Microbiome and gut as partners in the persistence of post-inflammatory visceral pain: insight into working mechanisms of faecal transplant for therapy advance

Microbiome and gut as partners in the persistence of post-inflammatory visceral pain: insight into working mechanisms of faecal transplant for therapy advance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Microbiome and gut as partners in the persistence of post-inflammatory visceral pain: insight into working mechanisms of faecal transplant for therapy advance Elena Lucarini, Alfonsina D’Amato, Vincenzo Di Pilato, Fabio Morecchiato, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6025304/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background. Gut dysbiosis is a common feature of patients complaining of chronic abdominal pain, including those in remission from inflammatory bowel diseases (IBDs). Although visceral sensitivity in animals can be modulated by faecal microbial transplant (FMT), controversy has emerged in clinical practice, drawing attention to the poor knowledge of the mechanisms underpinning host-microbiota crosstalk under pathological conditions. Here we sought to elucidate the mechanisms linking post-inflammatory dysbiosis and pain persistence, to improve the therapeutic strategies. Results. Colitis was induced in rats by intrarectal injection of 2,4-dinitrobenzenesulfonic acid (DNBS). Naïve rats subjected to FMT from viscerally hypersensitive DNBS-treated rats (FMT DNBS ) displayed a higher sensitivity than those receiving FMT from healthy donors (FMT CTR ). A reverse protocol highlighted the anti-hyperalgesic effect of FMT CTR in DNBS-treated rats. Difference in microbiota-to-gut signalling between dysbiotic or eubiotic conditions was investigated after the treatment with FMT CTR or FMT DNBS , in both the experimental protocols. Modification of pain threshold in the animals undergoing FMT correlated with changes in the composition of the gut microbiota and the metabolic profile, evaluated by 16S rRNA and proton Nuclear Magnetic Resonance ( 1 H NMR). Although a specific microbial community associated with the pain phenotype was not identified, significant differences between painful and painless conditions were detected in the faecal metabolome, involving fatty acids, purine metabolites, glutamate and lactate. Yet, proteomic analysis of colon tissues from FMT recipients revealed differential scenarios. Indeed, the proalgesic effect of FMT DNBS in healthy animals was accompanied by a detrimental modulation of pathways regulating cellular plasticity and clearance, cell-to-cell interaction and signaling, as well as epithelium growth and neuroplasticity phenomenon, besides metabolism and inflammation. Pain relief following FMT CTR in DNBS-treated rats was instead associated with a counteraction of inflammatory process, lymphocyte activation and submucosal mast cells infiltration. Conclusions. These results indicate the presence of signalling modulation within the gut rather than tissue alterations at the base of FMT-related effects on pain, providing novel insights into the mechanisms linking intestinal microbiota to visceral sensitivity which might be exploited to enhance the effectiveness of microbiota-targeted interventions in the treatment of chronic pain in IBD patients. FMT IBD visceral pain dysbiosis immune response neuroplasticity metabolic injury Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Abdominal pain is a hallmark of functional gastrointestinal disorders, such as irritable bowel syndrome (IBS), which often results from infections or prolonged inflammation, as seen in chronic inflammatory bowel diseases (IBDs) [ 1 , 2 ]. Colitis is characterized by relapsing and remitting phases and by the establishment of abdominal pain persisting even in a state of low disease activity [ 3 ]. Several gut-to-brain signalling factors play a significant role in the persistence of post-inflammatory hypersensitivity, such as peripheral and central sensitization, altered sympathovagal balance, hypothalamic–pituitary–adrenal axis activation, and psychosocial factors [ 3 ]. Due to its complex, multifactorial nature, post-inflammatory pain is often refractory to classical pain medications [ 4 , 5 ], hence the need for alternative approaches. The microbiota of IBD patients is substantially different from that of healthy controls in terms of diversity, volatility (change over time), taxonomy, and metabolism [ 6 – 10 ], even in a state of remission [ 11 ], suggesting that the microbiota might act as an important driver of chronicity. The gut microbiota represents a source of signalling molecules, including neurotransmitters and neuromodulators, which may play an important role in mediating neuroinflammation involved in peripheral and central sensitization [ 12 , 13 ]. There are numerous studies, both preclinical and clinical, reporting a close relationship between gut dysbiosis and visceral pain, [ 14 – 18 ] However, there are still inconsistencies to support the microbiota as a therapeutic target for visceral hypersensitivity. Furthermore, clinical practice, shows contradictory results on the efficacy of microbiota-targeted interventions, including faecal microbiota transplant (FMT), for pain associated with gastrointestinal diseases [ 19 – 25 ]. Mechanisms regulating microbiota-host interaction and its inflammation-mediated modifications remains to be determined. To delve deeper into post-inflammatory changes, we used an established model of colitis induced by intra-rectal injection of DNBS in rats, which recapitulates IBD in terms of pathophysiology and response to drugs [ 3 , 26 ]. In our previous work [ 27 ], we demonstrated that visceral hypersensitivity can be transferred from animals in the post-inflammatory phase of colitis induced by DNBS to naive control rats by means of FMT. Furthermore, a significant amelioration of persistent pain was achieved by the transfer of a healthy microbiota to DNBS-treated rats. In both cases, the FMT effect was long-lasting and reversible after FMT discontinuation, with animals returning to their initial condition after 2–3 weeks [ 27 ]. This evidence confirmed that FMT temporarily provides relief in an IBD rat model, which is also observed in the clinical practice [ 25 ], and underscores the importance of the intestinal condition of recipients in the outcomes of microbiota-targeted interventions. Current data suggests microbial engraftment is correlated with a positive response to FMT. However, the degree of engraftment, as well as its dynamics and determinants are not well understood, though they are likely dependent on donor and recipient factors, including but not limited to genetic, comorbidities, medication use, diet, lifestyle, and baseline microbiome factors [ 28 , 29 ]. Further research is therefore necessary to identify optimal conditions for the engraftment and biomarkers predictive of response, as well as potential adjunct therapies to enhance response. In the present work, we used the FMT-based experimental approach to investigate microbiota-to-gut signalling involved in post-inflammatory visceral pain persistence through a combined multi-omics approach. Results uncovered new mechanisms governing host-microbiota crosstalk in health and disease and provide new perspectives for improving the therapy of gastrointestinal pain associated with IBD. 2. Materials and Methods 2.1. Ethical approvals All animal manipulations were performed according to the Directive 2010/63/EU of the European parliament and of the European Union council (September 22, 2010) on the protection of animals used for scientific purposes. The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85 − 23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the described experiments was obtained from the Animal Subjects Review Board of the University of Florence (Italy) and the Italian Ministry of Health (388/2021-PR). Experiments involving animals have been reported according to the ARRIVE guidelines [ 30 ]. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2. Animals For all the experiments described below, 8-week-old male Sprague–Dawley rats (Envigo, Varese, Italy), weighing approximately 220 to 250 g at the beginning of the experimental procedure, were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least 1 week after their arrival. Before starting the treatments, 4 animals were housed per cage (size 26 × 41 cm). After starting the experimental procedures, animals were individually housed to avoid the damage of the electrodes implanted for electrophysiological measurements. Animals were fed a standard laboratory diet and purified water ad libitum and kept at 23°C ± 1°C with a 12-hour light or dark cycle, light at 7 am. 2.2. Induction of colitis Colitis was induced following the method described previously by Lucarini et al.[ 26 ]. In brief, during short anaesthesia with isoflurane (2%), 30 mg of DNBS (Merck Life Science, Milan, Italy) in 0.25 mL of 50% ethanol was administered intrarectally through a polyethylene PE-60 catheter inserted 8 cm proximal to the anus. Control rats received 0.25 mL of saline solution. 2.3. Faecal microbiota transplantation study design 2.3.1. Disease Protocol In the first experimental set-up, rats were randomized to four groups: 1) control (vehicle, no antibiotic treatment, no FMT; n = 4) 2) antibiotics (abx) + vehicle (antibiotic treatment followed by vehicle administration; n = 4) 3) abx + FMT (antibiotic treatment followed by FMT from controls; n = 8) 4) abx + FMT (antibiotic treatment followed by FMT from DNBS treated animals; n = 8) The animals underwent the following antibiotic or antifungal regimen to prepare them to the FMT: Day 0 to 6 rats received a daily oral gavage (10mL·kg − 1 ) of amphotericin B (1 mg·kg − 1 ) and metronidazole (100 mg·kg − 1 ) whereas an antibiotic mix (ceftazidime 1 g·L − 1 , vancomycin 0.5 g·L − 1 , and neomicin 1 g·L − 1 ) was added to the autoclaved drinking water and changed every 2 days. On day 7, 24 hours after the interruption of the antibiotic treatment, the animals underwent the FMT procedure. FMT was daily performed on days 7 to 11 (set I) and on days 21 to 25 (set II). The animals were singly housed also to improve reproducibility of data. Behavioural tests were performed at the end of the antibiotic treatment (before starting the FMT), 24 hours and 7 days after each FMT set. Faecal material collected from different controls (n = 4) or DNBS-treated animals (n = 4) (between 14 and 21 days after the intrarectal injection of the inflammatory agent) were combined and homogenized to generate a single solution for FMT CTR and FMT DNBS , respectively. To achieve this the tubes containing faecal pellets in sterile saline solution were left on ice for 60 minutes and then homogenized for 2 minutes on ice using a hand-held pellet pestle device with sterile, reuseable pestles. When fully homogenized, the suspended pellets were centrifuged, and the supernatant directly used for the FMT procedure. FMT was performed through oral gavage with a faecal suspension (50 mg·mL − 1 ) in a final volume of 3 mL. 2.3.2. Therapy Protocol In the second experimental set-up rats were randomized to four groups: 1) control (vehicle + vehicle; n = 4) 2) DNBS + vehicle (intrarectal injection of DNBS 30 mg followed by vehicle administration; n = 6) 3) DNBS + FMT (intrarectal injection of DNBS 30 mg followed by FMT from controls; n = 8) 4) DNBS + FMT DNBS (intrarectal injection of DNBS 30 mg followed by FMT from DNBS animals; n = 8) Colitis was induced in animals through intrarectal injection of DNBS (30 mg in 0.25 mL EtOH 50%) on day 1. The control group was intrarectally administered with saline solution. DNBS-treated animals did not receive any antibiotic pretreatment before the FMT due to the influence on the induction of colitis [ 31 ]. Seven days after DNBS injection, the animals were split into 3 groups receiving: 1) the vehicle, 2) the naive control–derived faecal microbiota suspensions (FMT CTR ) or 3) the DNBS–derived faecal microbiota suspensions (FMT DNBS ). FMT was performed for 5 consecutive days/week and the same protocol was repeated for 4 weeks (I-IV set of FMT): on days 7 to 11 (set I), 14 to 18 (set II), 21 to 25 (set III), and 28 to 32 (set IV) after DNBS injection as per [ 27 ]. Behavioural tests were conducted 7 days after DNBS injection (before starting FMT), 3 days after each FMT set, and 10 days after the last treatment. For the FMT, faecal material was processed as mentioned above. FMT was performed by oral gavage with a faecal suspension (100 mg·mL − 1 ) in a final volume of 3 mL. 2.4. Assessment of visceral sensitivity The behavioural responses to colorectal distension (CRD) were assessed in the animals by measuring the abdominal withdrawal reflex (AWR), a semiquantitative score described previously in conscious animals [ 32 ]. In brief, rats were anesthetized with isoflurane (2%), and a lubricated latex balloon (length: 4.5 cm), attached to polyethylene tubing, assembled to an embolectomy catheter, and connected to a syringe filled with water, was inserted through the anus into the rectum and descending colon of adult rats. The tubing was taped to the tail to hold the balloon in place. Then rats were allowed to recover from the anaesthesia for 30 minutes. AWR measurement consisted of visual observation of animal responses to graded CRD (0.5, 1, 2, and 3 mL) blinded observers who assigned AWR scores: no behavioural response to colorectal distention (0); immobile during colorectal distention and occasional head clinching at stimulus onset (1); mild contraction of the abdominal muscles but absence of abdomen lifting from the platform (2); observed strong contraction of the abdominal muscles and lifting of the abdomen off the platform (3); and arching of the body and lifting of the pelvic structures and scrotum (4). The time elapsed between 2 consecutive distension was 5 minutes. AWR measurements were conducted in all the animals (n = 8 per group ). 2.5. Assessment of thermal and tactile pain threshold The thermal pain threshold was assessed using the hot plate test as previously described with minor adjustments [ 33 ]. With minimal animal–handler interaction, rats were taken from their home cages and placed onto the surface of the hot plate (Ugo Basile, Varese, Italy) maintained at a constant temperature of 48 ◦C ± 1 ◦C (first evaluation) and 50° ± 1 ◦C (second evaluation after 1 h). Ambulation was restricted by a cylindrical Plexiglas chamber (diameter, 10 cm; height, 15 cm), with an open top. A timer controlled by a foot peddle began timing response latency from the moment the rat was placed onto the hot plate. Pain-related behavior (licking of the hind paw) was observed, and the time (seconds) of the first sign was recorded. The cutoff time of the latency of paw lifting or licking was set at 40 s. An analgesimeter (Ugo Basile, Varese, Italy) was used to measure the tactile pain threshold of rats according to [ 34 ]. We applied a constantly increasing pressure on the dorsal surface of the hind paw using a blunt conical probe by a mechanical device. The pressure was increased until vocalization or a withdrawal reflex occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were expressed in grams. Rats scoring below 40 g or over 75 g during the test before drug administration were rejected (25%). A cut-off of 200 g was adopted. Paw pressure test and Hot plate test were performed on day 32 (Disease Protocol) and on Day 42 (Therapy Protocol). 2.6. Profiling of the gut microbiota Fecal pellets (50 mg) and fecal suspensions (volume equivalent to 50 mg) were processed for the total DNA extraction using the DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany). Next-generation sequencing of 16S ribosomal RNA amplicons of the V3-V4 regions ((341F, 5’- CCTAYGGGRBGCASCAG-3’; 806R, 5’- GGACTACNNGGGTATCTAAT-3’) was performed using the Illumina NovaSeq 6000 Sequencing System (Novogene Co., Ltd.), using a 2 × 250 bp paired-end approach. Extraction and PCR amplification negative controls were also included. Sequencing results were analyzed using the QIIME 2 suite (Quantitative Insights Into Microbial Ecology)[ 35 ]. In brief, after raw reads denoising (ie, error correction, removal of chimeric and singleton sequences, and joining of denoised paired-end reads), DADA2 [ 36 ] was used to annotate genus-level feature tables, based on SILVA 16S reference database (release 138.2) ( https://www.arb-silva.de/documentation/release-138/ ). Functional annotation was performed using picrust2[ 37 ], after which neuroactive potential was estimated through the functional gut-brain module framework[ 38 ]. Microbial diversity was estimated by evaluating alpha diversity (Chao1, Simpson’s Index, Shannon Entropy) and beta diversity (Aitchison distance) metrics in R. Features were transformed into centered log-ratios (CLR) to account for compositionality as by recent recommendation [ 39 , 40 ]. R scripts are available online on github (URL: https://github.com/thomazbastiaanssen/FMT_pain_analysis/ ). 2.7. 1H NMR metabolomics Faecal metabolites were analysed and quantified by 1H NMR analysis. The preparation method was as previously described [ 41 , 42 ]. Briefly, frozen faecal pellets were thoroughly mixed at 5,000 rpm in a Precellys®24 (Bertin Technologies, France) and diluted to a faeces-to-buffer ratio of 13 (e.g., 50 mg faeces in 750 µL buffer) by adding deuterated phosphate buffer (1.9 mM Na2HPO4, 8.1 mM NaH2PO4, and 1 mM sodium 3-(trimethysilyl)-propionate-d4 in deuterated water (Goss Scientifics, Crewe, United Kingdom)). After mixing and centrifugation, 500 µl was transferred into a 5 mm NMR tube for spectral acquisition. High resolution 1H NMR spectra were recorded on a 600 MHz Bruker Avance spectrometer fitted with a 5 mm TCI proton-optimized triple resonance NMR inverse cryoprobe and a 24-slot autosampler (Bruker, Rheinstetten, Germany). Sample temperature was controlled at 300 K. Each spectrum consisted of 128 scans of 65,536 complex data points with a spectral width of 20 ppm (acquisition time 2.6 s). The noesypr1d presaturation sequence was used to suppress the residual water signal with low power selective irradiation at the water frequency during the recycle delay (D1 = 2 s) and mixing time (D8 = 0.01 s). A 90° pulse length of 11.4 µs was set for all samples. Spectra were transformed with a 0.1 Hz line broadening and zero filling, manually phased, baseline corrected and referenced by setting the trimethylsilylpropanoic acid methyl signal to 0 ppm. Metabolites were identified using information found in the literature or on the web (Human Metabolome Database, https://www.hmdb.ca/ ) and quantified using the software Chenomx® NMR Suite 8.6™. 2.8. Proteomic analysis Each colon sample was homogenized and tryptic digested as in D'Amato A, et al. (2020)[ 43 ]. The peptides were analyzed using a Dionex Ultimate 3000 nano-LC system (Sunnyvale CA, USA) connected to Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Thermo Scientific, Bremen, Germany) equipped with a nano-electrospray ion source (nESI). The elution gradient was from 96% buffer A (0.10.1% formic acid (FA) in water )to 40% buffer B (0.1% FA in water/acetonitrile with 2/8 ratio) for 110 min. MS spectra were collected over an m/z range of 375–1500 Da at 120,000 resolutions, operating in data dependent scan mode, cycle time 3 sec between master scans [ 44 ]. The instrumental raw files were processed by MaxQuant software v1.6.6.0 (Cox et al, 2014) set on Uniprot_Rat database against the Andromeda search engine (D’Amato A. et al Microbiome 2020). The quantification of peptides and related proteins for each control and treated sample in biological duplicate and technical triplicates was based on the LFQ intensities. The interpretation and visualization of results obtained from MaxQuant software were performed by a two-sample t-test using Perseus (v1.6.1.3, Max Planck Institute of Biochemistry, Germany). Statistical parameters (p < 0.05; q < 0.05, q = FDR adjusted p-value) were set to identify the differentially expressed proteins between samples (log2 ratio). Variability of biological replicates were measured using the scatter plot with Pearson correlation coefficient values of the LFQ intensities. (Zoanni et al., 2023). The network protein analysis related to significantly altered proteins was carried out by Ingenuity Pathways Analysis (last release; Qiagen) based on Gene Ontology database. The network protein analysis related to significantly altered proteins was carried out by Ingenuity Pathways Analysis (IPA) (last release; Qiagen) The statistical enrichment of involved pathways is performed by the right-tailed Fisher’s exact test, in correlation with QIAGEN Knowledge Base, assigning a p-value ( https://digitalinsights.qiagen.com/products/features/analysis-match/ ). The overall activation/inhibition states of canonical pathways are predicted based on a z-score algorithm. This z-score is used to statistically compare the uploaded dataset with the pathway patterns [ 45 ]. 2.9. Histochemical analysis The evaluation of colon damage was performed at the macroscopic level in accordance with the criteria previously reported by Antonioli et al.[ 46 ]. The macroscopic criteria included the presence of adhesions between the colon and other intra-abdominal organs (0–2); consistency of colonic faecal material (an indirect marker of diarrhoea; 0–2); thickening of the colonic wall (mm); presence and extension of hyperaemia and macroscopic mucosal damage (0–5). The colon length was measured after the explant of the tissue. For the histological analysis, the colon was fixed in 4% paraformaldehyde for 24 h, dehydrated in sucrose, frozen, and embedded in OCT for cryostat sectioning (5 µm sections), by using the “Swiss roll” technique to visualize all the colon length in each tissue section. Microscopic evaluations of colon damage (mucosal architecture loss, cellular infiltrate, muscle thickening, crypt abscess, and goblet cell depletion) were carried out on haematoxylin/eosin-stained sections. The infiltration of mast cells (MCs) and eosinophils was investigated on colon sections stained with GIEMSA (Sigma-Aldrich, Milan, Italy). Digitalized images were collected by a Leica DMRB light microscope equipped with a DFC480 digital camera (40× magnification; Leica Microsystems, Wetzlar, Germany). The quantitative analysis was carried out by two blind investigators with the software ImageJ. For each animal, the cellular density (cell number/respective arbitrary field) of 3–5 independent arbitrary optical fields (0.1 mm 2 ) collected from the submucosa was measured. The analysis was performed on 6 animals per group . 