Exercise training-induced modification of the gut microbiota persists after microbiota colonization and attenuates the response to chemically-induced colitis in gnotobiotic mice

Abstract

Exercise reduces the risk of inflammatory disease by modulating a variety of tissue and cell types, including those within the gastrointestinal tract. Recent data indicates that exercise can also alter the gut microbiota, but little is known as to whether these changes affect host function. Here, we use a germ-free (GF) animal model to test whether exercise-induced modifications in the gut microbiota can directly affect host responses to microbiota colonization and chemically-induced colitis. Donor mice (n = 19) received access to a running wheel (n = 10) or remained without access (n = 9) for a period of six weeks. After euthanasia, cecal contents were pooled by activity treatment and transplanted into two separate cohorts of GF mice. Two experiments were then conducted. First, mice were euthanized five weeks after the microbiota transplant and tissues were collected for analysis. A second cohort of GF mice were colonized by donor microbiotas for four weeks before dextran-sodium-sulfate was administered to induce acute colitis, after which mice were euthanized for tissue analysis. We observed that microbial transplants from donor (exercised or control) mice led to differences in microbiota β-diversity, metabolite profiles, colon inflammation, and body mass in recipient mice five weeks after colonization. We also demonstrate that colonization of mice with a gut microbiota from exercise-trained mice led to an attenuated response to chemical colitis, evidenced by reduced colon shortening, attenuated mucus depletion and augmented expression of cytokines involved in tissue regeneration. Exercise-induced modifications in the gut microbiota can mediate host-microbial interactions with potentially beneficial outcomes for the host.

Keywords: colitis; colonization inflammation; exercise; germ-free; gut; microbiome; microbiota; transplant; voluntary wheel running.

Figure 1.

Experimental Design. Graphical representation of the designs of Experiment 1 (colonization experiment) and Experiment 2 (acute colitis experiment). CMT = cecal microbial transplant, DSS = dextran sodium sulfate.

Figure 2.

Comparisons of gut microbiome β-diversity in donor and recipient mice. (Experiment 1). (A) Exercise training leads to differences in β-diversity of the fecal microbiome (Unweighted UniFrac) of donor mice after 6 weeks of exercise training; red circles = Donor-Control (D-CON), green circles = Donor-Exercise (D-EX); PERMANOVA p<0.05. (B) Community structure of the fecal microbiome (Unweighted UniFrac) of recipient, gnotobiotic mice 5 weeks after colonization by donor cecal microbiota; orange circles = Recipient-Control (R-CON), teal circles = Recipient-Exercise (R-EX); PERMANOVA p<0.01. (C) Abundance (% of total bacteria) of bacterial genera that were differentially represented by exercise group in donor mice and that remained differentially abundant (*) in recipient mice after transplant. *FDR p < 0.05.

Figure 3.

Distal colon cytokine gene expression in recipient mice after colonization (Experiment 1). (A) Pro-inflammatory and, (B) Anti-inflammatory cytokines after 5 weeks of colonization (n = 15-16/group). *denotes significant difference, ns = not significantly different at p < 0.05.

Figure 4.

Distal colon histology of recipient mice after colonization (Experiment 1). (A) Representative hemotoxylin and eosin (H&E) stains (20x) of distal colons from i) R-CON and ii) R-EX after 5 weeks of colonization. Open arrows indicate areas of goblet cell (GC) depletion in R-CON vs R-EX mice. Closed arrows indicate areas of inflammatory cell infiltrate. (B) Histological scoring of distal colons from recipient mice after 5 weeks of colonization: i) inflammatory cell infiltration (range 0–4), ii) GC mucus depletion (range 0–4) and, iii) overall histology score (range 0–16); includes aggregate scores of inflammatory cell infiltration, GC mucus depletion, destruction of architecture and crypt abscesses (ranges 0–4; not significant, data not shown). *denotes significant difference at p < 0.05.

Figure 5.

Body weight and cecal SCFA profiles of recipient mice after colonization (Experiment 1). (A) Body weight after 5 weeks of colonization in recipient mice.(B-D) Short chain fatty acid concentrations (μmole/gram) of cecal content dry matter. (E) Relative gene abundance (per 16S rRNA) of the butyrate producing enzyme butyryl CoA: acetate CoA transferase (BCoAT) in the feces of recipient mice.(F) Ratio of [Butyrate]: [Acetate] strongly correlates to body weight. (G) Pathway schematic of acetate to butyrate conversion through BCoAT. *p < 0.05

Figure 6.

Gut microbiome β-diversity in recipient mice after colonization and an acute, DSS-colitis insult (Experiment 2). Transfer of cecal contents from EX or CON donor groups led to differences in community structure of the fecal microbiota (Unweighted UniFrac) in recipient, gnotobiotic mice after acute DSS colitis insult; dark blue circles = Recipient-Exercise-DSS, pink circles = Recipient-Control-DSS. PERMANOVA, p < 0.01.

Figure 7.

DSS-induced clinical symptoms in recipient mice (Experiment 2). (A) Percent (%) body weight change, (B) percent (%) diarrhea incidence, and (C) percent (%) rectal bleeding incidence of recipient mice during eight days of acute colitis insult. (D) Colon lengths of recipient mice after sacrifice on Day 8 of colitis insult. *denotes significant difference at p < 0.05.

Figure 8.

Distal colon gene expression in DSS-treated recipient mice (Experiment 2). Colonic gene expression of (A) pro-inflammatory and (B) anti-inflammatory cytokines in recipient mice that received 5 days of DSS (2%) followed by 3 days of H₂O after a 4 week colonization period. *denotes significant difference at p < 0.05.

Figure 9.

Distal colon histology in DSS-treated recipient mice (Experiment 2). (A) Representative H&E stains of distal colons from i) R-CON-DSS (20x) and ii) R-EX-DSS (20x) after 4 weeks of colonization followed by DSS colitis insult. Open arrows indicate areas of goblet cell depletion. Closed arrows indicate areas of inflammatory cell infiltrate. (B) Histological scoring of distal colons: i) inflammatory cell infiltration (range 0–4), ii) GC mucus depletion (range 0–4), and iii) overall histology score (range 0–16); includes aggregate scores of inflammatory cell infiltration, GC mucus depletion, destruction of architecture and crypt abscesses (ranges 0–4; not significant, data not shown). *denotes significant difference at p < 0.05.