Abstract
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.
Highlights
Knowledge of the endocannabinoid system (ECS) has immensely grown in the past few years
in situ hybridization (ISH) RNAscope® and ISH/IHC were performed to determine detailed cellular localizations of mRNA and to investigate, if changes in gene expression by dextran sulfate sodium (DSS) and LPS treatment can be detected on a cellular level in situ
To better distinguish between ISH and IHC stainings, we switched to the red detection kit for some selected targets
Summary
Knowledge of the endocannabinoid system (ECS) has immensely grown in the past few years. Much of the research progress on the ECS is hampered by a lack of specific tools, especially of antibodies against GPR55 and C B2, to detect their localization in different cell populations of the gut. Available antibodies against C B1 have been evaluated, showing that unspecific staining may be an issue with CB1 antibodies as well (Grimsey et al 2008). Commercially available GPR55 antibodies have been used for immunohistochemistry with more or less strict controls (Galiazzo et al 2018; Li et al 2013; Lin et al 2011). The strongest indication for antibody specificity, is the absence of staining in the respective knockout mouse
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