Celiac Disease (CD) represents an autoimmune disorder triggered by the exposure to gluten in genetically susceptible individuals. Recent studies suggest the involvement of macrophages in CD pathogenesis. Macrophages are immune cells, present as pro-inflammatory classically activated macrophages (M1) or as anti-inflammatory alternatively activated macrophages (M2). The Cannabinoid Receptor 2 (CB2) has important anti-inflammatory and immunoregulatory properties. We previously demonstrated that a common CB2 functional variant, Q63R, causing CB2 reduced function, is associated with several inflammatory and autoimmune diseases The first aim of this study was to investigate the phenotype of macrophages isolated from peripheral blood of CD patients and CB2 expression. The second aim was to evaluate the effects of CB2 pharmacological modulation on CD macrophage polarization. Moreover, by an in vitro model of “immunocompetent gut” we investigated the role of CD macrophages in inducing intestinal barrier damage and the possibility to restore its functionality modulating their polarization. We found an increased expression of M1 macrophages and a CB2 reduced expression. We also demonstrated CD M1 macrophages in inducing the typical mucosal barrier damage of CD. CB2 stimulation switches macrophage polarization towards the anti-inflammatory M2 phenotype thus reducing inflammation but also limiting the epithelial dysfunction. Therefore, we suggest CB2 receptor as a possible novel therapeutic target for CD by regulating macrophages polarization and by preventing mucosal barrier damage.

Background The reduction of bone mineral density and osteoporosis have high impacts on the health of patients with inflammatory bowel diseases (IBD). We have previously shown that a dysregulated iron metabolism occurs in IBD and leads to a decrease in circulating iron concentration and excessive intracellular sequestration of iron. Studies suggest that iron overload significantly affects the bone, accelerating osteoclast (OC) differentiation and activation, promoting bone resorption. Moreover, we demonstrated that iron overload causes OC overactivity. The cannabinoid receptor type 2 (CB2) and the transient receptor potential vanilloid type-1 (TRPV1) are potential therapeutic targets for bone diseases. The aim of this study was to evaluate the roles of CB2 and TRPV1 receptors and of iron in the development of osteoporosis in pediatric IBD. Methods We differentiated OCs from peripheral blood mononuclear cells of patients with IBD and healthy donors and evaluated CB2 and TRPV1 receptor expression; OC activity, and iron metabolism by Western blot, TRAP assays, bone resorption assays, and iron assays. Moreover, we analyzed the effects of the pharmacological modulation of CB2 and TRPV1 receptors on OC activity and on the iron metabolism. Results We confirmed the well-known roles of CB2 and TRPV1 receptors in bone metabolism and suggested that their stimulation can reduce the OC overactivity induced by iron, providing new insights into the pathogenesis of pediatric IBD-related bone resorption. Conclusions Stimulation of CB2 and TRPV1 could reduce IBD-related osteoporosis due to their direct effects on OC activity and to modulating the iron metabolism.

