Su1731 Novel Nongenomic Bacterial Component Sensing in Duodenum via Pattern Recognition Receptors

Abstract

Duodenal brush border dual oxidase 2 (Duox2), a NADPH oxidase, generates luminal H2O2 by luminal ATP, P2Y receptor activation and intracellular Ca2+ signal. Rapid production of antimicrobial H2O2 from the duodenal surface via the ATP-P2Y-Duox2 pathway suggests the presence of luminal bacterial sensing in the duodenum. We hypothesized that the pattern recognition receptors (PRRs) Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain 2 (NOD2) are involved in the regulation of duodenal antimicrobial defenses. We measured duodenal HCO3 secretion (DBS) with pH and CO2 electrodes in the perfused duodenum of anesthetized rats. The TLR2 ligand Pam3CSK4 (Pam3, 1 μg/ml), the TLR4 ligand lipopolysaccharide (LPS, 1 μg/ml), the NOD2 ligand muramyl dipeptide (MDP, 10 μg/ml) or its control peptide murabutide (10 μg/ml) were perfused. H2O2 in the perfusate was measured with the fluorogenic substrate Amplex Red. TLR2, TLR4 and NOD2 expression on the villous brush borders, with NOD2 also present in Brunner's glands was detected by immunofluorescence. Luminal perfusion of Pam3, LPS or MDP alone had little effect on DBS or H2O2 output. Co-perfusion of Pam3 or LPS with MDP, but not with murabutide, increased DBS with a parallel increase of luminal H2O2 output. In Vivo x-z confocal images demonstrated that luminal LPS +MDP rapidly increased Amplex Red fluorescence, equivalent to H2O2 generation, from the villous surface. Furthermore, the IkB kinase inhibitor TCPA1 (1 μM) or the proteasome/NFkB inhibitor MG132 (10 μM) reduced the augmented DBS and H2O2 output, suggesting the involvement of NFkB signals. These results suggest that bacteria in the duodenal lumen are sensed by PRRs, which acutely signal the release of antimicrobial H2O2, partially via NFkB signaling. Rapid protective responses to luminal bacterial attachment, rather than genomic mechanisms, may be a highly novel component of the foregut antimicrobial defense system.