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Clinical Trial
. 2022 Jan 6:12:781147.
doi: 10.3389/fimmu.2021.781147. eCollection 2021.

Cholinergic Signaling Attenuates Pro-Inflammatory Interleukin-8 Response in Colonic Epithelial Cells

Isabelle Müller  1 , Urs Kym  1 , Virginie Galati  1 , Sasha Tharakan  2 , Ulrike Subotic  1   2 , Thomas Krebs  3 , Eleuthere Stathopoulos  4 , Peter Schmittenbecher  5 , Dietmar Cholewa  6 , Philipp Romero  7 , Bertram Reingruber  8 ; NIGStudy Group; Stefan Holland-Cunz  1 , Simone Keck  1
Collaborators, Affiliations
Clinical Trial

Cholinergic Signaling Attenuates Pro-Inflammatory Interleukin-8 Response in Colonic Epithelial Cells

Isabelle Müller et al. Front Immunol. .

Abstract

Infants affected by Hirschsprung disease (HSCR), a neurodevelopmental congenital disorder, lack ganglia of the intrinsic enteric nervous system (aganglionosis) in a variable length of the colon, and are prone to developing severe Hirschsprung-associated enterocolitis (HAEC). HSCR patients typically show abnormal dense innervation of extrinsic cholinergic nerve fibers throughout the aganglionic rectosigmoid. Cholinergic signaling has been reported to reduce inflammatory response. Consequently, a sparse extrinsic cholinergic innervation in the mucosa of the rectosigmoid correlates with increased inflammatory immune cell frequencies and higher incidence of HAEC in HSCR patients. However, whether cholinergic signals influence the pro-inflammatory immune response of intestinal epithelial cells (IEC) is unknown. Here, we analyzed colonic IEC isolated from 43 HSCR patients with either a low or high mucosal cholinergic innervation density (fiber-low versus fiber-high) as well as from control tissue. Compared to fiber-high samples, IEC purified from fiber-low rectosigmoid expressed significantly higher levels of IL-8 but not TNF-α, IL-10, TGF-β1, Muc-2 or tight junction proteins. IEC from fiber-low rectosigmoid showed higher IL-8 protein concentrations in cell lysates as well as prominent IL-8 immunoreactivity compared to IEC from fiber-high tissue. Using the human colonic IEC cell line SW480 we demonstrated that cholinergic signals suppress lipopolysaccharide-induced IL-8 secretion via the alpha 7 nicotinic acetylcholine receptor (a7nAChR). In conclusion, we showed for the first time that the presence of a dense mucosal cholinergic innervation is associated with decreased secretion of IEC-derived pro-inflammatory IL-8 in the rectosigmoid of HSCR patients likely dependent on a7nAChR activation. Owing to the association between IL-8 and enterocolitis-prone, fiber-low HSCR patients, targeted therapies against IL-8 might be a promising immunotherapy candidate for HAEC treatment.

