Pasteurized akkermansia muciniphila improves irritable bowel syndrome-like symptoms and related behavioral disorders in mice.

Gut - brain communications disorders in irritable bowel syndrome (IBS) are associated with intestinal microbiota composition, increased gut permeability, and psychosocial disturbances. Symptoms of IBS are difficult to medicate, and hence much research is being made into alternative approaches. This study assesses the potential of a treatment with pasteurized Akkermansia muciniphila for alleviating IBS-like symptoms in two mouse models of IBS with different etiologies. Two clinically relevant animal models were used to mimic IBS-like symptoms in C57BL6/J mice: the neonatal maternal separation (NMS) paradigm and the Citrobacter rodentium infection model. In both models, gut permeability, colonic sensitivity, fecal microbiota composition and colonic IL-22 expression were evaluated. The cognitive performance and emotional state of the animals were also assessed by several tests in the C. rodentium infection model. The neuromodulation ability of pasteurized A. muciniphila was assessed on primary neuronal cells from mice dorsal root ganglia using a ratiometric calcium imaging approach. The administration of pasteurized A. muciniphila significantly reduced colonic hypersensitivity in both IBS mouse models, accompanied by a reinforcement of the intestinal barrier function. Beneficial effects of pasteurized A. muciniphila treatment have also been observed on anxiety-like behavior and memory defects in the C. rodentium infection model. Finally, a neuroinhibitory effect exerted by pasteurized A. muciniphila was observed on neuronal cells stimulated with two algogenic substances such as capsaicin and inflammatory soup. Our findings demonstrate novel anti-hyperalgesic and neuroinhibitory properties of pasteurized A. muciniphila, which therefore may have beneficial effects in relieving pain and anxiety in subjects with IBS.

Antihyperalgesic properties of gut microbiota: Parabacteroides distasonis as a new probiotic strategy to alleviate chronic abdominal pain.

ABSTRACT: The potential role of gut microbiota in pain modulation is arousing an emerging interest since recent years. This study investigated neuromodulatory properties of gut microbiota to identify next-generation probiotics to propose alternative therapies for visceral pain management. Neuromodulation ability of 10 bacterial strains isolated from a healthy donor was assessed both on ND7/23 immortalized cell line and primary neuronal cells from rat dorsal root ganglia. This screening highlighted the neuroinhibitory property of Parabacteroides distasonis (F1-2) strain, supported both by its intracellular content and membrane fraction, which was further investigated in visceral pain mouse models. Oral administration of F1-2 resulted in a significant decrease of colonic hypersensitivity (CHS) in dextran sulfate sodium (0.5%) model associated with low-grade inflammation and a significant decrease of CHS in Citrobacter rodentium postinfectious models. No effect of F1-2 oral administration on CHS was observed in a neonatal maternal separation stress model. Antihyperalgesic effect unlikely involved modulation of inflammatory processes or restoration of intestinal barrier. Exploration of direct dialogue mechanisms between this strain and nervous system, assessed by calcium imaging experiments, revealed that F1-2 interacts directly with nociceptors by reducing activation level on capsaicin, inflammatory soup, and bradykinin stimulations. Our study provides new insights about bacteria-host interaction and places P distasonis as a potential therapeutic strategy in the treatment of visceral pain observed in leaky gut-associated pathologies.

Beneficial Effects of Lactobacilli Species on Intestinal Homeostasis in Low-Grade Inflammation and Stress Rodent Models and Their Implication in the Modulation of the Adhesive Junctional Complex.

Intestinal barrier integrity is essential in order to maintain the homeostasis of mucosal functions and efficient defensive reactions against chemical and microbial challenges. An impairment of the intestinal barrier has been observed in several chronic diseases. The gut microbiota and its impact on intestinal homeostasis is well described and numerous studies suggest the ability of some probiotic strains to protect the intestinal epithelial integrity and host homeostasis. In this work, we aimed to assess the beneficial effects of three Lactobacillus strains (Lacticaseibacillus rhamnosus LR04, Lacticaseibacillus casei LC03, and Lactiplantibacillus plantarum CNCM I-4459) and their mechanism of action in low-grade inflammation or neonatal maternal separation models in mice. We compared the impact of these strains to that of the well-known probiotic Lacticaseibacillus rhamnosus GG. Our results demonstrated that the three strains have the potential to restore the barrier functions by (i) increasing mucus production, (ii) restoring normal permeability, and (iii) modulating colonic hypersensitivity. Moreover, gene expression analysis of junctional proteins revealed the implication of Claudin 2 and Cingulin in the mechanisms that underlie the interactions between the strains and the host. Taken together, our data suggest that LR04, CNCM I-4459, and LC03 restore the functions of an impaired intestinal barrier.