2.10. Immunofluorescence The colon was cut into 30 µm slices (by using the “Swiss roll” technique)[ 47 ], which were collected in wells containing PBS 1x. Immunolabelling was performed in the same wells by free-floating technique according to standard protocols[ 48 ]. Selected slices from each animal, were incubated overnight at 4°C with the following combination of primary antibodies, diluted in T-PBS/5% BSA (Sigma-Aldrich, Milan, Italy): 1) rabbit anti-panaxonal marker PGP9.5 (ab108986, Abcam, UK; 1:500) + mouse major histocompatibility complex II (MHC II, ab23990, Abcam, UK; 1:100); 2) mouse anti-UCH-L1/PGP9.5 (Novus Biologicals-31A3, Bio-Techne Ltd., Abingdon, UK; 1:500) + rabbit anti-glial fibrillary acidic protein (GFAP, DAKO-Z0334, Agilent Technologies Italia, Milan, Italy; 1:500); 3) mouse anti-UCH-L1/PGP9.5 (Novus Biologicals-31A3, Bio-Techne Ltd., Abingdon, UK; 1:500) + rabbit anti-Iba-1 (Wako Chemicals, Richmond, VA, USA; 1:250). The day after, slices were incubated for 2 h with secondary antibodies (1:500) labelled with Alexa Fluor 488, 568 or 647 (Invitrogen-Thermo Fisher Scientific, Milan, Italy), and then with DAPI to stain the nuclei. The slices were finally transferred onto slides and mounted with Fluoromount-G™ Mounting Medium (Thermo Fisher Scientific, Milan, Italy). Digitalized images were collected at 400× (myenteric plexus and mucosa) and 200× (colon wall) total magnification, based on the type of analysis performed, using a motorized Leica microscope DM6 B equipped with a DFC9000 GT camera, supported by a THUNDER Workstation 3D DCV and by the software LAS X (Leica Biosystems, Milan, Italy). The quantitative analysis of colon PGP9.5- MHC II and Iba-1-related immunofluorescence intensity (Disease and Therapy Protocols) was performed by collecting independent fields (200×; 4–6 for each animal) from the colon wall and by analysing selected ROI for mucosa, submucosa and muscular layers with the software FIJI (NIH, Bethesda, MD, USA). Quantitative analysis of IENF density (fibers/mm) was performed by collecting 4–6 independent fields in the skin of each animal and counting the number of single PGP9.5-positive fibers crossing the mucosa-submucosa boundary and dividing them for the analysed mucosal length, measured at the base of the crypt by using FIJI. Secondary branching is excluded from this quantification. The quantitative analysis of colon PGP9.5- and GFAP-related immunofluorescence intensity (Disease and Therapy Protocols) was performed by collecting independent fields (400×; 4–6 for each animal) from the myenteric plexus and analysing them with FIJI. The number of PGP9.5- and GFAP-positive cells for each myenteric plexus was counted and normalized to the plexus area. The immunofluorescence relative to the expression of PGP9.5 and GFAP was quantified (arbitrary units), normalized to the area of the myenteric plexus. The value relative to the background was subtracted from the value obtained from the analysed area. 2.11. Statistical analysis All the experimental procedures were performed by a researcher blind to the treatment. Behavioural and histological results were expressed as mean ± SEM. The analysis of variance (ANOVA) was performed by one-way ANOVA with Bonferroni's significant difference procedure used for post-hoc comparisons. P values of less than 0.05 were considered significant. Data were analyzed using the “Origin 9” software (OriginLab, Northampton, MA). Statistical analysis on 16S rRNA amplicon sequencing data was performed in R using the Rstudio GUI. using (generalized) linear models and mixed models. The permutational multivariate ANOVA (PERMANOVA) test was applied to Aitchison distance matrices generated to assess whole-composition level differences between treatment groups and timepoints. R scripts are available online on github ( https://github.com/thomazbastiaanssen/FMT_pain_analysis/ ). Spearman correlation coefficients between relative abundances of microbial taxa and levels of SCFAs were computed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). Statistical analysis of metabolomics data was carried out using MetaboAnalystR Package. Data was normalised by median, log10 transformed and scaled by Pareto scaling (mean-centered and divided by the square root of the standard deviation of each variable). Sparse Partial Least-Squares Discriminant Analysis (sPLS-DA) was employed to illustrate the clustering of different metabolites across groups. Univariate Analysis was carried out by Wilcoxon rank-sum tests. Dendrogram and heatmaps were created with Euclidean distances and Ward clustering methods. Heatmap shows the top metabolites based upon Wilcoxon rank-sum test results. P values of less than 0.05 were considered statistically significant. Correlation analysis between metabolomics data and microbiome data was conducted using both the anansi framework [ 49 ] with the KEGG database as a reference, and M2IA [ 50 ]. Missing values were filtered if present in more than 80% of samples or the relative standard deviation was smaller than 30% [ 51 ]. The remaining missing data values were handled using random forest. Data was transformed using total sum scaling. All other correlation analyses were conducted using Spearman’s rank-order correlation analysis [ 52 ]. Correlation analysis of the microbiota and metabolome datasets was performed by DIABLO (Data Integration Analysis for Biomarker discovery using Latent cOmponents)[ 53 ] using the R package “mixOmics”, a multiomics integrative method based on a variant of the multivariate statistical technique generalised canonical correlation analysis 3. Results 3.1. Parallel changes of visceral pain threshold and microbiota composition caused by faecal microbiota transplantation (FMT). 3.1.1 Modification of visceral sensitivity and microbiota composition caused FMT in healthy animals. We adopted two opposite, complementary FMT-based approaches as per our previous work [ 27 ] with the aim to study: 1) the mechanisms governing pain caused by post-inflammatory dysbiosis, referred to as the Disease Protocol (Fig. 1 A); and 2) the mechanisms underlying pain relief mediated by shifts in the gut microbiota composition-metabolism modification in DNBS-treated animals, referred to as the Therapy Protocol (Fig. 2 A). Body weight, regarded as a disease index, was monitored throughout the experiment (Fig. 1 B and 2 B). Visceral sensitivity was assessed by evaluating abdominal withdrawal reflex (AWR) to colorectal distension (CRD) after abx treatment and FMT (Fig. 1 C and 2 C; Figure S1 for time course of visceral pain). Preventive antibiotic treatment in rats caused a transitory increase of visceral sensitivity (Figure S1 C, day 7). Starting on day 7, FMT was performed using faeces from control (FMT CTR ) or animals manifesting with severe visceral pain in the post-inflammatory phase of colitis induced by the intrarectal injection of DNBS (FMT DNBS ). Although no differences in the body weight were detected between the experimental groups, FMT DNBS induced a persistent increase in the AWR to CRD, while FMT CTR accelerated the restoration of a normal visceral sensitivity in abx-treated animals (Fig. 1 C, day 32), as previously observed [ 27 ]. The impact of FMT DNBS induced visceral sensitivity exclusively, as neither the antibiotic treatment nor FMT altered thermal and tactile somatic pain threshold of naive animals (Figure S2 A and S2B, respectively). In terms of the microbiome (Fig. 1D i − iii ), we did not observe statistically significant effects of time or treatment on alpha diversity nor in functional neuroactive potential. However, we did observe statistically significant effects of both experimental timepoint and treatment in several genera as well as overall ecosystem composition (Beta diversity), indicating that DNBS exposure substantially affects the microbiome, in a manner likely independent of host-microbial communication through co-metabolism. 3.1.2 Modification of visceral sensitivity and microbiota composition caused FMT in post-inflammatory conditions. In the Therapy Protocol we confirmed that post-inflammatory visceral pain in DNBS-treated animals can be relieved by means of FMT from healthy donors (Fig. 2 C). Seven days after the induction of the damage, the abdominal response of animals to CRD was significantly higher in both groups treated with DNBS (Figure S1 F, day 7). The FMT CTR led to a progressive reduction of visceral hypersensitivity in DNBS-treated animals, DNBS + FMT CTR group reached the values of controls (vehicle + vehicle group) after set IV of FMT, and pain relief has been maintained 10 days after the discontinuation of treatment (Fig. 2 C, day 42). Moreover, DNBS animals receiving FMT CTR showed a significant increase in the thermal and tactile somatic pain threshold with respect of those transplanted with FMT DNBS , displaying somatic hypersensitivity in addition to visceral hypersensitivity (Figure S2 C and S2D, respectively). No differences were observed between DNBS animals receiving vehicle or FMT DNBS at any time during the trial (Fig. 2 C), confirming previous evidence collected in our laboratory [ 27 ]. The body weight loss resulting from colitis and the body weight recovery in the remission phase were equivalent and followed the same trend in all the experimental groups treated with DNBS, irrespective from the FMT or the donor (Fig. 2 B). In terms of the microbiome (Fig. 2D i − iii ), we did observe a subtle positive association of treatment and alpha diversity indices, shannon entropy reaching the critical value for significance. Furthermore, we observed changes in the genetic potential of the microbiome to metabolize neuroactive compounds, most notably in tryptophan synthesis and degradation which change in opposite directions, suggesting a dramatic shift in host-microbial co-metabolism of tryptophan. We further observed differences in the composition of the microbiome (Beta diversity) that could be explained by experimental timepoint and treatment group, indicating that DNBS exposure impacts microbial composition as well as functional neuroactive host-microbial co-metabolism. Conversely, we did not observe statistically significant effects in terms of genus-level abundance. 3.2. Metabolome profiling and correlation analysis identified common effects of FMT in healthy or post-inflammatory conditions. Considering the microbial shifts mediated in both experimental models, we extended our line of investigation to encompass the gut metabolomic profile. Sparse Partial least squares-discriminant analysis (sPLS-DA) plots showed a clear separation of experimental groups indicating a metabolomic shift in response to pathological treatments (Fig. 3 A) or therapeutic interventions (Fig. 3 C) the extent to which can be seen through the heatmap provided which displays all significantly altered metabolites (Fig. 3 B and 3 D); p < 0.05; FDR < 0.2). The interplay across gut microbiota composition and metabolites was explored with DIABLO, a generalized and supervised version of PLS-DA. The DIABLO analysis identified a clear discrimination between samples of the experimental groups from the Disease (abx + FMT CTR , abx + FMT DNBS , collected at day 32) and Therapy (DNBS + FMT CTR , DNBS + FMT DNBS , collected at day 42) protocols (Figure S3 , A-B), suggesting that the two omics (i.e. microbiota and metabolomics) can likely separate the two conditions within each group; a robust correlation between the microbial and the metabolomic profile in each study protocols was observed (Figure S3 , C-D). The optimally selected key predictors included several microbial taxa, a few of which were concomitantly identified as contributors to discriminate the Disease (abx + FMT) and Therapy (DNBS + FMT) experimental groups, including Romboutsia , Turicibacter , Clostrodium sensu stricto 1, Akkermansia (Fig. 4 A-B). Among the Disease protocol, few metabolites including malate, malonate, proprionate, arabinose were strongly associated with the abx + FMT DNB group, being overall positively correlated (mean r = 0.84 ± 0.07) with Corpobacillus , Parabacteroides , Erysipelatoclostridium and Gastranaerophilales (Fig. 4 A-C); no metabolic signatures associated with the abx + FMT CTR group were found (Fig. 4 A). Interestingly, several metabolites were associated with the DNBS + FMT DNBS group from the Therapy protocol; overall, the most relevant products included alpha-ketoisovaletate, nicotinate, 2-oxoglutarate, 2-oxoisocaproate, 3-methyl-2-oxovalerate and were positively correlated (mean r = 0.79 ± 0.07) with Turicibacter , Ruminococcus , Romboutsia and Clostridium sensu strictu 1 (Fig. 4 D). Conversely, few metabolic products were identified as contributors to the DNBS + FMT CTR group (Fig. 4 B); in that case, correlation analysis showed that cis-aconitate was negatively correlated with Turicibacter , Romboutsia and Ruminococcus (mean r=-0.75 ± 0.03), while ethanol wa negatively correlated with Ruminococcus (r=-0.76) (Fig. 5 D). 3.3. Disease activity in the colon after FMT-based Disease or Therapeutic protocol. The animals receiving FMT from CTR or DNBS donors were sacrificed at the peak of the effect (7 or 10 days after the discontinuation of FMT, according to Disease or Therapy Protocol) to examine the relationship between pain threshold and colon damage (Fig. 5 ). No difference was observed in the colon length, specific weight, macroscopic and microscopic damage in abx-treated animals receiving FMT CTR or FMT DNBS (Fig. 5 A and 6 B. In particular, the ratio of colon weight to colon length (colon specific weight) demonstrated no difference in oedema. Also, the density of mast cell and eosinophils in the submucosa of abx-treated animals was unaffected by FMT from the different donors (Fig. 5 C). Immunofluorescence analysis performed on the colon specimens showed the apparent integrity of epithelium (E-CAD; Figure S4 B-C) and its regenerative capacity (Ki67; Figure S4 A-C) in the experimental groups. The same analysis highlighted a decrease immunoreactivity for MHC-II (marker for antigen presenting cells) in the submucosa of abx + FMT DNBS group compared with abx + FMT CTR group (Figure S4 B). MHC-II immunoreactivity did not change in either mucosa or muscularis (Figure S4 A and S4C), neither Iba-1 (marker for macrophages) immunoreactivity (Fig. 6 A-C). At the same time, a lowered muscular immunoreactivity for the pan-neuronal marker PGP 9.5 and the glial marker GFAP was observed in animals receiving FMT DNBS (Figure S4 G and S4N). PGP 9.5 immunoreactivity in the submucosa and myenteric plexus of abx-treated animals was unaffected by FMT (Figure S4 F, S4I and S4F). The tendency to increase in the mucosal PGP 9.5 immunoreactivity in abx + FMT DNBS group with respect of abx + FMT CTR group was independent from intraepithelial nerve fibres (IENFs) density (Figure S4 D). Similarly, we found that the different pain-threshold resulting from FMT CTR or FMT DNBS in DNBS-treated animals was not explained by significant modifications of colon length, specific weight, macroscopic and microscopic damage to the colon. Although the colon muscle layer appeared thickened because of the inflammatory insult resulting from DNBS injection (Fig. 5 D) [ 54 ], the mucosa and submucosa were fully restored in both experimental groups (Fig. 5 E). These outcomes are consistent with colon remission state 42 days after the injection of DNBS [ 26 , 55 ]. However, a significant reduction in the number of mast cells infiltrating the submucosa was detected in DNBS-treated animals receiving FMT CTR with respect of those receiving FMT DNBS . The infiltration of eosinophils was instead unaffected by the different treatment (Fig. 5 F), which suggests specific immunomodulatory mechanisms underlying FMT effects on pain. Immunofluorescence analysis performed on the same colon specimens highlighted a decrease immunoreactivity for Iba1 in the submucosa of DNBS + FMT DNBS group compared with DNBS + FMT CTR group (Fig. 6 F) which was concomitant with a reduced immunoreactivity for the pan-neuronal marker PGP 9.5 (Figure S5 F). No significant difference was detected for MHC-II (Figure S5 A-C) and for Iba-1 (Fig. 6 E-G) and PGP 9.5 in the other intestinal compartments analysed (Figure S5 D-E-G-I-L). 3.4. Pathways selectively modulated by FMT in abx-treated animals. In parallel to the study of changes occurring within the lumen, we investigated the response of colon to FMT by proteomic analysis, looking for correlation with pain phenotype. First, we investigated the alterations occurring in the colon of naïve animals developing hypersensitivity after FMT DNBS , comparing them with those receiving FMT CTR , not showing sensitivity alterations (Fig. 7 A). Then, we explored the mechanisms underlying the therapeutic effect of FMT by comparing DNBS animals subjected to FMT CTR , showing pain relief, with those receiving FMT DNBS , which was unable to modify pain threshold (Fig. 7 B). Colon samples were analysed by one shot label free proteomics. 3880 proteins were identified and quantified of which 94 were upregulated and 63 downregulated in abx + FMT DNBS vs abx + FMT CTR (Disease Protocol) while 29 were upregulated and 18 downregulated in DNBS + FMT CTR vs DNBS + FMT DNBS (Therapy Protocol), considering a fold change threshold of 1.5 (Fig. 7 – 11 ). Network analyses performed on differentially regulated proteins showed several modulated pathways according to Disease Protocol (Table 1) or Therapy Protocol (Table 2). From the network analysis of canonical pathways, it emerged that the proalgesic effect of FMT DNBS was related to a negative induction of the signalling pathway of Integrins, CLEAR (Zscore = -1,63), GNRH (Zscore = -1,00), Ephrin receptor (Zscore = -1,00), Protein kinase A (Zscore = -0,82), Neurovascular coupling (Zscore = -0,45) and EIF2 (Zscore = -0,38), with a concomitant positive induction of Sirtuin pathway (Zscore = 0,45) (Table 1). The negatively induced integrin signalling (Zscore = -1.63; pvalue = 7.8 x 10 − 3 ) in abx + FMT DNBS vs abx + FMT CTR attested the modulation and the deactivation of important cellular functions, such as cell cycle, cellular growth, proliferation, and development. Actin gamma 1 (ACTG1, log2 ratio = -3.85), ADP ribosylation factor 4 (ARF4, log2 ratio = 0.50), Actin related protein 23 complex subunit 5 (ARPC5, log2 ratio = -0.51), protein tyrosine kinase 2 (PTKS, log2 ratio = -0.60) and paxillin (PXN, log2 ratio = 1.05) were involved in the negatively regulation of integrin signalling (Figure S6 ). In addition, NADH: ubiquinone oxidoreductase subunit B9 (NDUFB9, log2 ratio = -0.50) and NADH: ubiquinone oxidoreductase core subunit S3 were down regulated (NDUFS3, log2 ratio = -0.70). These enzymes are involved in mitochondrial activities, such as electron transfer, ATP synthesis and integrin signalling. The Actin cytoskeleton signalling was also negatively induced (Zscore = -0.45; pvalue = 4.9 x 10 − 2 ) attesting impaired cell development (Table 1). Looking at non-canonical pathways significantly modulated by FMT DNBS , we observed a negative induction of pathways involved in the organization of cytoplasm and cytoskeleton (Zscore = -1,18), microtubule dynamics (Zscore = -0,84), which was correlated with a positive induction of cellular protrusions formation (Zscore = 0,12) (Table 1). The concomitant positive induction of pathways involved in the expression, translation, and metabolism of proteins (Zscore = 1,45; Z score = 1,17; Z-score = 0,82) (Table 1) indicates that DNBS microbial transfer in abx-treated rats induced a disease phenotype characterized by variation of cell-cell and cell-ECM interactions, and increased trafficking of immune cells, a result also corroborated by significant changes in the expression of single proteins (Fig. 8 A). At the same time, FMT DNBS -related downregulation of epithelium neoplasm pathway (Zscore = -1,53) suggests structural alterations in the mucosal barrier, a hypothesis which matches with the positive induction of pathways involved in the transport of molecules and Diarrhea (Zscore = 0,76) (Table 1). Noteworthy, FMT DNBS effects on visceral sensitivity also correlated with increased Neurite branching (Zscore = 1,14) and Axonogenesis (Zscore = 0,61), as regard of non-canonical pathways (Table 1). Beyond network analysis, several proteins involved in neuroplasticity and neuromodulation were found to be differentially expressed after FMT DNBS in the Disease Protocol. Among them we were able to identify structural and functional proteins participating to sensitization, synaptic plasticity, and glial physiology, such as GFAP (Fig. 8 B). We also observed a significant reduction in AH receptor-interacting protein, Tryptophan-tRNA ligase (cytoplasmic) and Kynurenine-oxoglutarate transaminase 3 (Fig. 8 B), involved in tryptophan metabolism in the intestinal mucosa, which is known to contribute to IBS pathophysiology [ 56 ]. Moreover, several proteins significantly up- and down-regulated in abx + FMT DNBS group emerged to be involved in energy metabolism and redox balance (Fig. 9 A). Furthermore, proteins related to immune response and inflammatory process regulation were found differentially expressed after FMT DNBS or FMT CTR in abx-treated animals (Figure S8B). The combination of evidence might indicate the presence a low-grade colon inflammation related to a dysregulated mitochondrial dynamics and bioenergetics [ 57 ], not detectable with histological investigations. In fact, proteins regulating mitochondria function, as cytochrome c oxidase, mitochondrial fission protein 1, NDUFA4, NADH ubiquinone oxidoreductase, ATP synthase, and glutaredoxin are the most impacted by FMT DNBS (Fig. 9 A). Of note, glutathione-glutaredoxin system is also a crucial thiol-dependent redox system involved in cellular redox balance regulation along with thioredoxin system [ 58 ]. The expression of proteins involved in iron metabolism, (ceruloplasmin, ferrochelatase and hephaestin) was also significantly affected by FMT DNBS treatment (Fig. 9 A). Other relevant proteins upregulated in abx + FMT DNBS group are kininogen-1, Prothymosin alpha, Complement C1q, LPS-responsive beige-like anchor protein, Thymosin beta 4, Serpine1 mRNA binding protein 1, Glucocorticoid receptor, Leukocyte elastase inhibitor, trefoil factor 3, prostaglandins, immunoglobulins, annexins and proteins involve in mucus production (Fig. 9 B), attesting a different modulation of immune response mediated by FMT DNBS with respect of FMT CTR . Table 1 3.5. Pathways selectively modulated by FMT in post-inflammatory colon. Network analysis comparing DNBS + FMT CTR vs DNBS + FMT DNBS group correlated the improvement of the pain symptomatology with negative induction of inflammatory response (Zscore = -2.41; pvalue = 1.3 x 10 − 2 , Table 2) involving modulated proteins, such as Apolipoprotein A1 (APOA1, log2 ratio = 0.56), Alpha-1-antiproteinase (SERPINA1, log2 ratio = 0.78), Chymotrypsin C (CTRC, log2 ratio = 1.71) and Clusterin (CLU, log2 ratio = 0,63) (Figure S9). Moreover, the activation of immune system was inhibited (Zscore = -2.21; pvalue = 8.0 x 10 − 3 , Table 2) showing a decreasing of leukocyte migration by modulation of Thymosin B10/4X (TMB10/4X, log2 ratio = 0.71), Integrin subunit alpha 1 (ITGA1, log2 ratio = -0.40) and Tetraspanin (CD81, log2 ratio = -0.68). A further confirmation of the immunological matrix underlying