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Background The endocannabinoid (EC) system has been recently indicated as a possible therapeutic target in inflammatory bowel disease patients (IBD). G-protein-coupled cannabinoid receptors type 1 and 2 (CB1 and CB2) are the two most important components of the EC system. In our previous study, we demonstrated that a specific CB2 variant contributes to the risk for pediatric IBD, especially UC, and that this variant is associated with a more severe phenotype in both UC and CD. The aim of this study was to directly evaluate on intestinal biopsies of pediatric IBD patients the expression of EC system receptors and of several molecules associated with inflammatory pathway in IBD. Methods In this preliminary study, we analyzed the CB1 and CB2 expression in the intestinal mucosa of paediatric patients affected by Crohn’s disease (CD) or ulcerative colitis (UC), recruited at diagnosis, and of non-IBD healthy controls (HC). Three patients were enrolled for each group. In particular, we analyzed biopsies taken from the ileum and rectum, which are the tract often more inflamed in CD and UC respectively. Moreover, we evaluated Toll-like receptor 4 (TLR 4), interleukin- 6 (IL6), interleukin-1 β (IL1-β), prokineticin receptor 2 (PKR2) expression, which are known as pro-inflammatory elements. The expression of these receptors and pro- inflammatory cytokines was analyzed by western blot and immunofluorescence. Results We found an increased expression of CB2 and TLR4 in UC rectum biopsies compared to CD and HC children. Accordingly, there was also a significant increase of IL-6 expression in UC rectum biopsies compared to CD and HC children (p≤0.05 UC vs HC), as well as, we identified a high expression of IL-6 in CD ileum compared to UC and HC patients. Moreover, in CD ileum, we found an increase in TLR4 compared to UC and HC ileum. We did not find statistical difference for the low number of analyzed samples, but to be a preliminary work we are confident to reach statistical significance increasing the samples enrolled. Conclusion In according with our previous data, our results confirm that the EC system and in particular the receptor CB2 is more expressed in the intestine of IBD patients, especially in the rectum of UC patients, which is the tract with greater inflammatory involvement. We can speculate that CB2 receptor is involved in the pathogenesis of paediatric IBD, but additional functional studies are required to understand how the EC system contributes to disease development in pediatric IBD, and if these cannabinoid receptors might represent a new disease marker and could be used as a potential therapeutic target. Expression of CB2 receptor in rectum biopsies

Background The reduction of bone mineral density and osteoporosis (OP) has an high impact on inflammatory bowel disease (IBD) patients’ health. We have previously shown that a dysregulated iron metabolism occurs in IBD and leads to a decrease in circulating iron concentration and an excessive intracellular sequestration of iron. Studies suggest that iron overload affects the bone, accelerating osteoclasts (OCs) differentiation and activation with consequent bone resorption. The aim of this study was to evaluate the role of Cannabinoid Receptor type 2 (CB2) and transient receptor potential vanilloid type-1 (TRPV1) and of the iron in the development of osteoporosis in pediatric IBD. Methods We enrolled 21 young subjects aged less than 18 years stratified into 3 groups on the basis of the clinical diagnosis: 7 with Ulcerative Colitis (UC) 7 with Crohn’s disease (CD) and 7 were non-IBD controls (CTR). We evaluated the role of CB2 and TRPV1 receptors and of iron in the development of OP in pediatric IBD at diagnosis and analyzed the effects of the pharmacological modulation of CB2 and TRPV1 receptors on osteoclast activity and on iron metabolism by Western Blotting, Tartrate-resistant acid phosphatase assay (TRAP assay) and Iron Assay Kit. We evaluated bone resorption through a commercially available kit visualizing and counting the resorption pits with the optical microscope. Results We observed higher levels of TRPV1 and lower levels of CB2 in the OCs from UC and CD patients compared to CTR. In IBD patients we found a significant up-regulation of osteoclast markers TRAP and Cathepsin K and a consequent osteoclast hyper-activation. The treatments of OCs from IBD patients with the CB2 agonist JWH-133 and the TRPV1 agonist Resinferatoxin, RTX, led to a significant reduction of osteoclastic hyper-activation affecting bone markers expression, number and size of active OCs, and bone resorption area. We also analyzed the [Fe3+] concentration and the expression of iron metabolism modulators, ferroportin1(FPN-1) and divalent metal transporter1(DMT1), in OCs derived from IBD patients. We demonstrated an increase of [Fe3+] and accordingly a decrease of FPN-1 in both groups of patients and an increase of DMT1 levels. After 48h of exposure to JWH-133 and RTX we observed a significant reduction of DMT1 expression in OCs from IBD patients together with a strong increase of FPN-1. Conclusion Our data confirm the well-known role of CB2 and TRPV1 receptors in bone metabolism and suggest that their stimulation can reduce the osteoclast overactivity induced by iron, thus providing new insights into the pathogenesis and in the treatment of pediatric IBD-related bone resorption.