From In Vitro to In Vivo: A Rational Flowchart for the Selection and Characterization of Candidate Probiotic Strains in Intestinal Disorders.

Experimental and clinical evidence has demonstrated the potential of probiotic strains in the prevention or treatment of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, there is little data on what the methodology leading to the identification of such strains should be. In this work, we propose a new flowchart to identify strains with probiotic potential for the management of IBS and IBD, which we tested on a collection of 39 lactic acid bacteria and Bifidobacteria strains. This flowchart included in vitro tests of immunomodulatory properties on intestinal and peripheral blood mononuclear cells (PBMCs), assessment of the barrier-strengthening effect by measuring transepithelial electric resistance (TEER) and quantification of short-chain fatty acids (SCFAs) and aryl hydrocarbon receptor (AhR) agonists produced by the strains. The in vitro results were then combined in a principal component analysis (PCA) to identify strains associated with an anti-inflammatory profile. To validate our flowchart, we tested the two most promising strains identified in the PCA in mouse models of post-infectious IBS or chemically induced colitis to mimic IBD. Our results show that this screening strategy allows the identification of strains with potential beneficial effects on colonic inflammation and colonic hypersensitivity.

Role of T CD4+ cells, macrophages, C-low threshold mechanoreceptors and spinal Cav 3.2 channels in inflammation and related pain-like symptoms in murine inflammatory models.

BACKGROUND AND PURPOSE: T-type calcium channels, mainly the Cav 3.2 subtype, are important contributors to the nociceptive signalling pathway. We investigated their involvement in inflammation and related pain-like symptoms. EXPERIMENTAL APPROACH: The involvement of Cav 3.2 and T-type channels was investigated using genetic and pharmacological inhibition to assess mechanical allodynia/hyperalgesia and oedema development in two murine inflammatory pain models. The location of Cav 3.2 channels involved in pain-like symptoms was studied in mice with Cav 3.2 knocked out in C-low threshold mechanoreceptors (C-LTMR) and the use of ABT-639, a peripherally restricted T-type channel inhibitor. The anti-oedema effect of Cav 3.2 channel inhibition was investigated in chimeric mice with immune cells deleted for Cav 3.2. Lymphocytes and macrophages from either green fluorescent protein-targeted Cav 3.2 or KO mice were used to determine the expression of Cav 3.2 protein and the functional status of the cells. KEY RESULTS: Cav 3.2 channels contributed to the development of pain-like symptoms and oedema in the two murine inflammatory pain models. Our results provided evidence of the involvement of Cav 3.2 channels located on C-LTMRs and spinal cord in inflammatory pain. Cav 3.2 channels located in T cells and macrophages contribute to the inflammatory process. CONCLUSION AND IMPLICATIONS: Cav 3.2 channels play crucial roles in inflammation and related pain, implying that targeting of Cav 3.2 channels with pharmacological agents could be an attractive and readily evaluable strategy in clinical trials, to relieve chronic inflammatory pain in patients.

Serpin-positive Bifidobacterium breve CNCM I-5644 improves intestinal permeability in two models of irritable bowel syndrome.