the therapeutic effect of FMT CTR , was the significant downregulation of both subunit alpha and beta of beta-hexosaminidase (Figure S9), a protein involved in mast cell degranulation [ 59 ], as a result of FMT CTR . In addition to the negative induction of these pathways, a differential expression of proteins involved in either fibrosis, energy metabolism, mucosal barrier, neuroendocrine signalling and neuroplasticity was associated with the pain-relieving effect of CTR microbial transfer in DNBS animals (Fig. 10 , Suppl. Table?). In particular, the analysis of single proteins highlighted a significant down-regulation of proteins potentially involved in fibrotic disorders (LTBP4 - latent transforming growth factor beta binding protein 4, EGF Containing Fibulin Extracellular Matrix Protein and Collagen type 1 alpha), as well as neuroplasticity (Liprin-beta-1, Trefoil factor 3, Calcium-binding mitochondrial carrier protein Aralar1, Synapse Associated Protein 1, Voltage-dependent calcium channel subunit alpha-2/delta-1, Calcineurin B homologous protein 1) in the DNBS + FMT CTR group. Besides, it has been observed a positive effect of FMT CTR on different partners involved in lipid transport, synthesis and metabolism (NDUFA4 Mitochondrial Complex Associated, acyl-CoA dehydrogenase family of enzymes (ACADs), Protein-glucosylgalactosylhydroxylysine glucosidase activity, Myristoylated alanine-rich C-kinase substrate, Beta-enolase, Fatty acid-binding protein 4, Fatty acid-binding protein 2, Steroid Delta-isomerase, Apolipoprotein E and Palmitoyl-protein thioesterase 1; Fig. 10 , Suppl. Table?). This finding confirms the presence of an impairment of lipid metabolism in DNBS animals, which can be corrected by the transfer of “healthy” microbiota. The expression of epithelial carbonic anhydrase 9, a hypoxia-induced catalytic component of pH regulating machinery, and Trefoil factor 3, mainly secreted by intestinal epithelium with potential regulatory effect of nervous and endocrine systems [ 60 ], was also reduced because of FMT CTR (Fig. 10 ), while a significant upregulation of secernin-2 (exocytosis) was detected. Finally, an increase in the expression of protein disulfide-isomerase, essential for in vivo production of the intestinal mucin [ 61 ], and secretogranin-1 was found in DNBS animals receiving FMT CTR with respect of those receiving FMT DNBS (Fig. 10 ). This last protein, also known as Chromogranin B, is a widespread constituent of neuroendocrine secretory granules [ 62 , 63 ]. Table 2 3.6. Pathways modulated by FMT irrespective of the donor. By comparing the Disease and Therapy Protocols, it emerged that certain proteins were similarly modulated by FMT DNBS in abx-treated animals and by FMT CTR in DNBS animals (Fig. 11 ). Therefore, changes involving these proteins are apparently unrelated to either FMT donor or pain phenotype. The up- and down-regulation of these protein are likely triggered by sensing a different microbial environment, irrespectively of the healthy/disease status of either the donor or the recipient. Among the proteins upregulated, the largest difference was found in the expression of seminal vesicles secretory proteins 2, 4 and 6, whose expression was about 10-fold higher in abx and DNBS animals receiving the FMT from the opposite donor (DNBS and CTR donors, respectively), with respect of those receiving the microbiota related to their pre-FMT condition (Fig. 11 ). The presence of this class of proteins has not been described in the colon before. In the reproductive tract, Svs-proteins were reported as immunomodulatory substances that inhibit cell-mediated cytotoxicity as well as lymphocyte response to allogenic cells [ 64 , 65 ]. Structurally analogous proteins produced by epithelium might assume a similar role in the gut. Related upregulation, albeit to a lesser extent, was observed for chymotrypsin-C, alfa-1-antiproteinase, thymosin beta-4, apolipoprotein A1, thioredoxin, ZO-2 tight junction protein, lypd8 and protein S100-A6 (Fig. 11 ), which might have implication in the nonspecific response of gut microbiota perturbation. This gut instinct of self-preservation might also explain the upregulation of keratin type 2 and keratin type I 18–20, the major type of keratins expressed also by the human colon crypt epithelial cells. keratins provide a structural and mechanical scaffold to support cellular stability, integrity, and stress protection in this rapidly regenerating tissue. They participate in colonocyte processes including barrier function, ion transport, differentiation, proliferation and inflammatory signalling [ 66 ]. In the same experimental conditions, the downregulation of prostaglandin G/H synthase, monoamine oxidase B and immunoglobulin light chain variable region suggest again a fine regulation of gut immune response to FMT (Fig. 11 ). Noteworthy, immunoglobulin light chain variable region resulted negatively induced with a fold change of about 4 in the comparison DNBS + FMT CTR vs DNBS + FMT DNBS , whereas the same protein was negatively induced with a fold change of about 1.5 in abx + FMT DNBS vs abx + FMT CTR (Fig. 11 ). The difference in the ratio between Disease and Therapy Protocols suggests a peculiar deregulation of Immunoglobulin light chain variable region expression in post-inflammatory conditions, with implication in the pain-relieving effect of FMT CTR in DNBS-treated rats. 4. Discussion The present work contributes to the elucidation of the molecular mechanisms linking microbiome disruption and persistent abdominal pain after colitis remission. We started from the dual evidence that post-inflammatory visceral hypersensitivity can be transferred from DNBS-treated donors (FMT DNBS ) to naïve recipients, and vice versa an effective pain relief can be achieved by transplanting a healthy microbiota (FMT CTR ) into DNBS-treated animals [ 67 ]. After reproducing both behavioural patterns in the present study referred to as the “Disease Protocol” and the “Therapy Protocol”, the changes induced by FMT DNBS or FMT CTR on the colon of abx- and DNBS-treated animals were investigated. The 16S rRNA-amplicon sequencing performed on fecal samples revealed significant taxonomic difference in the microbiota composition between animals receiving FMT DNBS and those receiving FMT CTR , in both the experimental arms. Although we were unable to identify specific microbial communities associated with pain phenotype, we found that this latter was correlated with a specific signature in the fecal metabolome. Indeed, when comparing the two arms of the study, we found an opposite trend in the relative abundance of 3-hydrobutyrrate, purines (adenosine, xanthosine, AMP and hypoxanthine), glutamate and lactate, between the pain and the pain-free condition. Yet, we observed changes in the genetic potential of the microbiome to metabolize neuroactive compounds, most notably in tryptophan synthesis and degradation, which changed in opposite directions after FMT CTR in DNBS animals. Although tryptophan levels within the lumen were not significantly modified by the treatments, we observed a different expression of factors contributing to tryptophan metabolism and signalling at mucosal level (AH receptor-interacting protein, Tryptophan-tRNA ligase and Kynurenine-oxoglutarate transaminase 3) after FMT DNBS in abx-treated animals. Noteworthy, tryptophan and its metabolites are known to contribute to IBS pathophysiology [ 52 ]. Results from the DIABLO analysis identified correlating (or co-expressed) variables between the two omics datasets generated from samples from the Disease and the Therapy protocols, and showed a strong contribution of five metabolites and five microbial species to the discrimination between the abx + FMT CTR and abx + FMT DNBS groups, as well as a contribution of eight metabolites and four bacterial genera to the discrimination between the DNBS + FMT CTR , DNBS + FMT DNBS groups. Since we primarily observed taxonomic differences in the first (Disease) arm of this study, while observing more functional differences in the second (Therapy) arm, it could be the case that the pain related to FMT DNBS is not transferred through the microbiome per se , but that DNBS exposure does result in a perturbed microbiome that exacerbates, throughout the colon response, the negative effects on visceral sensitivity. Conversely, FMT from a non-DNBS-exposed microbiome does present a distinct functional profile. It is thus possible that the ameliorative effect of the FMT can be seen in part as a strengthening of certain microbial functions important for gut homeostasis. This evidence suggests two distinct but probably related mechanisms through which DNBS-exposed microbiome can convey a chronic negative effect on the gut and how a fresh microbiome can help restore a physiological condition. Another possibility is that microbial colonization after FMT is driven by metabolic independence. According to this theory, FMT might serve as an environmental filter that favours populations with higher independence in the synthesis of critical metabolites, including amino acids, nucleotides, and vitamins. In other words, increased microbial abundance in disease does not necessitate causal involvement of the microbe in disease, it could be one of few taxa able to grow in the diseased gut environment [ 68 ], which might provide an explanation for 16S rRNA-sequencing and metabolomics data on fecal samples. A further correlation analysis between fecal microbiota and metabolome confirmed that, under pain conditions, the abundance of purines within the lumen was directly correlated with bacterial nucleosidase. Under healthy conditions, purine levels were instead independent of bacterial genomic structure (Figure S7 ). This is interesting as the microbiota contributes to maintain integrity of the mucosal barrier by supporting epithelial purine metabolism [ 69 ], whose alteration has been recently associated with pain in IBS patients [ 70 ]. In parallel to the analysis of the changes induced by FMT on the lumen content, we investigated the response of the colon to the transplant using proteomic approach, which revealed two different scenarios according to the different experimental paradigm considered. Indeed, pain induction mediated by FMT DNBS in naïve animals was related to the alteration of several signalling pathways, including integrins, GnRH, and sirtuin, and to phenomenon of cellular plasticity involving the epithelium and the nervous system. Besides, the pain-relieving effect of FMT CTR in DNBS animals was related to a negative induction of inflammatory response and leukocytes activity, with a significant decrease in the number of submucosal mast cells. Regarding the Disease protocol, network analysis on proteome highlighted a negative induction of epithelial neoplasm in abx + FMT DNBS compared to abx + FMT CTR group, encountering 170 differentially expressed proteins. Although neoplasm is beyond the scope of the current work, this finding suggests a close relationship between epithelium dysfunction and visceral hypersensitivity. In agreement with this hypothesis, pain in abx + FMT DNBS group was associated with decreased levels of fecal adenosine, whose production support epithelium barrier function [ 71 ]. Besides, pain in DNBS + FMT DNBS group (Therapy protocol) was associated with a significant increase of fecal hypoxanthine, which has an altered reactivity in an anaerobic environment, such as the lumen, and has been reported to cause an oxidative damage on the epithelium [ 72 , 73 ]. Moreover, alterations in the metabolism might prevent epithelial cells from a proper use of microbiota-derived rescue energy sources, such as fatty acids and purines, as suggested in other pathological contexts [ 74 ]. This observation might explain the increase of these metabolites in the feces of animals showing pain phenotype. If the theory that epithelial alterations caused by dysbiosis have a downstream impact on the activation of sensory nerve endings is confirmed, nociception might be configured as a wake-up call for the brain of an altered homeostasis in the microbiota-host interaction. Another pathway affected by FMT DNBS in abx-treated animals (Disease protocol) involved integrins, adhesion molecules which modulate a variety of epithelial functions. Noteworthy, integrin signalling impinges on pathways downstream of other receptors, creating elaborate intracellular signalling networks [ 75 ]. Actin gamma 1 (G-actin) is the driving protein in the negative induction of the integrins pathway mediated by FMT DNBS , which is concomitant to negative induction of Actin cytoskeleton Protein kinase A signalling. It has been recently observed that microbial metabolites induce actin cytoskeletal rearrangement and protect the blood-brain barrier function [ 76 ]. A similar mechanism might be involved in the maintenance of the intestinal epithelial barrier [ 77 , 78 ]. Moreover, integrins, actin cytoskeleton and protein kinase A signalling are all involved in processes requiring localized cell protrusion, such as cell migration and axonal path finding [ 79 ]. Integrins are also expressed on primary afferent neurons, where they can interact with signalling systems involved in hyperalgesia. Indeed, plasticity in cytoskeleton-anchored signal transduction elements within sensory neuron terminals has been observed to mediate altered pain plasticity in sensory neurons primed by injury [ 80 , 81 ]. Beyond directly affecting the functionality of both epithelium and neurons innervating the colon, the detrimental signalling associated with post-inflammatory dysbiosis and conveyed by FMT DNBS might also influence the activity of enteric glia cells inhabiting the mucosa. These cells have been reported to promote intestinal mucosal healing via activation of focal adhesion kinase and release of pro-EGF [ 82 ]. Intriguingly, glial dysfunction caused by the systemic injection of LPS was associated with actin cytoskeleton reorganization, along with Ca 2+ signalling alterations, Na + and glutamate transporters downregulation, and pro-inflammatory cytokines release [ 83 ], which can influence the response of neighbouring neurons. A similar phenomenon might occur in the enteric glia exposed to bacteria products post-colitis, contributing to enteric neuroplasticity and visceral pain chronicity. This hypothesis is reinforced by the positive induction of pathways related to neurite branching and axonogenesis together with the altered expression of peculiar proteins (i.e., GFAP, ProSAAS, volt-gated Ca2 + channel alpha-2/delta-1, tubulin beta-3 chain, and neuroplastin) in the colon of animals receiving FMT DNBS . In addition to proteomics, histological analysis confirmed a slight increase of PGP 9.5-related mucosal immunoreactivity, which suggests an altered homeostasis in nerve endings pruning and tuning. The remodelling of sensory neurons, crucial for the maintenance of receptive endings shape and functionality, is controlled by glia- and macrophages-mediated phagocytosis [ 84 – 87 ]. Under post-inflammatory conditions, alteration in the activity of microbiota, which physiologically contribute to the colonization and homeostasis of glial [ 88 ] and immune cells [ 87 ], might interfere with these mechanisms. In line with this hypothesis, we observed a reduction in the number of submucosal MHC-II positive cells, concomitantly with reduced glia-related GFAP expression in the colon of abx + FMT DNBS group. Furthermore, it is interesting to note that neurodegenerative disorders involving both neuronal and non-neuronal cells, such as microglia (tissue-resident macrophages) and astrocytes (physiologically similar to enteric glial cells ) in the CNS, are commonly associated with lysosomal dysfunction [ 89 ]. In the abx + FMT DNBS group we observed a negative induction of CLEAR (Coordinated Lysosomal Expression and Regulation) network, which regulates lysosome-associated processes, including phagocytosis and immune response [ 90 , 91 ]. Dysfunction of lysosomes, the key cellular hub for macromolecule catabolism, recycling and signalling, generates a cascade of events that impair cellular trafficking, cell signalling, autophagic flux, mitochondria functionality and calcium homeostasis [ 91 ]. It is noteworthy that most of these processes were found to be affected by FMT DNBS along with a predicted negative induction of CLEAR signalling pathway. Interestingly, the most common lysosomal storage disease, namely Fabry disease, is characterized by neuropathic pain and functional gastrointestinal disorders [ 92 – 94 ]. Mice affected by Fabry disease manifest both visceral hypersensitivity and gut dysbiosis, combined with alterations in faecal SCFAs [ 95 ], which parallels our findings and suggest a link between lysosome dysfunction and pain persistence. Lysosome damage has a negative impact on mitochondrial function, which is also affected by the treatment with FMT DNBS (see the expression of cytochrome c oxidase, mitochondrial fission protein 1, NDUFA4, glutaredoxin-1 and ATP synthase). Combined alterations in lysosome and mitochondria functioning driven by post-inflammatory dysbiosis might have a great impact especially on highly metabolically epithelial cells and neurons [ 96 – 98 ]. In the same cells, lysosome dysfunction might also affect the signaling of integrins, which is finely regulated by a circular process of internalization, recycling, and degradation [ 99 ], and which we observed to be involved in the pathogenic effects of FMT DNBS , including neuroplasticity. However, metabolism and function of epithelial cells under dysbiosis condition might be also influenced by the positive induction of SIRT1 [ 100 , 101 ], which promotes epithelial-mesenchymal transition [ 102 , 103 ]. Altogether these mechanisms, and not just a single one, likely contribute to gut homeostasis disruption caused by FMT DNBS . In search of further mechanisms for FMT DNBS -induced pain, we focused on the negative induction of gonadotropin-releasing hormone (GnRH) pathway, since the therapeutic efficacy of GnRH analogues, used to treat pre-menopausal pain in women [ 104 ], has been argued for several years in IBS patients, who express serum antibodies against GnRH [ 105 ]. The connection between the presence of antibodies and IBS disease is still unclear, but it has been demonstrated that GnRH and its receptor are expressed in both enteric neurons and intestinal epithelium [ 105 ]. Our data from the colon sums up to previous evidence reporting GnRH to be associated to the largest proportion of differentially expressed genes in the hippocampus of viscerally-hypersensitive germ-free mice [ 106 ], reinforcing the link between microbiota, GnRH signalling and pain regulation, both peripherally and centrally. Pain induced by FMT DNBS was also accompanied by the upregulation of colon kininogen, fibrinogen, stool trefoil factor 3 (TFF3). The first can interact with kallikrein to produce bradykinin, a peptide implicated in inflammatory pain [ 107 ], or can alternatively release antimicrobial and antifungal peptides for immune functions [ 108 ], which suggests a circularity between pain and dysbiosis maintenance within the gut. Intriguingly, kininogen-1 and fibrinogen plasma levels have emerged to be directly correlated with pain intensity in women with chronic widespread pain [ 109 ] and higher TFF3 levels were instead detected in patients with IBS [ 110 ]. Interestingly, pain relief mediated by FMT CTR in DNBS-treated animals correlated with a significant reduction of TFF3 levels, reinforcing the link between this protein and pain. Finally, the upregulation of serpine1, prothymosin alpha, thymosin β(4) and other partners of immune response in the colon abx + FMT DNBS group, without overt changes emerging at histological analysis, confirm the presence of a low-grade inflammation sustained by the immune response to microbes, which likely contribute to dysbiosis-related pain persistence [ 111 – 113 ]. In addition, the increased expression of APOA1 common to the different pathways modulated by FMT CTR , might link the effects of microbiota manipulation on lipid metabolism to those on immune response [ 114 – 116 ], both associated with pain relief. Although specific bacterial products are known to trigger immune activation [ 117 – 119 ], evidence supporting a cause-and-effect relationship between dysbiosis, altered immune response and post-inflammatory pain has been based on indirect associations [ 120 , 121 ]. Indeed, no study has ever before explored the effect of manipulating the microbiota on the immune response which, moreover, is compromised due to chronic disease [ 122 ]. In our Therapy protocol, we demonstrated that the pain-relieving effect of FMT CTR in DNBS animals was supported by the inhibition of inflammatory response, leucocytes migration e mast cell activation. By analyzing colonic proteins, we found that this immunosuppressive effect could be related to an increase in seminal vesicle secretory proteins [ 123 ], which are involved in suppressing the immune response in the female reproductive tract but have an unknown role in the intestine. Besides, the dampening of mast cells activation following FMT CTR treatment, confirmed by beta-hexasominidase levels [ 59 ], could be linked to the concomitant reduction in the production and release of derived immunoglobulin free light chains by lymphocytes. Indeed these proteins can trigger mast cell-mediated hypersensitivity [ 124 ], which play a key role in the pathophysiology of abdominal pain in IBS [ 120 , 125 , 126 ]. Restoring mast cells homeostasis might account for the pain-relieving effects of FMT CTR in DNBS animals, tough the escalation of immune events linking microbiota perturbation to hypersensitivity needs to be further elucidated. In this regard, it is interesting to note that the anti-hyperalgesic effect of FMT CTR was associated with a facilitated infiltration of macrophages in the submucosa of DNBS-treated animals, which are in remission from colitis. In addition to having an active role in damage resolution and in the maintenance of tolerogenic environment [ 87 ], macrophages can transfer mitochondria to the sensory neurons of the DRG to resolve inflammatory pain [ 127 ]. As discussed above, the presence of an intestinal “metabolic injury”, involving mitochondria, might represent the link between the microbiota and the different partners apparently contributing to the disease. High levels of microbial-derived energy source such as SCFAs, particularly 3-hydroxybutyrate, directly correlated with pain persistence in DNBS-treated animals. Since these metabolites physiologically contribute to strengthen intestinal barrier and to prevent harmful inflammatory responses [ 128 ], it is possible that the capacity of the gut to properly absorb and use them might be impaired under post-inflammatory condition, causing localized (epithelium) or diffuse (immune cells and neurons) alterations in the metabolism, therefore contributing to visceral