Adenosine-mediated enteric neuromuscular function is affected during herpes simplex virus type 1 infection of rat enteric nervous system.

Adenosine plays an important role in regulating intestinal motility and inflammatory processes. Previous studies in rodent models have demonstrated that adenosine metabolism and signalling are altered during chronic intestinal inflammatory diseases. However, the involvement of the adenosinergic system in the pathophysiology of gut dysmotility associated to a primary neurodysfunction is still unclear. Recently, we showed that the neurotropic Herpes simplex virus type-1 (HSV-1), orally inoculated to rodents, infects the rat enteric nervous system (ENS) and affects gut motor function without signs of systemic infection. In this study we examined whether changes in purinergic metabolism and signaling occur during permanent HSV-1 infection of rat ENS. Using isolated organ bath assays, we found that contraction mediated by adenosine engagement of A1 or A2A receptors was impaired at 1 and 6 weeks post-viral administration. Immunofluorescence studies revealed that viral infection of ENS led to a marked redistribution of adenosine receptors: A1 and A2B receptors were confined to the muscle layers whereas A2A and A3 receptors were expressed mainly in the myenteric plexus. Viral-induced ENS neurodysfunction influenced adenosine metabolism by increasing adenosine deaminase and CD73 levels in longitudinal muscle-myenteric plexus with no sign of frank inflammation. This study provides the first evidence for involvement of the adenosinergic system during HSV-1 infection of the ENS. As such, this may represent a valid therapeutic target for modulating gut contractility associated to a primary neurodysfunction.

Toll-like receptor 2 regulates intestinal inflammation by controlling integrity of the enteric nervous system.

BACKGROUND & AIMS: In the intestines, Toll-like receptor 2 (TLR2) mediates immune responses to pathogens and regulates epithelial barrier function; polymorphisms in TLR2 have been associated with inflammatory bowel disease phenotype. We assessed the effects of TLR2 signaling on the enteric nervous system (ENS) in mice. METHODS: TLR2 distribution and function in the ileal neuromuscular layer of mice were determined by immunofluorescence, cytofluorimetric analysis, immunoprecipitation, and immunoblot analyses. We assessed morphology and function of the ENS in Tlr2(-/-) mice and in mice with wild-type Tlr2 (wild-type mice) depleted of intestinal microbiota, using immunofluorescence, immunoblot, and gastrointestinal motility assays. Levels and signaling of glial cell line-derived neurotrophic factor (GDNF) were determined using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and immunoprecipitation analyses. Colitis was induced by administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid to Tlr2(-/-) mice after termination of GDNF administration. RESULTS: TLR2 was expressed in enteric neurons, glia, and smooth muscle cells of the intestinal wall. Tlr2(-/-) mice had alterations in ENS architecture and neurochemical profile, intestinal dysmotility, abnormal mucosal secretion, reduced levels of GDNF in smooth muscle cells, and impaired signaling via Ret-GFRα1. ENS structural and functional anomalies were completely corrected by administration of GDNF to Tlr2(-/-) mice. Wild-type mice depleted of intestinal microbiota had ENS defects and GDNF deficiency, similar to Tlr2(-/-) mice; these defects were partially restored by administration of a TLR2 agonist. Tlr2(-/-) mice developed more severe colitis than wild-type mice after administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid; colitis was not more severe if Tlr2(-/-) mice were given GDNF before dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid. CONCLUSIONS: In mice, TLR2 signaling regulates intestinal inflammation by controlling ENS structure and neurochemical coding, along with intestinal neuromuscular function. These findings provide information as to how defective TLR2 signaling in the ENS affects inflammatory bowel disease phenotype in humans.

Herpes simplex virus type 1 infection of the rat enteric nervous system evokes small-bowel neuromuscular abnormalities.

BACKGROUND & AIMS: Infectious agents, such as neurotropic viruses, are proposed to disrupt the enteric neuromuscular system, leading to dysmotility, although the mechanisms are unknown. Our purpose was to assess whether herpes simplex virus type-1 (HSV-1) establishes an enteric-neuronal infection and induces gut dysmotility. METHODS: Rats were inoculated with HSV-1 intranasally and after 4 weeks intragastrically. After 1-10 weeks, infection was determined by molecular analysis whereas neuromuscular function was evaluated by pharmacologic/electrical stimulation of longitudinal ileal segments and by gastrointestinal transit and by [(3)H]acetylcholine release measurements. Inflammation in the neuromuscular layer was assessed by myeloperoxidase and cytokine levels and by anti-CD3(+) immunohistochemistry. RESULTS: After 1-10 weeks of intragastric inoculation, HSV-1 latency-associated messenger RNA transcripts were detected in the brain and in ileal neurons with no signs of illness or histologic gut abnormalities. By using a recombinant HSV-1 carrying the lacZ gene, HSV-1 virions were localized in myenteric ganglia by in situ X-gal staining. Interleukin-2 and IFN-gamma levels were increased significantly 1 and 6 weeks after inoculation. CD3(+) cells were found around the myenteric ganglia 6 weeks after inoculation. Smooth muscle responses to carbachol, CaCl(2), and gut transit were increased significantly after 1 and 6 weeks, whereas KCl- and electrical field stimulation-mediated contractions were modified significantly only 1-2 weeks after HSV-1 administration. The release of [(3)H]acetylcholine was reduced significantly in ileum segments after 1 and 6 weeks. CONCLUSIONS: After intragastric inoculation, HSV-1 establishes a latent infection in the rat myenteric ganglia, which leads to gut dysmotility.