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  • In order for cytokines to exert any

    2021-10-25

    In order for cytokines to exert any effect on the NSC population, they must first bind to specific receptors on the cell surface. Numerous pro- and anti-inflammatory cytokines are able to exert effects on the NSC population, including IL-1β through IL-1R1 and IL-1R2, TNFα through TNFR1 and TNFR2, IL-6 through the surface co-receptor IL-6st (gp130), and the anti-inflammatory cytokine IL-10 through IL-10Rα (Green and Nolan, 2012, Ben-Hur et al., 2003, Chen and Palmer, 2013, Kotasova et al., 2014, Perez-Asensio et al., 2013). To investigate if activation of GPR55 had any effect on the acute immune response by NSCs to IL-1β, we measured mRNA expression of known cytokine receptors expressed by NSCs (IL-1R1, IL-1R2, TNFR1, TNFR2, IL-6st, IL-10Rα) at 4 and 24 h post insult. We found that activation of GPR55 reduced increases in pro-inflammatory receptor mRNA (IL-1R1, IL-6st) while further promoting anti-inflammatory receptor mRNA (IL-10Rα, TNFR2) during insult with IL-1β. It is interesting that activation of GPR55 induced effects on some specific cytokine receptors while not affecting others. IL-1R1 is the primary receptor through which IL-1β signals and GPR55 significantly attenuated increases due to IL-1β treatment while having no effect on IL-1R2, a decoy receptor with no signaling capacity (Wang et al., 2007, Koo and Duman, 2008, Colotta et al., 1993). Activation of TNFR1 is known to induce detrimental effects on neurogenesis. GPR55 agonist treatment of NSCs during insult did not alter TNFR1 mRNA levels; however, TNFR2, which has been shown to be neuroprotective, was significantly upregulated as compared to IL-1β treatment alone (Chen and Palmer, 2013). IL-6st increases were also attenuated by GPR55 activation yet this signal transducer functions not only for IL-6 mediated signaling, but also for IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OSM), which are all part of the IL-6 family of cytokines (Kwak et al., 2010). Insult with IL-1β upregulated IL-10Rα mRNA in human samples and pre-treatment with ML184 significantly increased this upregulation. It should be noted that we could not determine expression of IL-10Rα on murine hippocampal NSC samples so analysis of effects of GPR55 activation could not be performed. Results from inflammatory response experiments are indicative of an anti-inflammatory, or neuroprotective, mechanism through which GPR55 is actively attenuating the ability of pro-inflammatory cytokines (IL-1β, IL-6) to signal while increasing the effect of anti-inflammatory and neuroprotective signals (TNFα via TNFR2, IL-10). It is unclear how activation of GPR55 affected changes to mRNA levels. The genes for these receptors, either human or mouse, are on different Capsaicin (except IL-1R1 and IL-1R2; human-chromosome 2, mouse-chromosome 1) so it is unlikely that there is only one specific loci or promoter where these effects are taking place, yet treatment with GPR55 may be inducing upregulation of transcription factors necessary for cytokine receptor transcription. Another possibility is that GPR55 signaling increases post-transcriptional degradation of IL-1R1 and IL-6st mRNA through a yet unknown mechanism. IL-1R1 mRNA degradation has been shown during inflammatory states by microRNAs through targeting of the 3′UTR suggesting that the process by which GPR55 activation regulates inflammatory cytokine receptor mRNA expression could be microRNA-dependent (Skinner et al., 2017, Halappanavar et al., 2013). Regulation of microRNA by the cannabinoid system, specifically the CB2 receptor, results in protective effects against acute liver failure caused by d-galactosamine/LPS, further supporting the possibility that GPR55 may act in a similar fashion (Tomar et al., 2015). Further study is needed to fully elucidate these mechanisms. A major facet of neurodegenerative disorders and viral infection is a chronic upregulation of neuroinflammatory mediators and subsequent reductions in hippocampal neurogenesis leading to cognitive impairment (Ferguson et al., 2016, Zonis et al., 2015, Ferrell and Giunta, 2014, Belarbi et al., 2012). We wanted to understand if low levels of chronic, systemic inflammation could exert negative effects on neurogenesis similar to studies utilizing acute, high dose, or direct administration of inflammatory mediators to the CNS. We therefore utilized osmotic mini-pumps to deliver LPS (0.2 mg/kg/day) sub-dermally for 14 days and found significant reductions in NSC survival and immature neuron formation within the hippocampus. These results indicate that even very low levels of systemic inflammation can, over time, induce negative regulation of hippocampal neurogenesis. Interestingly, NSC proliferation within the SGZ (Ki67+) was not significantly reduced 14 days after pump implantation. Taken together with data showing significant reduction of BrdU+ cells (BrdU was administered on days 1–4 of LPS treatment), these data suggest that the animals may be acclimating to the levels of LPS being administered and attenuating their immune responses. Tolerance of LPS-induced signals impairs the production of pro-inflammatory cytokines without inhibiting the expression of anti-inflammatory mediators and occurs through numerous mechanisms including reduction in TLR4, MyD88, IRAK-1, and TRAF6 (Nomura et al., 2000, Oak et al., 2006, Li et al., 2006, Xiong et al., 2011). Indeed, we did see trends in downregulation of mRNA for MyD88 and TRAF6 in the hippocampus of C57BL/6 animals chronically treated with LPS suggesting an increase in tolerance. We did not see changes in mRNA expression of TLR4 or IRAK-1 in C57BL/6 animals, but that does not rule out changes in protein levels because LPS tolerance has been shown to decrease protein levels while maintaining mRNA expression (Li et al., 2006). Moreover, we observed a trend in reduction of mRNA for major cytokines including IL1β, TNFα, IL6, IL12A, and IL12B, further suggesting LPS tolerance in C57BL/6 mice. GPR55−/− mice showed some reduction of TLR4 and MyD88, yet these animals also displayed higher levels of TLR4 in the hippocampus compared to C57BL/6 animals. All other tolerance-like effects due to LPS treatment in C57BL/6 mice were altered in GPR55−/− animals. GPR55−/− mice also had increased mRNA expression of pro-inflammatory cytokines (IL1β, TNFα, IL12A) and increased IRAK-1 and TRAF6, suggesting that lack of GPR55 in these animals is altering the chronic immune response due to LPS administration. It is important to note that the animals tested for hippocampal mRNA expression did not receive cannula implantation nor any agonist treatment. The exact contribution GPR55 has on this immune response is still not fully understood and requires further study.