hypersensitivity. In support to this, a recent multi-omics analysis of gut microbiome in IBS patients highlighted peculiar transcriptional modifications of enzymes involved in the metabolism of carbohydrate and lipids [ 129 ]. The same authors explain the increased levels of free fatty acids, particularly in IBS-C, by altered epithelial lipid metabolism [ 129 ]. Although a direct association with pain was not drawn, epithelium dysfunction might be potentially implicated in shaping microbial environments [ 57 ], as well as sensory neurons activity [ 130 , 131 ], creating again a vicious circle between dysbiosis and pain. Epithelium involvement in the therapeutic effect of FMT CTR in DNBS-treated animals seems to be confirmed by the increased expression of secretogranin-1, a neuroendocrine secretory granule protein, which can be the precursor for other biologically active peptides [ 62 , 63 ]. We also observed that the effect of FMT on the expression of certain proteins (i.e. immunoglobulin free light chains and seminal vesicles secretory proteins, chymotrypsin-C) is unrelated to the donor, as same changes occur because of the transplant of either FMT DNBS in abx-treated animals or FMT CTR in DNBS-treated animals. This phenomenon, which deserve further investigation, implicates that the mechanisms by which FMT exerts its effects are strictly dependent on the healthy or disease state of the recipient. This could call into question the need to identify a super-donor to improve therapies and shift the spotlight to the need to better understand the pathophysiology of patients. Finally, though it is important to continue clarifying the peripheral mechanisms before defining central mechanisms involved in pain chronicity and FMT effects, behavioral results suggest that visceral and somatic pain relief in DNBS animals receiving FMT CTR might involve the modulation of common pain pathways at central level. Conclusion Overall, our results confirm that metabolic injury driven by post-inflammatory dysbiosis (FMT DNBS ) contributes to maintain a low-grade inflammation, epithelium dysfunctions, and enteric neuroplasticity phenomena, initiated by the inflammatory insult and underpinning visceral pain persistence after colitis remission. On the other hand, the therapeutic effect FMT CTR on post-inflammatory pain might be related to the suppression of immune response which is likely deregulated in the colon of DNBS animals. In this context, the inability to reprogram the immune system and to revert neuroplastic changes caused by inflammation might explain the reversibility of FMT effect on pain, suggesting a combined intervention on colon as a strategy to improve therapy efficacy. Indeed, though modification of microbiome represents the primum movens , the FMT effects are likely sustained by other cells within the gut. Since not necessarily all the pathways modulated by FMT are involved in pain regulation, further validation is needed to define the cellular partners and the order of events linking dysbiosis and pain. This comprehensive study is expected to enhance the understanding and treatment of gut dysmotility, a condition commonly linked to pain. Declarations Ethics approval and consent to participate All experiments were performed using rodents in accordance with the International Association for the Study of Pain, the European Union directives, and the National Institutes of Health guidelines on laboratory animal welfare and approved by the Animal Care committee at the University of Florence (Italy) and the Italian Ministry of Health (No. 388/2021-PR). The data sets generated and analysed during the current study are included as additional files. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Funding This research was supported by the Italian Ministry of Instruction, University and Research, by the University of Florence, by the “linea 2” of University of Milan, by the University of East Anglia and by the University College Cork. Author Contribution E.L., A.D.A., V.D.P., D.V., S.O.M., C.N. and L.D.C.M. conceptualized and designed the experiments and analytical approaches. V.D.P., F.M., L.M. and T.F.S.B. processed, analysed and interpreted 16S rRNA gene sequence data. D.V.. and G.L.G. processed, analysed and interpreted metabolomics data. A.D.A. and E.L, processed, analysed and interpreted proteomics data. E.L., L.M. and A.T. designed behaviour experiments and histological analysis. E.L., L.M., C.C. and A.T. carried out experiments and analysed data. E.L., V.D.P., T.F.S.B., D.V., A.D.A., wrote the paper with contributions from all authors. C.N., S.O.M., G.M.R., L.D.C.M. and C.G. revised the manuscript. L.D.C.M. and C.G. supervised the study and provided the experimental resources. All authors read and approved the final manuscript. Acknowledgement We acknowledge UNITECH OMICs, mass spectrometry platform of Università degli Studi di Milano, for running mass spectrometric analyses for the proteomic study. We are grateful to Università degli Studi di Milano which partially funded the research through Piano di Sostegno alla Ricerca, linea 2, (2022 and 2023). Data Availability All data generated or analysed during this study are included in this published article and its supplementary information files. The 16S rRNA sequence data have been deposited in the NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/bioproject/) under the BioProject accession number PRJNA1213328. References Drewes AM, Olesen AE, Farmer AD, Szigethy E, Rebours V, Olesen SS: Gastrointestinal pain. Nat Rev Dis Primers 2020, 6:1. 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Bayrer JR, Castro J, Venkataraman A, Touhara KK, Rossen ND, Morrie RD, Maddern J, Hendry A, Braverman KN, Garcia-Caraballo S, et al: Gut enterochromaffin cells drive visceral pain and anxiety. Nature 2023, 616:137–142. Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.tif Table2.tiff Supplementary.docx AdditionalFile1FMTDisease.xlsx AdditionalFile2FMTTherapy.xlsx AdditionalFile3Proteome.xlsx AdditionalFile4Metabolomics.xlsx AdditionalFile5Immunofluorescence.xlsx AdditionalFile6Comprehensivestatmicrobiomedata.zip floatimage14.jpeg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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14:26:07","extension":"html","order_by":74,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":379216,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/87adb441bd683ca29002849e.html"},{"id":93947102,"identity":"273b0967-252f-4699-bde0-d33cd4a1d55f","added_by":"auto","created_at":"2025-10-20 14:25:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":480022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFMT\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eDNBS\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e induces pain in naïve controls along with changes in gut microbiome.\u0026nbsp; \u003c/strong\u003eRats were treated as shown in the scheme (\u003cstrong\u003eA\u003c/strong\u003e). On day 7, the abx-treated animals were divided into 3 groups, and respectively administered FMT from CTR donors, FMT from DNBS donors or vehicle\u003cem\u003e per os\u003c/em\u003e for 5 consecutive days. One week after, the administrations were repeated. Animals body weight was monitored throughout the experiment (\u003cstrong\u003eB\u003c/strong\u003e). Behavioural tests were performed 7 days after the last FMT (\u003cstrong\u003eC\u003c/strong\u003e). Visceral sensitivity was assessed in the animals by measuring the AWR to CRD (0.5-3 mL). Each value is the mean ± SEM of 4 (veh + veh and abx + veh group) or 8 (abx + FMT\u003csup\u003eCTR\u003c/sup\u003e and abx + FMT\u003csup\u003eDNBS\u003c/sup\u003e) animals per group. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. *P\u0026lt;0.05 and **P\u0026lt;0.01 vs abx + vehicle–treated animals. ^P\u0026lt;0.05 and ^^P\u0026lt;0.01 vs abx + FMT\u003csup\u003eCTR\u003c/sup\u003e–treated animals.\u0026nbsp; \u003cstrong\u003e(D) \u003c/strong\u003eMicrobiome (16S) was assessed in the FMT donor samples and FMT recipient samples, on day 0 and day 32 (n=8). Alpha diversity and genus-level taxononomical differences were assessed using generalized linear mixed-effects models (glmer), while beta diversity was assessed using PERMANOVA. Comprehensive statistical description of microbiome data can be found in the supplementary files: *P\u0026lt; 0.05, **P\u0026lt; 0.01\u0026nbsp;and ***P\u0026lt; 0.001. abx, antibiotics; AWR, abdominal withdrawal reflex; CRD, colorectal distension; CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/a1c9f0c7aa46a843c6cb138f.png"},{"id":93947108,"identity":"37087f7b-bded-4e24-9bfe-0265c2ca7768","added_by":"auto","created_at":"2025-10-20 14:26:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":499954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFMT\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eCTR\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e counteracts pain in DNBS-animals along with changes in gut microbiome.\u0026nbsp; \u003c/strong\u003eRats were treated as shown in the scheme (\u003cstrong\u003eA\u003c/strong\u003e). On day 7, DNBS-injected animals were divided into 3 groups, respectively administered with FMT from CTR donors, FMT from DNBS donors or vehicle \u003cem\u003eper os\u003c/em\u003e for 5 consecutive days. The FMT set was weekly repeated for 4 times. Animals body weight was monitored throughout the experiment (\u003cstrong\u003eB\u003c/strong\u003e). Behavioral tests were performed 10 days after the last FMT (\u003cstrong\u003eC\u003c/strong\u003e). Visceral sensitivity was assessed in the animals by measuring the AWR to CRD (0.5-3 mL). Each value is the mean ± SEM of 4 (veh + veh and DNBS + veh group) or 8 (DNBS + FMT\u003csup\u003eCTR\u003c/sup\u003e and DNBS + FMT\u003csup\u003eDNBS\u003c/sup\u003e) animals per group. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. °°P\u0026lt;0.01 vs vehicle + vehicle–treated animals. *P\u0026lt;0.05 and **P\u0026lt;0.01 vs DNBS + vehicle–treated animals. ^P\u0026lt;0.05 and ^^P\u0026lt;0.01 vs DNBS + FMT\u003csup\u003eDNBS\u003c/sup\u003e–treated animals. \u003cstrong\u003e(D) \u003c/strong\u003eMicrobiome (16S) was assessed in the FMT donor samples and FMT recipient samples, on day 0 and day 42 (n=8). Alpha diversity and functional potential were assessed using generalized mixed-effects models (glmer), while beta diversity was assessed using PERMANOVA. Comprehensive statistical description of microbiome data can be found in the supplementary files: *P\u0026lt; 0.05, **P\u0026lt; 0.01\u0026nbsp;and ***P\u0026lt; 0.001. AWR, abdominal withdrawal reflex; CRD, colorectal distension; CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/02f85b78d0e5153aa9375c77.png"},{"id":93947140,"identity":"0c59e0d8-0cca-40f7-9157-0977a87e942f","added_by":"auto","created_at":"2025-10-20 14:26:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":768256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFaecal metabolomic profiles.\u003c/strong\u003e Sparse PLS-DA (sPLS-DA) plots showed separation of groups indicative of a metabolomic shift in the pathological model (Panel \u003cstrong\u003eA\u003c/strong\u003e) and the therapeutic model (Panel \u003cstrong\u003eC\u003c/strong\u003e) (n=8). Heatmaps depicting the 6 (Panel \u003cstrong\u003eB\u003c/strong\u003e) and 21 (Panel \u003cstrong\u003eD\u003c/strong\u003e) significantly altered metabolites as determined by Wilcoxon rank-sum test (P \u0026lt; 0.05) (n = 8). abx, antibiotics; CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/a166748b3b6aa5d877a4a8cd.png"},{"id":93947107,"identity":"6091118f-5ba0-4884-b68b-6fefdc2d9ee2","added_by":"auto","created_at":"2025-10-20 14:26:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":504104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation analysis between the metabolome and microbiome. \u003c/strong\u003eDIABLO integrative analysis of microbiota and metabolome datasets, including samples from the Disease (32 days) and Therapy (42 days) experimental protocols. (A-B) Loading weights of the top 15 features on component 1, ranked by loading weight, for each omics datasets from both experimental protocols. Colours indicate the group of discriminant microbial taxa (i.e ASVs) and metabolites in which the median relative abundance is maximum, and values indicate the contribution to the first component. Common taxa and metabolites are highlighted in bold. (C-D) Circos plots showing correlations (r=0.7) between the most discriminatory microbial taxa and metabolites. Positive and negative correlations are displayed by green and light red inner line-connectors, respectively. The outermost circles indicate to which group the feature is associated in samples from the Disease (i.e. abx + FMT) and Therapy (i.e. DNBS + FMT) experimental protocols\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/53b3a728c67868be7dde987c.png"},{"id":93947121,"identity":"26a225e8-b72d-4080-a87a-895be2904895","added_by":"auto","created_at":"2025-10-20 14:26:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":534367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisease activity in the colon of FMT recipients. \u003c/strong\u003eAnimals were sacrificed after behavioural tests. Abx-treated animals receiving the FMT\u003csup\u003eDNBS\u003c/sup\u003e or FMT\u003csup\u003eCTR\u003c/sup\u003e were sacrificed on day 32, 7 days after the last treatment. DNBS-treated animals receiving the FMT\u003csup\u003eDNBS\u003c/sup\u003e or FMT\u003csup\u003eCTR\u003c/sup\u003e were sacrificed on day 42, 10 days after the last treatment.\u003cstrong\u003e \u003c/strong\u003eColon length, specific weight and macroscopic damage were assessed at the time of colon explantation from abx- (\u003cstrong\u003eA\u003c/strong\u003e) or DNBS-treated animals (\u003cstrong\u003eD\u003c/strong\u003e) undergoing FMT. Microscopic damage was evaluated on H\u0026amp;E-stained colon slices from abx- (\u003cstrong\u003eB\u003c/strong\u003e) or DNBS-treated animals (\u003cstrong\u003eE\u003c/strong\u003e) undergoing FMT, as shown in the representative microphotographs (100x magnification). Mast cell- and Eosinophil- density in the submucosal layer was quantified on GIEMSA-stained colon slices from abx- (\u003cstrong\u003eC\u003c/strong\u003e) or DNBS-treated animals (\u003cstrong\u003eF\u003c/strong\u003e) undergoing FMT, as shown in the representative microphotographs (40x magnification). Each value represents the mean ± SEM of 7-8 animals per group. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. *P\u0026lt;0.01 vs FMT\u003csup\u003eDNBS\u003c/sup\u003e. abx, antibiotics; CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/3067f1c86ff10e56f185d15f.png"},{"id":93947924,"identity":"9b53818d-ec5e-4f9d-b812-9e51f13a19ba","added_by":"auto","created_at":"2025-10-20 14:34:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":582143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIba-1 expression in the colon of FMT recipients. \u003c/strong\u003eAnimals were sacrificed after behavioural tests. Abx-treated animals receiving the FMT\u003csup\u003eDNBS\u003c/sup\u003e or FMT\u003csup\u003eCTR\u003c/sup\u003e were sacrificed on day 32, 7 days after the last treatment. DNBS-treated animals receiving the FMT\u003csup\u003eDNBS\u003c/sup\u003e or FMT\u003csup\u003eCTR\u003c/sup\u003e were sacrificed on day 42, 10 days after the last treatment. The Immunoreactivity related to Iba-1 expression in abx- and DNBS-treated animals was measured in the mucosa (A-E), submucosa (B-F) and muscolaris (C-G), respectively, as represented in the images (D-H).\u0026nbsp; Each value represents the mean ± SEM of 6 animals per group. Statistical analysis was one-way analysis of variance followed by Bonferroni post hoc comparison. ^P\u0026lt;0.01 vs FMT\u003csup\u003eDNBS\u003c/sup\u003e. abx, antibiotics; CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation; Iba-1, ionized calcium-binding adapter molecule 1.\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/67fdedc90055c290c532e02b.png"},{"id":93949283,"identity":"6ac08007-159a-44d5-ab97-736f76678506","added_by":"auto","created_at":"2025-10-20 14:42:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":91917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of FMT effects on colon proteome. \u003c/strong\u003eScatter plots of log2 ratio on x-axis against -log10 p value on y-axis of significantly quantified proteins. (A) Quantified proteins in Disease FMT. (B) Quantified proteins in Therapy FMT. Green color indicates downregulation (log2 ratio ≤ −0.55), red color represents upregulation (log2 ratio ≥ 0.55). Statistical parameters (p \u0026lt; 0.05; q \u0026lt; 0.05, q = FDR adjusted + value) were set to identify the differentially expressed proteins between samples (n = 5). abx, antibiotics; CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/fcafa95f19c47ff7b64b1eb6.png"},{"id":93947930,"identity":"c8026eb5-46bc-4f16-8a8a-e5a5bce71b24","added_by":"auto","created_at":"2025-10-20 14:34:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":398241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle proteins modulated by FMT in the Disease Protocol I. \u003c/strong\u003e\u0026nbsp;Column charts showing log2 ratio of proteins which resulted differentially expressed in abx + FMT\u003csup\u003eDNBS\u003c/sup\u003e with respect of abx + FMT\u003csup\u003eCTR\u003c/sup\u003e group (P \u0026lt; 0.05; n=5). Proteins were clustered according to their involvement in cell shape, cell-adhesion, ECM-to-cell and cell-to-cell interaction (A), neuroplasticity and neuromodulation (B). Colors indicate the degree of protein downregulation or upregulation according to the scale on the right. abx, antibiotics; CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.\u003c/p\u003e","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/a355a4e64d8ed64dd428faf6.png"},{"id":93949311,"identity":"688607c2-74cd-4d3a-92a3-ef64a5b36962","added_by":"auto","created_at":"2025-10-20 14:42:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":623740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle proteins modulated by FMT in the Disease Protocol II. \u003c/strong\u003eColumn charts showing log2 ratio of proteins which resulted differentially expressed in abx + FMT\u003csup\u003eDNBS\u003c/sup\u003e with respect of abx + FMT\u003csup\u003eCTR\u003c/sup\u003e group (P \u0026lt; 0.05; n=5). Proteins were clustered according to their involvement in energy metabolism, iron metabolism and redox balance (A), immune response and inflammatory process regulation (B). Colors indicate the degree of protein downregulation or upregulation according to the scale on the right. abx, antibiotics; CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.\u003c/p\u003e","description":"","filename":"OnlineFigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/a27b64254f0da006e40d8634.png"},{"id":93947117,"identity":"350b14d0-82ea-443d-b10b-9d84428bb650","added_by":"auto","created_at":"2025-10-20 14:26:03","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":345776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle proteins modulated by FMT in the Therapy protocol I. \u003c/strong\u003eColumn charts showing log2 ratio of proteins which resulted differentially expressed in DNBS + FMT\u003csup\u003eCTR\u003c/sup\u003e with respect of DNBS + FMT\u003csup\u003eDNBS\u003c/sup\u003e group (P \u0026lt; 0.05; n=5). Colors indicate the degree of protein downregulation or upregulation according to the scale on the right. CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.\u003c/p\u003e","description":"","filename":"OnlineFigure10.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/fd03aafd94ba57c3eff61926.png"},{"id":93947115,"identity":"34f4b971-869b-466d-b30c-6163ea29b5b2","added_by":"auto","created_at":"2025-10-20 14:26:02","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":302610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle proteins modulated by FMT irrespective of donors. \u003c/strong\u003eColumn charts showing log2 ratio of proteins which resulted differentially expressed in the comparison between abx + FMT\u003csup\u003eDNBS\u003c/sup\u003e and abx + FMT\u003csup\u003eCTR\u003c/sup\u003e (orange; p \u0026lt; 0.05), as well as in the comparison between DNBS + FMT\u003csup\u003eCTR\u003c/sup\u003e with respect of DNBS + FMT\u003csup\u003eDNBS\u003c/sup\u003e (blue; P \u0026lt; 0.05). abx, antibiotics; CTR, control animals; DNBS, 2,4-dinitrobenzenesulfonic acid; FMT, faecal microbiota transplantation.\u003c/p\u003e","description":"","filename":"OnlineFigure11.png","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/eea0504447c1dd7c3cc88a08.png"},{"id":95801399,"identity":"ae87cfd2-677a-4fa4-968b-67b2c841fa00","added_by":"auto","created_at":"2025-11-13 08:25:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9803728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/e7885b76-e17b-4b47-a334-061f0d8da065.pdf"},{"id":93947217,"identity":"a39f9152-1a1a-4587-af8c-25e251117b41","added_by":"auto","created_at":"2025-10-20 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14:34:04","extension":"zip","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":155793,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile6Comprehensivestatmicrobiomedata.zip","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/bfe9584c7e11e4715b9a0239.zip"},{"id":93947128,"identity":"13856c95-7081-4e6c-b16b-bbb6a09c189b","added_by":"auto","created_at":"2025-10-20 14:26:04","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":454856,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage14.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6025304/v1/d7d42537d3ca05219fbc8675.