A novel microglia cell line expressing the human EP2 receptor

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A novel microglia cell line expressing the human EP2 receptor Asheebo Rojas, Avijit Banik, Di Chen, Kevin Flood, Thota Ganesh, and Raymond Dingledine ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00311 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 31, 2019

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ACS Chemical Neuroscience

ACS Chemical Neuroscience A novel microglia cell line expressing the human EP2 receptor Asheebo Rojas¶, Avijit Banik¶, Di Chen, Kevin Flood, Thota Ganesh* & Raymond Dingledine Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA 30322 ¶contributed

equally

Running title: EP2 regulates classical activation of BV2 *Correspondence to: Thota Ganesh, PhD Department of Pharmacology Emory University School of Medicine Atlanta, GA 30322 Fax: 404-727-0365 E-mail: [email protected] Number of text pages: 31 Number of tables: 2 Number of figures: 9 (3 supplemental figures) Number of words: Abstract (241), Introduction (449) Number of references: 51

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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 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 KB = 2.6 nM). By contrast, untransfected BV2 cells were unresponsive to selective EP2 agonists. Similar to 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 TNF 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.

KEYWORDS: RT-PCR, EP2, phagocytosis, proliferation, BV2, microglia, cAMP, TR-FRET, ONO-AE1-259-1, CP544326, TG4-155, lipopolysaccharide, inflammation, cytokine


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INTRODUCTION Inflammation is the immune system’s response to an injury. In the central nervous system microglia, the resident immune cells, play a critical role in homeostasis. In rodents, microglia help to initiate an inflammatory response in areas of cell damage in the days following an acute brain injury caused by focal ischemia, stroke and status epilepticus (SE) 1-11. The microglial response to an injury, characterized by an increased number of amoeboid microglia in the injury site, is termed “microgliosis”. In rodent models of SE microgliosis can persist for many days. Microgliosis is also apparent in chronic neurological disorders such as Alzheimer’s disease (AD) and Parkinson’s disease and is associated with exacerbation of these neurodegenerative diseases Phagocytosis, the process by which solid particles are engulfed by immune cells, is an important function carried out by microglia. Upon activation microglia produce and release numerous inflammatory cytokines, chemokines and enzyme mediators. However, depending on the disease and the progression of the disease the activation of microglia can be either beneficial or detrimental in neuropathological conditions 12-13. Cyclooxygenase 2 (COX-2) is induced in microglia following classical activation by lipopolysaccharide (LPS) 14-17. COX-2 is the rate limiting enzyme in the production of prostaglandins including PGE2, which are hormone-like substances participating in many physiological functions. PGE2 acts on four different G-protein coupled receptors (EP1, EP2, EP3, and EP4). The EP2 receptor activated by PGE2 is involved in COX-2 associated brain inflammation 6, 16, 18-23, microglial phagocytosis 24-26 and proliferation of some cancer cells 27-33. Our recent studies have shown that inhibition of the EP2 receptor by novel selective small molecule antagonists leads to a beneficial outcome in the days following SE 6, 22-23, 34. The effective therapeutic window of these novel EP2 antagonists coincides with the temporal induction of COX-2 in the brain 6, 23. EP2 receptor-stimulated signaling pathways (both protein kinase A and exchange protein directly activated by cAMP) regulate the complex immune



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response during classical activation and death of microglia in rat primary microglia 16, 35. Taken together, these studies provide evidence that EP2 is immunomodulatory and alters key features of microglial activation. Here, we created a novel murine microglial cell line that stably expresses human EP2 receptors to explore the consequences of both the presence and activation of EP2 in microglia. This novel microglia cell line (termed BV2-hEP2) shares many features with cultured primary rat microglia 36 and was used to overcome the technical limitations of culturing primary microglia, i.e., obtaining dense cultures rapidly and reliably. We show that the novel BV2-hEP2 microglia display similar inflammatory regulation by EP2 as cultured primary rodent microglia upon classical activation with LPS, and that phagocytosis and proliferation are suppressed by EP2. RESULTS AND DISCUSSION BV2-hEP2 microglia display a robust inflammatory response to LPS The EP2 receptor alters the expression of a host of pro- and anti-inflammatory mediators in cultured rat primary microglia during classical activation 16. Prolonged EP2 activation also induces microglial apoptosis 35. Here, a novel BV2 cell line that stably expresses human EP2 receptors was created to better explore the role of EP2 in microglia function. Prior to experiments with LPS, qRT-PCR was used to quantify mouse EP2 (mEP2) mRNA. Untransfected BV2 and BV2-hEP2 microglia showed similar low levels of mEP2 with average cycle threshold (CT) values of 34.5 ± 0.7 and 34.8 ± 0.3 from 3 independent experiments, respectively. The expression of mouse EP1, EP2, EP3 and EP4 receptors as well as human EP2 receptors and other inflammatory cytokines was compared in BV2-hEP2 and untransfected BV2 microglia in the presence and absence of LPS. RNA was isolated under resting conditions (no LPS) from three independent cultures. The cycle threshold value for the hEP2 receptor was 27 ± 0.2 for BV2-hEP2 cells and greater than 40 (undetectable by qRT-PCR) for untransfected BV2 microglia (Figure 1A). A CT value of 40 was assigned to genes that were undetectable by PCR as this was determined to be the cutoff limit for detection in our instrument. BV2 and BV2-


