Acetylcholinesterase Inhibitor Pyridostigmine Bromide Attenuates Gut Pathology and Bacterial Dysbiosis in a Murine Model of Ulcerative Colitis
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Ulcerative colitis (UC) is a Th2 inflammatory bowel disease characterized by increased IL-5 and IL-13 expression, eosinophilic/neutrophilic infiltration, decreased mucus production, impaired epithelial barrier, and bacterial dysbiosis of the colon. Acetylcholine and nicotine stimulate mucus production and suppress Th2 inflammation through nicotinic receptors in lungs but UC is rarely observed in smokers and the mechanism of the protection is unclear.
In order to evaluate whether acetylcholine can ameliorate UC-associated pathologies, we employed a mouse model of dextran sodium sulfate (DSS)-induced UC-like conditions, and a group of mice were treated with Pyridostigmine bromide (PB) to increase acetylcholine availability. The effects on colonic tissue morphology, Th2 inflammatory factors, MUC2 mucin, and gut microbiota were analyzed.
DSS challenge damaged the murine colonic architecture, reduced the MUC2 mucin and the tight-junction protein ZO-1. The PB treatment significantly attenuated these DSS-induced responses along with the eosinophilic infiltration and the pro-Th2 inflammatory factors. Moreover, PB inhibited the DSS-induced loss of commensal Clostridia and Flavobacteria, and the gain of pathogenic Erysipelotrichia and Fusobacteria.
Together, these data suggest that in colons of a murine model, PB promotes MUC2 synthesis, suppresses Th2 inflammation and attenuates bacterial dysbiosis therefore, PB has a therapeutic potential in UC.
KeywordsUlcerative colitis Pyridostigmine bromide Dextran sodium sulfate Bacterial dysbiosis Acetylcholine
Inflammatory bowel disease
Dextran sodium sulfate
Nicotinic acetylcholine receptors
Ulcerative colitis (UC) is a chronic idiopathic inflammatory bowel disease (IBD), where inflammation is mainly restricted to colon. The incidence of UC is steadily increasing particularly in the Western world . Although the exact etiology of UC is unclear, increasing evidence suggests that a combination of factors including genetics, environmental agents, immune dysregulation, gut barrier dysfunction, and dysbiosis of the gut microbiota influence the course of the disease [2, 3]. UC is rarely observed in smokers, as nicotine/nicotinic receptor agonists suppress colitis ; however, the mechanism by which cigarette smoke/nicotine suppresses the development of UC is unclear. UC is primarily a Th2-mediated IBD associated with eosinophilic infiltration and overproduction of Th2 chemokines/cytokines including eotaxin (CCL11), IL-5, and IL-13 [5, 6, 7]. In animal models, activation of pulmonary nicotinic acetylcholine receptors (nAChRs) moderates eosinophilic infiltration through suppression of Th2 responses . In mammals, acetylcholine (ACh) is the only known biological ligand for nAChRs, and increasing evidence suggests that ACh and PB—the latter of which increases ACh by inhibiting acetylcholine esterase (AChE)—tend to protect against tissue injury [9, 10]. We and others have shown that nicotine, ACh, and the AChE inhibitor neostigmine bromide (similar to PB) promote mucus formation in human lung epithelial cells through α7-nAChRs [11, 12]. MUC2, the predominant mucin in the gut, plays a critical role in gut homeostasis, and MUC2-deficient mice develop colitis spontaneously ; however, unlike MUC5AC in bronchial epithelial cells , IL-13 does not always stimulate MUC2 . UC also affects gut microbiota and the integrity of the epithelial barrier in the colon [2, 5, 6]; thus normal fecal microbial transplantation is a promising treatment for UC [16, 17]. As DSS has been found to induce UC-like conditions in a mouse model [18, 19], in this study we examined whether PB attenuated DSS-induced colon pathology in C57BL/6 mice.
Materials and Methods
Pathogen-free 5–6-week-old C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Animals were kept in chambers as described previously . All animal protocols were approved by the Institutional Animal Care and Use Committee.
DSS and PB Treatment
Mice (8–9 weeks old) were divided into three groups (control, DSS, and DSS+PB) with 6–8 animals/group. PB and DSS were purchased from Sigma-Aldrich, USA. Mice were given PB through intraperitoneally implanted Alzet mini-osmotic pumps  starting at day 7 prior to DSS treatment and continued until they were euthanized. Mice implanted with mini-osmotic pumps containing sterile saline served as the control. The pumps provided PB at a concentration of 2 mg/day/kg body weight. Colitis was induced by 3.0% (w/v) DSS-containing water. The treatment continued for 7 days; the animals were euthanized and colons harvested on day 8. Animals were weighed daily after DSS treatment. Where indicated, intestines were dissected to obtain ileum, cecum, and colon; tissues were frozen for RNA and protein, and fecal material for bacterial microbiome analysis.