Probiotic supplementation can help to mitigate the pathogenesis of irritable bowel syndrome (IBS) by reinforcing the intestinal barrier, and reducing both inflammation and proteolytic activity. Here, a combination of in vitro tests was performed on 33 Bifidobacterium strains as probiotic candidates for IBS. In addition to the classical tests performed, the detection of the serine protease inhibitor (serpin) enzyme capable of decreasing the high proteolytic activity found in IBS patients was included. Three serpin-positive strains were selected: Bifidobacterium breve CNCM I-5644, Bifidobacterium longum subsp. infantis CNCM I-5645 and B. longum CNCM I-5646 for their immunomodulation properties and protection of intestinal epithelial integrity in vitro. Furthermore, we found that B. breve CNCM I-5644 strain prevented intestinal hyperpermeability by upregulating Cingulin and Tight Junction Protein 1 mRNA levels and reducing pro-inflammatory markers. The ability of CNCM I-5644 strain to restore intestinal hyperpermeability (FITC-dextran) was shown in the murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS). This effect of this strain was corroborated in a second model of IBS, the neonatal maternal separation model in mice. Altogether, these data suggest that serpin-positive B. breve CNCM I-5644 may partially prevent disorders associated with increased barrier permeability such as IBS.

Involvement of toll-like receptor 5 in mouse model of colonic hypersensitivity induced by neonatal maternal separation.

BACKGROUND: Chronic abdominal pain is the most common cause for gastroenterology consultation and is frequently associated with functional gastrointestinal disorders including irritable bowel syndrome and inflammatory bowel disease. These disorders present similar brain/gut/microbiota trialogue alterations, associated with abnormal intestinal permeability, intestinal dysbiosis and colonic hypersensitivity (CHS). Intestinal dysbiosis can alter colon homeostasis leading to abnormal activation of the innate immunity that promotes CHS, perhaps involving the toll-like receptors (TLRs), which play a central role in innate immunity. AIM: To understand the mechanisms between early life event paradigm on intestinal permeability, fecal microbiota composition and CHS development in mice with TLRs expression in colonocytes. METHODS: Maternal separation model (NMS) CHS model, which mimics deleterious events in childhood that can induce a wide range of chronic disorders during adulthood were used. Colonic sensitivity of NMS mice was evaluated by colorectal distension (CRD) coupled with intracolonic pressure variation (IPV) measurement. Fecal microbiota composition was analyzed by 16S rRNA sequencing from weaning to CRD periods. TLR mRNA expression was evaluated in colonocytes. Additionally, the effect of acute intrarectal instillation of the TLR5 agonist flagellin (FliC) on CHS in adult naive wildtype mice was analyzed. RESULTS: Around 50% of NMS mice exhibited increased intestinal permeability and CHS associated with intestinal dysbiosis, characterized by a significant decrease of species richness, an alteration of the core fecal microbiota and a specific increased relative abundance of flagellated bacteria. Only TLR5 mRNA expression was increased in colonocytes of NMS mice with CHS. Acute intrarectal instillation of FliC induced transient increase of IPV, reflecting transient CHS appearance. CONCLUSION: Altogether, these data suggest a pathophysiological continuum between intestinal dysbiosis and CHS, with a role for TLR5.

AhR/IL-22 pathway as new target for the treatment of post-infectious irritable bowel syndrome symptoms.

Alterations in brain/gut/microbiota axis are linked to Irritable Bowel Syndrome (IBS) physiopathology. Upon gastrointestinal infection, chronic abdominal pain and anxio-depressive comorbidities may persist despite pathogen clearance leading to Post-Infectious IBS (PI-IBS). This study assesses the influence of tryptophan metabolism, and particularly the microbiota-induced AhR expression, on intestinal homeostasis disturbance following gastroenteritis resolution, and evaluates the efficacy of IL-22 cytokine vectorization on PI-IBS symptoms. The Citrobacter rodentium infection model in C57BL6/J mice was used to mimic Enterobacteria gastroenteritis. Intestinal homeostasis was evaluated as low-grade inflammation, permeability, mucosa-associated microbiota composition, and colonic sensitivity. Cognitive performances and emotional state of animals were assessed using several tests. Tryptophan metabolism was analyzed by targeted metabolomics. AhR activity was evaluated using a luciferase reporter assay method. One Lactococcus lactis strain carrying an eukaryotic expression plasmid for murine IL-22 (L. lactisIL-22) was used to induce IL-22 production in mouse colonic mucosa. C. rodentium-infected mice exhibited persistent colonic hypersensitivity and cognitive impairments and anxiety-like behaviors after pathogen clearance. These post-infectious disorders were associated with low-grade inflammation, increased intestinal permeability, decrease of Lactobacillaceae abundance associated with the colonic layer, and increase of short-chain fatty acids (SCFAs). During post-infection period, the indole pathway and AhR activity were decreased due to a reduction of tryptophol production. Treatment with L. lactisIL-22 restored gut permeability and normalized colonic sensitivity, restored cognitive performances and decreased anxiety-like behaviors. Data from the video-tracking system suggested an upgrade of welfare for mice receiving the L.lactisIL-22 strain. Our findings revealed that AhR/IL-22 signaling pathway is altered in a preclinical PI-IBS model. IL-22 delivering alleviate PI-IBS symptoms as colonic hypersensitivity, cognitive impairments, and anxiety-like behaviors by acting on intestinal mucosa integrity. Thus, therapeutic strategies targeting this pathway could be developed to treat IBS patients suffering from chronic abdominal pain and associated well-being disorders.