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hEP2 cells were incubated in the presence of LPS (100 ng/ml) for 16 hours to induce expression changes of inflammatory mediators and glial markers, since LPS induces inflammatory gene expression changes in cultured rat primary microglia. mRNA levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNFα were all robustly induced upon treatment with LPS to a similar degree in both BV2 and BV2-hEP2 microglia (Figure 1B, C). On the other hand, LPS reduced or did not alter mRNA of the mannose receptor C type 1 (CD206), arginase 1 (ARG1) and chitinase-3-like-3 (YM1) that are markers of M2 microglial activation in untransfected BV2 and BV2-hEP2 microglia (Figure 1C). The expression levels of the three housekeeping genes (Table 2) were unaffected by LPS. Mouse EP1 and EP3 mRNA levels were extremely low or undetectable in BV2 and BV2-hEP2 microglia. The level of EP4 mRNA was high in BV2 and BV2-hEP2 similarly however, LPS exposure reduced EP4 mRNA levels (Figure 1A, B). The mouse EP2 mRNA was also not affected by LPS (34.5 ± 0.7 cycle threshold for BV2 without LPS vs. 34.5 ± 0.8 for BV2 with LPS, n=3, Figure 1). In contrast, only a slight increase in expression of COX-2 (reduced CT value) was observed by 16 hr exposure to LPS in BV2-hEP2 microglia (Figure 1). The mRNA level of the microglial markers, ionized calcium-binding adapter molecule 1 (Iba1) and cluster of differentiation molecule 11B (CD11b) were similar in both BV2 and BV2-hEP2 regardless of whether or not the cells were exposed to LPS (Figure 1A, B). The astrocyte marker, glial fibrillary acidic protein (GFAP), was undetectable in both BV2 and BV2-hEP2 cells (Figure 1A, B). These data show that BV2-hEP2 cells have a high level of human EP2 mRNA but the parent BV2 cells do not, and that both cell lines respond similarly to LPS with cytokines induced to the same degree. Analysis of RT-PCR carried out on mRNA of genes that are associated with microglia phagocytosis revealed that cluster of differentiation molecules 14 (CD14) and 36 (CD36) as well as toll-like receptor 2 (TLR2) and NADPH oxidase 2 (NOX2) were upregulated in the presence of LPS in untransfected BV2 microglia (Figure 1D, E). In the presence of LPS, CD14, TLR2 and NOX2 were also upregulated in BV2-hEP2 microglia (Figure 1D, E).



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EP2 receptors are functional in BV2-hEP2 microglia The EP2 receptor is a G-protein coupled receptor. Binding of the natural agonist PGE2 to EP2 results in activation of a Gαs mediated signaling cascade that involves stimulation of adenylate cyclase, leading to elevated cellular cAMP. BV2-hEP2 microglia were exposed to 1 M EP2 receptor agonists (PGE2, butaprost, ONO-AE1-259-1, CP544326) for 40 min and the change in cellular cAMP was determined by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Compared to vehicle treated cells (control), all EP2 agonists decreased the TR-FRET signal to a similar degree as the adenylate cyclase activator, forskolin (Figure 2A). Selective EP2 agonists ONO-AE1-259-1 and CP544326 had no effect on untransfected BV2 microglia (Figure 2A) and BV2 microglia expressing pcDNA3.1(+) without hEP2 (Supplemental Figure 2C). Unlike untransfected BV2 microglia, the BV2-hEP2 cells displayed an increase in intracellular cAMP that was concentration dependent when exposed to the selective EP2 receptor agonist ONO-AE1-259-1, with an average half maximally effective concentration (EC50) of 0.7 ± 0.1 nM (4 independent experiments). A representative dose response curve is shown in Figure 2B. Similarly, the selective EP2 receptor agonist CP544326 caused an increase in intracellular cAMP in BV2-hEP2 microglia that was concentration dependent (Supplemental Figure 2D). Exposure of BV2-hEP2 microglia to the natural agonist PGE2 also resulted in a similar concentration dependent increase in cellular cAMP with an EC50 of 6 nM (data not shown). The parent BV2 cells were unresponsive to the EP2 agonist up to at least 300 nM (Figure 2B). Functional agonist−antagonist competition assays were performed to determine whether an EP2 antagonist (TG4-155) alters the activation of the EP2 receptor by ONO-AE1-259-1 in BV2-hEP2 microglia. The ONO-AE1-259-1 induced activation of BV2-hEP2 cells was antagonized competitively by TG4-155 (300 nM), causing an EC50 shift of the ONOAE1-259-1 cAMP response on average to 83 ± 15 nM (4 independent experiments), resulting in an average Schild KB of 2.6 ± 0.2 nM. The KB was calculated assuming a linear Schild plot with slope = 1. Thus the potency of TG4-155 as an EP2 antagonist is somewhat higher than that


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measured on endogenous mouse EP2 in primary microglia (KB = 10-14 nM) 16, but similar to that measured in C6 glioma cells overexpressing human EP2 (2.4 nM) 34. A representative shift in the ONO-AE1-259-1 cAMP dose response curve by TG4-155 (300 nM) is shown in Figure 2B. Phase-contrast images of BV2 microglia in the absence and presence of LPS were taken with a Zeiss Axio Observer A1 fluorescence microscope. Under normal culture conditions BV2 microglia displayed an elongated morphology (Figure 2C). Like cultured rat primary microglia, BV2 cells round up during activation by LPS (Figure 2D). Overexpression of hEP2 receptors or pcDNA3.1(+) does not change the morphology of BV2 microglia under normal culture conditions or alter rounding in the presence of LPS (Figure 2E, F; Supplemental Figure 2A, B). The BV2-hEP2 cells display the same high sensitivity to selective EP2 modulators measured by the functional TR-FRET assay at low and high passage numbers whereas the untransfected BV2 microglia show no response to ONO-AE1-259-1 and CP544326, suggesting stable expression of human EP2 receptors in the BV2-hEP2 microglia. Taken together, these data indicate that the human EP2 receptor is functional in BV2-hEP2 microglia and does not alter the morphology of the cells. EP2 activation modulates expression of inflammatory mediators in BV2-hEP2 microglia Rodent primary cultured microglia express the EP2 receptor subtype 16, 18, 37, and incubation with LPS (10 or 100 ng/ml) increased the level of mRNAs encoding EP2 and other inflammatory mediators 16, 35. To explore whether human EP2 activation has a similar effect in BV2-hEP2 cells, BV2-hEP2 microglia were exposed to LPS (100 ng/ml) for 2 hours followed by addition of various concentrations of ONO-AE1-259-1 for 1 hour. Like rat primary cultured microglia, BV2-hEP2 microglia also displayed a change in the mRNA level of a small group of inflammatory mediators (COX-2, IL-1β, TNFα, IL-6, iNOS and mouse EP2) following treatment with LPS. In the presence of LPS, ONO-AE1-259-1 concentration-dependently increased the mRNA level of IL-1β, IL-6, COX-2 and endogenous mouse EP2 (mEP2), but decreased that of