Keywords: Hirschsprung disease; Hirschsprung-associated enterocolitis; Interleukin-8; acetylcholine receptors; intestinal epithelial cell.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Neuronal innervation of the lower colon track in HSCR and non-HSCR patients. Schematic representation of extrinsic AChE+ nerve fiber innervation (A). Anatomically, extrinsic innervation derived from sacral roots S2–4 is limited to the rectosigmoid and does not reach the descending colon. Rec (1), rectum; SC (2), sigmoid colon; DC (3), descending colon; AChE, acetylcholinesterase; S2–4, sacral roots S2–4. Cryosections (5µm) of aganglionic and ganglionic colonic tissue from HSCR and non-HSCR patients showing anti-AChE immunohistochemistry and anti-ß3 tubulin immunofluorescence (B). Five different colonic tissue types were distinguished, and each showed different expression of ENS (aganglionic versus ganglionic) and mucosal nerve fiber innervation. Scale bar: 50µm. IHC, immunohistochemistry; IF, immunofluorescence; ENS, enteric nervous system.
Figure 2
Figure 2
Expression of cytokines, mucin, and tight junctions in patient-derived IEC. FACS analysis of isolated IEC. Cells were isolated by digestion and purified using a 20%/40% Percoll gradient. Purity was determined by flow cytometric analysis using antibodies against human CD45-PerCP and EpCam-Alexa488. Unstained controls were used to determine the negative population (A). Purified epithelial cells were immediately lysed, total RNA isolated and cDNA synthesized. Relative gene expression was calculated using the 2-ΔCT method, with β2-microglobulin as the housekeeping gene (B–E). Scatter plots with bar show mean ± SEM. Significance was determined using the Kruskal-Wallis test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Fiber-low (n=30); Fiber-high (n=13); DC AG (n=10); DC G, HSCR (n=12); DC G, non-HSCR (n=6). Not detectable samples: Fiber-low: TNF-α (n=2); IL-10 (n=6); TGF-β (n=1); Muc2 (n=2); ZO-1 (n=4); occludin (n=2). Fiber-high: IL-10 (n=4); ZO-1 (n=1). DC AG: IL-8 (n=1); TNF-α (n=1); IL-10 (n=3). DC G, HSCR: IL-8 (n=1); TNF-α (n=1); IL-10 (n=3); ZO-1 (n=2); occludin (n=2). DC G, non-HSCR: IL-10 (n=2); ZO-1 (n=1); occludin (n=1). DC, descending colon; AG, aganglionic; G, ganglionic.
Figure 3
Figure 3
IL-8 protein expression in patient-derived IEC. Immunofluorescence (5µm cryosections) of rectosigmoid using human antibodies against EpCAM (Alexa488, green) and IL-8 (Alexa555, red). DAPI (blue) shows cell nuclei. Secondary antibody goat anti-mouse IgG1 Alexa 555 was used as the negative control. Scale bars 50µm (A). Quantification of IL-8 using Cellprofiler-3.1.9 software. IL-8 expression in EpCAM+ IEC (n=3) was quantified in 4 to 8 epithelial regions (on average 1500 IEC) per patient from fiber-low and fiber-high rectosigmoid tissue. Frequencies of IL-8+ EpCam+ cells were shown as the percentage of total EpCam+ epithelial cells. (B). Patient-derived IEC were lysed, and IL-8 was determined using human IL-8 ELISA Ready-SET-Go Kit. (C) Significance was determined using the Kruskal-Wallis test (*p ≤ 0.05, **p ≤ 0.01). Scatter plots show means ± SEM. AG, aganglionic; G, ganglionic; DC, descending colon.
Figure 4
Figure 4
Expression of IEC-specific cholinergic receptors. Purified epithelial cells were immediately lysed, total RNA isolated and cDNA synthesized. Relative gene expression was calculated using the 2-ΔCT method, with β2-microglobulin as the housekeeping gene (A, B). Scatter plots with bar show mean ± SEM. Significance was determined using the Kruskal-Wallis test (*p ≤ 0.05, **p ≤ 0.01). Fiber-low (n=30); Fiber-high (n=13); DC AG (n=10); DC G, HSCR (n=12); DC G, non-HSCR (n=6). Not detectable samples: Fiber-low: CHRM1 (n=1); CHRNA3 (n=2); CHRNA4 (n=23); CHRNA5 (n=14); CHRNA7 (n=2). Fiber-high: CHRNA3 (n=6); CHRNA4 (n=10); CHRNA5 (n=8); CHRNA7 (n=1). DC AG: CHRNA3 (n=3); CHRNA4 (n=10); CHRNA5 (n=8); CHRNA7 (n=2). DC G, HSCR: CHRM1 (n=1); CHRM3 (n=1); CHRNA3 (n=6); CHRNA4 (n=9); CHRNA5 (n=8); CHRNA7 (n=4). DC G, non-HSCR: CHRNA3 (n=2); CHRNA4 (n=6); CHRNA5 (n=4); CHRNA7 (n=1). nd: not detectable. CHRNA7 Immunofluorescence (5µm cryosections) from fiber-low and fiber-high rectosigmoid using purified mouse IgG1 anti-alpha7nAchR antibody (green) (C). DAPI (blue) shows cell nuclei. Secondary antibody goat anti-mouse IgG1 Alexa 555 was used as the negative control. Scale bar 50µm.
Figure 5
Figure 5
Cholinergic stimulation attenuates LPS-induced IL-8 response in SW480 cells via a7nAChR. Expression of ACHRs in SW480 cells (A). SW480 cells (5 x 104/200µl) were stimulated with LPS (100ng/ml) in the presence of different agonist and antagonists. IL-8 was measured in cell-free supernatants 20h after LPS stimulation. Cells were stimulated with LPS 20min prior to acetylcholine (ACh, 10µM), nicotine (10µM), and muscarine (10µM) stimulation (B). Cells were pretreated (20min) with the non-selective nAChR antagonist Mecamylamine (Mec, 100µM) followed by treatment with ACh (10µM), 20min prior to LPS stimulation (C). Cells were pretreated (20min) with the selective a7nAChR antagonist alpha-Bungarotoxin (a-BTX, 100µM) followed by treatment with GTS-21 (100µM), 20min prior to LPS stimulation (D). Cells were pretreated (20min) with GTS-21 (100µM) followed by stimulation with LPS. Phospho (p)-NFĸB-p65 was measured 4h after LPS stimulation (E). Cells were pretreated (20min) with DMSO (0.01%, control) or tyrphostin AG490 (1µM) or wortmannin (1µM) followed by stimulation with LPS (100ng/ml), 20min prior to treatment with a7nAChR agonist GTS-21 (20min) (F). Cell viability was assessed by colorimetric MTS assay (G). Each point in the scatter plots with bar represent triplicates or quadruplicates ± SEM. Data are representative of three independent experiments. Significance was determined using one-way ANOVA multiple comparison analysis (A–D) and paired t test (E, F) with *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005. ns, not significant. n.d., not detectable.
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