The cholinergic anti-inflammatory pathway (CAIP) has been proposed as a key mechanism by which the brain, through the vagus nerve, modulates the immune system in the spleen. Vagus nerve stimulation (VNS) reduces intestinal inflammation and improves postoperative ileus. We investigated the neural pathway involved and the cells mediating the anti-inflammatory effect of VNS in the gut. The effect of VNS on intestinal inflammation and transit was investigated in wild-type, splenic denervated and Rag-1 knockout mice. To define the possible role of α7 nicotinic acetylcholine receptor (α7nAChR), we used knockout and bone marrow chimaera mice. Anterograde tracing of vagal efferents, cell sorting and Ca ²⁺ imaging were used to reveal the intestinal cells targeted by the vagus nerve. VNS attenuates surgery-induced intestinal inflammation and improves postoperative intestinal transit in wild-type, splenic denervated and T-cell-deficient mice. In contrast, VNS is ineffective in α7nAChR knockout mice and α7nAChR-deficient bone marrow chimaera mice. Anterograde labelling fails to detect vagal efferents contacting resident macrophages, but shows close contacts between cholinergic myenteric neurons and resident macrophages expressing α7nAChR. Finally, α7nAChR activation modulates ATP-induced Ca ²⁺ response in small intestine resident macrophages. We show that the anti-inflammatory effect of the VNS in the intestine is independent of the spleen and T cells. Instead, the vagus nerve interacts with cholinergic myenteric neurons in close contact with the muscularis macrophages. Our data suggest that intestinal muscularis resident macrophages expressing α7nAChR are most likely the ultimate target of the gastrointestinal CAIP.

Histone deacetylases (HDACs) regulate inflammatory gene expression, as indicated by the potent antiinflammatory activity of pan-HDAC inhibitors. However, the specific contribution of each of the 11 HDAC proteins to the inflammatory gene expression program is unknown. Using an integrated genomic approach, we found that Hdac3-deficient macrophages were unable to activate almost half of the inflammatory gene expression program when stimulated with LPS. A large part of the activation defect was attributable to loss of basal and LPS-inducible expression of IFN-β, which maintains Stat1 protein levels in unstimulated cells and acts in an autocrine/paracrine manner after stimulation to promote a secondary wave of Stat1-dependent gene expression. Loss of Hdac3-mediated repression of nuclear receptors led to hyperacetylation of thousands of genomic sites and associated gene derepression. The up-regulation of the constitutively expressed prostaglandin endoperoxide synthase, Ptgs1 (Cox-1), a nuclear receptor target, had a causative role in the phenotype because its chemical inhibition reverted, albeit partially, the Ifn-β activation defect. These data indicate a central role for Hdac3 in inflammation and may have relevance for the use of selective Hdac inhibitors as antiinflammatory agents.

Objective: In mice, a subpopulation of gut dendritic cells (DCs) expressing CD103 drives the development of regulatory T (T _reg ) 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 T _reg 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 T _reg cell differentiation experiments. Results: It was found that human IECs promoted the differentiation of tolerogenic DCs able to drive the development of adaptive Foxp3 ⁺ T _reg 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 T _reg 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 T _reg cell development.

Objective: CD103 ⁺ 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. Design: The 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. Results: We 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. Conclusions: We 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.