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TNFα and iNOS (Figure 3). IL-1 induction appeared to be most sensitive to EP2 activation, with an EC50 approximately 12-fold lower than that for IL-6 induction. The mRNA level of the same six inflammatory mediators was measured in untransfected BV2 microglia at the maximum concentration of ONO-AE1-259-1 (100 nM) following incubation with LPS (100 ng/ml for 2 hours). There was no effect of this dose of ONO-AE1-259-1 on the mRNA level of the inflammatory mediators in untransfected BV2 microglia (Figure 3 open circles), confirming the action of this agonist on EP2 receptors in BV2-hEP2 microglia. Experiments were performed to determine whether EP2 inhibition prevents the change in the mRNA levels of the inflammatory mediators induced by LPS and ONO-AE1-259-1. BV2hEP2 microglia were stimulated with a near-maximally-effective concentration of ONO-AE1-2591 (30 nM), LPS (100 ng/ml) or a combination of the two for 2 hours. The mRNA levels of IL-1β, IL-6, TNFα, COX-2, iNOS and mEP2 as measured by qRT-PCR were found to be significantly augmented in BV2-hEP2 cells upon treatment with LPS, ONO-AE1-259-1 or their combination (Figure 4). The EP2 agonist alone did not alter IL-6 levels (1.1 ± 0.3 fold of control, n= 4 independent experiments), but LPS enhanced IL-6 mRNA (5.5 ± 1.2 fold of control, n= 4 independent experiments). The action of LPS and EP2 activation appeared to be synergistic on IL-6 induction, reaching a level of 26.3 ± 6.9 fold of control (n= 4; Figure 4). This is similar to their interaction in primary microglia 16. TG4-155, a selective EP2 antagonist (0.3 µM or 1 µM) was applied to the BV2-hEP2 cells in the presence of ONO-AE1-259-1 and LPS. Administration of TG4-155 prevented the change in the expression of IL-1β, IL-6, TNFα, COX-2 and mEP2 induced by the combination of ONO-AE1-259-1 and LPS (Figure 4). Although there was a similar trend for TG4-155 to reverse the induction of iNOS by the combination of ONO-AE1-2591 and LPS, statistical significance was not attained (Figure 4). These data confirm that EP2 modulates the inflammatory response during classical activation in BV2-hEP2 microglia as it does in primary rodent microglial cultures. In particular, EP2 activation increases mRNA levels of IL-1, COX-2 and IL-6, but reduces levels of TNF. However, in contrast to the elevation of


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iNOS levels by EP2 activation in primary microglia 16, EP2 activation reduced iNOS level in BV2-hEP2 cells (Figures 3, 4). EP2 presence but not activation reduces the phagocytic ability of BV2 microglia Like cultured rodent primary microglia, the BV2 microglia also display an ability to phagocytose foreign substances, and phagocytosis is stimulated by LPS 38. Here, experiments were performed to compare the phagocytic ability of untransfected BV2 microglia with BV2hEP2 microglia to determine whether overexpression of EP2 alters phagocytosis. We first determined the fluorescence and flow cytometric characteristics of 1 µm fluorescent-conjugated polystyrene latex microspheres and that of BV2 cells following time dependent incubation with the microspheres in normal culture media. Preliminary experiments were performed with varying concentrations of the microspheres and incubation time to determine the minimum incubation time to obtain near-maximal phagocytic uptake of fluorescent microspheres by BV2 and BV2-hEP2 cells. Fluorescence was measured by flow cytometry 15, 30, 45 and 60 minutes after incubation with the microspheres in LPS activated untransfected BV2 and BV2-hEP2 microglia. On average 30 ± 6% (8 independent experiments) of all BV2 cells took up at least 1 fluorescent bead by 45 minutes (Figure 5A, K) and this appeared to be maximal uptake as the 60 minute exposure resulted in a similar percentage of phagocytic cells. On the other hand, phagocytosis was considerably lower at the 15 and 30-minute incubations. This time-dependent uptake of fluorescent microspheres in untransfected BV2 microglia was consistent with the results of a recent study 38. Flow cytometric analysis revealed a complex distribution of BV2 microglia that phagocytosed the microspheres. On average 70% of BV2 microglia did not exhibit any fluorescence indicating they did not phagocytose the fluorescent latex microspheres (Figure 5G). Of the cells that phagocytosed microspheres a large proportion phagocytosed one bead only, represented by a single peak in the histogram distribution (Figure 5G). The remaining cells phagocytosed two or more microspheres as multiple fluorescence peaks were observed in the histogram (Figure 5G). BV2 microglia were directly visualized individually by



