Abstract
BackgroundIn the last years, Wnt signaling was demonstrated to regulate inflammatory processes. In particular, an increased expression of Wnts and Frizzled receptors was reported in inflammatory bowel disease (IBD) and ulcerative colitis to exert both anti- and pro-inflammatory functions regulating the intestinal activated nuclear factor κB (NF-кB), TNFa release, and IL10 expression.MethodsTo investigate the role of Wnt pathway in the response of the enteric nervous system (ENS) to inflammation, neurons and glial cells from rat myenteric plexus were treated with exogenous Wnt3a and/or LPS with or without supporting neurotrophic factors such as basic fibroblast growth factor (bFGF), epithelial growth factor (EGF), and glial cell-derived neurotrophic factor (GDNF). The immunophenotypical characterization by flow cytometry and the protein and gene expression analysis by qPCR and Western blotting were carried out.ResultsFlow cytometry and immunofluorescence staining evidenced that enteric neurons coexpressed Frizzled 9 and toll-like receptor 4 (TLR4) while glial cells were immunoreactive to TLR4 and Wnt3a suggesting that canonical Wnt signaling is active in ENS.Under in vitro LPS treatment, Western blot analysis demonstrated an active cross talk between canonical Wnt signaling and NF-кB pathway that is essential to negatively control enteric neuronal response to inflammatory stimuli. Upon costimulation with LPS and Wnt3a, a significant anti-inflammatory activity was detected by RT-PCR based on an increased IL10 expression and a downregulation of pro-inflammatory cytokines TNFa, IL1B, and interleukin 6 (IL6). When the availability of neurotrophic factors in ENS cultures was abolished, a changed cell reactivity by Wnt signaling was observed at basal conditions and after LPS treatment.ConclusionsThe results of this study suggested the existence of neuronal surveillance through FZD9 and Wnt3a in enteric myenteric plexus. Moreover, experimental evidences were provided to clarify the correlation among soluble trophic factors, Wnt signaling, and anti-inflammatory protection of ENS.
Highlights
In the last years, Wnt family proteins (Wnts) signaling was demonstrated to regulate inflammatory processes
Growth factors modulate the expression of FZD9 and toll-like receptor 4 (TLR4) positive cells By flow cytometric analysis, freshly isolated ENS cells (ENSc) showed a heterogeneous immunophenotype with a specific stemness pattern (Figure 1A), as suggested at T0 by the expression of Nanog (26.9 ± 0.6%), Sox2 (64.9 ± 1.4%), Sox10 (17.7 ± 0.4%), and p75 (55.0 ± 3.2%)
Multidifferentiative potential of freshly isolated rat enteric nervous system (ENS) cells was confirmed by the expression of nerve/glial antigen 2 (NG2) (51.9 ± 4.3%), a proteoglycan typically observed on the membrane of multipotent neural stem cells
Summary
Wnt signaling was demonstrated to regulate inflammatory processes. An increased expression of Wnts and Frizzled receptors was reported in inflammatory bowel disease (IBD) and ulcerative colitis to exert both anti- and pro-inflammatory functions regulating the intestinal activated nuclear factor κB (NF-кB), TNFa release, and IL10 expression. As suggested by the increased expression of Wnts and Frizzled receptors in inflammatory bowel disease (IBD) and ulcerative colitis [17], Wnt signaling is involved in gut inflammation and exerts both anti- and pro-inflammatory functions. Β-catenin has shown to i) negatively regulate intestinal NF-кB activity in bacterial-induced epithelial inflammation [18], ii) reduce TNFa release [19], and iii) induce the expression of IL10 and TGFB [20] while the activation of proinflammatory mediators seems to be correlated to noncanonical Wnt signaling [21]
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