Fecal dysbiosis associated with colonic hypersensitivity and behavioral alterations in chronically Blastocystis-infected rats.

BACKGROUND: Infectious gastroenteritis is a risk factor for the development of post-infectious Irritable Bowel Syndrome (PI-IBS). Recent clinical studies reported a higher prevalence of the intestinal parasite Blastocystis in IBS patients. Using a rat model, we investigated the possible association between Blastocystis infection, colonic hypersensitivity (CHS), behavioral disturbances and gut microbiota changes. METHODS: Rats were orally infected with Blastocystis subtype 4 (ST4) cysts, isolated from human stool samples. Colonic sensitivity was assessed by colorectal distension and animal behavior with an automatic behavior recognition system (PhenoTyper), the Elevated Plus Maze test and the Forced Swimming tests. Feces were collected at different time points after infection to study microbiota composition by 16 S rRNA amplicon sequencing and for short-chain fatty acid (SFCA) analysis. RESULTS: Blastocystis-infected animals had non-inflammatory CHS with increased serine protease activity. Infection was also associated with anxiety- and depressive-like behaviors. Analysis of fecal microbiota composition showed an increase in bacterial richness associated with altered microbiota composition. These changes included an increase in the relative abundance of Oscillospira and a decrease in Clostridium, which seem to be associated with lower levels of SCFAs in the feces from infected rats. CONCLUSIONS: Our findings suggest that experimental infection of rats with Blastocystis mimics IBS symptoms with the establishment of CHS related to microbiota and metabolic shifts.

c-Jun/p38MAPK/ASIC3 pathways specifically activated by nerve growth factor through TrkA are crucial for mechanical allodynia development.

Mechanical allodynia is a cardinal sign of several inflammatory pain disorders where nerve growth factor, a prototypic neurotrophin, plays a crucial role by binding to TrkA receptors. Here, we took the advantage of our generated knock-in mouse model expressing a chimeric TrkA/TrkC receptor that seems to not specifically develop mechanical allodynia after inflammation, to identify the TrkA downstream pathways involved in this phenomenon. We confirmed and extended that disrupting TrkA-specific pathways leads to a specific deficit in mechanical hypersensitivity development after somatic (systemic nerve growth factor administration and paw incision) and, to a lesser extent, visceral injuries. Despite a deficit in thin, mainly peptidergic, fibre innervation in TrkAC mice, thermal hyperalgesia development was not different from WT mice. Inflammatory reaction (oedema, IL-6 content), pain behaviours after intraplantar capsaicin, as well as TRPV1 calcium imaging response of dorsal root ganglion neurons were similar between TrkAC and WT mice. This deficiency in mechanical allodynia development in TrkAC mice is likely due to the alteration of the expression of different TrkA transduction pathways (ie, Akt, p38 MAPK, and c-Jun) especially p38 MAPK, in the dorsal root ganglion cell bodies, ultimately leading to an alteration of at least, ASIC3 channel overexpression, known to participate in nociceptor mechanosensory function.

Microbiota: a novel regulator of pain.

