Animals and Experimental Design

The study subjects were all sourced from a multigenerational pedigreed colony of vervet monkeys (Chlorocebus aethiops sabeus; n = 326, 4–27 years, lifespan ≈ 26 years) which descended from 57 founder monkeys (the Wake Forest Vervet Research Colony)21. Most females are housed within their natal social group for the duration of their naturally determined lifespan21. Due to the nature of a breeding colony, aged males are not retained, so our study populations were all female. Females were classified as either young or old (>18 years), and evaluated them in a series of studies depicted in Fig. 1. Animals in the colon survey study (Study 1) were fed a commercial laboratory primate chow (Laboratory Diet 5038; LabDiet, St. Louis, MO) with daily supplemental fresh fruits and vegetables. This standard laboratory diet comprised of 13% of calories from fat; 69% of calories from carbohydrates; and 18% of calories from protein (Supplementary Table S5). These animals were housed in large indoor/outdoor enclosures that provide elevated perches and climbing structures within multigenerational social groups and ad libitum opportunities to socialize, eat, and exercise. Diet consumption patterns were evaluated in a representative selection of young and old monkeys from the same colony by measurement of diet fed and diet remaining over 24 hours. Measures were available on average over 3 different days (range of 1–10 consumption measures/monkey). Blood and tissue samples were collected from Study 1 at a scheduled euthanasia time point, which differed from studies 2 and 3 as anaesthesia was achieved with sodium pentobarbital and blood was terminally collected from the vena cava. A separate selection of old and young monkeys from the colony were challenged with a Western Diet (Study 2; Supplementary Table S5). The Western diet comprised of 35.5% total calories supplied as fat and 14% as saturated fat. Carbohydrates were 46% of total calories with 22% supplied as simple sugars and total caloric density was. Protein content was comparable to the chow diet at 19% of calories, however the protein source was predominantly from animal sources. Dietary fibre was comparable between diets and approximates recommended human consumption levels at 35 g/day. Assessments were performed pre-diet and 8-weeks post-diet initiation. At the 8 week time point, the old monkeys continued on Western diet to evaluate the effects of SBI supplementation (Study 3). Old monkeys (n = 6) were given placebo or 6 g/day of SBI daily added to 80 g of their daily food allotment for 2 weeks in a cross-over study with 4 weeks washout between treatment periods. This dosing duration has resulted in improved MT parameters in animal studies20,26. The SBI product used was EnteraGam® (Entera Health Inc., Cary NC), which is a medical food product available by prescription for the treatment of severe inflammatory bowel disease and HIV-associated enteropathy. The dose was chosen to deliver the paediatric dose (4 g/d), while allowing for waste. The dosed food was consumed fully before additional food was offered to the monkeys. Daily observations were made and all monkeys were compliant in consuming the SBI dose. Blood samples were collected at the end of each treatment period. One animal was euthanized such that 5 monkeys were assessed with placebo and 6 monkeys were assessed with SBI treatment. The euthanasia occurred during the washout period following SBI therapy and was necessitated due to a diagnosis severe pancreatitis. Pancreatitis was confirmed by diagnostic pathology on tissues collected at necropsy.

All animal procedures were performed on a protocol approved by the Wake Forest University Institutional Animal Care and Use Committee according to recommendations in the Guide for Care and Use of Laboratory Animals (Institute for Laboratory Animal Research) and in compliance with the USDA Animal Welfare Act and Animal Welfare Regulations (Animal Welfare Act as Amended; Animal Welfare Regulations).

MT and Metabolic Health Biomarkers

Animals were fasted overnight and anesthetized with intramuscular ketamine (10 to 15 mg/kg) to allow for sample and data collections in Study 2 and 3. Each animal was weighed and waist circumference measured with a flexible tape measure at the level of the umbilicus. Blood samples were obtained by venipuncture of the femoral vein into ethylenediaminetetraacetic acid (EDTA) and serum separator blood tubes. The EDTA anticoagulated blood was held on ice until it could be processed. After processing the plasma and whole blood, samples were stored at −80 °C until analysis. General metabolic health and biochemistry panels were measured as previously described21,34. Portal vein endotoxin levels were measured from samples collected in Study 1 coincident with colon tissue and measured by the Kinetic-QCL™ Kinetic Chromogenic LAL assay (Lonza Group Ltd, Walkersville, MD) and results characterized as high (>0.5 EU/mL) or low (<0.5EU/mL) endotoxin levels. In studies 1, 2, and 3, the biomarkers of MT used were plasma concentrations of lipopolysaccharide binding protein 1 (LBP-1) and soluble CD14 measured by ELISA as previously described10. Innate immune response to MT was estimated by ELISA detection of circulatory levels of secretory IgA and the antimicrobial peptide, α-defensin 5, which is primarily sourced from gut Paneth cells (eBioscience, Vienna Austria and MyBioSource Inc., San Diego CA). We included liver enzymes alanine aminotransferase and alkaline phosphatase (ALT, ALP) in Studies 2 and 3 as portal delivery of endotoxin is known to initiate hepatic damage in monkeys within this timeframe38.

