Inflammation and CB ₂ signaling drive novel changes in the ocular lipidome and regulate immune cell activity in the eye

Uveitis is inflammation of the uvea which consists of the iris, ciliary body and the choroid of the eye. Uveitis can lead to impaired vision and is responsible for 10% of all cases of blindness globally. Using an endotoxin-induced uveitis (EIU) rodent model, our previous data implicated the endogenous cannabinoid system (ECS) in the amelioration of many of the components of the inflammatory response. Here, we test the hypothesis that the reduction in inflammatory mediators in the EIU model by the CB ₂ agonist, HU308, is associated with changes in ECS endogenous ligands as well as related lipids, prostaglandins (PGs), 2-acyl glycerols, and lipoamines. Analysis of leukocytes and neutrophils, CB ₂ mRNA, and 26 lipids in the eye of WT mice after EIU induction and HU308 treatment were compared to the same analyses in the CB ₂ knock-out (CB ₂ KO) mouse. Endothelial leukocyte adhesion and neutrophil migration were significantly increased in both WT and CB ₂ KO after EIU. HU308 significantly reduced the leukocyte adhesion and neutrophil recruitment in the WT animals. HU308 also significantly reduced leukocyte adhesion in the CB ₂ KO mouse, yet, had no effect on neutrophil infiltration suggesting an important off-target effect of HU308. Lipidomics profiles revealed significant increases in 6 non-ECS lipids after EIU in the WT and that HU308 effectively reduced these back to control levels; in addition, HU308 increased levels of 2-acyl glycerols and decreased all N-acyl glycines. CB ₂ KOs with saline-injection compared to WT had significantly elevated levels of 2-acyl glycerols, whereas levels of N-oleoyl ethanolamine (OEA), N-stearoyl ethanolamine (SEA), and PGE ₂ were reduced. CB ₂ KOs with EIU had 13 lipids that were significantly lower than WT with EIU including 4 N-acyl glycines. HU308 had no effect on lipid concentrations in the CB ₂ KOs with EIU, however, it did cause further reductions on 3 additional lipids compared to saline controls. HU308 appears to be acting at a non-CB ₂ target for the reduction of leukocyte infiltration in the EIU model; however, our data suggest that HU308 is working through CB ₂ to reduce neutrophil migration and for the regulation of multiple lipid signaling pathways including PGs, lipoamines, and 2-acyl glycerols. These data implicate ocular CB ₂ as a key component of lipid signaling in the eye and part of the regulatory processes of inflammation.