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Microbiome and gut as partners in the persistence of post-inflammatory visceral pain: insight into working mechanisms of faecal transplant for therapy advance","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAbdominal pain is a hallmark of functional gastrointestinal disorders, such as irritable bowel syndrome (IBS), which often results from infections or prolonged inflammation, as seen in chronic inflammatory bowel diseases (IBDs) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Colitis is characterized by relapsing and remitting phases and by the establishment of abdominal pain persisting even in a state of low disease activity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Several gut-to-brain signalling factors play a significant role in the persistence of post-inflammatory hypersensitivity, such as peripheral and central sensitization, altered sympathovagal balance, hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal axis activation, and psychosocial factors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Due to its complex, multifactorial nature, post-inflammatory pain is often refractory to classical pain medications [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], hence the need for alternative approaches. The microbiota of IBD patients is substantially different from that of healthy controls in terms of diversity, volatility (change over time), taxonomy, and metabolism [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], even in a state of remission [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], suggesting that the microbiota might act as an important driver of chronicity. The gut microbiota represents a source of signalling molecules, including neurotransmitters and neuromodulators, which may play an important role in mediating neuroinflammation involved in peripheral and central sensitization [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. There are numerous studies, both preclinical and clinical, reporting a close relationship between gut dysbiosis and visceral pain, [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] However, there are still inconsistencies to support the microbiota as a therapeutic target for visceral hypersensitivity. Furthermore, clinical practice, shows contradictory results on the efficacy of microbiota-targeted interventions, including faecal microbiota transplant (FMT), for pain associated with gastrointestinal diseases [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Mechanisms regulating microbiota-host interaction and its inflammation-mediated modifications remains to be determined. To delve deeper into post-inflammatory changes, we used an established model of colitis induced by intra-rectal injection of DNBS in rats, which recapitulates IBD in terms of pathophysiology and response to drugs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our previous work [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], we demonstrated that visceral hypersensitivity can be transferred from animals in the post-inflammatory phase of colitis induced by DNBS to naive control rats by means of FMT. Furthermore, a significant amelioration of persistent pain was achieved by the transfer of a healthy microbiota to DNBS-treated rats. In both cases, the FMT effect was long-lasting and reversible after FMT discontinuation, with animals returning to their initial condition after 2\u0026ndash;3 weeks [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This evidence confirmed that FMT temporarily provides relief in an IBD rat model, which is also observed in the clinical practice [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and underscores the importance of the intestinal condition of recipients in the outcomes of microbiota-targeted interventions. Current data suggests microbial engraftment is correlated with a positive response to FMT. However, the degree of engraftment, as well as its dynamics and determinants are not well understood, though they are likely dependent on donor and recipient factors, including but not limited to genetic, comorbidities, medication use, diet, lifestyle, and baseline microbiome factors [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Further research is therefore necessary to identify optimal conditions for the engraftment and biomarkers predictive of response, as well as potential adjunct therapies to enhance response. In the present work, we used the FMT-based experimental approach to investigate microbiota-to-gut signalling involved in post-inflammatory visceral pain persistence through a combined multi-omics approach. Results uncovered new mechanisms governing host-microbiota crosstalk in health and disease and provide new perspectives for improving the therapy of gastrointestinal pain associated with IBD.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Ethical approvals\u003c/h2\u003e \u003cp\u003e All animal manipulations were performed according to the Directive 2010/63/EU of the European parliament and of the European Union council (September 22, 2010) on the protection of animals used for scientific purposes. The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the described experiments was obtained from the Animal Subjects Review Board of the University of Florence (Italy) and the Italian Ministry of Health (388/2021-PR). Experiments involving animals have been reported according to the ARRIVE guidelines [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. All efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animals\u003c/h2\u003e \u003cp\u003eFor all the experiments described below, 8-week-old male Sprague\u0026ndash;Dawley rats (Envigo, Varese, Italy), weighing approximately 220 to 250 g at the beginning of the experimental procedure, were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least 1 week after their arrival. Before starting the treatments, 4 animals were housed per cage (size 26 \u0026times; 41 cm). After starting the experimental procedures, animals were individually housed to avoid the damage of the electrodes implanted for electrophysiological measurements. Animals were fed a standard laboratory diet and purified water ad libitum and kept at 23\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a 12-hour light or dark cycle, light at 7 am.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Induction of colitis\u003c/h2\u003e \u003cp\u003eColitis was induced following the method described previously by Lucarini et al.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In brief, during short anaesthesia with isoflurane (2%), 30 mg of DNBS (Merck Life Science, Milan, Italy) in 0.25 mL of 50% ethanol was administered intrarectally through a polyethylene PE-60 catheter inserted 8 cm proximal to the anus. Control rats received 0.25 mL of saline solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Faecal microbiota transplantation study design\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Disease Protocol\u003c/h2\u003e \u003cp\u003eIn the first experimental set-up, rats were randomized to four groups:\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003e1) control (vehicle, no antibiotic treatment, no FMT; n = 4)\u003c/h3\u003e\n\n\u003ch3\u003e2) antibiotics (abx) + vehicle (antibiotic treatment followed by vehicle administration; n = 4)\u003c/h3\u003e\n\n\u003ch3\u003e3) abx + FMT (antibiotic treatment followed by FMT from controls; n = 8)\u003c/h3\u003e\n\n\u003ch3\u003e4) abx + FMT (antibiotic treatment followed by FMT from DNBS treated animals; n = 8)\u003c/h3\u003e\n\u003cp\u003eThe animals underwent the following antibiotic or antifungal regimen to prepare them to the FMT: Day 0 to 6 rats received a daily oral gavage (10mL\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of amphotericin B (1 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and metronidazole (100 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) whereas an antibiotic mix (ceftazidime 1 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, vancomycin 0.5 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and neomicin 1 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added to the autoclaved drinking water and changed every 2 days. On day 7, 24 hours after the interruption of the antibiotic treatment, the animals underwent the FMT procedure. FMT was daily performed on days 7 to 11 (set I) and on days 21 to 25 (set II). The animals were singly housed also to improve reproducibility of data. Behavioural tests were performed at the end of the antibiotic treatment (before starting the FMT), 24 hours and 7 days after each FMT set. Faecal material collected from different controls (n\u0026thinsp;=\u0026thinsp;4) or DNBS-treated animals (n\u0026thinsp;=\u0026thinsp;4) (between 14 and 21 days after the intrarectal injection of the inflammatory agent) were combined and homogenized to generate a single solution for FMT\u003csup\u003eCTR\u003c/sup\u003e and FMT\u003csup\u003eDNBS\u003c/sup\u003e, respectively. To achieve this the tubes containing faecal pellets in sterile saline solution were left on ice for 60 minutes and then homogenized for 2 minutes on ice using a hand-held pellet pestle device with sterile, reuseable pestles. When fully homogenized, the suspended pellets were centrifuged, and the supernatant directly used for the FMT procedure. FMT was performed through oral gavage with a faecal suspension (50 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in a final volume of 3 mL.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.3.2. Therapy Protocol\u003c/h2\u003e \u003cp\u003eIn the second experimental set-up rats were randomized to four groups:\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e1) control (vehicle + vehicle; n = 4)\u003c/h3\u003e\n\n\u003ch3\u003e2) DNBS + vehicle (intrarectal injection of DNBS 30 mg followed by vehicle administration; n = 6)\u003c/h3\u003e\n\n\u003ch3\u003e3) DNBS + FMT (intrarectal injection of DNBS 30 mg followed by FMT from controls; n = 8)\u003c/h3\u003e\n\u003cp\u003e4) DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e (intrarectal injection of DNBS 30 mg followed by FMT from DNBS animals; n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003cp\u003eColitis was induced in animals through intrarectal injection of DNBS (30 mg in 0.25 mL EtOH 50%) on day 1. The control group was intrarectally administered with saline solution. DNBS-treated animals did not receive any antibiotic pretreatment before the FMT due to the influence on the induction of colitis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Seven days after DNBS injection, the animals were split into 3 groups receiving: 1) the vehicle, 2) the naive control\u0026ndash;derived faecal microbiota suspensions (FMT\u003csup\u003eCTR\u003c/sup\u003e) or 3) the DNBS\u0026ndash;derived faecal microbiota suspensions (FMT\u003csup\u003eDNBS\u003c/sup\u003e). FMT was performed for 5 consecutive days/week and the same protocol was repeated for 4 weeks (I-IV set of FMT): on days 7 to 11 (set I), 14 to 18 (set II), 21 to 25 (set III), and 28 to 32 (set IV) after DNBS injection as per [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Behavioural tests were conducted 7 days after DNBS injection (before starting FMT), 3 days after each FMT set, and 10 days after the last treatment. For the FMT, faecal material was processed as mentioned above. FMT was performed by oral gavage with a faecal suspension (100 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in a final volume of 3 mL.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Assessment of visceral sensitivity\u003c/h2\u003e \u003cp\u003eThe behavioural responses to colorectal distension (CRD) were assessed in the animals by measuring the abdominal withdrawal reflex (AWR), a semiquantitative score described previously in conscious animals [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In brief, rats were anesthetized with isoflurane (2%), and a lubricated latex balloon (length: 4.5 cm), attached to polyethylene tubing, assembled to an embolectomy catheter, and connected to a syringe filled with water, was inserted through the anus into the rectum and descending colon of adult rats. The tubing was taped to the tail to hold the balloon in place. Then rats were allowed to recover from the anaesthesia for 30 minutes. AWR measurement consisted of visual observation of animal responses to graded CRD (0.5, 1, 2, and 3 mL) blinded observers who assigned AWR scores: no behavioural response to colorectal distention (0); immobile during colorectal distention and occasional head clinching at stimulus onset (1); mild contraction of the abdominal muscles but absence of abdomen lifting from the platform (2); observed strong contraction of the abdominal muscles and lifting of the abdomen off the platform (3); and arching of the body and lifting of the pelvic structures and scrotum (4). The time elapsed between 2 consecutive distension was 5 minutes. AWR measurements were conducted in all the animals (n\u0026thinsp;=\u0026thinsp;8 \u003cem\u003eper group\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Assessment of thermal and tactile pain threshold\u003c/h2\u003e \u003cp\u003eThe thermal pain threshold was assessed using the hot plate test as previously described with minor adjustments [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. With minimal animal\u0026ndash;handler interaction, rats were taken from their home cages and placed onto the surface of the hot plate (Ugo Basile, Varese, Italy) maintained at a constant temperature of 48 ◦C\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ◦C (first evaluation) and 50\u0026deg; \u0026plusmn; 1 ◦C (second evaluation after 1 h). Ambulation was restricted by a cylindrical Plexiglas chamber (diameter, 10 cm; height, 15 cm), with an open top. A timer controlled by a foot peddle began timing response latency from the moment the rat was placed onto the hot plate. Pain-related behavior (licking of the hind paw) was observed, and the time (seconds) of the first sign was recorded. The cutoff time of the latency of paw lifting or licking was set at 40 s. An analgesimeter (Ugo Basile, Varese, Italy) was used to measure the tactile pain threshold of rats according to [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We applied a constantly increasing pressure on the dorsal surface of the hind paw using a blunt conical probe by a mechanical device. The pressure was increased until vocalization or a withdrawal reflex occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were expressed in grams. Rats scoring below 40 g or over 75 g during the test before drug administration were rejected (25%). A cut-off of 200 g was adopted. Paw pressure test and Hot plate test were performed on day 32 (Disease Protocol) and on Day 42 (Therapy Protocol).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Profiling of the gut microbiota\u003c/h2\u003e \u003cp\u003eFecal pellets (50 mg) and fecal suspensions (volume equivalent to 50 mg) were processed for the total DNA extraction using the DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany). Next-generation sequencing of 16S ribosomal RNA amplicons of the V3-V4 regions ((341F, 5\u0026rsquo;- CCTAYGGGRBGCASCAG-3\u0026rsquo;; 806R, 5\u0026rsquo;- GGACTACNNGGGTATCTAAT-3\u0026rsquo;) was performed using the Illumina NovaSeq 6000 Sequencing System (Novogene Co., Ltd.), using a 2 \u0026times; 250 bp paired-end approach. Extraction and PCR amplification negative controls were also included. Sequencing results were analyzed using the QIIME 2 suite (Quantitative Insights Into Microbial Ecology)[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In brief, after raw reads denoising (ie, error correction, removal of chimeric and singleton sequences, and joining of denoised paired-end reads), DADA2 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] was used to annotate genus-level feature tables, based on SILVA 16S reference database (release 138.2) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arb-silva.de/documentation/release-138/\u003c/span\u003e\u003cspan address=\"https://www.arb-silva.de/documentation/release-138/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Functional annotation was performed using picrust2[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], after which neuroactive potential was estimated through the functional gut-brain module framework[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Microbial diversity was estimated by evaluating alpha diversity (Chao1, Simpson\u0026rsquo;s Index, Shannon Entropy) and beta diversity (Aitchison distance) metrics in R. Features were transformed into centered log-ratios (CLR) to account for compositionality as by recent recommendation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. R scripts are available online on github\u003c/p\u003e \u003cp\u003e(URL: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/thomazbastiaanssen/FMT_pain_analysis/\u003c/span\u003e\u003cspan address=\"https://github.com/thomazbastiaanssen/FMT_pain_analysis/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.7. 1H NMR metabolomics\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFaecal metabolites were analysed and quantified by 1H NMR analysis. The preparation method was as previously described [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Briefly, frozen faecal pellets were thoroughly mixed at 5,000 rpm in a Precellys\u0026reg;24 (Bertin Technologies, France) and diluted to a faeces-to-buffer ratio of 13 (e.g., 50 mg faeces in 750 \u0026micro;L buffer) by adding deuterated phosphate buffer (1.9 mM Na2HPO4, 8.1 mM NaH2PO4, and 1 mM sodium 3-(trimethysilyl)-propionate-d4 in deuterated water (Goss Scientifics, Crewe, United Kingdom)). After mixing and centrifugation, 500 \u0026micro;l was transferred into a 5 mm NMR tube for spectral acquisition. High resolution 1H NMR spectra were recorded on a 600 MHz Bruker Avance spectrometer fitted with a 5 mm TCI proton-optimized triple resonance NMR inverse cryoprobe and a 24-slot autosampler (Bruker, Rheinstetten, Germany). Sample temperature was controlled at 300 K. Each spectrum consisted of 128 scans of 65,536 complex data points with a spectral width of 20 ppm (acquisition time 2.6 s). The noesypr1d presaturation sequence was used to suppress the residual water signal with low power selective irradiation at the water frequency during the recycle delay (D1\u0026thinsp;=\u0026thinsp;2 s) and mixing time (D8\u0026thinsp;=\u0026thinsp;0.01 s). A 90\u0026deg; pulse length of 11.4 \u0026micro;s was set for all samples. Spectra were transformed with a 0.1 Hz line broadening and zero filling, manually phased, baseline corrected and referenced by setting the trimethylsilylpropanoic acid methyl signal to 0 ppm. Metabolites were identified using information found in the literature or on the web (Human Metabolome Database, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.hmdb.ca/\u003c/span\u003e\u003cspan address=\"https://www.hmdb.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and quantified using the software Chenomx\u0026reg; NMR Suite 8.6\u0026trade;.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Proteomic analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEach colon sample was homogenized and tryptic digested as in D'Amato A, et al. (2020)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The peptides were analyzed using a Dionex Ultimate 3000 nano-LC system (Sunnyvale CA, USA) connected to Orbitrap Fusion\u0026trade; Tribrid\u0026trade; Mass Spectrometer (Thermo Scientific, Bremen, Germany) equipped with a nano-electrospray ion source (nESI). The elution gradient was from 96% buffer A (0.10.1% formic acid (FA) in water )to 40% buffer B (0.1% FA in water/acetonitrile with 2/8 ratio) for 110 min. MS spectra were collected over an m/z range of 375\u0026ndash;1500 Da at 120,000 resolutions, operating in data dependent scan mode, cycle time 3 sec between master scans [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The instrumental raw files were processed by MaxQuant software v1.6.6.0 (Cox et al, 2014) set on Uniprot_Rat database against the Andromeda search engine (D\u0026rsquo;Amato A. et al Microbiome 2020). The quantification of peptides and related proteins for each control and treated sample in biological duplicate and technical triplicates was based on the LFQ intensities. The interpretation and visualization of results obtained from MaxQuant software were performed by a two-sample t-test using Perseus (v1.6.1.3, Max Planck Institute of Biochemistry, Germany). Statistical parameters (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; q\u0026thinsp;\u0026lt;\u0026thinsp;0.05, q\u0026thinsp;=\u0026thinsp;FDR adjusted p-value) were set to identify the differentially expressed proteins between samples (log2 ratio). Variability of biological replicates were measured using the scatter plot with Pearson correlation coefficient values of the LFQ intensities. (Zoanni et al., 2023). The network protein analysis related to significantly altered proteins was carried out by Ingenuity Pathways Analysis (last release; Qiagen) based on Gene Ontology database. The network protein analysis related to significantly altered proteins was carried out by Ingenuity Pathways Analysis (IPA) (last release; Qiagen) The statistical enrichment of involved pathways is performed by the right-tailed Fisher\u0026rsquo;s exact test, in correlation with QIAGEN Knowledge Base, assigning a p-value (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://digitalinsights.qiagen.com/products/features/analysis-match/\u003c/span\u003e\u003cspan address=\"https://digitalinsights.qiagen.com/products/features/analysis-match/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The overall activation/inhibition states of canonical pathways are predicted based on a z-score algorithm. This z-score is used to statistically compare the uploaded dataset with the pathway patterns [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Histochemical analysis\u003c/h2\u003e \u003cp\u003eThe evaluation of colon damage was performed at the macroscopic level in accordance with the criteria previously reported by Antonioli et al.