The present work highlights the relevance of microbiota in pain signalling from the gut. The FMT from animals affected by visceral pain to healthy rats was sufficient to induce visceral hypersensitivity. On the other hand, FMT from healthy donors induced a reduction of persistent visceral pain. A strong association between animals’ pain threshold and microbiota composition changes was demonstrated. Indeed, intestinal inflammation as well as gut permeability and gut monoamines were found not to be involved in pain transfer mediated by FMT.
Although the gut microbiota appeared to be clearly involved in visceral pain regulation [3–5], the evidence collected so far are circumstantial [16–18]. Moreover, little is known on the evolution of microbiota over time after transplantation from animals affected by a disease, like pain, to naïve recipients. Here, we demonstrated a distinct temporal correlation between the animals’ sensitivity and changes in the microbiota composition. As suggested by the analysis of the beta-diversity in samples from FMTDNBS animals, major modifications in the composition and structure of the gut microbiota occurred after the FMT set II (Day 32), correlating with a significantly increased response of these animals to CRD. At a later stage (Day 46), when the difference in visceral sensitivity perception disappeared, a less pronounced variation in beta-diversity was observed.
Evaluation of taxonomic profiles confirmed that marked differences between the animals that underwent the FMTCTR or FMTDNBS were identifiable on Day 32 at phylum and family level for major members of gut microbiota, including protective microbial taxa known to be associated with gastrointestinal health (i.e. Akkermansiaceae) [19, 20]. Akkermansia muciniphila is known to mediate a variety of host functions ranging from metabolism to immune regulation [21–23]. Regarding its role in pain syndromes or functional gastrointestinal disorders, A. muciniphila was found to be significantly reduced in children with IBS [24] as well as in animal models of post-inflammatory IBS [25]. Recently, Guida et al. reported that pain related to dysbiosis in vitamin D deficient mice were ameliorated by an anandamide congener, in concomitance with increased levels of Akkermansia, Eubacterium and Enterobacteriaceae [26]. However, there is little evidence for a pain-alleviating effect mediated by A. muciniphila. One hypothesis is that SCFAs, like propionate and acetate, both end-products of mucus degradation by Akkermansia, may modulate visceral nociception, as stimulation of the SCFA receptor GPR43 has been shown to decrease visceral pain sensitivity in healthy humans [27, 28]. The SCFAs have been associated to human gastrointestinal disorders [29] and proposed as regulators of visceral sensitivity since the activation of their receptors, FFAR2/3 regulates leucocyte functions, such as the production of cytokines (e.g. TNF-a, IL-2, IL-6, and IL-10), eicosanoids and chemokines (e.g. CCL2)[30]. Nevertheless, the role of SCFAs in visceral and somatic pain perception is still the subject of debate: depending on the context the effect of SCFAs signalling can be either analgesic or nociceptive [31]. Clinical evidence has shown that butyrate, a SCFAs found dramatically reduced in the faecal samples of IBS and IBD patients, is highly effective in relieving abdominal pain [32, 33]. The significant reduction of butyrate in the FMTDNBS group on Day 32 is in line with this evidence. Nevertheless, despite the reduction of butyrate on Day 32, the total amount of SCFAs increased in these animals both on Day 18 and 32 mainly contributed by an overproduction of acetate. Specific bacteria taxa were found to be negatively correlated with the increased acetate levels uniquely on Day 32, suggesting the presence of a defined microbiota profile at this stage. Of note, the recently identified bacterial genus Acetatifactor belonging to Lachnospiraceae, one of the most prominent bacterial family being expanded in samples from the FMTDNBS group, and showing strong acetate-producing capabilities [34], resulted overrepresented in FMTDNBS samples (Figure S4B). However, there are no consistent data in literature reporting a negative impact of acetate on gut physiology.
