Involvement of Toll-like receptor 4 on small intestine neuromuscular dysfunction in a mouse model of high-fat diet-induced obesity

Background and Aim. High fat diet (HFD)-induced obesity determines gut dysbiosis, delayed gastrointestinal (GI) transit and Toll-like receptor 4 (TLR4)-dependent neuronal loss in mice (Anitha et al., 2016). In this study, we aimed to assess TLR4 role in tuning small bowel contractility to identify the neurotransmitter pathways affected by HFD-induced obesity. Methods. TLR4-/- and wild-type (WT) C57BL/6J male mice were fed with standard-diet (SD; 18% kcal fat) or HFD (60% kcal fat) for 8 weeks. GI transit was measured by in vivo distribution of nonabsorbable-FITC-labeled dextran, fecal pellet frequency and water content. In ileal longitudinal muscle-myenteric plexus, neuronal HuC/D and neuronal NOS (nNOS), and glial S100β distribution was analyzed by confocal microscopy. Changes in ileal muscle tension were isometrically recorded following: i) electric field stimulation (EFS, 0-40 Hz), ii) 10 Hz-EFS in non-adrenergic non-cholinergic (NANC) condition (1 μM guanethidine + 1 μM atropine) with or without 10 μM 1400W (iNOS inhibitor) or 100 µM L-NAME (pan-NOS inhibitor) as previously described by Caputi et al (2017). Data are presented as mean±SEM. Results. In TLR4-/- mice, SD determined a reduced excitatory neurotransmission (-23±3%; n=6 animals, p<0.05) together with an increased NANC relaxation (+36±2%; n=5 animals, p<0.05) mediated by both iNOS- and nNOS-derived NO, which was found abolished in HFD-fed animals. In WT mice, HFD impaired GI transit (-20±4%; n=6 animals, p<0.05), increased tachykininergicmediated responses in NANC conditions while decreasing cholinergic-mediated contraction (31±3%; n=5 animals, p<0.05). After SD, increased S100β immunofluorescence (+82±6%; n=5, animals, p<0.05) and reduced number of nNOS+ neurons (-24±3%; n=5 animals, p<0.05) were found in TLR4-/- mice. HFD determined reactive enteric gliosis in WT mice whereas reduced S100β immunoreactivity in TLR4-/- mice. These findings were associated to a decreased number of nNOS+ neurons in WT and to a further decrease in TLR4-/- mice (by -11±2% and -23±4%, respectively; n=5 animals, p<0.05). Conclusion. HFD-induced chronic systemic low-grade inflammation caused complex morphofunctional neuromuscular rearrangements, characterized by intestinal dysmotility and enteric reactive gliosis. The absence of TLR4 appears to partially protect from HFD detrimental effects, suggesting the involvement of TLR4 signaling in HFD-mediated GI molecular events.