Acetylcholinesterase Inhibitor Pyridostigmine Bromide Attenuates Gut Pathology and Bacterial Dysbiosis in a Murine Model of Ulcerative Colitis
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Florida International University FIU Digital Commons HWCOM Faculty Publications Herbert Wertheim College of Medicine 10-23-2019 Acetylcholinesterase Inhibitor Pyridostigmine Bromide Attenuates Gut Pathology and Bacterial Dysbiosis in a Murine Model of Ulcerative Colitis SP Singh Hitendra S. Chand S. Banerjee H. Agarwal V. Raizada See next page for additional authors Follow this and additional works at: https://digitalcommons.fiu.edu/com_facpub Part of the Medicine and Health Sciences Commons This work is brought to you for free and open access by the Herbert Wertheim College of Medicine at FIU Digital Commons. It has been accepted for inclusion in HWCOM Faculty Publications by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. Authors SP Singh, Hitendra S. Chand, S. Banerjee, H. Agarwal, V. Raizada, S. Roy, and M. Sopori Digestive Diseases and Sciences (2020) 65:141–149 https://doi.org/10.1007/s10620-019-05838-6 ORIGINAL ARTICLE Acetylcholinesterase Inhibitor Pyridostigmine Bromide Attenuates Gut Pathology and Bacterial Dysbiosis in a Murine Model of Ulcerative Colitis Shashi P. Singh1 · Hitendra S. Chand2 · Santanu Banerjee3 · Hemant Agarwal4 · Veena Raizada4 · Sabita Roy3 · Mohan Sopori1 Received: 30 March 2019 / Accepted: 10 September 2019 / Published online: 23 October 2019 © The Author(s) 2019 Abstract Background Ulcerative colitis (UC) is a Th2 infammatory bowel disease characterized by increased IL-5 and IL-13 expres- sion, eosinophilic/neutrophilic infltration, decreased mucus production, impaired epithelial barrier, and bacterial dysbiosis of the colon. Acetylcholine and nicotine stimulate mucus production and suppress Th2 infammation through nicotinic receptors in lungs but UC is rarely observed in smokers and the mechanism of the protection is unclear. Methods 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 efects on colonic tissue morphology, Th2 infammatory factors, MUC2 mucin, and gut microbiota were analyzed. Results DSS challenge damaged the murine colonic architecture, reduced the MUC2 mucin and the tight-junction protein ZO-1. The PB treatment signifcantly attenuated these DSS-induced responses along with the eosinophilic infltration and the pro-Th2 infammatory factors. Moreover, PB inhibited the DSS-induced loss of commensal Clostridia and Flavobacteria, and the gain of pathogenic Erysipelotrichia and Fusobacteria. Conclusions Together, these data suggest that in colons of a murine model, PB promotes MUC2 synthesis, suppresses Th2 infammation and attenuates bacterial dysbiosis therefore, PB has a therapeutic potential in UC. Keywords Ulcerative colitis · Pyridostigmine bromide · Dextran sodium sulfate · Bacterial dysbiosis · Acetylcholine Abbreviations AChe Acetylcholine esterase UC Ulcerative colitis TJ Tight junction IBD Infammatory bowel disease ZO-1 Zona occludin-1 PB Pyridostigmine bromide DSS Dextran sodium sulfate nAChR Nicotinic acetylcholine receptors Introduction * Mohan Sopori Ulcerative colitis (UC) is a chronic idiopathic infamma- [email protected] tory bowel disease (IBD), where infammation is mainly 1 restricted to colon. The incidence of UC is steadily increas- Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr ing particularly in the Western world [1]. Although the exact SE, Albuquerque, NM 87108, USA 2 etiology of UC is unclear, increasing evidence suggests that Department of Immunology and Nano-Medicine, Herbert a combination of factors including genetics, environmental Wertheim College of Medicine, Florida International University, Miami, FL 33199, USA agents, immune dysregulation, gut barrier dysfunction, and dysbiosis of the gut microbiota infuence the course of the 3 Department of Surgery and Sylvester Cancer Center, Miller School of Medicine, University of Miami, Miami, FL 33101, disease [2, 3]. UC is rarely observed in smokers, as nico- USA tine/nicotinic receptor agonists suppress colitis [4]; however, 4 University of New Mexico Health Sciences Center, the mechanism by which cigarette smoke/nicotine sup- Albuquerque, NM 87131, USA presses the development of UC is unclear. UC is primarily a Vol.:(0123456789)1 3 142 Digestive Diseases and Sciences (2020) 65:141–149 Th2-mediated IBD associated with eosinophilic infltration Histochemical Staining and Scoring and overproduction of Th2 chemokines/cytokines including eotaxin (CCL11), IL-5, and IL-13 [5–7]. In animal models, Formalin-fxed and parafn-embedded tissue sections (5 activation of pulmonary nicotinic acetylcholine receptors μm) were stained with hematoxylin