By monitoring the time course modifications and evolution of the gut microbiota in this model, emerged that the effects of FMT were long-lasting but temporary, demonstrating the presence of a continuous bidirectional influence between the microbiota and the gut, as previously postulated [35, 36]. FMTDNBS-induced visceral hypersensitivity was evident already 24 h after the first set of FMT, suggesting the presence of pain mediators in the supernatant obtained from the faeces, as supposed in other studies [17, 37]. The presence of pain mediators in the faecal medium might explain the acute effect of FMT. By contrast, the long-lasting hyperalgesia observed in these animals is not attributable to the acute stimulation, nor to local intestinal irritation, since colon histological analysis revealed no damage on Day 32 (7 days after the last FMT). These findings support the idea of an active role of microbiota in pain induction mediated by the FMT. Besides, the apparent longer persistence of visceral hypersensitivity in the AWR test in respect to VMR test, suggested a possible central sensitization to the painful stimulus induced by CRD. Similarly, in the animals treated with DNBS, VMR response was significantly decreased after 4 weeks, while the augmented AWR response persisted up to 13 weeks after DNBS injection [38].
A slight increase in gut permeability occurred after the antibiotic treatment, with an increase in the expression of Zo-1, which has been positively correlated with a leaky gut [39], confirming the positive effect of microbiota on gut barrier integrity maintenance [40].
The bacterial community participates in maintaining intestinal homeostasis through the “training” of the immune system and inhibiting growth of pathogens [41]. The analysis of gene expression in the gut confirmed a derangement of cytokines as a result of both the antibiotic treatment and the FMT, but no significant correlations were found between the cytokines profiles among the groups and the observed differences in visceral pain.
Then we analysed the effect of FMT on tryptophan metabolism (Table S1-2), considering its relevance in the microbiota-gut-brain axis as well as in the pathophysiology of both IBS and IBDs [42]. A decrease in plasma tryptophan was found in DNBS donors, likely consequent to an increased biosynthesis of serotonin in the gut, as confirmed by the HPLC (Table S3-4). This phenomenon, which is a characteristic consequence of intestinal damages, can directly influence the neuronal firing of visceral afferents [43, 44]. A plasma tryptophan decrease was also detected in the animals underwent FMT and correlated to a massive and long-lasting increase of serotonin in the faeces (Table S6), but not in the gut tissue (Table S3). An increase of 5-HT in the gut lumen has been recently demonstrated to selectively modulate the colonization of bacteria species in the gut [45]. Nevertheless, no difference was found between the animals receiving the FMT from CTR or from DNBS donors, demonstrating again that 5-HT is not involved in the transfer of pain mediated by FMTDNBS.
The decrease of dopamine consequent to the antibiotic treatment and the FMT (Table S5 and S7) resulted in accordance to previous data reporting an antibiotic-mediated reduction of this neurotransmitter in the gut, where it plays a role in gastrointestinal motility regulation [46].
The anti-hyperalgesic effect obtained by transplanting a healthy microbiota in DNBS animals contributes further to sustain the role of microbiota in pain signalling. Currently, many discrepancies do exist on the therapeutic potential of FMT [47]. So far it has been considered a good protocol to maintain a low number of FMT and supplied by a single donor [48, 49]. However, recent clinical studies highlighted the efficacy of a multiple donor and repeated treatment-based therapy in patients affected by UC, chronic intestinal pseudo-obstruction and IBS [50–52]. In certain pathological conditions, the interactions between genetic, immunological, and environmental factors might determine a state more resistant to therapeutic microbial manipulation [53]. In the preclinical model of colitis-induced visceral hypersensitivity, we obtained a substantial reduction in pain after 4 cycles of FMT, utilizing different donors. It is likely that the “inflammatory” environment wherein the microbiota is transplanted may pose as an obstacle to the engraftment of some species of microbiota compared to others. In this case, the repetition of the FMT could “force” the host to accept the newly introduced microbiota. These findings suggest the possibility to enhance the therapeutic efficacy of FMT by developing different protocols according to the specific disorder to be treated.