P014 Impact of experimental ileitis and Toll-Like Receptor 4 signaling on enteric inhibitory neurotransmission
Faggin, S.(1);Cerantola, S.(1);Dal Prà, A.(1);Caldana, G.(1);Bosi, A.(2);Banfi, D.(2);Giaroni, C.(2);Savarino, E.(3);Giron, M.C.(1);
(1)University of Padova, Department of Pharmaceutical and Pharmacological Sciences, Padova, Italy;(2)University of Insubria, Department of Medicine and Surgery, Varese, Italy;(3)University of Padova, Department of Surgery- Oncology and Gastroenterology, Padova, Italy;
Alterations in Toll-like receptor 4 (TLR4) expression and changes in enteric neurotransmissions influence the onset and severity of inflammatory bowel disease (IBD). We aimed to assess the interaction between the enteric inhibitory pathways and TLR4 signaling in a mouse model of dinitrobenzene sulfonic acid (DNBS)-induced small intestine inflammation.
Male TLR4-/- mice (9±2 weeks old) were intrarectally treated with 2.5% DNBS. Pro-inflammatory cytokines (IL-6 and TNF-α) were measured in ileal samples. Changes in ileal muscle tension were recorded following exposure to: i) 10-Hz electric field stimulation in non-adrenergic-non-cholinergic (NANC) conditions (1 μM guanethidine + 1 μM atropine) with or without 10 µM 1400W (a inducible nitric oxide synthase (iNOS) inhibitor), or 0.1 µM Nω-nitro-L-arginine methyl ester (L-NAME; a pan-NOS inhibitor); ii) 30 μM dopamine or 30 μM SKF38393 (a dopamine 1 receptor (D1R) agonist) or bromocriptine (a D2R agonist). Immunoreactivity of neuronal (HuC/D) and glial (S100β) markers as well as nitrergic chemical coding (i.e. nNOS and iNOS) were determined in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs) by confocal microscopy.
No changes in IL-6 and TNF-α ileal mRNA transcripts were found after DNBS administration. The development of ileitis did not affect the NANC-inhibitory response, that was partially reduced in presence of 1400W or L-NAME, suggesting that TLR4 signaling influences NO-mediated inhibitory responses. DNBS-treated mice showed a significant increase in dopamine-induced relaxation (+31%) and in D1R- and D2R-mediated inhibitory responses (by +57% and +75%, respectively). A significant reduction of the total number of HuC/D+ neurons (-25%), with a proportional increase in nNOS+ neurons (+11%) was found in LMMPs of DNBS mice, associated with a significant reduction of S100β immunofluorescence (-12%) and no changes in iNOS immunoreactivity, highlighting a potential involvement of TLR4 signaling in the development of neuroplasticity and reactive gliosis.
The present data suggest that the severity of small intestine ileitis and neuronal dysfunction depend on the interplay occurring between TLR4 signaling and enteric inhibitory pathways. This evidence further strengthens the importance of characterizing neuroimmune interactions to identify potential molecular mechanism(s) representing putative pharmacological targets for the development of efficacious IBD therapies.