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Objective:In mice, a subpopulation of gut dendritic cells (DCs) expressing CD103 drives the development of regulatory T (Treg) cells. Further, it was recently described that the cross-talk between human intestinal epithelial cells (IECs) and DCs helps in maintaining gut immune homeostasis via the induction of non-inflammatory DCs. In this study, an analysis was carried out to determine whether IECs could promote the differentiation of CD103+ tolerogenic DCs, and the function of primary CD103+ DCs isolated from human mesenteric lymph nodes (MLNs) was evaluated.Methods:Monocyte-derived DCs (MoDCs) and circulating CD1c+ DCs were conditioned or not with supernatants from Caco-2 cells or IECs isolated from healthy donors or donors with Crohn’s disease and analysed for their ability to induce Treg cell differentiation. In some cases, transforming growth factor β (TGFβ), retinoic acid (RA) or thymic stromal lymphopoietin (TSLP) were neutralised before conditioning. CD103+ and CD103− DCs were sorted by fluorescence-activated cell sorting (FACS) from MLNs and used in Treg cell differentiation experiments.Results:It was found that human IECs promoted the differentiation of tolerogenic DCs able to drive the development of adaptive Foxp3+ Treg cells. This control was lost in patients with Crohn’s disease and paralleled a reduced expression of tolerogenic factors by primary IECs. MoDCs differentiated with RA or IEC supernatant upregulated the expression of CD103. Consistently, human primary CD103+ DCs isolated from MLNs were endowed with the ability to drive Treg cell differentiation. This subset of DCs expressed CCR7 and probably represents a lamina propria-derived migratory population.Conclusions:A population of tolerogenic CD103+ DCs was identified in the human gut that probably differentiate in response to IEC-derived factors and drive Treg cell development.

ObjectiveCD103+ gut dendritic cells (DCs) have been shown to be required for de novo conversion of adaptive T regulatory (Treg) cells. Indoleamine 2,3-dioxygenase (IDO) is an enzyme involved in tryptophan catabolism that is expressed by DCs isolated from tumour-draining lymph nodes. IDO-expressing DCs sustain and differentiate Tregs. The aim of this study was to investigate the expression and the possible physiological role of IDO in the tolerogenic properties of intestinal DCs.DesignThe expression level of IDO in CD103+ and CD103− DCs was analysed by qRT-PCR, western blot and immunofluorescence. CD103+ and CD103− DCs were sorted from mesenteric lymph nodes (MLNs) and the small intestinal lamina propria, and the role of IDO in the conversion of Tregs and Th effector cell development was evaluated via specific inhibition or gene deletion. Oral tolerance, experimental colitis and T cell differentiation in vivo were assessed upon IDO inactivation.ResultsWe show that, primarily, CD103+ but not CD103− gut DCs express IDO whose inhibition results in reduced CD4+Foxp3+ T regulatory cell conversion and enhanced T cell proliferation. When IDO was inhibited or genetically deleted there was an increase in Th1 and Th17 differentiation both in vitro and in vivo. Finally, in vivo IDO blockade affected the development of Tregs specific for orally administered antigens, impaired oral tolerance induction and exacerbated colitis.ConclusionsWe identified a new IDO-dependent pathway leading to acquisition of tolerogenic functions in mucosal CD103-expressing DCs, indicating IDO as a possible therapeutic target for gut disorders.

Intestinal dendritic cells (DCs) have been shown to display specialized functions, including the ability to promote gut tropism to lymphocytes, to polarize noninflammatory responses, and to drive the differentiation of adaptive Foxp3+ regulatory T (Treg) cells. However, very little is known about what drives the mucosal phenotype of DCs. Here, we present evidence that the local microenvironment, and in particular intestinal epithelial cells (ECs), drive the differentiation of Treg-cell-promoting DCs, which counteracts Th1 and Th17 development. EC-derived transforming growth factor-β (TGF-β) and retinoic acid (RA), but not thymic stromal lymphopoietin (TSLP), were found to be required for DC conversion. After EC contact, DCs upregulated CD103 and acquired a tolerogenic phenotype. EC-conditioned DCs were capable of inducing de novo Treg cells with gut-homing properties that when adoptively transferred, protected mice from experimental colitis. Thus, we have uncovered an essential mechanism in which EC control of DC function is required for tolerance induction.