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fluorescence microscopy, and a similar distribution of phagocytic cells found in flow analysis was detected by microscopy (Figure 5A). Incubation with the actin polymerization inhibitor, cytochalasin D (10 µM), for 3-4 hours prior to the fluorescent microsphere application strongly reduced the phagocytic activity of untransfected BV2 microglia (Figure 5I, K). The flow cytometric profile of BV2-hEP2 microglia following incubation with the fluorescent latex microspheres revealed that on average only 9 ± 3% (8 independent experiments) of all BV2-hEP2 cells took up at least 1 fluorescent bead by 45 minutes (Figure 5D). This was significantly lower than untransfected BV2 microglia (Figure 5K) as determined by one-way ANOVA and post hoc Bonferroni tests with selected pairs. Flow cytometric analysis revealed a similar distribution of phagocytic cells for untransfected BV2 and BV2-hEP2 microglia (Figure 5G, H). Fluorescence microscopy images (Figure 5A, D) of the cells and latex beads agreed with the distribution of phagocytic cells by FACS analysis, however the number of cells containing fluorescent microspheres was noticeably lower for BV2-hEP2 microglia (Figure 5D). Although the phagocytic ability of BV2-hEP2 microglia was lower than that of untransfected BV2 cells, Cytochalasin D was able to inhibit BV2-hEP2 phagocytosis (Figure 5J, E). Using a selective EP2 receptor agonist (ONO-AE1-259-1) and antagonist (TG4-155) we determined whether EP2 activation influences phagocytosis. Pre-incubation of the untransfected BV2 or BV2-hEP2 microglia with ONO-AE1-259-1 for 1 hour prior to microspheres did not alter phagocytosis (Figure 5B, E, L; 6 independent experiments for each). Importantly, the EP2 receptor antagonist TG4-155 at 1 M, a concentration that abolishes the effect of saturating concentrations of an EP2 agonist on induction of cAMP (Fig 2B) and cytokine modulation (Fig 4) also did not affect phagocytosis of untransfected BV2 or BV2-hEP2 microglia (Figure 5C, F, L; 6 independent experiments). It is unlikely that homeostatic adjustment to chronic EP2 activation is responsible for lower phagocytic activity in BV2-hEP2 compared to BV2 cells, because incubation of BV2-hEP2 cells for 1 or 16 hr with EP2 agonist or antagonist did not enhance phagocytic activity (Supplemental Figure 3). Taken together, these results confirm the


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phagocytic ability of BV2-hEP2 microglia and indicate that the presence but not activation of the EP2 receptor reduces phagocytosis in BV2-hEP2 cells. EP2 presence but not activation reduces cell proliferation in BV2-hEP2 cells Activation of EP2 promotes cell growth and proliferation of some cancer cells 27-33. Experiments were performed to determine whether EP2 receptor modulation alters BV2-hEP2 microglial growth and proliferation using a bromodeoxyuridine (BrdU) incorporation assay. Preliminary assay optimization experiments resulted in a 50% well confluence on day 3 in culture when BV2 and BV2-hEP2 cells were seeded at 1000 and 2000 cells per well in culture media, respectively. A similar cell density just prior to the incubation with BrdU minimizes the effect of cell density on proliferation rate as BV2-hEP2 microglia lag in early growth compared to untransfected BV2 microglia (Supplemental Figure 1). BV2 and BV2-hEP2 cultures were treated with ONO-AE1-259-1 (3 nM), TG4-155 (2 µM), a combination of ONO-AE1-259-1 and TG4-155 or the vehicle for 72 hours to obtain ~50% well confluence followed by incubation with BrdU (10 µM) for 1 hour. The cells were fixed and stained for BrdU using immunocytochemistry combined with Hoechst labeling to identify proliferating cells using an ImageXpress Micro system. BV2-hEP2 microglia stained positive for BrdU incorporation indicating cell proliferation (Figure 6A). The percent BrdU-positive cells of untransfected BV2 microglia was significantly higher than that of BV2-hEP2 microglia (p = .04, student’s t-test, 4 independent experiments) (Figure 6C). Incubation with ONO-AE1-259-1 (3 nM) appeared to increase the proliferation of BV2-hEP2 microglia to a small extent, but TG4-155 had no significant effect on proliferation compared to vehicle treated cells (Figure 6B, D). A higher concentration of ONO-AE1-259-1 (3 µM) resulted in a similar change in proliferation as 3nM. These results demonstrate that overexpression of the hEP2 receptor slows BV2 microglial proliferation in a manner unrelated to EP2 activation by agonist. Discussion



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Microglia play an important role in maintaining homeostasis in the central nervous system, where they exist in multiple states. During pathological conditions microglia enter an activated state in which they upregulate expression and release of inflammatory cytokines and chemokines. Activated microglia can reduce or exacerbate neuropathological conditions depending on the disease and disease state 12-13. To aid in the investigation of whether activated microglia play a detrimental or beneficial role in the response to injury, studies have made use of primary microglia cultures obtained from rats and mice. The prostaglandin PGE2 mediates brain inflammation 18-21 and the presence of the EP2 receptor reduces microglial phagocytosis 24-26. EP2 receptor signaling pathways regulate classical activation and death of microglia in rat primary microglial cultures 16, 35. Here, we have created a stable microglial cell line that expresses human EP2 receptors (BV2-hEP2) to overcome some of the logistical challenges of working with primary microglia. Our results show that EP2 activation elevates cAMP and influences immune regulation, however the presence of EP2 receptors is sufficient to reduce phagocytic ability and proliferation of BV2-hEP2 microglia; EP2 activation appears to be uninvolved in these processes. Murine BV2 microglia have been used to investigate microglial function since they were created in 1990 39. There is a wealth of literature comparing the characteristics of BV2-microglia to primary rodent microglial cultures as summarized in a review36. Although BV2 microglia share many features with primary rodent cultured microglia, we found a major difference between primary rodent cultured microglia and BV2 microglia, which is that primary rodent microglia in culture express functional EP2 receptors 16, 35 but the parent BV2 microglia do not. Furthermore, endogenous mouse EP2 mRNA was low under basal and activated conditions in parent BV2 cells (Figure 1) 21, 40. Thus a major COX-2 driven inflammatory signaling pathway, that mediated by EP2, is absent in the BV2 cell line. This is an important deficiency considering that EP2 alters microglia activation. Under basal conditions (no LPS), we demonstrated that BV2 and BV2-hEP2 microglia had a nearly identical pattern of expression of inflammatory


