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Background: Alterations of astrocyte function play a crucial role in neuroinflammatory diseases due to either the loss of their neuroprotective role or the gain of their toxic inflammatory properties. Accumulating evidence highlights that cannabinoids and cannabinoid receptor agonists, such as WIN55,212-2 (WIN), reduce inflammation in cellular and animal models. Thus, the endocannabinoid system has become an attractive target to attenuate chronic inflammation in neurodegenerative diseases. However, the mechanism of action of WIN in astrocytes remains poorly understood. Objective: We studied the immunosuppressive property of WIN by examining gene expression patterns that were modulated by WIN in reactive astrocytes. Materials and Methods: Transcriptomic analysis by RNA-seq was carried out using primary human astrocyte cultures stimulated by the proinflammatory cytokine interleukin 1 beta (IL1β) in the presence or absence of WIN. Real-time quantitative polymerase chain reaction analysis was conducted on selected transcripts to characterize the dose-response effects of WIN, and to test the effect of selective antagonists of cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptors (PPAR). Results: Transcriptomic analysis showed that the IL1β-induced inflammatory response is robustly inhibited by WIN pretreatment. WIN treatment alone also induced substantial gene expression changes. Pathway analysis revealed that the anti-inflammatory properties of WIN were linked to the regulation of kinase pathways and gene targets of neuroprotective transcription factors, including PPAR and SMAD (mothers against decapentaplegic homolog). The inhibitory effect of WIN was dose-dependent, but it was not affected by selective antagonists of CB1 or PPAR. Conclusions: This study suggests that targeting the endocannabinoid system may be a promising strategy to disrupt inflammatory pathways in reactive astrocytes. The anti-inflammatory activity of WIN is independent of CB1, suggesting that alternative receptors mediate the effects of WIN. These results provide mechanistic insights into the anti-inflammatory activity of WIN and highlight that astrocytes are a potential therapeutic target to ameliorate neuroinflammation in the brain.

Background and aimsIn clinical and experimental NASH, the origin of the scar-forming myofibroblast is the HSC. We used foz/foz mice on a Western diet to characterize in detail the phenotypic changes of HSCs in a NASH model.Approach and resultsWe examined the single-cell expression profiles (scRNA sequencing) of HSCs purified from the normal livers of foz/foz mice on a chow diet, in NASH with fibrosis of foz/foz mice on a Western diet, and in livers during regression of NASH after switching back to a chow diet. Selected genes were analyzed using immunohistochemistry, quantitative real-time PCR, and short hairpin RNA knockdown in primary mouse HSCs. Our analysis of the normal liver identified two distinct clusters of quiescent HSCs that correspond to their acinar position of either pericentral vein or periportal vein. The NASH livers had four distinct HSC clusters, including one representing the classic fibrogenic myofibroblast. The three other HSC clusters consisted of a proliferating cluster, an intermediate activated cluster, and an immune and inflammatory cluster. The livers with NASH regression had one cluster of inactivated HSCs, which was similar to, but distinct from, the quiescent HSCs.ConclusionsAnalysis of single-cell RNA sequencing in combination with an interrogation of previous studies revealed an unanticipated heterogeneity of HSC phenotypes under normal and injured states.