The interplay between diet, host genetics, microbiota, and immune system has a key role in the pathogenesis of inflammatory bowel disease (IBD). Although the causal pathophysiological mechanisms remain unknown, numerous dietary nutrients have been shown to regulate gut mucosal immune function, being effective in influencing innate or adaptive immunity. Here, we proved that transient receptor potential melastatin 8 (TRPM8), a non-selective cation channel, mediates LPS- evoked Ca 2+ influx in macrophages leading to their activation. Additionally, we showed that TRPM8 is selectively blocked by the dietary flavonoid luteolin, which induced a pro-tolerogenic phenotype in pro-inflammatory macrophages. Accordingly, genetic deletion of Trpm8 in macrophages caused a deficit in the activation of pro-inflammatory metabolic and transcriptional reprogramming, leading to reduced production of key pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. The TRPM8 anti-inflammatory effect was found to be dependent on lactate which in turn induces IL-10 gene expression. Adoptive transfer of TRPM8-deficient bone marrow in wild-type mice improved intestinal inflammation in a model of colitis. Accordingly, oral administration of luteolin protected mice against colitis through an impairment in the innate immune response. Our study reveals the potential of targeting TRPM8 through specific nutrient interventions to regulate immune function in sub-clinical scenarios or to treat inflammatory diseases, primarily driven by chronic immune responses, such as IBD.

Injury to the enteric nervous system (ENS) can cause several gastrointestinal (GI) disorders including achalasia, irritable bowel syndrome, and gastroparesis. Recently, a subpopulation of enteric glial cells with neuronal stem/progenitor properties (ENSCs) has been identified in the adult ENS. ENSCs have the ability of reconstituting the enteric neuronal pool after damage of the myenteric plexus. Since the estrogen receptor β (ERβ) is expressed in enteric glial cells and neurons, we investigated whether a selective ERβ agonist, LY3201, can influence neuronal and glial cell differentiation. Myenteric ganglia from the murine muscularis externa were isolated and cultured in either glial cell medium or neuronal medium. In glial cell medium, the number of glial progenitor cells (Sox10⁺) was increased by fourfold in the presence of LY3201. In the neuronal medium supplemented with an antimitotic agent to block glial cell proliferation, LY3201 elicited a 2.7-fold increase in the number of neurons (neurofilament⁺ or HuC/D⁺). In addition, the effect of LY3201 was evaluated in vivo in two murine models of enteric neuronal damage and loss, namely, high-fat diet and topical application of the cationic detergent benzalkonium chloride (BAC) on the intestinal serosa, respectively. In both models, treatment with LY3201 significantly increased the recovery of neurons after damage. Thus, LY3201 was able to stimulate glial-to-neuron cell differentiation in vitro and promoted neurogenesis in the damaged myenteric plexus in vivo. Overall, our study suggests that selective ERβ agonists may represent a therapeutic tool to treat patients suffering from GI disorders, caused by excessive neuronal/glial cell damage.

ObjectivesVagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human.DesignUsing Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1–5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI.ResultsEFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery.ConclusionEnteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI.Trial registration number NCT02425774.