and eosin (H&E) for (nAChRs) moderates eosinophilic infltration through sup- general histology. Histochemical staining with Alcian Blue pression of Th2 responses [8]. In mammals, acetylcholine and periodic acid–Schif reagent (AB–PAS) was carried out (ACh) is the only known biological ligand for nAChRs, and as described previously [21]. Eosinophils and goblet cells increasing evidence suggests that ACh and PB—the latter were enumerated microscopically. Tissue sections were of which increases ACh by inhibiting acetylcholine esterase coded and assessed in a blinded manner. Histological dam- (AChE)—tend to protect against tissue injury [9, 10]. We age was scored as described previously [18]. Briefy, sec- and others have shown that nicotine, ACh, and the AChE tions were scored for eosinophil infltration (0–3), with 0 inhibitor neostigmine bromide (similar to PB) promote representing less than three eosinophils per feld of view at mucus formation in human lung epithelial cells through 40× magnifcation in the lamina propria, 1 for greater than α7-nAChRs [11, 12]. MUC2, the predominant mucin in the three eosinophils per feld of view in the lamina propria, 2 gut, plays a critical role in gut homeostasis, and MUC2- representing confuence of eosinophils extending into the defcient mice develop colitis spontaneously [13]; however, submucosa, and 3 representing confuence of eosinophils unlike MUC5AC in bronchial epithelial cells [14], IL-13 present in all tissue layers. does not always stimulate MUC2 [15]. UC also afects gut microbiota and the integrity of the epithelial barrier in the Immunofuorescence Imaging colon [2, 5, 6]; thus normal fecal microbial transplantation is a promising treatment for UC [16, 17]. As DSS has been Deparafnized and hydrated tissue sections were washed in found to induce UC-like conditions in a mouse model [18, 0.05% v Brij-35 in phosphate-bufered saline (PBS; pH 7.4) 19], in this study we examined whether PB attenuated DSS- and immunostained for antigen expression as described pre- induced colon pathology in C57BL/6 mice. viously [22]. Briefy, the antigens were unmasked and incu- bated in a blocking solution. The sections were stained with antibodies to ZO-1 (Invitrogen Inc., Carlsbad, CA, USA) or Materials and Methods anti-eosinophil antibody (Novus Biologicals, Littleton, CO, USA), or isotype control antibodies. Immunofuorescence Animals images were captured with a BZX700 all-in-one microscope (Keyence Corp., Japan). Pathogen-free 5–6-week-old C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Ani- qRT‑PCR Analysis mals were kept in chambers as described previously [11]. All animal protocols were approved by the Institutional Animal Total RNA from frozen mouse colon tissue was isolated Care and Use Committee. using TRI Reagent (Molecular Research Center, Inc., Cin- cinnati, OH, USA). Quantitative reverse transcription poly- DSS and PB Treatment merase chain reaction (qRT-PCR) was performed using the StepOnePlus detection system (Applied Biosystems, Foster Mice (8–9 weeks old) were divided into three groups (con- City, CA, USA) and the TaqMan One-Step RT-PCR kit con- trol, DSS, and DSS+PB) with 6–8 animals/group. PB and taining AmpliTaq Gold® DNA polymerase [23]. Specifc DSS were purchased from Sigma-Aldrich, USA. Mice were primers and probes were obtained from Applied Biosystems. given PB through intraperitoneally implanted Alzet mini- Fold changes in qPCR expression were calculated by the osmotic pumps [20] starting at day 7 prior to DSS treatment 2(−ΔΔCT) method [24]. and continued until they were euthanized. Mice implanted with mini-osmotic pumps containing sterile saline served Western Blot Analysis as the control. The pumps provided PB at a concentration of 2 mg/day/kg body weight. Colitis was induced by 3.0% Western blot analysis of eotaxin, IL-5, IL-13 and MUC2 (w/v) DSS-containing water. The treatment continued for in mice colon homogenates was performed as described 7 days; the animals were euthanized and colons harvested previously [23]. Briefy, colon homogenates (70 μg) were on day 8. Animals were weighed daily after DSS treatment. fractionated on 10% sodium dodecyl sulfate polyacryla- Where indicated, intestines were dissected to obtain ileum, mide gel electrophoresis (SDS-PAGE) for eotaxin, IL-5, cecum, and colon; tissues were frozen for RNA and protein, and IL-13, and 5% SDS-PAGE for MUC2. After transfer to and fecal material for bacterial microbiome analysis. 1 3 Digestive Diseases and Sciences (2020) 65:141–149 143 nitrocellulose membranes, blots were probed with antibod- Results and Discussion ies to eotaxin, IL-5, IL-13, and MUC2 (Abcam), and after stripping were re-probed with anti–β actin antibody. PB Ameliorates the