Thrombin modifies growth, proliferation and apoptosis of human colon organoids: a protease-activated receptor 1- and protease-activated receptor 4-dependent mechanism

International audienceExperimental Approach: Crypts were isolated from human colonic resections and cultured for 6 days, forming human colon organoids. Cultured organoids were exposed to 10 and 50 mU·mL−1 of thrombin, in the presence or not of protease‐activated receptor (PAR) antagonists. Organoid morphology, metabolism, proliferation and apoptosis were followed.Key Results: Thrombin favoured organoid maturation leading to a decreased number of immature cystic structures and a concomitant increased number of larger structures releasing cell debris and apoptotic cells. The size of budding structures, metabolic activity and proliferation were significantly reduced in organoid cultures exposed to thrombin, while apoptosis was dramatically increased. Both PAR1 and PAR4 antagonists inhibited apoptosis regardless of thrombin doses. Thrombin‐induced inhibition of proliferation and metabolic activity were reversed by PAR4 antagonist for thrombin's lowest dose and by PAR1 antagonist for thrombin's highest dose.Conclusions and Implications: Overall, our data suggest that the presence of thrombin in the vicinity of human colon epithelial cells favours their maturation at the expense of their regenerative capacities. Our data point to thrombin and its two receptors PAR1 and PAR4 as potential molecular targets for epithelial repair therapies

5-oxoETE triggers nociception in constipation-predominant irritable bowel syndrome through MAS-related G protein-coupled receptor D.

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 patients with IBS and evaluate their role in pain. Quantification of PUFA metabolites by mass spectrometry in colonic biopsies showed an increased abundance of 5-oxoeicosatetraenoic 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 ganglion (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.BBSRC BB/R006210/1 to James R F Hockley and Ewan St John Smith Rosetrees 834 Postdoctoral Grant (A1296) awarded to James R F Hockley and Ewan St John Smit

A Roadmap for the Human Gut Cell Atlas

The number of studies investigating the human gastrointestinal tract using various single-cell profiling methods has increased substantially in the past few years. Although this increase provides a unique opportunity for the generation of the first comprehensive Human Gut Cell Atlas (HGCA), there remains a range of major challenges ahead. Above all, the ultimate success will largely depend on a structured and coordinated approach that aligns global efforts undertaken by a large number of research groups. In this Roadmap, we discuss a comprehensive forward-thinking direction for the generation of the HGCA on behalf of the Gut Biological Network of the Human Cell Atlas. Based on the consensus opinion of experts from across the globe, we outline the main requirements for the first complete HGCA by summarizing existing data sets and highlighting anatomical regions and/or tissues with limited coverage. We provide recommendations for future studies and discuss key methodologies and the importance of integrating the healthy gut atlas with related diseases and gut organoids. Importantly, we critically overview the computational tools available and provide recommendations to overcome key challenges

Activation of proteinase-activated receptor 2 activation, a signal to joint innate immune responses.

Nossa hipótese é de que os efeitos pró-inflamatórios iniciais da ativação do receptor ativado por protease 2 (PAR2) na articulação temporomandibular (ATM) sejam mediados por mecanismos neurogênicos. A análise por imunofluorescência revelou um alto grau de imunorreatividade ao PAR2 em aferentes primários trigeminais da ATM. Além do mais, a imunorreatividade ao PAR2 também foi observada na camada íntima da sinóvia, além de co-localizar com o marcador neuronal PGP9.5 e o neuropeptídeo substância P. A injeção intra-articular de agonistas PAR2 na ATM induziu um aumento dependente da dose no extravasamento plasmático, influxo de neutrófilos e indução de alodinia mecânica. O bloqueio farmacológico de receptors NK1 inibiu o aumento no extravasamento plasmático, influxo de neutrófilos e alodinia induzido pela ativação do PAR2. Em conclusão, a ativação do PAR2 é pró-inflamatório na ATM, via mecanismos neurogênicos envolvendo receptores NK1, sugerindo que o PAR2 é um importante componente da resposta imunológica inata na ATM.We hypothesised that the early pro-inflammatory effects of proteinase-activated receptor 2 (PAR2) activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR2 in retrogradely labelled trigeminal ganglion neurons. Furthermore, PAR2 immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance P-containing peripheral sensory nerve fibres. The intra-articular injection of PAR2 agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx and induction of mechanical allodynia. The pharmacological blockade of NK1 receptors abolished PAR2-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR2 activation is pro-inflammatory in the TMJ, through a neurogenic mechanism involving NK1 receptors. This suggests that PAR2 is an important component of innate neuro-immune response in the TMJ

The Interplay Between Genetic Risk Factors and Proteolytic Dysregulation in the Pathophysiology of Inflammatory Bowel Disease

Crohn's disease [CD] and ulcerative colitis [UC] are the two main forms of inflammatory bowel disease [IBD]. Previous studies reported increased levels of proteolytic activity in stool and tissue samples from IBD patients, whereas the re-establishment of the proteolytic balance abrogates the development of experimental colitis. Furthermore, recent data suggest that IBD occurs in genetically predisposed individuals who develop an abnormal immune response to intestinal microbes once exposed to environmental triggers. In this review, we highlight the role of proteases in IBD pathophysiology, and we showcase how the main cellular pathways associated with IBD influence proteolytic unbalance and how functional proteomics are allowing the unambiguous identification of dysregulated proteases in IBD, paving the way to the development of new protease inhibitors as a new potential treatment.status: publishe

Ephrin-B2 signaling in the spinal cord as a player in post-inflammatory and stress-induced visceral hypersensitivity

BACKGROUND: Ephrin-B2/EphB receptor signaling contributes to persistent pain states such as postinflammatory and neuropathic pain. Visceral hypersensitivity (VHS) is a major mechanism underlying abdominal pain in patients with irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD) in remission, but the underlying pathophysiology remains unclear. Here, we evaluated the spinal ephrin-B2/EphB pathway in VHS in 2 murine models of VHS, that is, postinflammatory TNBS colitis and maternal separation (MS). METHODS: Wild-type (WT) mice and mice lacking ephrin-B2 in Nav 1.8 nociceptive neurons (cKO) were studied. VHS was induced by: 1. intracolonic instillation of TNBS or 2. water avoidance stress (WAS) in mice that underwent maternal separation (MS). VHS was assessed by quantifying the visceromotor response (VMRs) during colorectal distention. Colonic tissue and spinal cord were collected for histology, gene, and protein expression evaluation. KEY RESULTS: In WT mice, but not cKO mice, TNBS induced VHS at day 14 after instillation, which returned to baseline perception from day 28 onwards. In MS WT mice, WAS induced VHS for up to 4 weeks. In cKO however, visceral pain perception returned to basal level by week 4. The development of VHS in WT mice was associated with significant upregulation of spinal ephrin-B2 and EphB1 mRNA expression or protein levels in the TNBS model and upregulation of spinal ephrin-B2 protein in the MS model. No changes were observed in cKO mice. VHS was not associated with persistent intestinal inflammation. CONCLUSIONS AND INFERENCES: Overall, our data indicate that the ephrin-B2/EphB1 spinal signaling pathway is involved in VHS and may represent a novel therapeutic target.status: publishe