Early-life exposure to environmental toxins like tobacco can permanently re-program body structure and function. Here, we investigated the long-term effects on mouse adult sleep phenotype exerted by early-life exposure to nicotine or to its principal metabolite, cotinine. Moreover, we investigated whether these effects occurred together with a reprogramming of the activity of the hippocampus, a key structure to coordinate the hormonal stress response. Adult male mice born from dams subjected to nicotine (NIC), cotinine (COT) or vehicle (CTRL) treatment in drinking water were implanted with electrodes for sleep recordings. NIC and COT mice spent significantly more time awake than CTRL mice at the transition between the rest (light) and the activity (dark) period. NIC and COT mice showed hippocampal glucocorticoid receptor (GR) downregulation compared to CTRL mice, and NIC mice also showed hippocampal mineralocorticoid receptor downregulation. Hippocampal GR expression significantly and inversely correlated with the amount of wakefulness at the light-to-dark transition, while no changes in DNA methylation were found. We demonstrated that early-life exposure to nicotine (and cotinine) concomitantly entails long-lasting reprogramming of hippocampal activity and sleep phenotype suggesting that the adult sleep phenotype may be modulated by events that occurred during that critical period of life.

Duodenal hyperpermeability and low-grade inflammation in functional dyspepsia is potentially related to duodenal acid exposure. We aimed to evaluate in healthy volunteers the involvement of mast cell activation on the duodenogastric reflex and epithelial integrity during duodenal acidification. This study consisted of 2 parts: (1) Duodenal infusion of acid or saline during thirty minutes in a randomized, double-blind cross-over manner with measurement of intragastric pressure (IGP) using high resolution manometry and collection of duodenal biopsies to measure epithelial barrier function and the expression of cell-to-cell adhesion proteins. Mast cells and eosinophils were counted and activation and degranulation status were assessed. (2) Oral treatment with placebo or mast cell stabilizer disodiumcromoglycate (DSCG) prior to duodenal perfusion with acid, followed by the procedures described above. Compared with saline, acidification resulted in lower IGP (P < 0.01), increased duodenal permeability (P < 0.01) and lower protein expression of claudin-3 (P < 0.001). Protein expression of tryptase (P < 0.001) was increased after acid perfusion. Nevertheless, an ultrastructural examination did not reveal degranulation of mast cells. DSCG did not modify the drop in IGP and barrier dysfunction induced by acid. Duodenal acidification activates an inhibitory duodenogastric motor reflex and, impairs epithelial integrity in healthy volunteers. However, these acid mediated effects occur independently from mast cell activation.

In this study, three structurally distinct disintegrins (flavoridin, echistatin, kistrin) were used as molecular probes to further characterize the molecular mechanisms underlying Yersinia enterocolitica infection of host cells. The activity of the three disintegrins on Y. enterocolitica uptake into fibronectin-adherent HeLa cells was evaluated at disintegrin doses which were non-cytotoxic and unable to induce cell detachment. Flavoridin resulted to be the most effective in inhibiting bacterial entry into host cells; echistatin was almost 50% less effective than flavoridin, whereas kistrin was definitely inactive. Our results suggest that α 5β 1 integrin receptor, which binds flavoridin with higher affinity than the other two disintegrins, plays a major role in Y. enterocolitica uptake into HeLa cells. Furthermore, flavoridin binding to this integrin prevented the disruption of the functional complex FAK–Cas, which occurs in the Y. enterocolitica uptake process.

Autism Spectrum Disorder (ASD) is characterized by substantial, yet highly heterogeneous abnormalities in functional brain connectivity. However, the origin and significance of this phenomenon remain unclear. To unravel ASD connectopathy and relate it to underlying etiological heterogeneity, we carried out a bi-center cross-etiological investigation of fMRI-based connectivity in the mouse, in which specific ASD-relevant mutations can be isolated and modeled minimizing environmental contributions. By performing brain-wide connectivity mapping across 16 mouse mutants, we show that different ASD-associated etiologies cause a broad spectrum of connectional abnormalities in which diverse, often diverging, connectivity signatures are recognizable. Despite this heterogeneity, the identified connectivity alterations could be classified into four subtypes characterized by discrete signatures of network dysfunction. Our findings show that etiological variability is a key determinant of connectivity heterogeneity in ASD, hence reconciling conflicting findings in clinical populations. The identification of etiologically-relevant connectivity subtypes could improve diagnostic label accuracy in the non-syndromic ASD population and paves the way for personalized treatment approaches.