Among the various regulators of the nervous system, the gut microbiota has been recently described to have the potential to modulate neuronal cells activation. While bacteria-derived products can induce aversive responses and influence pain perception, recent work suggests that "abnormal" microbiota is associated with neurological diseases such as Alzheimer's, Parkinson's disease or autism spectrum disorder (ASD). Here we review how the gut microbiota modulates afferent sensory neurons function and pain, highlighting the role of the microbiota/gut/brain axis in the control of behaviors and neurological diseases. We outline the changes in gut microbiota, known as dysbiosis, and their influence on painful gastrointestinal disorders. Furthermore, both direct host/microbiota interaction that implicates activation of "pain-sensing" neurons by metabolites, or indirect communication via immune activation is discussed. Finally, treatment options targeting the gut microbiota, including pre- or probiotics, will be proposed. Further studies on microbiota/nervous system interaction should lead to the identification of novel microbial ligands and host receptor-targeted drugs, which could ultimately improve chronic pain management and well-being.

Blocking α2δ-1 Subunit Reduces Bladder Hypersensitivity and Inflammation in a Cystitis Mouse Model by Decreasing NF-kB Pathway Activation.

Bladder pain is frequently associated with bladder inflammation, as in conditions like interstitial cystitis (IC), for which current analgesic therapies have limited efficacy. The antinociceptive effect of alpha-2-delta (α2δ) ligands on inflammation-associated visceral pain like that experienced in cystitis has been poorly investigated. To investigate the effect of pregabalin (PGB), an α2δ ligand, we evaluated its impact on mechanical hyperalgesia in a mouse model of cystitis induced by cyclophosphamide (CYP). We further studied its effect on inflammation and NF-kB pathway activation. Acute cystitis was induced by intraperitoneal injection of 150 mg kg-1 of CYP in C57Bl/6J male mice. PGB was subcutaneously injected (30 mg kg-1) 3 h after CYP injection. The effect of PGB on CYP-induced mechanical referred hyperalgesia (abdominal Von Frey test), inflammation (organ weight, cytokine production, α2δ subunit level, NF-kB pathway activation) were assessed 1 h after its injection. In parallel, its effect on cytokine production, α2δ subunit level and NF-kB pathway activation was assessed in vitro on peritoneal exudate cells (PECs) stimulated with LPS. PGB treatment decreased mechanical referred hyperalgesia. Interestingly, it had an anti-inflammatory effect in the cystitis model by reducing pro-inflammatory cytokine production. PGB also inhibited NF-kB pathway activation in the cystitis model and in macrophages stimulated with LPS, in which it blocked the increase in intracellular calcium. This study shows the efficacy of PGB in hypersensitivity and inflammation associated with cystitis. It is therefore of great interest in assessing the benefit of α2δ ligands in patients suffering from cystitis.

Inhibition of Cav 3.2 calcium channels: A new target for colonic hypersensitivity associated with low-grade inflammation.

BACKGROUND AND PURPOSE: Abdominal pain associated with low-grade inflammation is frequently encountered in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) during remission. Current treatments are not very effective and new therapeutic approaches are needed. The role of CaV 3.2 channels, which are important in other chronic pain contexts, was investigated in a murine model of colonic hypersensitivity (CHS) associated with low-grade inflammation. EXPERIMENTAL APPROACH: Low doses of dextran sulfate sodium (DSS; 0.5%) were chronically administered to C57BL/6j mice in drinking water. Their inflammatory state was assessed by systemic and local measures of IL-6, myeloperoxidase, and lipocalin-2 using elisa. Colonic sensitivity was evaluated by the visceromotor responses to colorectal distension. Functional involvement of CaV 3.2 channels was assessed with different pharmacological (TTA-A2, ABT-639, and ethosuximide) and genetic tools. KEY RESULTS: DSS induced low-grade inflammation associated with CHS in mice. Genetic or pharmacological inhibition of CaV 3.2 channels reduced CHS. Cav3.2 channel deletion in primary nociceptive neurons in dorsal root ganglia (CaV 3.2Nav1.8 KO mice) suppressed CHS. Spinal, but not systemic, administration of ABT-639, a peripherally acting T-type channel blocker, reduced CHS. ABT-639 given intrathecally to CaV 3.2Nav1.8 KO mice had no effect, demonstrating involvement of CaV 3.2 channels located presynaptically in afferent fibre terminals. Finally, ethosuximide, which is a T-type channel blocker used clinically, reduced CHS. CONCLUSIONS AND IMPLICATIONS: These results suggest that ethosuximide represents a promising drug reposition strategy and that inhibition of CaV 3.2 channels is an attractive therapeutic approach for relieving CHS in IBS or IBD.