Histochemical Staining and Scoring
Formalin-fixed and paraffin-embedded tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) for general histology. Histochemical staining with Alcian Blue and periodic acid–Schiff reagent (AB–PAS) was carried out as described previously . Eosinophils and goblet cells were enumerated microscopically. Tissue sections were coded and assessed in a blinded manner. Histological damage was scored as described previously . Briefly, sections were scored for eosinophil infiltration (0–3), with 0 representing less than three eosinophils per field of view at 40× magnification in the lamina propria, 1 for greater than three eosinophils per field of view in the lamina propria, 2 representing confluence of eosinophils extending into the submucosa, and 3 representing confluence of eosinophils present in all tissue layers.
Deparaffinized and hydrated tissue sections were washed in 0.05% v Brij-35 in phosphate-buffered saline (PBS; pH 7.4) and immunostained for antigen expression as described previously . Briefly, the antigens were unmasked and incubated in a blocking solution. The sections were stained with antibodies to ZO-1 (Invitrogen Inc., Carlsbad, CA, USA) or anti-eosinophil antibody (Novus Biologicals, Littleton, CO, USA), or isotype control antibodies. Immunofluorescence images were captured with a BZX700 all-in-one microscope (Keyence Corp., Japan).
Total RNA from frozen mouse colon tissue was isolated using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using the StepOnePlus detection system (Applied Biosystems, Foster City, CA, USA) and the TaqMan One-Step RT-PCR kit containing AmpliTaq Gold® DNA polymerase . Specific primers and probes were obtained from Applied Biosystems. Fold changes in qPCR expression were calculated by the 2(−ΔΔCT) method .
Western Blot Analysis
Western blot analysis of eotaxin, IL-5, IL-13 and MUC2 in mice colon homogenates was performed as described previously . Briefly, colon homogenates (70 μg) were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for eotaxin, IL-5, and IL-13, and 5% SDS-PAGE for MUC2. After transfer to nitrocellulose membranes, blots were probed with antibodies to eotaxin, IL-5, IL-13, and MUC2 (Abcam), and after stripping were re-probed with anti–β actin antibody.
The H&E-stained sections were analyzed for histological grading to evaluate the severity of inflammation. The total score ranged from 0 to 14, representing the sum of scores from the following: (1) severity of inflammation, with 0 = none, 1 = mild, 2 = moderate, and 3 = severe; (2) mucosal damage, with 0 = none, 1 = mucosal layer, 2 = submucosa, and 3 = transmural; (3) crypt damage, with 0 = none, 1 = basal one-third damage, 2 = basal two-thirds damage, 3 = crypt lost with intact epithelium, and 4 = crypt and epithelium lost; and (4) percentage involvement, with 0 = none, 1 = ≤25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100%, as previously described .
Bacterial Microbiome Analysis: Sequencing and 16S DNA Analysis
Fecal contents were collected from the gut encompassing distal cecum and distal sigmoid colon and frozen on dry ice. The fecal matter was lysed using glass beads in a MagNA Lyser tissue disruptor (Roche Diagnostics, Indianapolis, IN, USA) and total DNA was isolated using the DNeasy Powersoil® or AllPrep PowerFecal® DNA isolation kits (MO BIO Laboratories, Carlsbad, CA, USA) and quantified as described elsewhere . Briefly, the bacterial 16S V4 rDNA region was amplified using fusion primers, and each sample was PCR-amplified with two differently barcoded V4 fusion primers and loaded into the MiSeq cartridge (Illumina Inc., San Diego, CA, USA). The amplicons were sequenced for 250 cycles with custom primers designed for paired-end sequencing. Using the QIIME platform, sequences were quality-filtered and demultiplexed using exact matches to the supplied DNA barcodes. The sequences were searched against the Greengenes reference database and clustered at 97% by UCLUST (closed-reference operational taxonomic unit [OTU] picking). The relative abundance of OTUs constituting a single phylum in treated animals was normalized with the OTUs in controls. The resulting ratios were analyzed using Prism software (GraphPad Software, LA Jolla, CA, USA) to identify changes between groups in the five major bacterial phyla in the microbiome.