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mediators as well as markers of M1 and M2 activation as shown in Figure 1. The expression levels of the inflammatory mediators measured by qRT-PCR were higher in BV2-hEP2 microglia compared to untransfected BV2 microglia when exposed to LPS. This is consistent with the LPS induced inflammatory gene expression changes found in rat primary microglial cultures 16. Elevation of cAMP and modulation of cytokine expression by EP2 activation were similar in BV2-hEP2 and primary microglia 16; both responses were prevented by the EP2 receptor antagonist, TG4-155. We have demonstrated that untransfected BV2 microglia display an increase in intracellular cAMP upon treatment with nonselective EP2 agonists butaprost (1 µM) and PGE2 (1 µM), but not with exposure to ONO-AE1-259 and CP544326 that are highly selective for EP2. The increase in intracellular cAMP in BV2 cells exposed to butaprost and PGE2 is likely not a result of EP2 activation (Figure 2A, B; Supplemental Figure 2C, D). Perhaps butaprost and PGE2 are acting on other Gαs coupled receptors expressed in BV2 microglia. In fact, PGE2 could potentially activate EP4 in BV2 microglia as EP4 mRNA is abundant in parent BV2 as demonstrated in the current study (Figure 1) as well as in a published study 40. Nevertheless, with the recent emergence of new selective EP2 receptor modulators it is imperative to use highly selective agonists and antagonist to demonstrate the effects of EP2 on inflammatory pathways and phagocytosis in BV2-cells. Phagocytosis is an important function of activated microglia, especially in neuropathological conditions in which this process clears dead cells and debris. Here, we show that the phagocytic ability of BV2-hEP2 microglia was lower than in parent BV2 microglia as measured by FACS following incubation with fluorescent latex microspheres. However, the ability of the cells to phagocytose latex microspheres was not altered by EP2 activation, as neither a selective EP2 receptor agonist nor antagonist had any effect on the ability of the cells to phagocytose. Similarly, cultured mouse microglia lacking the EP2 receptor displayed enhanced Aβ phagocytosis compared to cultured microglia obtained from wildtype mice 25. The



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nonselective EP2 agonist butaprost had no effect on phagocytic ability of wildtype mouse microglial cultures consistent with findings in the current study using BV2-hEP2 microglia. The study using EP2 lacking microglia25 could not rule out the possibility that EP2 receptors on wildtype microglia were already saturated by exposure to endogenous PGE2 considering the authors of this study did not have access at the time to a selective EP2 antagonist. Our findings exclude this possibility because a selective EP2 antagonist, TG4-155, was unable to increase phagocytic ability in BV2-hEP2 cells. Thus, the presence of EP2 reduces phagocytic ability. The EP2 receptor protein might bridge directly to scavenger receptors, the actin cytoskeleton or other proteins involved in recognizing and engulfing particles 41. Unfortunately, a selective EP2 receptor antibody is currently unavailable. Elucidation of the subcellular location of EP2 and the mechanism by which EP2 receptor protein regulates phagocytosis by microglia awaits the creation of a selective EP2 antibody. However, our results indicate that pharmacologic inhibition of EP2 is unlikely to enhance phagocytosis by microglia. On the other hand, there is evidence that activation of EP2 by PGE2 suppresses phagocytosis by peritoneal macrophages 42, suggesting that different mechanisms might mediate phagocytosis by microglia and peripheral macrophages. Enhanced expression of COX-2 and increased levels of PGE2 are found in tumor tissues 43-44.

Although many GPCRs can transactivate epidermal growth factor receptor (EGFR) by a

cAMP independent mechanism it was demonstrated that PGE2, via PKA-mediated EGFR activation, can promote cancer cell growth 45. Furthermore, genetic ablation of the EP2 receptor attenuates tumor growth in mouse tumor models and this is accompanied by defective dendriticcell proliferation 17. Here, we have found that BV2 microglia expressing functional human EP2 receptors display reduced proliferation compared to untransfected BV2 microglia, yet this effect was not influenced much by activation or block of EP2. This demonstration of altered cellular proliferation in BV2-hEP2 cells supports the idea that the EP2 receptor regulates microglial


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proliferation in a manner that does not strictly depend on classical EP2 signaling pathways involving cAMP, protein kinase A, Epac, or receptor-activated -arrestin. CONCLUSION BV2-hEP2 microglia display similar features as rodent primary microglia in culture, in that EP2 activation plays a role in immune regulation. By contrast, the presence of EP2 reduces both phagocytosis and to a lesser extent, proliferation. It would be impractical to use primary microglia in high throughput screens designed to identify novel anti-inflammatory compounds that target EP2 receptors. However, BV2-hEP2 microglia could be used to carry out high throughput screening to identify small molecules that alter inflammatory consequences of microglial EP2 activation, and these cells will also be useful for investigating other important functions of microglia.



