Tu2114 Visceral Pain-Related Responses Are Attenuated in Mice With Antibiotic-Induced Intestinal Dysbiosis

Abstract

BACKGROUND: Toll-like Receptors (TLRs) are effector components in host-bacterial interactions. Within the gut, TLRs participate microbial-related local neuroimmune control, likely including the modulation of epithelial barrier function (EBF). AIMS: To assess changes in colonic EBF associated to the local stimulation of TLR7, with the selective agonist imiquimod, in rats. METHODS: Adult male SD rats were treated intracolonically with the TLR7 agonist imiquimod (100-300 μg/rat, 0.2 ml) or vehicle (0.2 ml/rat). 5 h later colonic samples were obtained to assess EBF in vitro (Ussing chambers). Basal epithelial electrical parameters (short-circuit current; potential difference and conductance) and effects of neural blockade with tetrodotoxin (TTX basolateral, 1 μM) on epithelial secretion were assessed. Paracellular permeability was determined assessing the flux of 4kD fluorescein isothiocyanate-dextran (FD4). Expression of tight-junction-related proteins (occludin, zona occludens-1 [ZO-1], claudin-3, tricellulin and Jam-1), the barrier-related marker proglucagon (precursor of Glucagon-like Peptide-2 [GLP-2]); cytokines (IL-6 and IFN α) and TLR7 was assessed by RTqPCR. Gene expression (RT-qPCR) and distribution (immunohistochemistry) of the poreforming protein claudin-2 were also assessed. RESULTS: Imiquimod (300 μg/rat) showed a tendency to decrease conductance (Table). TTX reduced transepithelial ionic fluxes and conductance, in a similar proportion in either non-treated or imiquimod-treated animals. Imiquimod reduced paracellular flux of FD4 by 17% and 65% at 100 and 300 μg, respectively, vs. vehicle-treated controls (Table). Gene expression of tight-junction-related proteins was not affected by imiquimod. Similarly, expression of proglucagon, the precursor of the barrierenhancer GLP-2, was not modified by imiquimod. Neither gene expression levels nor distribution of the pore-forming protein claudin-2 along the colonic crypts were affected by imiquimod. No inflammatory-like changes associated to imiquimod administration were observed either at the macroscopical, microscopical (histopathology) or molecular (gene expression of cytokines) levels. CONCLUSIONS: In rats, activation of colonic TLR7 enhanced epithelial barrier function, as indicated by the reduction in conductance and permeability to macromolecules. These effects are mediated through yet to defined mechanisms, but were independent of changes in the expression of the main tight-junction-related proteins, the GLP-2 precursor, proglucagon, or the pore-forming protein, claudin-2. Since TLRs are activated by the gut microbiota, increased epithelial tightness associated to their activation might represent a local defensive mechanism generated by dysbiosis and directed towards the prevention of bacterial translocation and the penetration of luminal antigens. Table Effects of activation of TLR7 with imiquimod on colonic EBF