title: Toll like receptor-2 regulates production of glial-derived neurotrophic factors in murine intestinal smooth muscle cells.
abstract: Gut microbiota-innate immunity axis is emerging as a key player to guarantee the structural and functional integrity of the enteric nervous system (ENS). Alterations in the composition of the gut microbiota, derangement in signaling of innate immune receptors such as Toll-like receptors (TLRs), and modifications in the neurochemical coding of the ENS have been associated with a variety of gastrointestinal disorders. Indeed, TLR2 activation by microbial products controls the ENS structure and regulates intestinal neuromuscular function. However, the cellular populations and the molecular mechanisms shaping the plasticity of enteric neurons in response to gut microbes are largely unexplored. In this study, smooth muscle cells (SMCs), enteric glial cells (EGCs) and macrophages/dendritic cells (MΦ/DCs) were isolated and cultured from the ileal longitudinal muscle layer of wild-type (WT) and Toll-like receptor-2 deficient (TLR2(-/-)) mice. Quantification of mRNA levels of neurotrophins at baseline and following stimulation with TLR ligands was performed by RT-PCR. To determine the role of neurotrophins in supporting the neuronal phenotype, we performed co-culture experiments of enteric neurons with the conditioned media of cells isolated from the longitudinal muscle layer of WT or TLR2(-/-) mice. The neuronal phenotype was investigated evaluating the expression of βIII-tubulin, HuC/D, and nNOS by immunocytochemistry. As detected by semi-quantitative RT-PCR, SMCs expressed mRNA coding TLR1-9. Among the tested cell populations, un-stimulated SMCs were the most prominent sources of neurotrophins. Stimulation with TLR2, TLR4, TLR5 and TLR9 ligands further increased Gdnf, Ngf, Bdnf and Lif mRNA levels in SMCs. Enteric neurons isolated from TLR2(-/-) mice exhibited smaller ganglia, fewer HuC/D(+ve) and nNOS(+ve) neurons and shorter βIII-tubulin axonal networks as compared to neurons cultured from WT mice. The co-culture with the conditioned media from WT-SMCs but not with those from WT-EGCs or WT-MΦ/DCs corrected the altered neuronal phenotype of TLR2(-/-) mice. Supplementation of TLR2(-/-) neuronal cultures with GDNF recapitulated the WT-SMC co-culture effect whereas the knockdown of GDNF expression in WT-SMCs using shRNA interference abolished the effect on TLR2(-/-) neurons. These data revealed that by exploiting the repertoire of TLRs to decode gut-microbial signals, intestinal SMCs elaborate a cocktail of neurotrophic factors that in turn supports neuronal phenotype. In this view, the SMCs represent an attractive target for novel therapeutic strategies.
authors.affiliation: Department of Molecular Medicine, University of Padova, via A. Gabelli 63, 35121 Padova, Italy. Electronic address: paola.brun.1@unipd.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Brun
authors.affiliation: Department of Molecular Medicine, University of Padova, via A. Gabelli 63, 35121 Padova, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Gobbo
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Largo E. Meneghetti 2, 35131 Padova, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Caputi
authors.affiliation: Department of Molecular Medicine, University of Padova, via A. Gabelli 63, 35121 Padova, Italy.
authors.forename: Lisa
authors.initials: L
authors.lastname: Spagnol
authors.affiliation: Department of Molecular Medicine, University of Padova, via A. Gabelli 63, 35121 Padova, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Schirato
authors.affiliation: Department of Molecular Medicine, University of Padova, via A. Gabelli 63, 35121 Padova, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Pasqualin
authors.affiliation: Department of Molecular Medicine, University of Padova, via A. Gabelli 63, 35121 Padova, Italy.
authors.forename: Elia
authors.initials: E
authors.lastname: Levorato
authors.affiliation: Department of Molecular Medicine, University of Padova, via A. Gabelli 63, 35121 Padova, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Palù
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Largo E. Meneghetti 2, 35131 Padova, Italy.
authors.forename: Maria Cecilia
authors.initials: MC
authors.lastname: Giron
authors.affiliation: Department of Molecular Medicine, University of Padova, via A. Gabelli 63, 35121 Padova, Italy.
authors.forename: Ignazio
authors.initials: I
authors.lastname: Castagliuolo
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D000067778:ELAV-Like Protein 3 / Q000378:metabolism; D000067779:ELAV-Like Protein 4 / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism*; D007421:Intestine, Small / Q000166:cytology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009457:Neuroglia / Q000502:physiology; D009474:Neurons / Q000502:physiology; D011804:Quinolines / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism
chemical_list: D000199:Actins; D000067778:ELAV-Like Protein 3; D000067779:ELAV-Like Protein 4; C490263:Elavl3 protein, mouse; D051100:Glial Cell Line-Derived Neurotrophic Factor; D008070:Lipopolysaccharides; D011804:Quinolines; C495966:TOTO-3 compound; D013844:Thiazoles; D051195:Toll-Like Receptor 2; D014404:Tubulin; C462941:beta3 tubulin, mouse
keywords: Enteric nervous system; Glial derived neurotrophic factor; Neuronal integrity; Neurotrophin; Smooth muscle cell; Toll-like receptor