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MATERIALS AND METHODS Cell culture The BV2 cell line is a well established cell line that is commercially available. The BV2microglial cell line was originally created by infecting primary microglial cultures isolated from the brains of C57BL/6 mice with a v-raf/v-myc oncogene carrying retrovirus (J2) 39. The J2 virus infection immortalized the primary mouse microglia and the immortalized cells retained the same morphological, phenotypical and functional properties of microglia 39. The BV2 microglia were generously provided by Dr. Malu Tansey (Emory University, Atlanta, GA). BV2 microglia were grown and maintained in media containing: Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Invitrogen, Carlsbad, CA), supplemented with 10% (v/v) fetal bovine serum (Invitrogen), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). The cultures were incubated at 37 °C in 95% air/5% CO2, and the culture medium was replaced every 3−4 days with fresh medium. Transfection Murine derived BV2 microglia were transfected with human EP2 (Accession no. AY275471) under control of the CMV promoter in the pcDNA3.1(+) vector (University of Missouri-Rolla cDNA resource center) or the pcDNA3.1(+) vector alone using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. Following transfection the cells were allowed to grow and proliferate for 48 hours prior to incubation in complete media supplemented with 400 µg/mL G418 (Sigma-Aldrich, St. Louis, MO). After a week of incubation in the presence of G418 the culture was subcloned by limiting dilution. BV2hEP2 subclones were expanded from a single cell and maintained in complete medium supplemented with 800 µg/mL G418 at 37 °C in 95% air/5% CO2 for 8 weeks. The culture medium was replaced every 3−4 days with fresh medium and fresh G418. The cells were passaged once per week and the lowest passage number of BV2-hEP2 cells used in experiments was passage 12 (P12), with P35 the highest passage number used. It should be


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noted that expression of the human EP2 in murine BV2-hEP2 microglia is not controlled by the endogenous genomic regulatory sequences found in humans. Nevertheless, human EP2 mRNA and functional protein is abundant in BV2-hEP2 microglia. cAMP TR-FRET assay cAMP was measured with a homogeneous TR-FRET method (Cisbio Bioassay, Bedford, MA). BV2 and BV2-hEP2 microglia were seeded in 384-well plates at 6000 cells per well in 40μL complete medium. After incubation for 48 hours the media was replaced with 10 μl Hank’s Buffered Salt Solution (HBSS) (Fisher Scientific, Hampton, NH) containing the phosphodiesterase inhibitor rolipram (20 μM) for 0.5 h. Cells were then treated with EP2 antagonists or vehicle for 60 min before addition of EP2 agonists at various concentrations (PGE2, ONO-AE1-259-1 or butaprost) or the adenylyl cyclase activator forskolin (100 μM) for 40 min. The cells were lysed with 10 μL lysis buffer containing the FRET acceptor cAMP-d2 and 1 min later 10 μL of lysis buffer with the anti-cAMP-cryptate antibody was added. After incubation at room temperature for 90 minutes the TR-FRET signal was detected by an Envision 2103 Multimode Reader (Perkin Elmer, Waltham, MA). Cryptate excitation was at 337 nm, and emission from cryptate donor and d2 acceptor was measured at 620 nm and 665 nm, respectively, with the dichroic mirror set at 380 nm. All FRET signals were expressed as F665/F620 × 10,000. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated with Trizol and purified using the Zymo Research Quick-RNA miniprep kit according to the manufacturer’s protocol (Genesee Scientific, San Diego, CA) from BV2 and BV2-hEP2 cultures. RNA concentration and purity were measured by a SmartSpec 3000 spectrophotometer (Biorad, Hercules, CA) using the A260 value and the A260/A280 ratio, respectively. First-strand cDNA synthesis was performed with 1 µg of total RNA, 200 units of SuperScript II Reverse Transcriptase (Invitrogen), and 0.5 µg random primers in a reaction volume of 20 µl at 42 ºC for 50 min. The reaction was terminated by heating at 70 ºC for 15 min.



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qRT-PCR was performed by using 8 µl of 10 x diluted cDNA, 0.1-0.5 µM primers (Table 1), and B-R iQ SYBR Green Supermix (Quanta, Gaithersburg, MD) with a final volume of 20 µl in the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). Cycling conditions were as follows: 95 ºC for 2 min followed by 40 cycles of 95 ºC for 15 sec and 60 ºC for 1 min. Melting curve analysis was used to verify specificity of the primers by single-species PCR product. Fluorescent data were acquired at the 60 ºC step. The geometric mean of cycle thresholds for β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phosphoribosyltransferase1 (HPRT1) (Table 2) was used as an internal control for relative quantification of the inflammatory response to LPS in BV2 and BV2-hEP2 cells (Figure 1). Samples without cDNA template served as the negative controls. Analysis of quantitative real time PCR data was performed by subtracting the geometric mean of the three internal control genes from the measured cycle threshold value obtained from the log phase of each amplification curve of each gene of interest. The fold increase of each gene of interest was estimated for each treatment relative to the amount of RNA found in the untransfected BV2 microglia using the 2ΔΔCT method 46. All conditions for qRT-PCR were the same. Cytokine induction assay BV2 and BV2-hEP2 cells were grown overnight on poly-D-lysine (Sigma-Aldrich) coated 12 wells plates at 200,000 cells per well in culture media. The cells were exposed to the compounds by adding TG4-155 (0.3 µM or 1 µM) for 1 hr, ONO-AE1-259-1 (30 nM) for an additional hour and subsequently LPS (100 ng/ml) for 2 hrs. All compounds were dissolved in DMSO and diluted in media just prior to cell treatment. Following incubation all media was removed from the wells and the cells were subjected to RNA extraction and purification using Trizol and the Zymo Research Quick-RNA miniprep kit according to the manufacturer’s protocol (Genesee Scientific). First-strand cDNA synthesis, qRT-PCR and analysis was performed as described above using the primers listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a single internal control for relative quantification to


