Visceral sensitivity modulation by faecal microbiota transplantation: the active role of gut bacteria in pain persistence

Abstract

Recent findings linked gastrointestinal disorders characterized by abdominal pain to gut microbiota composition. The present work aimed to evaluate the power of gut microbiota as a visceral pain modulator and, consequently, the relevance of its manipulation as a therapeutic option in reversing postinflammatory visceral pain persistence. Colitis was induced in mice by intrarectally injecting 2,4-dinitrobenzenesulfonic acid (DNBS). The effect of faecal microbiota transplantation from viscerally hypersensitive DNBS-treated and naive donors was evaluated in control rats after an antibiotic-mediated microbiota depletion. Faecal microbiota transplantation from DNBS donors induced a long-lasting visceral hypersensitivity in control rats. Pain threshold trend correlated with major modifications in the composition of gut microbiota and short chain fatty acids. By contrast, no significant alterations of colon histology, permeability, and monoamines levels were detected. Finally, by manipulating the gut microbiota of DNBS-treated animals, a counteraction of persistent visceral pain was achieved. The present results provide novel insights into the relationship between intestinal microbiota and visceral hypersensitivity, highlighting the therapeutic potential of microbiota-targeted interventions.

Figure 1.

Effect of antibiotic treatment and FMT from DNBS-treated animals on visceral sensitivity of naive recipients. As shown in the scheme (A), rats were treated, with a combination of antibiotics for 7 days; the control group was treated with vehicle. On day 7, the abx-treated animals were divided into 3 groups, FMT from CTR donors, FMT from DNBS donors or vehicle, and were, respectively, administered per os for 5 consecutive days. One week after, the administrations were repeated. Behavioural tests were performed at the end of the antibiotic treatment, 24 hours and 7 days after each cycle of FMT and once week after the last treatment. Visceral sensitivity was assessed in the animals by measuring the AWR (B,D) and the VMR (C,E) to CRD (0.5-3 mL). Each value is the mean ± SEM of 5 rats per group in the VMR test and 10 or 14 (abx + FMT^DNBS) rats per group for the AWR test. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. *P < 0.05 and **P < 0.01 vs vehicle or vehicle + vehicle–treated animals. §P < 0.05 and §§P < 0.01 vs abx + FMT^CTR–treated animals. abx, antibiotics; AWR, abdominal withdrawal reflex; CRD, colorectal distension; CTR, control animals; DNBS, dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation; VMR, visceromotor response.

Figure 2.

Analysis of beta diversity and taxonomic profiles. Principal coordinate analysis plots based on the Bray–Curtis beta diversity metric showing (A) the clustering pattern (left) among samples from CTR and from antibiotic-treated animals after vehicle administration (abx + vehicle); (B) the clustering pattern (right) among samples from antibiotic-treated animals subjected to FMT from controls (abx + FMT^CTR) or from DNBS-treated animals (abx + FMT^DNBS). Samples obtained from control (FMT^CTR) or DNBS-treated (FMT^DNBS) animals and used for the I and II set of FMT are also shown. The percentage of total variance explained is shown for each component. Taxonomic composition at the phylum (C) and family (D) level of samples belonging to the abx + FMT^CTR and to the abx + FMT^DNBS group; microbial taxa showing a statistically significant difference (Kruskal–Wallis P < 0.05) in their relative abundance between groups are marked with a star (*) in each legend box. abx, antibiotics; CTR, control animals; DNBS, dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.

Figure 3.

Effect of FMT on faecal SCFAs concentrations. HPLC analysis of (A) total SCFAs, (B) acetic acid, (C) propionic acid, (D) butyric acid, and (E) other fatty acids concentration in faecal pellets. Each value represents the mean ± SEM of 9 (day 18) or 5 (day 32 and 46) animals per group. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. **P < 0.01 and *P < 0.05 vs abx + FMT^CTR treated animals. abx, antibiotics; CTR, control animals; DNBS, dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation; SCFA, short-chain fatty acid.

Figure 4.

Histological evaluation of colon damage after the antibiotic regimen and the FMT. Macroscopic damage score (left panel) and representative microphotograph of haematoxylin or eosin-stained colon slices. Each value represents the mean ± SEM of 6 (A - day 7 and B - day 32) or 4 (C - day 46) animals per group. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. **P < 0.01 vs vehicle-treated animals. abx, antibiotics; CTR, control animals; DNBS, dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.

Figure 5.

Effect of antibiotic regimen and FMT on gut permeability. (A) Elisa assay for LBP in plasma samples. Analysis of occludin (B) and Zo-1(C) gene expression on colon samples by RT-quantitative PCR. The mRNA expression was normalized to β-actin, and fold changes were expressed in comparison with the control group. (A) Each value is the mean ± SEM of 3 or 5 (vehicle + vehicle group) animals per group; (B and C) Each value is the mean ± SEM of 6 (donors, day 7 and day 32) or 4 (day 46) animals per group. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. *P < 0.05 and **P < 0.01 vs vehicle-treated animals. abx, antibiotics; CTR, control animals; DNBS, dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation LBP, lipopolysaccharide-binding protein; mRNA, messenger RNA.

Figure 6.

Effect of antibiotic regimen and FMT on the gut cytokines profile. Analysis of TNF-α (A), IL-6 (B), IL-10 (C), and TGF-β (D) gene expression on colon samples by Real Time PCR. The mRNA expression was normalized to β-actin, and fold changes were expressed in comparison with the control group. Each value is the mean ± SEM of 6 (donors, day 7 and day 32) or 4 (day 46) animals per group. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. *P < 0.05 and **P < 0.01 vs vehicle-treated animals. abx, antibiotics; CTR, control animals; DNBS, dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation; IL, interleukin; mRNA, messenger RNA; TGF, transforming growth factor; TNF, tumor necrosis factor.

Figure 7.

Therapeutic effect of FMT on DNBS-induced postinflammatory visceral pain. As shown in the scheme (A), rats were intrarectally injected with DNBS (30 mg in 0.25 mL EtOH 50%); on day 7, DNBS-injected animals were divided into 2 groups, respectively, administered with the vehicle or the FMT from CTR donors per os for 5 consecutive days. The FMT set was weekly repeated for 4 times. Behavioral tests were performed at the end of the antibiotic treatment, 3 days after each cycle of FMT, and once a week afterwards (B-D). Visceral sensitivity was assessed in the animals by measuring the AWR to CRD (0.5-3 mL). Each value is the mean ± SEM of 5 animals per group. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. *P < 0.05 and **P < 0.01 vs vehicle or vehicle + vehicle–treated animals. ^P < 0.05 and ^^P < 0.01 vs DNBS + vehicle–treated animals. abx, antibiotics; AWR, abdominal withdrawal reflex; CRD, colorectal distension; CTR, control animals; DNBS, dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.