CB2 AGONISM IS NEUROPROTECTIVE AND PROTECTS AGAINST IMMUNE DYSREGULATION POST-STROKE

Abstract

CNS injury-induced immunodepression syndrome (CIDS) significantly contributes to increased susceptibility to infections such as pneumonia. This immunodepression results from maladaptive neuroimmune interactions following stroke. Given its expression on microglia and immune cells, cannabinoid receptor 2 (CB2) represents a promising therapeutic target for modulating neuroinflammation. We tested the hypothesis that early, selective CB2 activation would reduce stroke severity, preserving peripheral immune function by attenuating the immunodepressive cascade associated with CIDS. The objectives were to (1) validate the photothrombotic stroke (PTS) model in the context of CIDS; (2) confirm the role of the endocannabinoid system (ECS) in neuroprotection using the non-selective CB1/2 agonist WIN-55,212-2 with selective receptor antagonists; (3) determine specific contribution of CB2 activation via HU-308; (4) characterize effects of HU-308 on microglial phenotype and function in-vitro; and (5) assess systemic immune consequences of early CB2 activation. Focal ischemia was induced using a mouse PTS model. Infarct size was assessed using 2,3,5-triphenyltetrazolium chloride staining, and neurological deficits were scored with a composite Neuroscore. Inflammatory signaling in the brain was analyzed using RT-qPCR. Cultured microglial phenotype and function were assessed using ELISA and flow cytometry. Intestinal intravital microscopy was used to assess leukocyte–endothelial interactions in response to Lipopolysaccharide (LPS) challenge as a readout for peripheral immune competence, and splenic immune populations were characterized by flow cytometry. Initial proof-of-concept studies indicated ECS activation by WIN-55,212-2 pretreatment significantly reduced infarct size and improved neurological outcomes. CB1 antagonist AM281 did not abolish these effects unlike CB2 blockade with AM630. Based on this, pretreatment with the selective CB2 agonist HU-308 in subsequent experiments significantly reduced infarct size, improved neurological function, suppressed pro-inflammatory cytokines and promoted anti- inflammatory microglial polarization. Post-stroke immune suppression evidenced by a ~60% reduction in leukocyte adhesion in PTS+LPS and an increase in splenic regulatory T cells animals relative to SHAM+LPS controls were prevented by early CB2 activation, suggesting that immune function is preserved due to a reduction in stroke severity. These findings validate the PTS model in the context of CIDS and demonstrate that early CB2 activation improves stroke severity and CIDS, supporting the ECS as a viable therapeutic target.