Efficient and reproducible experimental infections of rats with Blastocystis spp.

Although Blastocystis spp. infect probably more than 1 billion people worldwide, their clinical significance is still controversial and their pathophysiology remains poorly understood. In this study, we describe a protocol for an efficient and reproducible model of chronic infection in rats, laying the groundwork for future work to evaluate the pathogenic potential of this parasite. In our experimental conditions, we were unable to infect rats using vacuolar forms of an axenically cultivated ST4 isolate, but we successfully established chronic infections of 4 week-old rats after oral administration of both ST3 and ST4 purified cysts isolated from human stool samples. The infection protocol was also applied to 4 week-old C57BL/9, BALB/C and C3H mice, but any mouse was found to be infected by Blastocystis. Minimal cyst inoculum required for rat infection was higher with ST3 (105) than with ST4 (102). These results were confirmed by co-housing experiments highlighting a higher contagious potential of ST4 in rats compared to ST3. Finally, experiments mimicking fecal microbiota transfer from infected to healthy animals showed that Blastocystis spp. could easily infect a new host, even though its intestinal microbiota is not disturbed. In conclusion, our results provide a well-documented and robust rat model of Blastocystis chronic infection, reproducing "natural" infection. This model will be of great interest to study host parasite interactions and to better evaluate clinical significance of Blastocystis.

Adherent-Invasive E. coli enhances colonic hypersensitivity and P2X receptors expression during post-infectious period.

Irritable Bowel Syndrome (IBS) and Inflammatory Bowel Disease (IBD) are related gastrointestinal disorders characterized by abdominal pain associated with colonic hypersensitivity (CHS). Studies in humans have reported an abnormal colonization of Adherent-Invasive E. coli (AIEC) in the ileum of Crohn's disease (CD) patients associated with overexpression of the bacterial colonizing receptor CEACAM6. The aim of the present study was to investigate whether AIEC reference strain LF82 could induce intestinal impairment during infectious and/or post-infectious periods and subsequently the development of CHS. Transgenic mice overexpressing human CEACAM6 protein (TG) and their wild-type littermates were gavaged by CD-associated AIEC bacteria (reference strain LF82) or PBS for 3 d. Colonic hypersensitivity was assessed by colorectal distension (CRD) test during infectious (D4) and post-infectious periods (D21). Several markers of intestinal inflammation were monitored and the colonic expression of purinergic P2X receptors was quantified. At D4, an increased visceromotor response (VMR) to the CRD test was observed in TG mice infected with CD-associated AIEC LF82 in comparison with non-infected TG mice and persisted in a subgroup of infected animals at D21 after bacteria clearance. Increased VMR was associated with low-grade intestinal inflammation, increased intestinal permeability and expression of P2X 3, 4 and 7. This study shows that certain susceptible hosts infected with CD-associated AIEC bacteria can develop persistent CHS associated with low-grade inflammation and increased P2X receptors expression. Thus, CD-associated AIEC infection in CEACAM6 transgenic mice could be used as a novel post-infectious mouse model mimicking quiescent IBD with IBS-like symptoms such as visceral pain.

Comparative effects of α2δ-1 ligands in mouse models of colonic hypersensitivity.

