Novel Microglia Cell Line Expressing the Human EP2 Receptor

Abstract

Recently, EP2 signaling pathways were shown to regulate the classical activation and death of microglia in rat primary microglial culture. The study of microglial cells has been challenging because they are time-consuming to isolate in culture, they are demanding in their growth requirements, and they have a limited lifespan. To circumvent these difficulties, we created a murine BV2 microglial cell line stably expressing human EP2 receptors (BV2-hEP2) and further explored EP2 modulation of microglial functions. The BV2-hEP2 cells displayed cAMP elevation when exposed to the selective EP2 receptor agonists (ONO-AE1-259-1 and CP544326), and this response was competitively inhibited by TG4-155, a selective EP2 antagonist (Schild K B = 2.6 nM). By contrast, untransfected BV2 cells were unresponsive to selective EP2 agonists. Similar to the case of rat primary microglia, BV2-hEP2 microglia treated with lipopolysaccharide (LPS) (100 ng/mL) displayed rapid and robust induction of the inflammatory mediators COX-2, IL-1β, TNFα, and IL-6. EP2 activation depressed TNFα induction but exacerbated that of the other inflammatory mediators. Like primary microglia, classically activated BV2 microglia phagocytose fluorescent-labeled latex microspheres. The presence of EP2, but not its activation by agonists, in BV2-hEP2 microglia reduced phagocytosis and proliferation by 65% and 32%, respectively, compared to BV2 microglia. Thus, BV2-hEP2 is the first microglial cell line that retains the EP2 modulation of immune regulation and phagocytic ability of native microglia. Suppression of phagocytosis by the EP2 protein appears unrelated to classical EP2 signaling pathways, which has implications for therapeutic development of EP2 antagonists.

Prostaglandin EP2 receptor antagonist ameliorates neuroinflammation in a two-hit mouse model of Alzheimer’s disease

“…Although there was no effect found on the amyloid pathology in single-hit—genetic 5xFAD females upon TG11-77.HCl treatment. Nonetheless, the downstream mechanisms by which the EP2 receptor modulates the phagocytic activity of microglia is not clear, because either stimulation of EP2 receptors with an agonist or antagonist had no impact on the phagocytic activity of microglia BV2 cell line that overexpressed human EP2 receptors [ 48 ], in comparison to enhanced phagocytosis found in primary mouse microglia lacking EP2 receptors [ 44 ].…”

“…This antagonist showed a brain-to-plasma ratio ranging from 9A, B), and no adverse toxicity by chronic dosing ≥ 100 mg/kg (Rawat et al in preparation). It also displayed anti-inflammatory properties in vitro in a BV2 cell line overexpressing human EP2 receptor [48]. Moreover, this compound is soluble in water at concentration of 1.1 mg/mL, which facilitates chronic oral dosing in drinking water.…”

“…Lipopolysaccharide (LPS) (Sigma; L2880) was dissolved in sterile saline and injected intraperitoneally (IP) weekly once at 0.5 mg/kg of body weight. TG11-77.HCl, an EP2 antagonist recently developed and synthesized in our laboratory [ 48 ], was prepared fresh every week by dissolving in rodent drinking water at 0.5 mg/mL. For optimum solubility of the drug, the solution was adjusted to pH 3.5 using 1 M HCl.…”

“…For further analysis, blood and brain were harvested before and after perfusion, respectively. B Table showing all the groups and their sex, treatment and number (n) used in the study antagonist recently developed and synthesized in our laboratory [48], was prepared fresh every week by dissolving in rodent drinking water at 0.5 mg/mL. For optimum solubility of the drug, the solution was adjusted to pH 3.5 using 1 M HCl.…”

“…Moreover, a selective PGE 2 receptor EP2 antagonist also enhances Aβ-phagocytosis in vitro in a macrophage cell line [ 47 ]. Furthermore, our recent in vitro study revealed that in an activated state, a murine microglial cell line stably transfected with human EP2 receptors (BV2-hEP2), when challenged with a specific EP2 receptor agonist, produced inflammation that was competitively inhibited by a selective EP2 antagonist [ 48 , 49 ]. With these rationales, here we investigated whether a selective and potent EP2 antagonist, ameliorates neuroinflammation in 5xFAD model in the presence (two-hit), and absence (single-hit—genetic) of a second peripheral inflammatory insult by LPS.…”