Probes for INS

Preclinical studies have demonstrated that the endocannabinoid system (ECS) plays an important role in the protection against intestinal inflammation and colorectal cancer (CRC); however, human data are scarce. We determined members of the ECS and related components of the 'endocannabinoidome' in patients with inflammatory bowel disease (IBD) and CRC, and compared them to control subjects. Anandamide (AEA) and oleoylethanolamide (OEA) were increased in plasma of ulcerative colitis (UC) and Crohn's disease (CD) patients while 2-arachidonoylglycerol (2-AG) was elevated in patients with CD, but not UC. 2-AG, but not AEA, PEA and OEA, was elevated in CRC patients. Lysophosphatidylinositol (LPI) 18:0 showed higher levels in patients with IBD than in control subjects whereas LPI 20:4 was elevated in both CRC and IBD. Gene expression in intestinal mucosal biopsies revealed different profiles in CD and UC. CD, but not UC patients, showed increased gene expression for the 2-AG synthesizing enzyme diacylglycerol lipase alpha. Transcripts of CNR1 and GPR119 were predominantly decreased in CD. Our data show altered plasma levels of endocannabinoids and endocannabinoid-like lipids in IBD and CRC and distinct transcript profiles in UC and CD. We also report alterations for less known components in intestinal inflammation, such as GPR119, OEA and LPI.

Surveys suggest that Cannabis provides benefit for people with inflammatory bowel disease. However, mechanisms underlying beneficial effects are not clear. We performed in situ hybridization RNAscope^® combined with immunohistochemistry to show cell-specific distribution and regulation of cannabinoid receptor 1 and 2 (CB1, CB2), G protein-coupled receptor 55 (GPR55), and monoacylglycerol lipase (MGL) mRNA in immune cells using murine models of intestinal and systemic inflammation. In healthy animals, the presence in enteric ganglia is high for CB1 mRNA, but low for CB2 and GPR55 mRNAs. MGL mRNA is predominant throughout the intestinal wall including myenteric neurons, epithelium, circular and longitudinal muscular layers, and the lamina propria. Within the immune system, B220+ cells exhibit high gene expression for CB2 while the expression of CB2 in F4/80+ and CD3+ cells is less prominent. In contrast, GPR55 mRNA is highly present in F4/80+ and CD3+ cells. qRT-PCR of total colonic segments shows that the expression of GPR55 and MGL genes drops during intestinal inflammation. Also at cellular levels, GPR55 and MGL gene expression is reduced in F4/80+, but not CD3+ cells. As to systemic inflammation, reduced gene expression of MGL is observed in ileum by qRT-PCR, while at cellular levels, altered gene expression is also seen for CB1 and GPR55 in CD3+ but not F4/80+ cells. In summary, our study reveals changes in gene expression of members of the endocannabinoid system in situ attesting particularly GPR55 and MGL a distinct cellular role in the regulation of the immune response to intestinal and systemic inflammation.

Interleukin 10 receptor (IL10R)-deficient mice develop spontaneous colitis and, similarly, patients with loss-of-function mutations in IL10R develop severe infant-onset inflammatory bowel disease. Loss of IL10R signaling in mouse and human macrophages is associated with increased production of interleukin 1β. We demonstrated that innate immune production of IL1β mediates colitis in IL10R-deficient mice. Transfer of Il1r1-/- CD4+ T cells into Rag1-/-/Il10rb-/- mice reduced the severity of their colitis (compared to mice that received CD4+ T cells that express IL1R), accompanied by decreased production of interferon gamma, tumor necrosis factor-α, and IL17A. In macrophages from mice without disruption of IL10R signaling or from healthy humans (controls), incubation with IL10 reduced canonical activation of the inflammasome and production of IL1β through transcriptional and post-translational regulation of NLRP3. Lipopolysaccharide and adenosine triphosphate stimulation of macrophages from Il10rb-/- mice or IL10R-deficient patients resulted in increased production of IL1β. Moreover, in human IL10R-deficient macrophages, lipopolysaccharide stimulation alone triggered IL1β secretion via non-canonical, caspase 8-dependent activation of the inflammasome. We treated 2 IL10R-deficient patients with severe and treatment-refractory infant-onset inflammatory bowel disease with the IL1-receptor antagonist anakinra. Both patients had marked clinical, endoscopic, and histologic responses after 4-7 weeks. This treatment served as successful bridge to allogeneic hematopoietic stem cell transplantation in 1 patient. Our findings indicate that loss of IL10 signaling leads to intestinal inflammation, at least in part, through increased production of IL1 by innate immune cells, leading to activation of CD4+ T cells. Agents that block IL1 signaling might be used to treat patients with inflammatory bowel disease resulting from IL10R deficiency.