Histology

Full thickness sections collected from the midpoint between the cecum and of the transverse colon (ascending colon) were collected after euthanasia from each monkey in Study 1. Colon tissue was formalin fixed and paraffin-embedded prior to sectioning and staining with hematoxylin and eosin. Mucus content was estimated by co-staining with Alcian Blue and periodic acid Schiff staining for total proteoglycan content. After initial observation for quality, a blinded observer measured the crypt depth and number of goblet cells along 10 intact villi. Goblet cells are expressed as number per 100 microns. Mucus content was estimated by outlining 10 crypts of intact villi as a region of interest and analysing the percent area stained blue/purple using imaging software (Supplementary Fig. S3; Visiopharm, Broomfield CO). The thickness of the muscularis layer was measured in transection at its widest point. Immunohistochemical staining of the tight junction proteins occludin and claudin-1 was evaluated by color pixilation quantitation by imaging software and expressed as the average area for occludin (Image Pro Plus, Version 5.1; Media Cybernetics), and by scoring staining intensity as 1–5 for claudin-1. Antibodies for occludin and claudin-1 were sourced from Abcam (Cambridge, MA #ab15098 at 1:500) and Santa Cruz Biotechnology (Dallas, TX #133255 at 1:200) respectively, and immunostaining protocols have been previously described4. Example images are shown in Supplementary Fig. S3.

Microbiome

Samples from the 19 monkeys included in Study 1 were collected for microbiome characterization. Feces, colonic lumen content, and a mucosal scraping from the same region of ascending colon were collected from each monkey was used as material for DNA extraction. DNA was quantitated and sequenced by a commercial laboratory (HudsonAlpha Institute for Biotechnology, Huntsville AL). Briefly, DNA was extracted from tissue samples (Qiagen), the quality of DNA determined by gel electrophoresis, and then the amplified DNA was sequenced (Illumina Miseq PE250). Sequences are available at the SRA via accession SRP139357. Mucosally-sourced DNA had total bacterial gene content estimated in duplicate by PCR and was normalized to the tissue weight used as the starting material46. Old monkeys had variable 16 s abundance in the mucosa however all values, including outliers, were retained in the dataset as review of laboratory processes and data support these values are being real. Taxonomic assignments from 16 S rRNA sequencing were performed using the RDP classifier (version 2.6) with default parameters with a confidence score of 80%47. An additional taxonomic classification using QIIME (version 1.8) for closed-reference operational taxonomic unit (OTU) picking with default parameters against the Green Genes database version 13.5 at a 97% identity to test for consistency between classification methods was performed48. The resulting count tables were log normalized as previously described49. Ordination by principal coordinates analysis was performed using the Bray-Curtis dissimilarity via the capscale function of the vegan R package. The related R scripts are publicly available through GitHub at https://github.com/mcbtBINF/IntestinalAging/.

Statistical models in R were made for each of the response variables of log normalized bacterial abundance, sample Shannon diversity (representing within-sample alpha-diversity) and ordination axes (representing between-sample beta-diversity). The response variables were investigated on each of the three sample types (feces, lumen, and mucosa) as well as pooling the tissue types together in a mixed linear model. The mixed linear model nested the sample types within the associated animal using the lme function from the R package nlme, in addition to the explanatory variables of “age-group” and sample sequencing depth. Additional models consisted of the nonparametric Wilcoxon test with animals split by “old” and “young” status on each sample type, and a simple linear model with explanatory variables for “age-group” and sample sequencing depth for each sample type. The Benjamini-Hochberg method for multiple hypothesis correction was used with the false discovery rate set to 0.05. A detailed set of microbiome tables and graphs from these analyses is available in the Supplementary Data files.

Data analysis

Data are presented as means ± SEM for each group. Group sizes for each measure are indicated in figures and tables. Data were analyzed for normality and logarithmically transformed where necessary before AN(C)OVA (with adjustment for baseline value if available as in Study 2) was performed to assess for group differences. Differences in proportions in Study 1 endotoxin levels were analyzed by Fisher’s exact test. Study 3 was a cross-over design and repeated measures ANOVA was used with time, treatment sequence, and baseline values used in the statistical model. Post hoc determinations of group differences were done using Tukey’s honestly significant difference tests. Pairwise associations between variables were evaluated by using Pearson’s correlation coefficient if normally distributed. Statistical analysis was performed with Statistica v10 software (StatSoft Inc., Tulsa OK). Significance was set at α < 0.05 for group differences and α < 0.10 for trends.