[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The macroscopic criteria included the presence of adhesions between the colon and other intra-abdominal organs (0\u0026ndash;2); consistency of colonic faecal material (an indirect marker of diarrhoea; 0\u0026ndash;2); thickening of the colonic wall (mm); presence and extension of hyperaemia and macroscopic mucosal damage (0\u0026ndash;5). The colon length was measured after the explant of the tissue. For the histological analysis, the colon was fixed in 4% paraformaldehyde for 24 h, dehydrated in sucrose, frozen, and embedded in OCT for cryostat sectioning (5 \u0026micro;m sections), by using the \u0026ldquo;Swiss roll\u0026rdquo; technique to visualize all the colon length in each tissue section. Microscopic evaluations of colon damage (mucosal architecture loss, cellular infiltrate, muscle thickening, crypt abscess, and goblet cell depletion) were carried out on haematoxylin/eosin-stained sections. The infiltration of mast cells (MCs) and eosinophils was investigated on colon sections stained with GIEMSA (Sigma-Aldrich, Milan, Italy). Digitalized images were collected by a Leica DMRB light microscope equipped with a DFC480 digital camera (40\u0026times; magnification; Leica Microsystems, Wetzlar, Germany). The quantitative analysis was carried out by two blind investigators with the software ImageJ. For each animal, the cellular density (cell number/respective arbitrary field) of 3\u0026ndash;5 independent arbitrary optical fields (0.1 mm\u003csup\u003e2\u003c/sup\u003e) collected from the submucosa was measured. The analysis was performed on 6 animals \u003cem\u003eper group\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Immunofluorescence\u003c/h2\u003e \u003cp\u003eThe colon was cut into 30 \u0026micro;m slices (by using the \u0026ldquo;Swiss roll\u0026rdquo; technique)[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], which were collected in wells containing PBS 1x. Immunolabelling was performed in the same wells by free-floating technique according to standard protocols[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Selected slices from each animal, were incubated overnight at 4\u0026deg;C with the following combination of primary antibodies, diluted in T-PBS/5% BSA (Sigma-Aldrich, Milan, Italy): 1) rabbit anti-panaxonal marker PGP9.5 (ab108986, Abcam, UK; 1:500)\u0026thinsp;+\u0026thinsp;mouse major histocompatibility complex II (MHC II, ab23990, Abcam, UK; 1:100); 2) mouse anti-UCH-L1/PGP9.5 (Novus Biologicals-31A3, Bio-Techne Ltd., Abingdon, UK; 1:500)\u0026thinsp;+\u0026thinsp;rabbit anti-glial fibrillary acidic protein (GFAP, DAKO-Z0334, Agilent Technologies Italia, Milan, Italy; 1:500); 3) mouse anti-UCH-L1/PGP9.5 (Novus Biologicals-31A3, Bio-Techne Ltd., Abingdon, UK; 1:500)\u0026thinsp;+\u0026thinsp;rabbit anti-Iba-1 (Wako Chemicals, Richmond, VA, USA; 1:250). The day after, slices were incubated for 2 h with secondary antibodies (1:500) labelled with Alexa Fluor 488, 568 or 647 (Invitrogen-Thermo Fisher Scientific, Milan, Italy), and then with DAPI to stain the nuclei. The slices were finally transferred onto slides and mounted with Fluoromount-G\u0026trade; Mounting Medium (Thermo Fisher Scientific, Milan, Italy). Digitalized images were collected at 400\u0026times; (myenteric plexus and mucosa) and 200\u0026times; (colon wall) total magnification, based on the type of analysis performed, using a motorized Leica microscope DM6 B equipped with a DFC9000 GT camera, supported by a THUNDER Workstation 3D DCV and by the software LAS X (Leica Biosystems, Milan, Italy). The quantitative analysis of colon PGP9.5- MHC II and Iba-1-related immunofluorescence intensity (Disease and Therapy Protocols) was performed by collecting independent fields (200\u0026times;; 4\u0026ndash;6 for each animal) from the colon wall and by analysing selected ROI for mucosa, submucosa and muscular layers with the software FIJI (NIH, Bethesda, MD, USA). Quantitative analysis of IENF density (fibers/mm) was performed by collecting 4\u0026ndash;6 independent fields in the skin of each animal and counting the number of single PGP9.5-positive fibers crossing the mucosa-submucosa boundary and dividing them for the analysed mucosal length, measured at the base of the crypt by using FIJI. Secondary branching is excluded from this quantification. The quantitative analysis of colon PGP9.5- and GFAP-related immunofluorescence intensity (Disease and Therapy Protocols) was performed by collecting independent fields (400\u0026times;; 4\u0026ndash;6 for each animal) from the myenteric plexus and analysing them with FIJI. The number of PGP9.5- and GFAP-positive cells for each myenteric plexus was counted and normalized to the plexus area. The immunofluorescence relative to the expression of PGP9.5 and GFAP was quantified (arbitrary units), normalized to the area of the myenteric plexus. The value relative to the background was subtracted from the value obtained from the analysed area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the experimental procedures were performed by a researcher blind to the treatment. Behavioural and histological results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The analysis of variance (ANOVA) was performed by one-way ANOVA with Bonferroni's significant difference procedure used for post-hoc comparisons. \u003cem\u003eP\u003c/em\u003e values of less than 0.05 were considered significant. Data were analyzed using the \u0026ldquo;Origin 9\u0026rdquo; software (OriginLab, Northampton, MA). Statistical analysis on 16S rRNA amplicon sequencing data was performed in R using the Rstudio GUI. using (generalized) linear models and mixed models. The permutational multivariate ANOVA (PERMANOVA) test was applied to Aitchison distance matrices generated to assess whole-composition level differences between treatment groups and timepoints. R scripts are available online on github (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/thomazbastiaanssen/FMT_pain_analysis/\u003c/span\u003e\u003cspan address=\"https://github.com/thomazbastiaanssen/FMT_pain_analysis/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Spearman correlation coefficients between relative abundances of microbial taxa and levels of SCFAs were computed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). Statistical analysis of metabolomics data was carried out using MetaboAnalystR Package. Data was normalised by median, log10 transformed and scaled by Pareto scaling (mean-centered and divided by the square root of the standard deviation of each variable). Sparse Partial Least-Squares Discriminant Analysis (sPLS-DA) was employed to illustrate the clustering of different metabolites across groups. Univariate Analysis was carried out by Wilcoxon rank-sum tests. Dendrogram and heatmaps were created with Euclidean distances and Ward clustering methods. Heatmap shows the top metabolites based upon Wilcoxon rank-sum test results. P values of less than 0.05 were considered statistically significant. Correlation analysis between metabolomics data and microbiome data was conducted using both the anansi framework [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] with the KEGG database as a reference, and M2IA [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Missing values were filtered if present in more than 80% of samples or the relative standard deviation was smaller than 30% [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The remaining missing data values were handled using random forest. Data was transformed using total sum scaling. All other correlation analyses were conducted using Spearman\u0026rsquo;s rank-order correlation analysis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Correlation analysis of the microbiota and metabolome datasets was performed by DIABLO (Data Integration Analysis for Biomarker discovery using Latent cOmponents)[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] using the R package \u0026ldquo;mixOmics\u0026rdquo;, a multiomics integrative method based on a variant of the multivariate statistical technique generalised canonical correlation analysis\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Parallel changes of visceral pain threshold and microbiota composition caused by faecal microbiota transplantation (FMT).\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Modification of visceral sensitivity and microbiota composition caused FMT in healthy animals.\u003c/h2\u003e \u003cp\u003eWe adopted two opposite, complementary FMT-based approaches as per our previous work [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] with the aim to study: 1) the mechanisms governing pain caused by post-inflammatory dysbiosis, referred to as the Disease Protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA); and 2) the mechanisms underlying pain relief mediated by shifts in the gut microbiota composition-metabolism modification in DNBS-treated animals, referred to as the Therapy Protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Body weight, regarded as a disease index, was monitored throughout the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Visceral sensitivity was assessed by evaluating abdominal withdrawal reflex (AWR) to colorectal distension (CRD) after abx treatment and FMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for time course of visceral pain). Preventive antibiotic treatment in rats caused a transitory increase of visceral sensitivity (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, day 7). Starting on day 7, FMT was performed using faeces from control (FMT\u003csup\u003eCTR\u003c/sup\u003e) or animals manifesting with severe visceral pain in the post-inflammatory phase of colitis induced by the intrarectal injection of DNBS (FMT\u003csup\u003eDNBS\u003c/sup\u003e). Although no differences in the body weight were detected between the experimental groups, FMT\u003csup\u003eDNBS\u003c/sup\u003e induced a persistent increase in the AWR to CRD, while FMT\u003csup\u003eCTR\u003c/sup\u003e accelerated the restoration of a normal visceral sensitivity in abx-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, day 32), as previously observed [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The impact of FMT\u003csup\u003eDNBS\u003c/sup\u003e induced visceral sensitivity exclusively, as neither the antibiotic treatment nor FMT altered thermal and tactile somatic pain threshold of naive animals (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA and S2B, respectively). In terms of the microbiome (Fig.\u0026nbsp;1D\u003csub\u003ei\u0026thinsp;\u0026minus;\u0026thinsp;iii\u003c/sub\u003e), we did not observe statistically significant effects of time or treatment on alpha diversity nor in functional neuroactive potential. However, we did observe statistically significant effects of both experimental timepoint and treatment in several genera as well as overall ecosystem composition (Beta diversity), indicating that DNBS exposure substantially affects the microbiome, in a manner likely independent of host-microbial communication through co-metabolism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Modification of visceral sensitivity and microbiota composition caused FMT in post-inflammatory conditions.\u003c/h2\u003e \u003cp\u003eIn the Therapy Protocol we confirmed that post-inflammatory visceral pain in DNBS-treated animals can be relieved by means of FMT from healthy donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Seven days after the induction of the damage, the abdominal response of animals to CRD was significantly higher in both groups treated with DNBS (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF, day 7). The FMT\u003csup\u003eCTR\u003c/sup\u003e led to a progressive reduction of visceral hypersensitivity in DNBS-treated animals, DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e group reached the values of controls (vehicle\u0026thinsp;+\u0026thinsp;vehicle group) after set IV of FMT, and pain relief has been maintained 10 days after the discontinuation of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, day 42). Moreover, DNBS animals receiving FMT\u003csup\u003eCTR\u003c/sup\u003e showed a significant increase in the thermal and tactile somatic pain threshold with respect of those transplanted with FMT\u003csup\u003eDNBS\u003c/sup\u003e, displaying somatic hypersensitivity in addition to visceral hypersensitivity (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC and S2D, respectively). No differences were observed between DNBS animals receiving vehicle or FMT\u003csup\u003eDNBS\u003c/sup\u003e at any time during the trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), confirming previous evidence collected in our laboratory [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The body weight loss resulting from colitis and the body weight recovery in the remission phase were equivalent and followed the same trend in all the experimental groups treated with DNBS, irrespective from the FMT or the donor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In terms of the microbiome (Fig.\u0026nbsp;2D\u003csub\u003ei\u0026thinsp;\u0026minus;\u0026thinsp;iii\u003c/sub\u003e), we did observe a subtle positive association of treatment and alpha diversity indices, shannon entropy reaching the critical value for significance. Furthermore, we observed changes in the genetic potential of the microbiome to metabolize neuroactive compounds, most notably in tryptophan synthesis and degradation which change in opposite directions, suggesting a dramatic shift in host-microbial co-metabolism of tryptophan. We further observed differences in the composition of the microbiome (Beta diversity) that could be explained by experimental timepoint and treatment group, indicating that DNBS exposure impacts microbial composition as well as functional neuroactive host-microbial co-metabolism. Conversely, we did not observe statistically significant effects in terms of genus-level abundance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Metabolome profiling and correlation analysis identified common effects of FMT in healthy or post-inflammatory conditions.\u003c/h2\u003e \u003cp\u003eConsidering the microbial shifts mediated in both experimental models, we extended our line of investigation to encompass the gut metabolomic profile. Sparse Partial least squares-discriminant analysis (sPLS-DA) plots showed a clear separation of experimental groups indicating a metabolomic shift in response to pathological treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) or therapeutic interventions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) the extent to which can be seen through the heatmap provided which displays all significantly altered metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD); p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.2).\u003c/p\u003e \u003cp\u003eThe interplay across gut microbiota composition and metabolites was explored with DIABLO, a generalized and supervised version of PLS-DA. The DIABLO analysis identified a clear discrimination between samples of the experimental groups from the Disease (abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e, abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e, collected at day 32) and Therapy (DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e, DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e, collected at day 42) protocols (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, A-B), suggesting that the two omics (i.e. microbiota and metabolomics) can likely separate the two conditions within each group; a robust correlation between the microbial and the metabolomic profile in each study protocols was observed (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, C-D). The optimally selected key predictors included several microbial taxa, a few of which were concomitantly identified as contributors to discriminate the Disease (abx\u0026thinsp;+\u0026thinsp;FMT) and Therapy (DNBS\u0026thinsp;+\u0026thinsp;FMT) experimental groups, including \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eClostrodium sensu stricto\u003c/em\u003e 1, \u003cem\u003eAkkermansia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). Among the Disease protocol, few metabolites including malate, malonate, proprionate, arabinose were strongly associated with the abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNB\u003c/sup\u003e group, being overall positively correlated (mean r\u0026thinsp;=\u0026thinsp;0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) with \u003cem\u003eCorpobacillus\u003c/em\u003e, \u003cem\u003eParabacteroides\u003c/em\u003e, \u003cem\u003eErysipelatoclostridium\u003c/em\u003e and \u003cem\u003eGastranaerophilales\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C); no metabolic signatures associated with the abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e group were found (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Interestingly, several metabolites were associated with the DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group from the Therapy protocol; overall, the most relevant products included alpha-ketoisovaletate, nicotinate, 2-oxoglutarate, 2-oxoisocaproate, 3-methyl-2-oxovalerate and were positively correlated (mean r\u0026thinsp;=\u0026thinsp;0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) with \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e and \u003cem\u003eClostridium sensu strictu\u003c/em\u003e 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Conversely, few metabolic products were identified as contributors to the DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB); in that case, correlation analysis showed that cis-aconitate was negatively correlated with \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e and \u003cem\u003eRuminococcus\u003c/em\u003e (mean r=-0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), while ethanol wa negatively correlated with \u003cem\u003eRuminococcus\u003c/em\u003e (r=-0.76) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Disease activity in the colon after FMT-based Disease or Therapeutic protocol.\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e The animals receiving FMT from CTR or DNBS donors were sacrificed at the peak of the effect (7 or 10 days after the discontinuation of FMT, according to Disease or Therapy Protocol) to examine the relationship between pain threshold and colon damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). No difference was observed in the colon length, specific weight, macroscopic and microscopic damage in abx-treated animals receiving FMT\u003csup\u003eCTR\u003c/sup\u003e or FMT\u003csup\u003eDNBS\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. In particular, the ratio of colon weight to colon length (colon specific weight) demonstrated no difference in oedema. Also, the density of mast cell and eosinophils in the submucosa of abx-treated animals was unaffected by FMT from the different donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Immunofluorescence analysis performed on the colon specimens showed the apparent integrity of epithelium (E-CAD; Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB-C) and its regenerative capacity (Ki67; Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA-C) in the experimental groups. The same analysis highlighted a decrease immunoreactivity for MHC-II (marker for antigen presenting cells) in the submucosa of abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group compared with abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e group (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB). MHC-II immunoreactivity did not change in either mucosa or muscularis (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA and S4C), neither Iba-1 (marker for macrophages) immunoreactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). At the same time, a lowered muscular immunoreactivity for the pan-neuronal marker PGP 9.5 and the glial marker GFAP was observed in animals receiving FMT\u003csup\u003eDNBS\u003c/sup\u003e (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eG and S4N). PGP 9.5 immunoreactivity in the submucosa and myenteric plexus of abx-treated animals was unaffected by FMT (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eF, S4I and S4F). The tendency to increase in the mucosal PGP 9.5 immunoreactivity in abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group with respect of abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e group was independent from intraepithelial nerve fibres (IENFs) density (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eSimilarly, we found that the different pain-threshold resulting from FMT\u003csup\u003eCTR\u003c/sup\u003e or FMT\u003csup\u003eDNBS\u003c/sup\u003e in DNBS-treated animals was not explained by significant modifications of colon length, specific weight, macroscopic and microscopic damage to the colon. Although the colon muscle layer appeared thickened because of the inflammatory insult resulting from DNBS injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], the mucosa and submucosa were fully restored in both experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These outcomes are consistent with colon remission state 42 days after the injection of DNBS [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, a significant reduction in the number of mast cells infiltrating the submucosa was detected in DNBS-treated animals receiving FMT\u003csup\u003eCTR\u003c/sup\u003e with respect of those receiving FMT\u003csup\u003eDNBS\u003c/sup\u003e. The infiltration of eosinophils was instead unaffected by the different treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), which suggests specific immunomodulatory mechanisms underlying FMT effects on pain. Immunofluorescence analysis performed on the same colon specimens highlighted a decrease immunoreactivity for Iba1 in the submucosa of DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group compared with DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) which was concomitant with a reduced immunoreactivity for the pan-neuronal marker PGP 9.5 (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eF). No significant difference was detected for MHC-II (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e A-C) and for Iba-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-G) and PGP 9.5 in the other intestinal compartments analysed (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eD-E-G-I-L).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Pathways selectively modulated by FMT in abx-treated animals.\u003c/h2\u003e \u003cp\u003eIn parallel to the study of changes occurring within the lumen, we investigated the response of colon to FMT by proteomic analysis, looking for correlation with pain phenotype. First, we investigated the alterations occurring in the colon of na\u0026iuml;ve animals developing hypersensitivity after FMT\u003csup\u003eDNBS\u003c/sup\u003e, comparing them with those receiving FMT\u003csup\u003eCTR\u003c/sup\u003e, not showing sensitivity alterations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Then, we explored the mechanisms underlying the therapeutic effect of FMT by comparing DNBS animals subjected to FMT\u003csup\u003eCTR\u003c/sup\u003e, showing pain relief, with those receiving FMT\u003csup\u003eDNBS\u003c/sup\u003e, which was unable to modify pain threshold (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Colon samples were analysed by one shot label free proteomics. 3880 proteins were identified and quantified of which 94 were upregulated and 63 downregulated in abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e vs abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e (Disease Protocol) while 29 were upregulated and 18 downregulated in DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e vs DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e (Therapy Protocol), considering a fold change threshold of 1.