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determine whether EP2 activation modulates expression of inflammatory mediators in BV2hEP2 microglia (Figure 3, 4). PCR gene expression data are presented as the mean fold change of each gene of interest in the compound treated groups compared to vehicle. Phagocytosis assay with fluorescent latex microspheres Prior to the cell-based phagocytosis assay the fluorescence profiles of the fluorescent polystyrene microspheres (Sigma-Aldrich) alone and BV2 cells alone were first determined by flow cytometry. The microspheres gave the greatest signal with the Phycoerythrin (PE) filter (578 nm peak emission). Phycoerythrin is a red photosynthetic pigment found in red algae. For the cell based assay BV2 and BV2-hEP2 cells were plated in a 24-well plate at 200,000 cells per well in culture media (supplemented with 800 µg/ml G418 for BV2-hEP2). Twenty-four hours later LPS (100 ng/ml) was added and the cells were cultured overnight (16 hours). The next morning BV2 cells were pretreated with cytochalasin D (10 μM) in some wells for 2 hours at 37°C to specifically inhibit actin-dependent phagocytosis. The cells were incubated in the presence of the EP2 receptor agonist (ONO-AE1-259-1), the EP2 antagonist (TG4-155), vehicle only or a combination of ONO-AE1-259-1 and TG4-155 (added at the same time) for 1 hour or 16 hours. Fluorescent polystyrene microspheres (0.002%, 5 µl) were added to the culture media and the cells were incubated for 45 min. Following incubation with the microspheres the media was removed and 1 ml of cold FACS buffer (containing: 1x PBS, 5 mM EDTA, 1% sodium azide and 1% bovine serum albumin) was added to each well, and the cells were harvested by gentle pipetting (3-4 times). The cells were finally collected in 5 ml polystyrene flow cytometry compatible tubes. Greater than 90% cell viability was confirmed using optimal gating of live cells in FlowJo with the forward and side scatter (FSC and SSC) profiles. Cells were analyzed within 1 hour of collection on a FACSCanto II (BD Biosciences, Franklin Lakes, NJ) running FACS Diva6.0 (BD Biosciences). Data were analyzed with FlowJo software (FlowJo, Ashland, OR). BrdU cell proliferation assay



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Prior to the bromodeoxyuridine (BrdU) incorporation assay the growth of untransfected BV2 and BV2-hEP2 microglia was determined. Untransfected BV2 and BV2-hEP2 microglia were incubated at 1000 cells/well in a 96-well clear-bottom plate in normal BV2 growth media and media supplemented with G418 (800 µg/ml) for BV2-hEP2. The growth of the cells was measured spectrophotometrically by taking the absorbance at 450 nm daily using a Spectramax M2 plate reader (Molecular Devices, San Jose, CA). The absorbance readings were normalized to the measurement taken on day 0. Each treatment was carried out in triplicate on the plate and the experiment was repeated twice to obtain three independent experiments. The BrdU incorporation assay is often used as a measure of cell proliferation, as BrdU is incorporated into newly synthesized DNA of replicating cells. Anecdotally, we first noticed that within 24 hours of plating the untransfected BV2 cells appeared to divide more rapidly than BV2hEP2 cells. BV2 and BV2-hEP2 microglia were plated in a 96 well plate, 1000 and 2000 cells per well. The next day ONO-AE1-259-1 (3 nM), TG4-155 (2 µM), ONO-AE1-259-1 (3 nM) + TG4-155 (2 µM) and vehicle in culture media were added to labelled wells and cells were incubated in the presence of these compounds for 72 hours. On day 4 the media was replaced with fresh complete media containing BrdU (10 µM). Following a 1 hour incubation in the presence of BrdU the cells were washed three times with PBS (1x) then fixed in 4% paraformaldehyde for 10 minutes at room temperature. Paraformaldehyde was removed and the cells were exposed to 0.2% triton-X 100 in PBS for 20 minutes followed by three washes in PBS. Acidolysis was induced by incubating the cells in 1N HCl on ice for 10 minutes followed by 2N HCl at room temperature for an additional 10 minutes. PBS (1x) was added to dilute the HCl at room temperature for 20 minutes then cells were washed three times with PBS containing 0.2% triton-X 100. The cells were incubated in blocking buffer (containing: 1% BSA, 20% normal serum, 0.5% Triton X-100 in PBS) at room temperature for 2 hours. The blocking buffer was replaced with an antibody dilution solution (ADS, containing: 0.5% Triton X-100, 0.1% gelatin, 0.1% sodium azide in PBS) with the primary antibody (goat anti-BrdU, at 2 µg/mL,


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Roche, Basel, Switzerland) and incubated overnight at 4°C. The cells were washed three times with ADS followed by incubation in ADS containing an Alexa Fluor goat anti-rabbit 488 fluorophore, diluted at 1:1000 (Molecular Probes, Eugene, OR) and the blue-fluorescent Hoechst 33342 dye (at 10 µg/mL, Molecular Probes) for 2 hours at 25°C. Hoechst is used to label all DNA as it binds to the minor groove of double stranded DNA. The cells were washed three times with PBS. Fluorescence images were taken using an ImageXpress Micro system equipped with a FITC filter (Molecular Devices, San Jose, CA). The number of positive labelled cells was counted in 4 fields per well using Metamorph 6.2.1.704 software (Molecular Devices). Proliferation was quantified in Metamorph as the average number of BrdU positive cells (green fluorescence) divided by the average number of total cells (Hoechst positive cells). For each independent experiment a total of three wells were sampled per treatment group. On average 96 total BV2 and 702 total BV2-hEP2 microglia were counted per experiment. The experiments were repeated three times using different cultures to obtain four independent experiments. Chemicals and drugs PGE2, butaprost, rolipram, lipopolysaccharide, BSA, Cytochalasin D and forskolin were purchased from Sigma-Aldrich.

