Colonic Diverticula Are Not Associated With Mucosal Inflammation or Chronic Gastrointestinal Symptoms

Anne F. Peery; Temitope O. Keku; Cassandra Addamo; Amber N. McCoy; Christopher F. Martin; Joseph A. Galanko; Robert S. Sandler

Disclosures

Clin Gastroenterol Hepatol. 2018;16(6):884-891. 

In This Article

Methods

Study Design

Data for this analysis came from a prospective study to assess risk factors for colonic diverticulosis (National Institutes of Health R01DK094738). The study recruited outpatients undergoing a first-time screening colonoscopy between 2013 and 2015 at the University of North Carolina Hospital in Chapel Hill, North Carolina. The study included patients age 30 years and older with at least a satisfactory preparation for colonoscopy and a complete examination to the cecum. The study excluded any patient with a prior colonoscopy, familial polyposis syndrome (defined as >100 polyps or positive for a familial adenomatous polyposis gene test), evidence of colitis, previous colon resection, previous colon cancer or adenomas, or indication other than screening. Informed consent was obtained from all participants. For this analysis, we excluded patients with a history of diverticulitis (n = 1) or overt diverticular inflammation (n = 4). The University of North Carolina School of Medicine Institutional Review Board approved this study.

Participant Interviews

Each participant completed a detailed telephone interview before the colonoscopy. The interview captured diet, physical activity, tobacco and alcohol use, nonaspirin nonsteroidal anti-inflammatory drug use, aspirin use, bowel habits, and gastrointestinal symptoms. Dietary intake was collected using a 124-item food frequency questionnaire[10] and covered a 12-month period to avoid seasonal variation. Physical activity was assessed using a validated instrument and was expressed as metabolic equivalents in minutes.[11] Race and bowel movement frequency were self-reported. Participants identified the stool form (using the Bristol Stool Scale) they had most of the time. Gastrointestinal symptoms were assessed using Rome III diagnostic criteria questions for irritable bowel syndrome. Chronic abdominal pain was defined as pain or discomfort at least 2 to 3 days a month for at least 6 months.

Colonoscopy and Biopsy

A research assistant measured the participant's height and weight the day of the colonoscopy. During the colonoscopy, the gastroenterologist assessed the colon for diverticula either on insertion or withdrawal. A research assistant was present during the entire examination to document the procedure on a standard data collection form. The gastroenterologist counted the number of diverticula in the colon by segment: cecum, ascending, hepatic flexure, transverse, splenic flexure, descending, and sigmoid colon and reported the findings to the research assistant. Biopsy specimens were taken from normal-appearing mucosa in the midsigmoid in cases and controls for assessment of inflammatory markers. The biopsy specimens (diameter, approximately 3–4 mm) were obtained using standard (8-mm wing) disposable, fenestrated, colonoscopy forceps (Olympus, Center Valley, PA). Laboratory personnel were blinded to clinical information and the diverticulosis status of the subjects.

Intraepithelial Lymphocytes

To evaluate the role of the mucosal immune system in the etiology of diverticulosis, we assessed immune markers and cytokine levels that have been implicated in inflammatory bowel disease and irritable bowel syndrome. We evaluated the following immune markers: CD4, CD8, CD57, and mast cell tryptase. Mouse monoclonal antibodies against human CD4 (NCL-L-CD4-368), CD8 (CD8-4B11-L-CE-S), and CD57 (NCL-NK1) were purchased from Leica Microsystems, Inc (Norwell, MA). Mouse monoclonal anti-human mast cell tryptase (M7052) was purchased from Dako (Carpinteria, CA).

Immunohistochemistry was performed using the Bond fully automated slide staining system (Leica Microsystems, Inc). Tissue sections were deparaffinized in Bond dewax solution (AR9222) and hydrated in Bond wash solution (AR9590). Heat-induced antigen retrieval was performed for 30 minutes at 100°C in Bond-Epitope Retrieval solution 1, pH 6.0 (AR9961; CD4, CD57, and mast cell tryptase) or for 20 minutes in Bond-Epitope Retrieval solution 2, pH 9.0, (AR9640; CD8). Slides were incubated in anti-CD4 or anti-CD8 for 30 minutes at a dilution of 1:200. Incubation in anti-CD57 was performed for 1 hour at a dilution of 1:100, and mast cell tryptase was used at a dilution of 1:3000 with an incubation time of 30 minutes. Detection of all antibodies was performed using the Bond Polymer Refine Detection System (DS9800). Stained slides were dehydrated and cover slipped. Positive and negative controls (no primary antibody) were included for each antibody.

Immunohistochemistry stained sections were imaged digitally (objective, ×20) using the Aperio ScanScope XT (Aperio Technologies, Vista, CA). Analysis of biomarkers was performed using Definiens Tissue Studio software (version 3.6.1; Munich, Germany) with the Composer_Nuclei_and_Simulated_Cells algorithm. The algorithm was trained to recognize cells digitally from the stromal compartment of the colon tissue samples, greatly reducing the number of epithelial cells included in the analysis. The percentage of cells with strong (+3), medium (+2), and weak (+1) positive 3,3′-diaminobenzidine tetrahydrochloride signal was used to compare biomarker levels among tissue samples. The H score was calculated for each immune marker. The H score is a composite score that is based on the intensity of staining. The stained cells are counted and then multiplied by a constant based on the intensity of the stain (eg, most intensely stained cells are multiplied by 3). The H-score is computed as follows: (% at 0) × 0 + (% at 1+) × 1 + (% at 2+) × 2 + (% at 3+) × 3. The H score is a continuous variable that ranges from 0 to 300.

We assessed messenger RNA (mRNA) expression levels of interleukin (IL)6, IL10, and tumor necrosis factor α (TNF-α) by quantitative reverse-transcriptase polymerase chain reaction using an established protocol.[12] Briefly, RNA was extracted from mucosal biopsy specimens stored in RNAlater using the RNEasy Mini Kit (Qiagen, Valencia, CA). RNA was quantified by Nanodrop spectrophotometry (ThermoScientific, Wilmington, DE) and the quality was assessed on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). To ensure that the RNA was free of DNA contamination, we performed DNase digestion using RQ1 RNase Free DNase (Promega, Madison, WI) following the manufacturer's protocol. One microgram of RNA was reverse-transcribed to complementary DNA using Invitrogen cloned AMV (Invitrogen, Carlsbad, CA). Quantitative reverse-transcriptase polymerase chain reaction was performed in duplicate using SYBR Green (BioRad, Hercules, CA) and RT2 profiler (Qiagen) ready-to-use optimized primer sets specific for each gene. The expression of each inflammatory cytokine was evaluated relative to hydroxymethylbilane synthase, which served as a housekeeping gene.

Statistical Analysis

Means and SDs were calculated for continuous variables, medians were calculated for skewed distributions of continuous variables, and proportions were calculated for categoric data. For analysis in the models, the immune markers and mRNA expression levels were converted into categories (quartiles). The 10% change-in estimate approach was used to identify confounding variables.[13] To assess the association between colonic diverticula and markers of mucosal inflammation or gastrointestinal symptoms, proportional odds models[14] were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). The models were adjusted for age, sex, and body mass index. We performed analyses with all diverticulosis cases and cases stratified by number of diverticula. All tests of significance were 2-tailed and P values less than .05 were considered significant. The analysis was performed using SAS 9.4 (SAS, Cary, NC).

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