Abstract
Background&Aim: Small intestinal Paneth cells contnbute to mucosal innate immunity by sensing bactena and secreting microbicidaI peptides, particularly a-defensins (Nature Immunol l: 113, 2000). Our aims were to investigate the expression and function of Toll-like receptor (TLR)s and related molecules in crypts of human small intestine. Methods: Ileal mucosa were obtained from surgical specimens resected from patients with colon cancer with normal ileum under informed consent. Intact crypts were isolated by standard EDTA dissociation, and further collagenase dissociation was conducted to prepare crypt single cell s0spensions. RT-PCR experiments were performed on individual crypts or isolated Paneth cells to assay for TLRI-10, CD14, MD2 and MyD88 mRNAs. Specific TLRs were immunocolocalized in isolated crypts and in Paneth cells with a-defensin HD5 using anti-TLR-2 and TLR-4 antibodies and confocal microscopic analysis. Isolated intact crypts were exposed to I x l03 S. typhimurium CFU/crypt to induce Paneth cell secretion. Secretions were assayed for bactericidal activity against defensin sensitive S. typhimurium phoPand for HD5 secretion by western blot analysis. Inhibition assay was performed using anti-TLR2 neutralizing antibody in the secretion assay system Results: TLR1-8, TLR10, CD14 and MyD88, but not M02 mRNAs were detected in single isolated crypts and isolated Paneth cells. TLR2 and ]-LR4 expression was restricted to Paneth cells in the isolated crypts. TLR4 staining in single Paneth cells was localized at the membrane surface and also near secretory granules, aDefensin secretion by Paneth cells in response to bacteria was inhibited by TLR2 antibody pre-treatment as judged by the diminished bactencidal activity and reduced secretion of HD5 in western blots. Conclusion: Paneth cells in human small intestinal crypts express Toll-like receptors that appear to be implicated in ex vivo responses to bacterial exposure.
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