Up to 20% of the global population develops gastrointestinal symptoms following a meal 1 , leading to decreased quality of life, significant morbidity and high medical costs. Although the interest of both the scientific and lay community has increased dramatically with the worldwide introduction of gluten-free and other diets, the underlying mechanisms leading to food-induced abdominal complaints remain largely unknown. Here we show that a bacterial infection and bacterial toxins can trigger an immune response leading to the production of dietary antigen-specific IgE antibodies in mice, a mechanism confined to the intestine. Subsequent oral ingestion of the respective dietary antigen results in increased visceral pain via an IgE-and mast cell-dependent mechanism. This aberrant pain signaling results from histamine receptor H1 (H1R)-mediated sensitization of visceral afferents. Moreover, in patients with irritable bowel syndrome (IBS), we show that injection of food antigens (gluten, wheat, soy and milk) into the rectosigmoid induces local edema and mast cell activation. Hence, we have unveiled and characterized a novel peripheral mechanism underlying food-induced abdominal pain, which creates new opportunities for the treatment of IBS and related abdominal pain disorders. MAIN TEXT:The mucosal immune system provides a balanced response to pathogens and harmless commensal bacteria or food antigens, thereby limiting unnecessary inflammation and concomitant tissue damage 2 . This is achieved by an active suppression of cellular and humoral responses to orally administered antigens, a mechanism referred to as oral tolerance 3 . Viral and bacterial infections can, however, interfere with tolerance to dietary antigens, thereby perturbing intestinal homeostasis 4 . An infectious gastroenteritis is a significant risk factor to develop IBS, defined as a constellation of abdominal pain and altered bowel patterns. Between 3 and 36% of enteric infections lead to new onset IBS 5 , while up to 17% of IBS patients report that their symptoms started Supplementary information included as a separate pdf file and videos (Supplementary Information Video 1-4). EXTENDED DATA LEGENDS: Extended Data Fig. 1. Extended analysis of the OVA-specific immune response and VHS in postinfectious mice. a, b, diarrhea development quantification by (a) water content in feces and (b) whole-gut transit time upon gavage of carmine red dye in OVA/sham + OVA, OVA/infected + OVA (n = 10/group) mice. c, quantification of OVA-specific IgE in intestinal homogenates of OVA/sham + OVA, saline/infected + OVA,

ObjectivesProteases are key mediators of pain and altered enteric neuronal signalling, although the types and sources of these important intestinal mediators are unknown. We hypothesised that intestinal epithelium is a major source of trypsin-like activity in patients with IBS and this activity signals to primary afferent and enteric nerves and induces visceral hypersensitivity.DesignTrypsin-like activity was determined in tissues from patients with IBS and in supernatants of Caco-2 cells stimulated or not. These supernatants were also applied to cultures of primary afferents. mRNA isoforms of trypsin (PRSS1, 2 and 3) were detected by reverse transcription-PCR, and trypsin-3 protein expression was studied by western blot analysis and immunohistochemistry. Electrophysiological recordings and Ca2+ imaging in response to trypsin-3 were performed in mouse primary afferent and in human submucosal neurons, respectively. Visceromotor response to colorectal distension was recorded in mice administered intracolonically with trypsin-3.ResultsWe showed that stimulated intestinal epithelial cells released trypsin-like activity specifically from the basolateral side. This activity was able to activate sensory neurons. In colons of patients with IBS, increased trypsin-like activity was associated with the epithelium. We identified that trypsin-3 was the only form of trypsin upregulated in stimulated intestinal epithelial cells and in tissues from patients with IBS. Trypsin-3 was able to signal to human submucosal enteric neurons and mouse sensory neurons, and to induce visceral hypersensitivity in vivo, all by a protease-activated receptor-2-dependent mechanism.ConclusionsIn IBS, the intestinal epithelium produces and releases the active protease trypsin-3, which is able to signal to enteric neurons and to induce visceral hypersensitivity.

While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD). Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and thrombin were overactive in supernatants from IBD patient tissues compared to healthy controls. Gene expression analysis highlighted the transcription of genes encoding these proteases into intestinal mucosae. The functional ABP-targeted proteomic approach that we have used to identify active proteases in human colonic samples bears directly on the understanding of the role these enzymes may play in the pathophysiology of IBD.

Background and purpose: Recent findings suggest that the noxious gas H2S is produced endogenously, and that physiological concentrations of H2S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H2S to modulate carrageenan-induced synovitis in the rat knee. Experimental approach: Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H2S formation (DL-propargylglycine) or an H2S donor [Lawesson's reagent (LR)]. Key results: Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters. Conclusions and implications:Whereas exogenous H2S delivered to the knee joint can produce a significant antiinflammatory and anti-nociceptive effect, locally produced H2S exerts little immunomodulatory effect. These data further support the development and use of H2S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.

Temporomandibular disorders represent one of the major challenges in dentistry therapeutics. This study was undertaken to evaluate the time course of carrageenan-induced inflammation in the rat temporomandibular joint (TMJ) and to investigate the role of tachykinin NK(1) receptors. Inflammation was induced by a single intra-articular (i.art.) injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) and TNFalpha and IL-1beta concentrations were measured in the TMJ lavages at selected time-points. The carrageenan-induced responses were also evaluated after treatment with the NK(1) receptor antagonist SR140333. The i.art. injection of carrageenan into the TMJ caused a time-dependent plasma extravasation associated with mechanical allodynia, and a marked neutrophil accumulation between 4 and 24h. Treatment with SR140333 substantially inhibited the increase in plasma extravasation and leukocyte influx at 4 and 24h, as well as the production of TNFalpha and IL-1beta into the joint cavity, but failed to affect changes in head-withdrawal threshold. The results obtained from the present TMJ-arthritis model provide, for the first time, information regarding the time course of this experimental inflammatory process. In addition, our data show that peripheral NK(1) receptors mediate the production of both TNFalpha and IL-1beta in the TMJ as well as some of the inflammatory signs, such as plasma extravasation and leukocyte influx, but not the nociceptive component.

Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is characterized by chronic abdominal pain concurrent with altered bowel habit. Polyunsaturated fatty acid (PUFA) metabolites are increased in abundance in IBS and are implicated in the alteration of sensation to mechanical stimuli, which is defined as visceral hypersensitivity. We sought to quantify PUFA metabolites in IBS patients and evaluate their role in pain. Quantification of PUFA metabolites by mass spectrometry in colonic biopsies showed an increased abundance of 5-oxo-eicosatetraenoic acid (5-oxoETE) only in biopsies taken from patients with IBS with predominant constipation (IBS-C). Local administration of 5-oxoETE to mice induced somatic and visceral hypersensitivity to mechanical stimuli without causing tissue inflammation. We found that 5-oxoETE directly acted on both human and mouse sensory neurons as shown by lumbar splanchnic nerve recordings and Ca2+-imaging of dorsal root ganglia (DRG) neurons. We showed that 5-oxoETE selectively stimulated nonpeptidergic, isolectin B4 (IB4)-positive DRG neurons through a phospholipase C (PLC)– and pertussis toxin–dependent mechanism, suggesting that the effect was mediated by a G protein–coupled receptor (GPCR). The MAS-related GPCR D (Mrgprd) was found in mouse colonic DRG afferents and was identified as being implicated in the noxious effects of 5-oxoETE. Together, these data suggest that 5-oxoETE, a potential biomarker of IBS-C, induces somatic and visceral hyperalgesia without inflammation in an Mrgprd-dependent manner. Thus, 5-oxoETE may play a pivotal role in the abdominal pain associated with IBS-C.

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