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Network analyses performed on differentially regulated proteins showed several modulated pathways according to Disease Protocol (Table\u0026nbsp;1) or Therapy Protocol (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eFrom the network analysis of canonical pathways, it emerged that the proalgesic effect of FMT\u003csup\u003eDNBS\u003c/sup\u003e was related to a negative induction of the signalling pathway of Integrins, CLEAR (Zscore = -1,63), GNRH (Zscore = -1,00), Ephrin receptor (Zscore = -1,00), Protein kinase A (Zscore = -0,82), Neurovascular coupling (Zscore = -0,45) and EIF2 (Zscore = -0,38), with a concomitant positive induction of Sirtuin pathway (Zscore\u0026thinsp;=\u0026thinsp;0,45) (Table\u0026nbsp;1). The negatively induced integrin signalling (Zscore = -1.63; pvalue\u0026thinsp;=\u0026thinsp;7.8 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) in abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e vs abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e attested the modulation and the deactivation of important cellular functions, such as cell cycle, cellular growth, proliferation, and development. Actin gamma 1 (ACTG1, log2 ratio = -3.85), ADP ribosylation factor 4 (ARF4, log2 ratio\u0026thinsp;=\u0026thinsp;0.50), Actin related protein 23 complex subunit 5 (ARPC5, log2 ratio = -0.51), protein tyrosine kinase 2 (PTKS, log2 ratio = -0.60) and paxillin (PXN, log2 ratio\u0026thinsp;=\u0026thinsp;1.05) were involved in the negatively regulation of integrin signalling (Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). In addition, NADH: ubiquinone oxidoreductase subunit B9 (NDUFB9, log2 ratio = -0.50) and NADH: ubiquinone oxidoreductase core subunit S3 were down regulated (NDUFS3, log2 ratio = -0.70). These enzymes are involved in mitochondrial activities, such as electron transfer, ATP synthesis and integrin signalling. The Actin cytoskeleton signalling was also negatively induced (Zscore = -0.45; pvalue\u0026thinsp;=\u0026thinsp;4.9 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) attesting impaired cell development (Table\u0026nbsp;1). Looking at non-canonical pathways significantly modulated by FMT\u003csup\u003eDNBS\u003c/sup\u003e, we observed a negative induction of pathways involved in the organization of cytoplasm and cytoskeleton (Zscore = -1,18), microtubule dynamics (Zscore = -0,84), which was correlated with a positive induction of cellular protrusions formation (Zscore\u0026thinsp;=\u0026thinsp;0,12) (Table\u0026nbsp;1). The concomitant positive induction of pathways involved in the expression, translation, and metabolism of proteins (Zscore\u0026thinsp;=\u0026thinsp;1,45; Z score\u0026thinsp;=\u0026thinsp;1,17; Z-score\u0026thinsp;=\u0026thinsp;0,82) (Table\u0026nbsp;1) indicates that DNBS microbial transfer in abx-treated rats induced a disease phenotype characterized by variation of cell-cell and cell-ECM interactions, and increased trafficking of immune cells, a result also corroborated by significant changes in the expression of single proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). At the same time, FMT\u003csup\u003eDNBS\u003c/sup\u003e-related downregulation of epithelium neoplasm pathway (Zscore = -1,53) suggests structural alterations in the mucosal barrier, a hypothesis which matches with the positive induction of pathways involved in the transport of molecules and Diarrhea (Zscore\u0026thinsp;=\u0026thinsp;0,76) (Table\u0026nbsp;1). Noteworthy, FMT\u003csup\u003eDNBS\u003c/sup\u003e effects on visceral sensitivity also correlated with increased Neurite branching (Zscore\u0026thinsp;=\u0026thinsp;1,14) and Axonogenesis (Zscore\u0026thinsp;=\u0026thinsp;0,61), as regard of non-canonical pathways (Table\u0026nbsp;1). Beyond network analysis, several proteins involved in neuroplasticity and neuromodulation were found to be differentially expressed after FMT\u003csup\u003eDNBS\u003c/sup\u003e in the Disease Protocol. Among them we were able to identify structural and functional proteins participating to sensitization, synaptic plasticity, and glial physiology, such as GFAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). We also observed a significant reduction in AH receptor-interacting protein, Tryptophan-tRNA ligase (cytoplasmic) and Kynurenine-oxoglutarate transaminase 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), involved in tryptophan metabolism in the intestinal mucosa, which is known to contribute to IBS pathophysiology [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, several proteins significantly up- and down-regulated in abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group emerged to be involved in energy metabolism and redox balance (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Furthermore, proteins related to immune response and inflammatory process regulation were found differentially expressed after FMT\u003csup\u003eDNBS\u003c/sup\u003e or FMT\u003csup\u003eCTR\u003c/sup\u003e in abx-treated animals (Figure S8B). The combination of evidence might indicate the presence a low-grade colon inflammation related to a dysregulated mitochondrial dynamics and bioenergetics [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], not detectable with histological investigations. In fact, proteins regulating mitochondria function, as cytochrome c oxidase, mitochondrial fission protein 1, NDUFA4, NADH ubiquinone oxidoreductase, ATP synthase, and glutaredoxin are the most impacted by FMT\u003csup\u003eDNBS\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Of note, glutathione-glutaredoxin system is also a crucial thiol-dependent redox system involved in cellular redox balance regulation along with thioredoxin system [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The expression of proteins involved in iron metabolism, (ceruloplasmin, ferrochelatase and hephaestin) was also significantly affected by FMT\u003csup\u003eDNBS\u003c/sup\u003e treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Other relevant proteins upregulated in abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group are kininogen-1, Prothymosin alpha, Complement C1q, LPS-responsive beige-like anchor protein, Thymosin beta 4, Serpine1 mRNA binding protein 1, Glucocorticoid receptor, Leukocyte elastase inhibitor, trefoil factor 3, prostaglandins, immunoglobulins, annexins and proteins involve in mucus production (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB), attesting a different modulation of immune response mediated by FMT\u003csup\u003eDNBS\u003c/sup\u003e with respect of FMT\u003csup\u003eCTR\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Pathways selectively modulated by FMT in post-inflammatory colon.\u003c/h2\u003e \u003cp\u003eNetwork analysis comparing DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e vs DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group correlated the improvement of the pain symptomatology with negative induction of inflammatory response (Zscore = -2.41; pvalue\u0026thinsp;=\u0026thinsp;1.3 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, Table\u0026nbsp;2) involving modulated proteins, such as Apolipoprotein A1 (APOA1, log2 ratio\u0026thinsp;=\u0026thinsp;0.56), Alpha-1-antiproteinase (SERPINA1, log2 ratio\u0026thinsp;=\u0026thinsp;0.78), Chymotrypsin C (CTRC, log2 ratio\u0026thinsp;=\u0026thinsp;1.71) and Clusterin (CLU, log2 ratio\u0026thinsp;=\u0026thinsp;0,63) (Figure S9). Moreover, the activation of immune system was inhibited (Zscore = -2.21; pvalue\u0026thinsp;=\u0026thinsp;8.0 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, Table\u0026nbsp;2) showing a decreasing of leukocyte migration by modulation of Thymosin B10/4X (TMB10/4X, log2 ratio\u0026thinsp;=\u0026thinsp;0.71), Integrin subunit alpha 1 (ITGA1, log2 ratio = -0.40) and Tetraspanin (CD81, log2 ratio = -0.68). A further confirmation of the immunological matrix underlying the therapeutic effect of FMT\u003csup\u003eCTR\u003c/sup\u003e, was the significant downregulation of both subunit alpha and beta of beta-hexosaminidase (Figure S9), a protein involved in mast cell degranulation [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], as a result of FMT\u003csup\u003eCTR\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to the negative induction of these pathways, a differential expression of proteins involved in either fibrosis, energy metabolism, mucosal barrier, neuroendocrine signalling and neuroplasticity was associated with the pain-relieving effect of CTR microbial transfer in DNBS animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, Suppl. Table?). In particular, the analysis of single proteins highlighted a significant down-regulation of proteins potentially involved in fibrotic disorders (LTBP4 - latent transforming growth factor beta binding protein 4, EGF Containing Fibulin Extracellular Matrix Protein and Collagen type 1 alpha), as well as neuroplasticity (Liprin-beta-1, Trefoil factor 3, Calcium-binding mitochondrial carrier protein Aralar1, Synapse Associated Protein 1, Voltage-dependent calcium channel subunit alpha-2/delta-1, Calcineurin B homologous protein 1) in the DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e group. Besides, it has been observed a positive effect of FMT\u003csup\u003eCTR\u003c/sup\u003e on different partners involved in lipid transport, synthesis and metabolism (NDUFA4 Mitochondrial Complex Associated, acyl-CoA dehydrogenase family of enzymes (ACADs), Protein-glucosylgalactosylhydroxylysine glucosidase activity, Myristoylated alanine-rich C-kinase substrate, Beta-enolase, Fatty acid-binding protein 4, Fatty acid-binding protein 2, Steroid Delta-isomerase, Apolipoprotein E and Palmitoyl-protein thioesterase 1; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, Suppl. Table?). This finding confirms the presence of an impairment of lipid metabolism in DNBS animals, which can be corrected by the transfer of \u0026ldquo;healthy\u0026rdquo; microbiota. The expression of epithelial carbonic anhydrase 9, a hypoxia-induced catalytic component of pH regulating machinery, and Trefoil factor 3, mainly secreted by intestinal epithelium with potential regulatory effect of nervous and endocrine systems [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], was also reduced because of FMT\u003csup\u003eCTR\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), while a significant upregulation of secernin-2 (exocytosis) was detected. Finally, an increase in the expression of protein disulfide-isomerase, essential for in vivo production of the intestinal mucin [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], and secretogranin-1 was found in DNBS animals receiving FMT\u003csup\u003eCTR\u003c/sup\u003e with respect of those receiving FMT\u003csup\u003eDNBS\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). This last protein, also known as Chromogranin B, is a widespread constituent of neuroendocrine secretory granules [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Pathways modulated by FMT irrespective of the donor.\u003c/h2\u003e \u003cp\u003eBy comparing the Disease and Therapy Protocols, it emerged that certain proteins were similarly modulated by FMT\u003csup\u003eDNBS\u003c/sup\u003e in abx-treated animals and by FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Therefore, changes involving these proteins are apparently unrelated to either FMT donor or pain phenotype. The up- and down-regulation of these protein are likely triggered by sensing a different microbial environment, irrespectively of the healthy/disease status of either the donor or the recipient. Among the proteins upregulated, the largest difference was found in the expression of seminal vesicles secretory proteins 2, 4 and 6, whose expression was about 10-fold higher in abx and DNBS animals receiving the FMT from the opposite donor (DNBS and CTR donors, respectively), with respect of those receiving the microbiota related to their pre-FMT condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The presence of this class of proteins has not been described in the colon before. In the reproductive tract, Svs-proteins were reported as immunomodulatory substances that inhibit cell-mediated cytotoxicity as well as lymphocyte response to allogenic cells [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Structurally analogous proteins produced by epithelium might assume a similar role in the gut. Related upregulation, albeit to a lesser extent, was observed for chymotrypsin-C, alfa-1-antiproteinase, thymosin beta-4, apolipoprotein A1, thioredoxin, ZO-2 tight junction protein, lypd8 and protein S100-A6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e), which might have implication in the nonspecific response of gut microbiota perturbation. This gut instinct of self-preservation might also explain the upregulation of keratin type 2 and keratin type I 18\u0026ndash;20, the major type of keratins expressed also by the human colon crypt epithelial cells. keratins provide a structural and mechanical scaffold to support cellular stability, integrity, and stress protection in this rapidly regenerating tissue. They participate in colonocyte processes including barrier function, ion transport, differentiation, proliferation and inflammatory signalling [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. In the same experimental conditions, the downregulation of prostaglandin G/H synthase, monoamine oxidase B and immunoglobulin light chain variable region suggest again a fine regulation of gut immune response to FMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Noteworthy, immunoglobulin light chain variable region resulted negatively induced with a fold change of about 4 in the comparison DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e vs DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e, whereas the same protein was negatively induced with a fold change of about 1.5 in abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e vs abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The difference in the ratio between Disease and Therapy Protocols suggests a peculiar deregulation of Immunoglobulin light chain variable region expression in post-inflammatory conditions, with implication in the pain-relieving effect of FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS-treated rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe present work contributes to the elucidation of the molecular mechanisms linking microbiome disruption and persistent abdominal pain after colitis remission. We started from the dual evidence that post-inflammatory visceral hypersensitivity can be transferred from DNBS-treated donors (FMT\u003csup\u003eDNBS\u003c/sup\u003e) to na\u0026iuml;ve recipients, and \u003cem\u003evice versa\u003c/em\u003e an effective pain relief can be achieved by transplanting a healthy microbiota (FMT\u003csup\u003eCTR\u003c/sup\u003e) into DNBS-treated animals [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. After reproducing both behavioural patterns in the present study referred to as the \u0026ldquo;Disease Protocol\u0026rdquo; and the \u0026ldquo;Therapy Protocol\u0026rdquo;, the changes induced by FMT\u003csup\u003eDNBS\u003c/sup\u003e or FMT\u003csup\u003eCTR\u003c/sup\u003e on the colon of abx- and DNBS-treated animals were investigated.\u003c/p\u003e \u003cp\u003eThe 16S rRNA-amplicon sequencing performed on fecal samples revealed significant taxonomic difference in the microbiota composition between animals receiving FMT\u003csup\u003eDNBS\u003c/sup\u003e and those receiving FMT\u003csup\u003eCTR\u003c/sup\u003e, in both the experimental arms. Although we were unable to identify specific microbial communities associated with pain phenotype, we found that this latter was correlated with a specific signature in the fecal metabolome. Indeed, when comparing the two arms of the study, we found an opposite trend in the relative abundance of 3-hydrobutyrrate, purines (adenosine, xanthosine, AMP and hypoxanthine), glutamate and lactate, between the pain and the pain-free condition. Yet, we observed changes in the genetic potential of the microbiome to metabolize neuroactive compounds, most notably in tryptophan synthesis and degradation, which changed in opposite directions after FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS animals. Although tryptophan levels within the lumen were not significantly modified by the treatments, we observed a different expression of factors contributing to tryptophan metabolism and signalling at mucosal level (AH receptor-interacting protein, Tryptophan-tRNA ligase and Kynurenine-oxoglutarate transaminase 3) after FMT\u003csup\u003eDNBS\u003c/sup\u003e in abx-treated animals. Noteworthy, tryptophan and its metabolites are known to contribute to IBS pathophysiology [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResults from the DIABLO analysis identified correlating (or co-expressed) variables between the two omics datasets generated from samples from the Disease and the Therapy protocols, and showed a strong contribution of five metabolites and five microbial species to the discrimination between the abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e and abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e groups, as well as a contribution of eight metabolites and four bacterial genera to the discrimination between the DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e, DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e groups.\u003c/p\u003e \u003cp\u003eSince we primarily observed taxonomic differences in the first (Disease) arm of this study, while observing more functional differences in the second (Therapy) arm, it could be the case that the pain related to FMT\u003csup\u003eDNBS\u003c/sup\u003e is not transferred through the microbiome \u003cem\u003eper se\u003c/em\u003e, but that DNBS exposure does result in a perturbed microbiome that exacerbates, throughout the colon response, the negative effects on visceral sensitivity. Conversely, FMT from a non-DNBS-exposed microbiome does present a distinct functional profile. It is thus possible that the ameliorative effect of the FMT can be seen in part as a strengthening of certain microbial functions important for gut homeostasis. This evidence suggests two distinct but probably related mechanisms through which DNBS-exposed microbiome can convey a chronic negative effect on the gut and how a fresh microbiome can help restore a physiological condition. Another possibility is that microbial colonization after FMT is driven by metabolic independence. According to this theory, FMT might serve as an environmental filter that favours populations with higher independence in the synthesis of critical metabolites, including amino acids, nucleotides, and vitamins. In other words, increased microbial abundance in disease does not necessitate causal involvement of the microbe in disease, it could be one of few taxa able to grow in the diseased gut environment [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], which might provide an explanation for 16S rRNA-sequencing and metabolomics data on fecal samples.\u003c/p\u003e \u003cp\u003eA further correlation analysis between fecal microbiota and metabolome confirmed that, under pain conditions, the abundance of purines within the lumen was directly correlated with bacterial nucleosidase. Under healthy conditions, purine levels were instead independent of bacterial genomic structure (Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). This is interesting as the microbiota contributes to maintain integrity of the mucosal barrier by supporting epithelial purine metabolism [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], whose alteration has been recently associated with pain in IBS patients [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn parallel to the analysis of the changes induced by FMT on the lumen content, we investigated the response of the colon to the transplant using proteomic approach, which revealed two different scenarios according to the different experimental paradigm considered. Indeed, pain induction mediated by FMT\u003csup\u003eDNBS\u003c/sup\u003e in na\u0026iuml;ve animals was related to the alteration of several signalling pathways, including integrins, GnRH, and sirtuin, and to phenomenon of cellular plasticity involving the epithelium and the nervous system. Besides, the pain-relieving effect of FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS animals was related to a negative induction of inflammatory response and leukocytes activity, with a significant decrease in the number of submucosal mast cells.\u003c/p\u003e \u003cp\u003eRegarding the Disease protocol, network analysis on proteome highlighted a negative induction of epithelial neoplasm in abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e compared to abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eCTR\u003c/sup\u003e group, encountering 170 differentially expressed proteins. Although neoplasm is beyond the scope of the current work, this finding suggests a close relationship between epithelium dysfunction and visceral hypersensitivity. In agreement with this hypothesis, pain in abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group was associated with decreased levels of fecal adenosine, whose production support epithelium barrier function [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Besides, pain in DNBS\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group (Therapy protocol) was associated with a significant increase of fecal hypoxanthine, which has an altered reactivity in an anaerobic environment, such as the lumen, and has been reported to cause an oxidative damage on the epithelium [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Moreover, alterations in the metabolism might prevent epithelial cells from a proper use of microbiota-derived rescue energy sources, such as fatty acids and purines, as suggested in other pathological contexts [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. This observation might explain the increase of these metabolites in the feces of animals showing pain phenotype. If the theory that epithelial alterations caused by dysbiosis have a downstream impact on the activation of sensory nerve endings is confirmed, nociception might be configured as a wake-up call for the brain of an altered homeostasis in the microbiota-host interaction.