The cholinergic anti-inflammatory pathway is an efferent vagus nerve-based mechanism that regulates immune responses and cytokine production through α7nicotinic-acetylcholinereceptor (α7nAChR) signaling. Decreased efferent vagus nerve activity is observed in inflammatory bowel disease (IBD). We determined whether central activation of this pathway alters inflammation in mice with colitis and the mediating role of a vagus nerve-to spleen circuit and α7nAChR signaling. Two experimental models of colitis were used in C57BL/6 mice. Central cholinergic activation induced by the acetylcholinesterase inhibitor galantamine or a muscarinic acetylcholine receptor agonist treatments resulted in reduced mucosal inflammation associated with decreased MHC II level and pro-inflammatory cytokine secretion by splenic CD11c+ cells mediated by α7nAChR signaling. The cholinergic anti-inflammatory efficacy was abolished in mice with vagotomy, splenic neurectomy or splenectomy. In conclusion, central cholinergic activation of a vagus nerve–to spleen circuit controls intestinal inflammation and this regulation can be explored to develop novel therapeutic strategies.

The development of colitis in DSS model was accompanied with reduced microbial diversity and dysbiosis of gut microbiota at lower taxonomical levels.

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BackgroundThe cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4+/CD25−T cell activation in the context of experimental colitis.MethodsThe dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4+CD25−T cell co-culture were determined.ResultsMcN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4+CD25−T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy.ConclusionsSuppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.

BackgroundMany animal models have been developed to characterize the complexity of colonic inflammation. In dextran sodium sulfate (DSS) experimental colitis in mice the choice of reference genes is critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR). No studies have addressed the performance of reference genes in mice DSS-experimental colitis. This study aimed to determine the stability of reference genes expression (RGE) in DSS-experimental murine colitis.MethodsColitis was induced in male C57BL/6 mice using DSS5% for 5 days, control group received water. RNA was extracted from inflamed and non-inflamed colon. Using RT-qPCR, comparative analysis of 13 RGE was performed according to predefined criteria and relative colonic TNF-α and IL-1β gene expression was determined by calculating the difference in the threshold cycle.ResultsColitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest variability within the inflamed and control groups. Conversely, TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. Normalization of colonic TNF-α and IL-1β mRNA levels was dependent on the reference gene used. Depending on the genes used to normalize the data, statistical significance varied from significant when TBP / Eef2 were used to non-significant when Gapdh, Actb or β2m were used.ConclusionsThis study highlights the appropriate choice of RGE to ensure adequate normalization of RT-qPCR data when using this model. Suboptimal RGE may explain controversial results from published studies. We recommend using Tbp and Eef2 instead of Gapdh, Actb or β2m as reference genes.