AIM: To investigate anti-hypersensitive effects of α2δ-1 ligands in non-inflammatory and inflammation-associated colonic hypersensitivity (CHS) mouse models. METHODS: To induce an inflammation-associated CHS, 1% dextran sulfate sodium (DSS) was administered to C57Bl/6J male mice, in drinking water, for 14 d. Regarding the non-inflammatory neonatal maternal separation (NMS) -induced CHS model, wild-type C57BI/6J pups were isolated from their mother from day 2 to day 14 (P2 to P14), three hours per day (from 9:00 a.m. to 12:00 p.m.). Colorectal distension was performed by inflating distension probe from 20 µL to 100 µL by 20 µL increment step every 10 s. After a first colorectal distension (CRD), drugs were administered subcutaneously, in a cumulative manner, (Gabapentin at 30 mg/kg and 100 mg/kg; Pregabalin at 10 mg/kg and 30 mg/kg; Carbamazepine at 10 mg/kg and 30 mg/kg) and a second CRD was performed one hour after each injection. RESULTS: The visceromotor response (VMR) to CRD was increased by our NMS paradigm protocol in comparison to non-handled (NH) mice, considering the highest distension volumes (80 µL: 0.783 ± 0.056 mV/s vs 0.531 ± 0.034 mV/s, P < 0.05 and 100 µL: 1.087 ± 0.056 mV/s vs 0.634 ± 0.038 mV/s, P < 0.05 for NMS and NH mice, respectively). In the inflammation-associated CHS, DSS-treated mice showed a dramatic and significant increase in VMR at 60 and 80 µL distension volumes when compared to control mice (60 µL: 0.920 ± 0.079 mV/s vs 0.426 ± 0.100 mV/s P < 0.05 and 80 µL: 1.193 ± 0.097 mV/s vs 0.681 ± 0.094 mV/s P < 0.05 for DSS- and Water-treated mice, respectively). Carbamazepine failed to significantly reduce CHS in both models. Gabapentin significantly reduced CHS in the DSS-induced model for both subcutaneous injections at 30 or 100 mg/kg. Pregabalin significantly reduced VMR to CRD in the non-inflammatory NMS-induced CHS model for the acute subcutaneous administration of the highest cumulative dose (30 mg/kg) and significantly reduced CHS in low-dose DSS-treated mice in a dose-dependent manner. Finally, the percent decrease of AUC induced by acute GBP or Pregabalin treatment were higher in the inflammatory DSS-induced CHS model in comparison to the non-inflammatory NMS-induced CHS model. CONCLUSION: This preclinical study demonstrates α2δ-1 ligands efficacy on inflammation-associated CHS, highlighting their potential clinical interest in patients with chronic abdominal pain and moderate intestinal inflammation.

Proneness of TLR5 deficient mice to develop colitis is microbiota dependent.

Alterations in the gut microbiota have been implicated to play a role in potentiating inflammatory bowel diseases in both humans and mice. Mice lacking the flagellin receptor, toll-like receptor 5 (TLR5), are prone to develop spontaneous gut inflammation, but are significantly protected when treated with antibiotics or maintained in germ-free conditions. However, given that the incidence of spontaneous inflammation in TLR5KO mice is quite variable in conventional conditions (typically ∼10% show clear colitis), this result is far from definitive and does not rule out that TLR5KO mice might be prone to develop inflammation even in the absence of a microbiota. Herein, we demonstrate that neutralization of IL10 signaling induces colitis in 100% of TLR5KO mice which provide a more rigorous approach to evaluate the role of microbiota in gut inflammation. Mice treated with antibiotics or maintained in germ-free condition are substantially protected against IL-10R neutralization-induced colitis, underscoring that gut inflammation in TLR5KO mice is dependent upon the presence of a gut microbiota.

TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination.

Systems biological analysis of immunity to the trivalent inactivated influenza vaccine (TIV) in humans revealed a correlation between early expression of TLR5 and the magnitude of the antibody response. Vaccination of Trl5(-/-) mice resulted in reduced antibody titers and lower frequencies of plasma cells, demonstrating a role for TLR5 in immunity to TIV. This was due to a failure to sense host microbiota. Thus, antibody responses in germ-free or antibiotic-treated mice were impaired, but restored by oral reconstitution with a flagellated, but not aflagellated, strain of E. coli. TLR5-mediated sensing of flagellin promoted plasma cell differentiation directly and by stimulating lymph node macrophages to produce plasma cell growth factors. Finally, TLR5-mediated sensing of the microbiota also impacted antibody responses to the inactivated polio vaccine, but not to adjuvanted vaccines or the live-attenuated yellow fever vaccine. These results reveal an unappreciated role for gut microbiota in promoting immunity to vaccination.