorectally exposed to flagellin for 7 days. (A) Total protein prepared from colonic tissue extracts was subjected to Western blot analysis to determine MEK1/2 activation by using a phospho-MEK1/2 antibody. OD values obtained from x-ray films were expressed as relative ratio of phosphorylated to inactive MEK1/2. Data are expressed as mean ± SEM of five mice per group. *, P < 0.01. (B) Frozen colonic tissue sections obtained from the same mouse groups were exposed to a primary antibody recognizing phosphorylated MEK1/2, followed by a FITC-conjugated anti-rabbit antibody (green). The sections were counterstained with DAPI to localize cell nuclei (blue). Shown here are representative images from each experimental group.
Fig. 3.
Fig. 3.
Flagellin stimulates cytokine production in colonic explants obtained from DSS-treated mice. C3H/HeJ mice received either water alone or water containing 2.5% DSS for 7 days. Colon segments (1 × 0.5 cm) were then taken and incubated with flagellin (500 ng/ml) for 15 h. The level of IL-6, keratinocyte-derived chemokine (KC), and TNF-α was measured in the supernatants by ELISA. Data are expressed as mean ± SEM (n = 4 mice per group, each with triplicate determinations). *, P < 0.01; **, P < 0.05.
Fig. 4.
Fig. 4.
Flagellin aggravates clinical symptoms of colonic inflammation in DSS-induced colitis. C3H/HeJ mice received either water alone or water containing 2.5% DSS for the entire experimental period. Four days after the beginning of DSS administration, mice were injected intracolonically with flagellin (0.8-1.0 μg per mouse) daily for 14 days. Kaplan-Meier survival analysis showed that flagellin administration increased mortality of C3H/HeJ (C3H/HeJ, P < 0.001; log rank). Several clinical parameters of colonic inflammation were also increased by flagellin exposure to injured colon by DSS administration [C3H/HeJ mice: water alone (n = 22), water plus flagellin (n = 24), DSS alone (n = 21), and DSS plus flagellin (n = 26).
Fig. 5.
Fig. 5.
Flagellin exposed to injured colon causes severe histopathological damage in colonic mucosa. C3H/HeJ mice received either water alone or water containing 2.5% DSS through the entire experimental period. Four days after initiation of DSS treatment, mice were injected intracolonically with flagellin (0.8-1.0 μg per mouse) daily for 7 days. (A) Mice exposed to flagellin and DSS showed severe rectal bleeding and hemorrhagic colon (representative of ≈20 mice per experimental condition). (B) Colonic tissue sections obtained from mice at day 7 after flagellin administration were fixed in formalin, paraffin embedded, and stained with hematoxylin/eosin (×200, Upper; ×600, Lower) (C) Histopathologic scoring for several parameters of colonic inflammation was evaluated as described in Materials and Methods [water alone (n = 14), water plus flagellin (n = 14), DSS alone (n = 15), DSS plus flagellin (n = 19)]. *, P < 0.001; **, P < 0.01.
Fig. 6.
Fig. 6.
Flagellin induces apoptosis in the compromised colonic mucosa. C3H/HeJ mice received either water alone or water containing 2.5% DSS through the entire experimental period. Four days after initiation of DSS treatment, mice were colorectally exposed to flagellin for 7 days. (A) Total protein prepared from colon tissues of each mice group was subjected to Western blot analysis with an antibody recognizing cleaved Caspase-3. OD values obtained from the x-ray films were expressed as relative ratio of cleaved Caspase-3 to inactive ERK1/2 serving as a loading control. Data are expressed as mean ± SEM of six mice per group. (B) Frozen colonic tissue sections obtained from the same mice groups were stained with DAPI to examine formation of apoptotic bodies, indicated by red arrows. (C) TUNEL assays were performed in frozen tissue sections followed by DAPI counterstaining of the nuclei. Slides were visualized and photographed under a fluorescent microscope. Values represent mean scores for the TUNEL-positive stain (overlapped TUNEL with DAPI-stained nuclei) ± SEM. *, P < 0.001; **, P < 0.01.
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