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Intestinal mucosal cells, such as resident macrophages and epithelial cells, express adrenergic receptors and are receptive to norepinephrine, the primary neurotransmitter of the sympathetic nervous system (SNS). It has been suggested that the SNS affects intestinal immune activity in conditions, such as inflammatory bowel disease; however, the underlying mechanisms remain ambiguous. Here, we investigated the effect of SNS on mucosal immune and epithelial cell functions. We employed 6-OHDA-induced sympathetic denervation (cSTX) to characterize muscularis-free mucosal transcriptomes by RNA-seq and qPCR, and quantified mucosal immune cells by flow cytometry. The role of norepinephrine and cytokines on epithelial functions was studied using small intestinal organoids. cSTX increased the presence of activated CD68+CD86+ macrophages and monocytes in the mucosa. In addition, through transcriptional profiling, the proinflammatory cytokines IL-1β, TNF-α, and IFN-γ were induced, while Arg-1 and CD163 expression was reduced. Further, cSTX increased intestinal permeability in vivo and induced genes involved in barrier integrity and antimicrobial defense. In intestinal organoids, similar alterations were observed after treatment with proinflammatory cytokines, but not norepinephrine. We conclude that a loss in sympathetic input induces a proinflammatory mucosal state, leading to reduced epithelial barrier functioning and enhanced antimicrobial defense. This implies that the SNS might be required to maintain intestinal immune functions during homeostasis.

Upregulation of Wnt activity in the hippocampus and the spinal cord correlates with disease severity. (a) Iba1 immunostaining (red) of spinal cord sections from Axin2lacZ/+ EAE mice at day 20. The white dashed line highlights lesion areas; white matter tissue is outlined by yellow dashed line. Nuclei were counterstained with Hoechst (blue). Scale bar, 100 μm. (b) Histogram represents the average number of lesions per section of each EAE time point as mean + SEM. Spinal cord: day 20 (n = 4; 15–35 sections/mouse), day 30 (n = 3; 10–23 sections/mouse), day 50 (n = 3; 10–17 sections/mouse). (c) Representative image of lesion site in the DG. Iba1 (red) immunostaining in hippocampal section from EAE mouse at day 30. Nuclei (grey). Scale bar, 50 μm. (d) Representative image of neural stem cells located in the SGZ. SOX2 (green) and GFAP (red). Arrowheads indicate SOX2+/GFAP+ radial glia-like cells and arrows indicate SOX2+/GFAP+ horizontal progenitors. Nuclei (grey). Scale bar, 25 μm. (e+f) β-gal assay of cortical and cerebellar EAE tissue revealed no changes in Wnt activity. Histograms represent mean + SEM of fold-changes of β-gal activity in EAE mice relative to controls (CFA) set as 1. Day 20 (EAE, n = 6; CFA, n = 3); day 30 (EAE, n = 4; CFA, n = 3); day 50 (EAE, n = 8; CFA, n = 4). (g) qPCR analysis of Axin2 in different CNS parts in acute EAE (day 20). Data represent mean of fold-changes + SEM of gene expression in EAE mice (n = 6) relative to controls (CFA; n = 6) set as 1. Expression of Axin2 was normalized to Gapdh. (d) Regression analysis shows correlation between β-gal activity in inflamed CNS tissues and clinical disease score in EAE animals. Pearson correlation, r = 0.58; p = 0.018 (hippocampus) and r = 0.52, p = 0.036 (spinal cord). Statistics: Two-tailed, unpaired Student’s t-test,* * p

Effect of TNFα on gene expression of Axin2 and Wnt ligands in hippocampal cells. (a) The distribution pattern of LacZ expression in Axin2lacZ/+ hippocampal slice cultures. X-gal staining of hippocampal slices (postnatal day 10) shows LacZ expression predominantly in the granular cell layer (gcl). Scale bar, 50 μm. (b) qPCR analysis of Axin2, Wnt3, Wnt8b and Wnt5a gene expression in hippocampal progenitors after 6 h treatment with TNFα (0.2 ng/ml or 1.0 ng/ml; n = 3) or Wnt3a (20 ng/ml; n = 3). Histogram represents mean + SEM of fold-changes of gene expression relative to controls (PBS, n = 3) set as 1. (c) qPCR analysis of gene expression of selected Wnt ligands in hippocampal astrocytes (GLAST+) and microglia (CD11b+) cells treated with TNFα (0.2 ng/ml or 1.0 ng/ml; n = 3) for 6 h. Histogram represents mean + SEM of fold-changes of gene expression relative to controls (PBS, n = 3) set as 1. (c) Histological analysis of proliferating cells in the DG of hippocampal NestineGFP OSCs. Hippocampal slices were cultured for seven days followed by 6 h treatment with Wnt3a (20 ng/ml) or IFNγ (100 U/ml) and additional incubation in cytokine-free medium for 24 h. BrdU was administered for 24 h prior to treatment with cytokines. Representative images show BrdU (red) and eGFP (green). Arrow heads indicate BrdU+/eGFP+ co-labeled hippocampal progenitors. Scale bar, 50 μm. (d) The frequency of BrdU label-retaining cells in the DG of hippocampal NestineGFP OSCs is increased after treatment with Wnt3a. Data are shown as mean + SEM of cells per mm of the DG. Control (PBS, n = 15–20 slices); IFNγ (n = 4–11 slices) and Wnt3a (n = 8–14 slices). Statistics: Two-tailed, unpaired Student’s t-test,* * p

Gene expression analysis in the hippocampal tissue of EAE mice. (a) qPCR analysis of selected Wnt ligands in the hippocampus at early (day 20) and chronic (day 50) stages of passive EAE. Histogram represents mean + SEM of fold-changes relative to control group (PBS) set as 1. Gene expression was normalized to Gapdh. Day 20 (EAE, n = 4–6; control, n = 4); day 50 (EAE, n = 8–13; control, n = 7–11). (b) qPCR analysis of selected Wnt ligands in the cortex, cerebellum and spinal cord at early stages of passive EAE (day 20). Histogram represents mean + SEM of fold-changes relative to control group (PBS) set as 1. Gene expression was normalized to Gapdh. Six control and six EAE mice were analysed for each part of the CNS. n.d. not detectable. (c) A regression analysis shows correlation between the TNFα and the Axin2 gene expression levels examined in hippocampal tissue of individual EAE animals. Pearson correlation, r = 0.8458; p