Results and Discussion
PB Ameliorates the DSS-Induced Increase in IL-5 and Eotaxin
PB Inhibits DSS-Induced IL-13 Expression and Improves Colonic Epithelial Integrity and MUC2 Mucin Expression
PB Attenuates DSS-Induced Dysbiosis of Microbiota in the Colon
The intestines, particularly the colon, are inhabited by a large number of microorganisms that normally provide symbiotic benefits to the host; however, barrier dysfunction in UC causes unfavorable changes in the composition of gut microbiota (dysbiosis) that facilitates the penetration of colonic mucus layers by these bacteria to induce gut inflammation [13, 38, 54]. Normal gut bacterial microbial community is typically dominated (nearly 95%) by phyla Firmicutes, Bacteroidetes, and Proteobacteria , and each phylum has members that are beneficial or pathogenic in the gut. Imbalances in gut microbiota are seen in the DSS-induced UC in mice , and recent results suggest that fecal transplants containing normal bacterial microbiota attenuate UC symptoms in patients [17, 57] and in experimental animal models .
Clostridia difficile infection is known to recur in UC patients and is associated with increased morbidity and mortality in this population ; however, organisms within Clostridia genera are higher in normal gut flora and normal fecal microbiota is used to treat recurrent Clostridia difficile infection . Both Clostridia and Erysipelotrichia belong to phylum Firmicutes, but commensal Clostridia maintains gut homeostasis and moderates Clostridia difficile infection [60, 61]. Commensal Clostridia promotes the development of anti-inflammatory IL-10-producing Fox3p+ T-reg cells in the gut [62, 63, 64], and defects in the IL-10 or IL-10 receptor are known to promote early onset of UC . On the other hand, Erysipelotrichia increases mucosal permeability and stimulates inflammatory immune responses in the gut . The role of Flavobacteria in the human gut has not been clearly defined; however, Flavobacteria are less abundant in human IBD . Similarly, the function of Fusobacteria in the gut are unknown, but their numbers increase in colorectal cancer , and UC increases susceptibility to colon cancer . Therefore, it is likely that PB, through increased acetylcholine levels, (a) increases mucus formation and (b) inhibits the DSS-induced loss of the epithelial barrier function by suppressing IL-13 production. Together, this inhibits the DSS-induced migration of pathogenic bacteria and bacterial dysbiosis in the gut. Thus, PB has the potential to stabilize gut flora and attenuate inflammation associated with UC.
Cholinergic stimuli involving ACh are important in the regulation of gut function, and ACh regulates both motility and mucosal responses in the gut. Recent evidence suggests that ACh and PB tend to be protective against experimental tissue injury. PB restored cardiac autonomic balance in mice and rats after experimental myocardial infarction [9, 70, 71], and heart-specific overexpression of the ACh-synthesizing enzyme choline acetyltransferase was found to protect the myocardium against ischemia-induced injury in mice . PB has also been shown to help some patients with spinal cord injuries  and diabetic patients with gastrointestinal disorders . Moreover, release of ACh through electrical stimulation of the vagus nerve ameliorates gut inflammation through nAChRs . We have shown that ACh and neostigmine bromide induce mucus production in airway epithelial cells through α7-nAChRs , and nicotine suppresses both inflammatory and adaptive immune responses [76, 77]; interestingly, tobacco smokers were found to be very resistant to UC . In mammals, ACh is the only known biological ligand that reacts with nicotinic as well as muscarinic receptors, and nAChRs on non-neuronal cells respond to very low concentrations of nicotine [11, 79]. Therefore, it is likely that at lower concentrations, ACh reacts primarily with nAChRs thereby protecting the gut from colitis and bacterial dysbiosis and has the potential to suppress UC. Because nAChRs are present on many different cell types, including T cells, monocytes/macrophages, and epithelial cells, at present the identity of the cell type(s) that is/are the primary target of acetylcholine in the gut is unclear. In the lung, bronchial epithelial cells express nAChRs, and acetylcholine, neostigmine bromide, and nicotine promote mucus production from these cells [11, 80]; however, nicotine is known to inhibit Th2 immune responses through its effects on T cells and macrophages [8, 81]. It is therefore likely that the effects of acetylcholine/PB on UC severity involve multiple cell types. Further experiments are needed to identify the target cell type(s) that is/are critical in moderating UC in this model.
We thank Ruben Castro and Shah Hussain for help with staining and imaging of the tissue sections. This work was supported by a grant from CDMRP (GW150022) and NIH (RO1 HL 125000).
Compliance with Ethical Standards
Conflict of interest
The authors have no financial conflicts of interest.
- 11.Gundavarapu S, Wilder JA, Mishra NC, et al. Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia, and airway mucus formation in vitro and in vivo. J Allergy Clin Immunol. 2012;130:e711.Google Scholar
- 17.Moayyedi P, Surette MG, Kim PT, et al. Fecal microbiota transplantation induces remission in patients with active ulcerative colitis in a randomized controlled trial. Gastroenterology. 2015;149:e106.Google Scholar
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