Gastroenterology

Gastroenterology

Volume 132, Issue 4, April 2007, Pages 1359-1374
Gastroenterology

Basic–alimentary tract
Toll-Like Receptor 2 Controls Mucosal Inflammation by Regulating Epithelial Barrier Function

https://doi.org/10.1053/j.gastro.2007.02.056Get rights and content

Background & Aims: Toll-like receptors (TLRs) represent a class of transmembrane pattern recognition receptors essential for microbial recognition and control of innate immune responses. Commensal bacteria play an important role in maintaining tolerance and active stability of the intestinal epithelial barrier by suppressing intestinal inflammation, yet the mechanisms of action are unknown. The aim of this study was to determine the functional relevance of TLR2 to control tight junction (TJ)-associated intestinal epithelial barrier integrity to balance mucosal homeostasis against inflammatory stress-induced damage. Methods: TLR2 ligand (synthetic Pam3Cys-SK4 [PCSK])-induced activation of signaling cascades and TJ-associated distribution was assessed by using Western blotting and confocal microscopy combined with functional transfection and inhibitor studies in model intestinal epithelial cell (IEC) lines (IEC-6, Caco-2) or primary IEC cultured short-term ex vivo. DSS colitis was induced by standard protocol in wild-type, TLR2−/−, and MyD88−/− mice. Spontaneous apoptosis was assessed by terminal deoxinucleotidyl-transferase-mediated dUTP-biotin nick end-labeling. Results: Data from in vitro and ex vivo models of intestinal epithelial cells revealed that TLR2 stimulation effectively preserves TJ-associated barrier assembly against stress-induced damage through promotion of PI3K/Akt-mediated cell survival via MyD88. Furthermore, in vivo studies underscored that TLR2-mediated TJ regulation critically determines susceptibility to intestinal injury and inflammation. Inflammatory stress in mice deficient of TLR2 or MyD88 induced early TJ-associated disruption interrelated with anti-apoptotic failure of the intestinal epithelial barrier. Oral treatment of colitis with the TLR2 ligand PCSK significantly suppressed mucosal inflammation and apoptosis by efficiently restoring TJ-associated integrity of the intestinal epithelium in vivo. Conclusion: TLR2 may provide a target to pharmacologically modulate mucosal injury and intestinal inflammation.

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Reagents and Antibodies

Synthetic lipopeptide Pam3Cys-SKKKKx3HCl (Pam3CysSK4 [PCSK]; Lots #E08/02, L08, L08/02) was obtained from EMC Microcollections GmbH (Tübingen, Germany) and prepared as previously described.21 Purified lipopolysaccharide (LPS) from Escherichia coli (R515) was obtained from Alexis Biochemicals (Grünberg, Germany). LY294002 was from Cell Signaling (Beverly, MA) and wortmannin from Calbiochem (Schwalbach, Germany). Monoclonal antibodies (mAbs) to CD45, pan-cytokeratin, phospho-JNK, phospho-cJun,

TLR2-induced PI3K-Akt Limits Immune Responsiveness and Promotes IEC Survival

The PI3K/Akt-pathway controls cell survival, which plays a critical role in maintaining barrier homeostasis. We therefore first investigated the functional role of the PI3K/Akt-pathway in TLR2-mediated signaling via MyD88 in IEC. Stimulation of 2 different IEC lines (differentiated: Caco-2; nondifferentiated: IEC-6 [data not shown]) with TLR2 ligands (synthetic PCSK, PGN) resulted in significant phosphorylation of Akt and activation of downstream p70S6K and ribosomal protein S6 in a time- and

Discussion

TLRs are involved in recognition and response to gram-negative and gram-positive bacterial cell wall components and play a key role in innate immunity.4 Individual TLRs interact with different combinations of adaptor proteins and functionally activate NFκB as central transcription factor, driving TLR-specific immune responses of defense in the intestinal epithelium,5, 16 yet the molecular functions of TLRs in the intestinal mucosa are not well understood.3 Here we characterize a novel

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    Supported by DFG grants Ca226/4-1, Ca226/5-1, Ca226/4-2 (E.C.); IFORES award (E.C.); and NIH grants DK60049, DK43351, and DK41557 (D.K.P.).

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