The EP2 receptor antagonist TG4-155 was synthesized in our

laboratory as previously reported 34, 47-48. TG4-155 was

>97% pure as analyzed by nuclear

magnetic resonance (NMR), liquid chromatography mass spectrometry (LC/MS) and elemental composition. ONO-AE1-259-1 was generously provided by ONO Pharmaceuticals (Osaka, Japan). The EP2 receptor selective agonist CP544326 compound was purchased from Cayman chemical. The selectivity of ONO-AE1-259-1 was shown using both binding assays and functional measurement of cAMP in Chinese hamster ovary cells (CHO) individually expressing the mouse EP1, EP2, EP3 and EP4 49. Data analysis and statistics Concentration−response curves were fitted using Origin 7.0 (Origin-Lab, Northampton, MA) to a four-parameter logistic equation to determine EC50 or IC50 values. Statistical analysis



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of the TR-FRET, phagocytosis and cell proliferation assays was performed using GraphPad Prism software with one-way ANOVA and post hoc Bonferroni with selected pairs or by unpaired t-test (BRDU incorporation). For the cytokine induction assay, ΔΔCT values were used for statistical analysis while fold changes are represented in the graphs. Repeated measures ANOVA with Holm-Sidak multiple comparisons test for post hoc analysis was performed using GraphPad Prism. Differences were considered to be statistically significant if p < 0.05. A Grubb’s test was performed in GraphPad to identify outliers; none were found. All data are presented as mean ± SEM. ABBREVIATIONS: PGE2, prostaglandin-E2; EP2, prostaglandin-E2 receptor 2; SE, GFAP, glial fibrillary acidic protein; Iba1, ionized calcium binding adaptor molecule 1; CA1, CT, cycle threshold; COX-2, cyclooxygenase 2; qRT-PCR, quantitative real time polymerase chain reaction; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; IL-1β, interleukin -1β, TNFα, tumor necrosis factor alpha, IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; HPRT1, hypoxanthine phosphoribosyltransferase 1; CD206, mannose receptor C type 1 (MRC1); ARG1, arginase 1; YM1, chitinase-3-like-3; Veh, vehicle; TG4-155, potent and selective EP2 receptor antagonist; AD, alzheimer’s disease; cAMP, cyclic adenosine monophosphate; BV2, and immortalized murine microglial cell; BV2-hEP2, and immortalized murine microglial cell stably expressing the human EP2 receptor; LPS, lipopolysaccharide; TLR, toll-like receptor; Epac, exchange protein directly activated by cAMP; EGFR, epidermal growth factor receptor AUTHOR CONTRIBUTIONS: Rojas, Dingledine and Ganesh participated in the research design. Rojas, Banik, Chen and Flood conducted experiments and performed data analysis. Rojas, Banik, Chen, Dingledine and Ganesh wrote or contributed to the writing of the manuscript. FUNDING SOURCES AND INTERESTS This work was supported by NIH U01 AG052460 (TG), R21 NS10167 (TG) and R01 NS097776 (RD). The authors declare no competing financial interests. ACKNOWLEDGEMENTS: We thank Dr. Malu Tansey for providing the murine BV2 microglia and ONO Pharmaceuticals for kindly providing ONO-AE1-259-1. We thank Dr. Yuhong Du in the Emory Chemical Biology Discovery Center for technical help with cell proliferation assays. We thank Drs. Lawrence Boise and Shannon Matulis in the Emory Winship Cancer Institute for technical help with FACS and the phagocytosis experiments. We thank Ms. Chunxiang Jiang for help with cell growth assays. SUPPORTING INFORMATION: Supplemental Figure 1: growth rates of BV2 and BV2-hEP2 microglia


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Supplemental Figure 2: the effect of BV2 expressing pcDNA3.1(+) and the concentration response of CP544326 on BV2-hEP2 microglia Supplemental Figure 3: the effect of prolonged exposure to ONO-AE1-259 and TG4-155 on the phagocytosis of fluorescent latex microspheres in activated BV2 and BV2-hEP2 microglia



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FIGURE LEGENDS Figure 1. Induction of inflammatory mediators in BV2-hEP2 microglia following LPS administration. A, Basal expression of housekeeping genes (β-actin, GAPDH and HPRT1), glial markers (GFAP, Iba1, CD11b) and inflammatory mediators (IL-1β, IL-6, TNFα, COX-2 and mEP2) was similar in untransfected BV2 and BV2-hEP2 microglia as determined by the cycle threshold (CT) value following qRT-PCR. The level of mouse EP2 mRNA was low in BV2 parent cells. B, Most of the inflammatory mediators underwent induction following LPS treatment (100 ng/ml for 16 hours). Transcripts with strong expression changes in untransfected BV2 and BV2hEP2 microglia following LPS treatment are represented by blue font. The astrocyte protein GFAP was undetectable in both untransfected BV2 and BV2-hEP2 microglia in the presence or absence of LPS. Symbols represent mean and SEM. The slope of the lines fitted to the data are 1.03 in the absence of LPS, and 0.95 in the presence of LPS. Arrows indicate the detection limit of our assay (CT = 40). N = 3 independent cultures. hEP2 = human EP2. mEP2 = mouse EP2. C, untransfected BV2 and BV2-hEP2 microglia were incubated in the presence and absence of LPS (100 ng/ml) for 16 hours. mRNA levels of inflammatory mediators (COX-2, IL6, TNFα and IL-1β) and markers of the M2 phase of microglial activation (CD206, ARG1 and YM1) were measured by quantitative real time PCR. The mRNA changes were normalized to the mean of the vehicle treated BV2 (white bars in C) and BV2-hEP2 microglia (solid gray bars in D). Data are expressed as mean ± SEM (error bars), n = 3 independent experiments. The fold change is plotted but the ΔΔCT values were used for statistical analysis. * p

A novel microglia cell line expressing the human EP2 receptor

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