Role of Toll-Like receptor 2 in intestinal immune response and dysfunction induced by herpes symplex virus type 1 infection of murine enteric nervous system

The neurotropic virus herpes simplex virus type 1 (HSV-1) is a potential candidate pathogen involved in gastrointestinal motor disorders. Recently, our group has shown that HSV-1 infects the enteric nervous system (ENS) after intragastric (IG) inoculum leading to intestinal dysmotility. Aim. Since the ENS has the molecular machinery to recognize and react to viral infections we planned to assess the role of toll-like receptor (TLR) 2 during HSV-1 infection on ENS functional integrity and immune responses. Methods. Adult C57Bl/6 (WT) and TLR2 knock-out (KO) mice were inoculated with HSV-1 intranasally and after 4 weeks (W) by IG inoculum. After 1-8 W post IG administration, in isolated distal ileum segments, mounted vertically in organ baths, changes in muscle tension were recorded isometrically, after electric field stimulation (EFS, 1-40 Hz) in the presence or absence of 1 μM tetrodotoxin or atropine. ENS integrity was analyzed by immunofluorescence on whole mount preparations. Immune cells from mucosa and longitudinal muscle myenteric plexus (LMMP) were characterized by fluorescent activated flow cytometry (FACS). Results. At the early stage of infection, a mucosal infiltrate of CD11+F4/80+CD80+ macrophage was found together with resident F4/80+CD80- adherent cells with reduced phagocytic activity. At 8W post IG inoculum the proportion of CD11+ cells and CD8+ lymphocytes of LMMP producing INFgamma increased following HSV-1 challange. At 1 and 8W In Vitro contractility studies have shown a reduced neuronal excitability associated to an altered ENS architecture (i.e. decrease of neuronal HuC/D and glial S100beta immunoreactivity). By performing peripherin and beta III-tubulin immunofluorescence a morphological disorganization was found for both proteins at 2 and 6 W. A reduced staining of acetylcholine esterase-positive neurons, large fibers or small fibers, was revealed at 1W post IG inoculum in WT mice. In TLR2KO mice, mucosal infiltrate of CD11+F4/80+CD80+ macrophage was scant and the phagocytic activity of resident mucosal macrophages was preserved. During the infection time-course, TLR2KO mice failed to develop a strong anti HSV-1 adaptive immune response. Neurally-mediated contractions induced by EFS were significantly reduced only at 1-3W. Moreover, peripherin immunoreactivity increased at 1-6 W but disappeared at 8 W post IG inoculum whereas beta III-tubulin immunofluorescence was found altered during all time-course of the infection. An increased staining of acetylcholine esterase-positive neurons was found at 1W post IG inoculum in TLR2KO mice. Conclusion. Intestinal HSV-1 infection causes a time-dependent dysmotility and an altered ENS architecture. TLR2 activation influences the onset of gut dysmotility and ENS dysfunction, and the development of virus-specific adaptive immune responses.