\u003c/p\u003e \u003cp\u003eAnother pathway affected by FMT\u003csup\u003eDNBS\u003c/sup\u003e in abx-treated animals (Disease protocol) involved integrins, adhesion molecules which modulate a variety of epithelial functions. Noteworthy, integrin signalling impinges on pathways downstream of other receptors, creating elaborate intracellular signalling networks [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Actin gamma 1 (G-actin) is the driving protein in the negative induction of the integrins pathway mediated by FMT\u003csup\u003eDNBS\u003c/sup\u003e, which is concomitant to negative induction of Actin cytoskeleton Protein kinase A signalling. It has been recently observed that microbial metabolites induce actin cytoskeletal rearrangement and protect the blood-brain barrier function [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. A similar mechanism might be involved in the maintenance of the intestinal epithelial barrier [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Moreover, integrins, actin cytoskeleton and protein kinase A signalling are all involved in processes requiring localized cell protrusion, such as cell migration and axonal path finding [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Integrins are also expressed on primary afferent neurons, where they can interact with signalling systems involved in hyperalgesia. Indeed, plasticity in cytoskeleton-anchored signal transduction elements within sensory neuron terminals has been observed to mediate altered pain plasticity in sensory neurons primed by injury [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Beyond directly affecting the functionality of both epithelium and neurons innervating the colon, the detrimental signalling associated with post-inflammatory dysbiosis and conveyed by FMT\u003csup\u003eDNBS\u003c/sup\u003e might also influence the activity of enteric glia cells inhabiting the mucosa. These cells have been reported to promote intestinal mucosal healing via activation of focal adhesion kinase and release of pro-EGF [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Intriguingly, glial dysfunction caused by the systemic injection of LPS was associated with actin cytoskeleton reorganization, along with Ca\u003csup\u003e2+\u003c/sup\u003e signalling alterations, Na\u003csup\u003e+\u003c/sup\u003e and glutamate transporters downregulation, and pro-inflammatory cytokines release [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e], which can influence the response of neighbouring neurons. A similar phenomenon might occur in the enteric glia exposed to bacteria products post-colitis, contributing to enteric neuroplasticity and visceral pain chronicity. This hypothesis is reinforced by the positive induction of pathways related to neurite branching and axonogenesis together with the altered expression of peculiar proteins (i.e., GFAP, ProSAAS, volt-gated Ca2\u0026thinsp;+\u0026thinsp;channel alpha-2/delta-1, tubulin beta-3 chain, and neuroplastin) in the colon of animals receiving FMT\u003csup\u003eDNBS\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to proteomics, histological analysis confirmed a slight increase of PGP 9.5-related mucosal immunoreactivity, which suggests an altered homeostasis in nerve endings pruning and tuning. The remodelling of sensory neurons, crucial for the maintenance of receptive endings shape and functionality, is controlled by glia- and macrophages-mediated phagocytosis [\u003cspan additionalcitationids=\"CR85 CR86\" citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Under post-inflammatory conditions, alteration in the activity of microbiota, which physiologically contribute to the colonization and homeostasis of glial [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e] and immune cells [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e], might interfere with these mechanisms. In line with this hypothesis, we observed a reduction in the number of submucosal MHC-II positive cells, concomitantly with reduced glia-related GFAP expression in the colon of abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group.\u003c/p\u003e \u003cp\u003eFurthermore, it is interesting to note that neurodegenerative disorders involving both neuronal and non-neuronal cells, such as microglia (tissue-resident macrophages) and astrocytes (physiologically similar to enteric glial cells\u003cb\u003e)\u003c/b\u003e in the CNS, are commonly associated with lysosomal dysfunction [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. In the abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group we observed a negative induction of CLEAR (Coordinated Lysosomal Expression and Regulation) network, which regulates lysosome-associated processes, including phagocytosis and immune response [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. Dysfunction of lysosomes, the key cellular hub for macromolecule catabolism, recycling and signalling, generates a cascade of events that impair cellular trafficking, cell signalling, autophagic flux, mitochondria functionality and calcium homeostasis [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. It is noteworthy that most of these processes were found to be affected by FMT\u003csup\u003eDNBS\u003c/sup\u003e along with a predicted negative induction of CLEAR signalling pathway. Interestingly, the most common lysosomal storage disease, namely Fabry disease, is characterized by neuropathic pain and functional gastrointestinal disorders [\u003cspan additionalcitationids=\"CR93\" citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. Mice affected by Fabry disease manifest both visceral hypersensitivity and gut dysbiosis, combined with alterations in faecal SCFAs [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e], which parallels our findings and suggest a link between lysosome dysfunction and pain persistence.\u003c/p\u003e \u003cp\u003eLysosome damage has a negative impact on mitochondrial function, which is also affected by the treatment with FMT\u003csup\u003eDNBS\u003c/sup\u003e (see the expression of cytochrome c oxidase, mitochondrial fission protein 1, NDUFA4, glutaredoxin-1 and ATP synthase). Combined alterations in lysosome and mitochondria functioning driven by post-inflammatory dysbiosis might have a great impact especially on highly metabolically epithelial cells and neurons [\u003cspan additionalcitationids=\"CR97\" citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. In the same cells, lysosome dysfunction might also affect the signaling of integrins, which is finely regulated by a circular process of internalization, recycling, and degradation [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e], and which we observed to be involved in the pathogenic effects of FMT\u003csup\u003eDNBS\u003c/sup\u003e, including neuroplasticity. However, metabolism and function of epithelial cells under dysbiosis condition might be also influenced by the positive induction of SIRT1 [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e], which promotes epithelial-mesenchymal transition [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e, \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e]. Altogether these mechanisms, and not just a single one, likely contribute to gut homeostasis disruption caused by FMT\u003csup\u003eDNBS\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn search of further mechanisms for FMT\u003csup\u003eDNBS\u003c/sup\u003e-induced pain, we focused on the negative induction of gonadotropin-releasing hormone (GnRH) pathway, since the therapeutic efficacy of GnRH analogues, used to treat pre-menopausal pain in women [\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e], has been argued for several years in IBS patients, who express serum antibodies against GnRH [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. The connection between the presence of antibodies and IBS disease is still unclear, but it has been demonstrated that GnRH and its receptor are expressed in both enteric neurons and intestinal epithelium [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. Our data from the colon sums up to previous evidence reporting GnRH to be associated to the largest proportion of differentially expressed genes in the hippocampus of viscerally-hypersensitive germ-free mice [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e], reinforcing the link between microbiota, GnRH signalling and pain regulation, both peripherally and centrally.\u003c/p\u003e \u003cp\u003ePain induced by FMT\u003csup\u003eDNBS\u003c/sup\u003e was also accompanied by the upregulation of colon kininogen, fibrinogen, stool trefoil factor 3 (TFF3). The first can interact with kallikrein to produce bradykinin, a peptide implicated in inflammatory pain [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e], or can alternatively release antimicrobial and antifungal peptides for immune functions [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e], which suggests a circularity between pain and dysbiosis maintenance within the gut. Intriguingly, kininogen-1 and fibrinogen plasma levels have emerged to be directly correlated with pain intensity in women with chronic widespread pain [\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e] and higher TFF3 levels were instead detected in patients with IBS [\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]. Interestingly, pain relief mediated by FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS-treated animals correlated with a significant reduction of TFF3 levels, reinforcing the link between this protein and pain. Finally, the upregulation of serpine1, prothymosin alpha, thymosin β(4) and other partners of immune response in the colon abx\u0026thinsp;+\u0026thinsp;FMT\u003csup\u003eDNBS\u003c/sup\u003e group, without overt changes emerging at histological analysis, confirm the presence of a low-grade inflammation sustained by the immune response to microbes, which likely contribute to dysbiosis-related pain persistence [\u003cspan additionalcitationids=\"CR112\" citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e]. In addition, the increased expression of APOA1 common to the different pathways modulated by FMT\u003csup\u003eCTR\u003c/sup\u003e, might link the effects of microbiota manipulation on lipid metabolism to those on immune response [\u003cspan additionalcitationids=\"CR115\" citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e], both associated with pain relief. Although specific bacterial products are known to trigger immune activation [\u003cspan additionalcitationids=\"CR118\" citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e], evidence supporting a cause-and-effect relationship between dysbiosis, altered immune response and post-inflammatory pain has been based on indirect associations [\u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e, \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e]. Indeed, no study has ever before explored the effect of manipulating the microbiota on the immune response which, moreover, is compromised due to chronic disease [\u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e122\u003c/span\u003e]. In our Therapy protocol, we demonstrated that the pain-relieving effect of FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS animals was supported by the inhibition of inflammatory response, leucocytes migration e mast cell activation. By analyzing colonic proteins, we found that this immunosuppressive effect could be related to an increase in seminal vesicle secretory proteins [\u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e], which are involved in suppressing the immune response in the female reproductive tract but have an unknown role in the intestine. Besides, the dampening of mast cells activation following FMT\u003csup\u003eCTR\u003c/sup\u003e treatment, confirmed by beta-hexasominidase levels [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], could be linked to the concomitant reduction in the production and release of derived immunoglobulin free light chains by lymphocytes. Indeed these proteins can trigger mast cell-mediated hypersensitivity [\u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e124\u003c/span\u003e], which play a key role in the pathophysiology of abdominal pain in IBS [\u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e, \u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e, \u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e]. Restoring mast cells homeostasis might account for the pain-relieving effects of FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS animals, tough the escalation of immune events linking microbiota perturbation to hypersensitivity needs to be further elucidated. In this regard, it is interesting to note that the anti-hyperalgesic effect of FMT\u003csup\u003eCTR\u003c/sup\u003e was associated with a facilitated infiltration of macrophages in the submucosa of DNBS-treated animals, which are in remission from colitis. In addition to having an active role in damage resolution and in the maintenance of tolerogenic environment [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e], macrophages can transfer mitochondria to the sensory neurons of the DRG to resolve inflammatory pain [\u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs discussed above, the presence of an intestinal \u0026ldquo;metabolic injury\u0026rdquo;, involving mitochondria, might represent the link between the microbiota and the different partners apparently contributing to the disease. High levels of microbial-derived energy source such as SCFAs, particularly 3-hydroxybutyrate, directly correlated with pain persistence in DNBS-treated animals. Since these metabolites physiologically contribute to strengthen intestinal barrier and to prevent harmful inflammatory responses [\u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e], it is possible that the capacity of the gut to properly absorb and use them might be impaired under post-inflammatory condition, causing localized (epithelium) or diffuse (immune cells and neurons) alterations in the metabolism, therefore contributing to visceral hypersensitivity. In support to this, a recent multi-omics analysis of gut microbiome in IBS patients highlighted peculiar transcriptional modifications of enzymes involved in the metabolism of carbohydrate and lipids [\u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e]. The same authors explain the increased levels of free fatty acids, particularly in IBS-C, by altered epithelial lipid metabolism [\u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e]. Although a direct association with pain was not drawn, epithelium dysfunction might be potentially implicated in shaping microbial environments [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], as well as sensory neurons activity [\u003cspan citationid=\"CR130\" class=\"CitationRef\"\u003e130\u003c/span\u003e, \u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e131\u003c/span\u003e], creating again a vicious circle between dysbiosis and pain. Epithelium involvement in the therapeutic effect of FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS-treated animals seems to be confirmed by the increased expression of secretogranin-1, a neuroendocrine secretory granule protein, which can be the precursor for other biologically active peptides [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe also observed that the effect of FMT on the expression of certain proteins (i.e. immunoglobulin free light chains and seminal vesicles secretory proteins, chymotrypsin-C) is unrelated to the donor, as same changes occur because of the transplant of either FMT\u003csup\u003eDNBS\u003c/sup\u003e in abx-treated animals or FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS-treated animals. This phenomenon, which deserve further investigation, implicates that the mechanisms by which FMT exerts its effects are strictly dependent on the healthy or disease state of the recipient. This could call into question the need to identify a super-donor to improve therapies and shift the spotlight to the need to better understand the pathophysiology of patients.\u003c/p\u003e \u003cp\u003eFinally, though it is important to continue clarifying the peripheral mechanisms before defining central mechanisms involved in pain chronicity and FMT effects, behavioral results suggest that visceral and somatic pain relief in DNBS animals receiving FMT\u003csup\u003eCTR\u003c/sup\u003e might involve the modulation of common pain pathways at central level.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e "},{"header":"Conclusion","content":"\u003cp\u003eOverall, our results confirm that metabolic injury driven by post-inflammatory dysbiosis (FMT\u003csup\u003eDNBS\u003c/sup\u003e) contributes to maintain a low-grade inflammation, epithelium dysfunctions, and enteric neuroplasticity phenomena, initiated by the inflammatory insult and underpinning visceral pain persistence after colitis remission. On the other hand, the therapeutic effect FMT\u003csup\u003eCTR\u003c/sup\u003e on post-inflammatory pain might be related to the suppression of immune response which is likely deregulated in the colon of DNBS animals. In this context, the inability to reprogram the immune system and to revert neuroplastic changes caused by inflammation might explain the reversibility of FMT effect on pain, suggesting a combined intervention on colon as a strategy to improve therapy efficacy. Indeed, though modification of microbiome represents the \u003cem\u003eprimum movens\u003c/em\u003e, the FMT effects are likely sustained by other cells within the gut. Since not necessarily all the pathways modulated by FMT are involved in pain regulation, further validation is needed to define the cellular partners and the order of events linking dysbiosis and pain. This comprehensive study is expected to enhance the understanding and treatment of gut dysmotility, a condition commonly linked to pain.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All experiments were performed using rodents in accordance with the International Association for the Study of Pain, the European Union directives, and the National Institutes of Health guidelines on laboratory animal welfare and approved by the Animal Care committee at the University of Florence (Italy) and the Italian Ministry of Health (No. 388/2021-PR). The data sets generated and analysed during the current study are included as additional files.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the Italian Ministry of Instruction, University and Research, by the University of Florence, by the \u0026ldquo;linea 2\u0026rdquo; of University of Milan, by the University of East Anglia and by the University College Cork.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE.L., A.D.A., V.D.P., D.V., S.O.M., C.N. and L.D.C.M. conceptualized and designed the experiments and analytical approaches. V.D.P., F.M., L.M. and T.F.S.B. processed, analysed and interpreted 16S rRNA gene sequence data. D.V.. and G.L.G. processed, analysed and interpreted metabolomics data. A.D.A. and E.L, processed, analysed and interpreted proteomics data. E.L., L.M. and A.T. designed behaviour experiments and histological analysis. E.L., L.M., C.C. and A.T. carried out experiments and analysed data. E.L., V.D.P., T.F.S.B., D.V., A.D.A., wrote the paper with contributions from all authors. C.N., S.O.M., G.M.R., L.D.C.M. and C.G. revised the manuscript. L.D.C.M. and C.G. supervised the study and provided the experimental resources. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge UNITECH OMICs, mass spectrometry platform of Universit\u0026agrave; degli Studi di Milano, for running mass spectrometric analyses for the proteomic study. We are grateful to Universit\u0026agrave; degli Studi di Milano which partially funded the research through Piano di Sostegno alla Ricerca, linea 2, (2022 and 2023).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files. The 16S rRNA sequence data have been deposited in the NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/bioproject/) under the BioProject accession number PRJNA1213328.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDrewes AM, Olesen AE, Farmer AD, Szigethy E, Rebours V, Olesen SS: Gastrointestinal pain. Nat Rev Dis Primers 2020, 6:1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpiller R, Major G: IBS and IBD - separate entities or on a spectrum? 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Nature 2023, 616:137\u0026ndash;142.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"FMT, IBD, visceral pain, dysbiosis, immune response, neuroplasticity, metabolic injury","lastPublishedDoi":"10.21203/rs.3.rs-6025304/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6025304/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGut dysbiosis is a common feature of patients complaining of chronic abdominal pain, including those in remission from inflammatory bowel diseases (IBDs). Although visceral sensitivity in animals can be modulated by faecal microbial transplant (FMT), controversy has emerged in clinical practice, drawing attention to the poor knowledge of the mechanisms underpinning host-microbiota crosstalk under pathological conditions. Here we sought to elucidate the mechanisms linking post-inflammatory dysbiosis and pain persistence, to improve the therapeutic strategies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults.\u003c/strong\u003e Colitis was induced in rats by intrarectal injection of 2,4-dinitrobenzenesulfonic acid (DNBS). Naïve rats subjected to FMT from viscerally hypersensitive DNBS-treated rats (FMT\u003csup\u003eDNBS\u003c/sup\u003e) displayed a higher sensitivity than those receiving FMT from healthy donors (FMT\u003csup\u003eCTR\u003c/sup\u003e). A reverse protocol highlighted the anti-hyperalgesic effect of FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS-treated rats. Difference in microbiota-to-gut signalling between dysbiotic or eubiotic conditions was investigated after the treatment with FMT\u003csup\u003eCTR\u003c/sup\u003e or FMT\u003csup\u003eDNBS\u003c/sup\u003e, in both the experimental protocols. Modification of pain threshold in the animals undergoing FMT correlated with changes in the composition of the gut microbiota and the metabolic profile, evaluated by 16S rRNA and proton Nuclear Magnetic Resonance (\u003csup\u003e1\u003c/sup\u003eH NMR). Although a specific microbial community associated with the pain phenotype was not identified, significant differences between painful and painless conditions were detected in the faecal metabolome, involving fatty acids, purine metabolites, glutamate and lactate. Yet, proteomic analysis of colon tissues from FMT recipients revealed differential scenarios. Indeed, the proalgesic effect of FMT\u003csup\u003eDNBS\u003c/sup\u003e in healthy animals was accompanied by a detrimental modulation of pathways regulating cellular plasticity and clearance, cell-to-cell interaction and signaling, as well as epithelium growth and neuroplasticity phenomenon, besides metabolism and inflammation. Pain relief following FMT\u003csup\u003eCTR\u003c/sup\u003e in DNBS-treated rats was instead associated with a counteraction of inflammatory process, lymphocyte activation and submucosal mast cells infiltration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese results indicate the presence of signalling modulation within the gut rather than tissue alterations at the base of FMT-related effects on pain, providing novel insights into the mechanisms linking intestinal microbiota to visceral sensitivity which might be exploited to enhance the effectiveness of microbiota-targeted interventions in the treatment of chronic pain in IBD patients.\u003c/p\u003e","manuscriptTitle":"Microbiome and gut as partners in the persistence of post-inflammatory visceral pain: insight into working mechanisms of faecal transplant for therapy advance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 14:25:49","doi":"10.21203/rs.3.rs-6025304/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43f5017e-604d-449e-b567-02f96ad2da42","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-12T12:24:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-20 14:25:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6025304","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6025304","identity":"rs-6025304","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}