Prostaglandin E2 Receptor Subtype 2 Regulation of Scavenger Receptor CD36 Modulates Microglial Ab42 Phagocytosis Xianwu Li, Erica Melief, Nadia Postupna, Kathleen S

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provided by Elsevier - Publisher Connector The American Journal of Pathology, Vol. 185, No. 1, January 2015

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NEUROBIOLOGY

Prostaglandin E2 Receptor Subtype 2 Regulation of Scavenger Receptor CD36 Modulates Microglial Ab42 Phagocytosis Xianwu Li, Erica Melief, Nadia Postupna, Kathleen S. Montine, C. Dirk Keene, and Thomas J. Montine

From the Department of Pathology, University of Washington, Seattle, Washington

Accepted for publication September 18, 2014. Recent studies underline the potential relevance of microglial innate immune activation in Alzheimer disease. Primary mouse microglia that lack prostaglandin E2 receptor subtype 2 (EP2) show decreased Address correspondence to Xianwu Li, Ph.D., Department innate immune-mediated neurotoxicity and increased amyloid b (Ab) peptide phagocytosis, features that of Pathology, University of were replicated in vivo. Here, we tested the hypothesis that scavenger receptor CD36 is an effector of EP2- Washington, Box 359645, regulated Ab phagocytosis. CD36 expression was 143-fold greater in mouse primary microglia than in Seattle, WA 98104. E-mail: primary astrocytes. Three different means of suppressing EP2 signaling increased and an agonist of EP2 [email protected]. decreased CD36 expression in primary wild-type microglia. Activation of Toll-like receptor (TLR) 3, TLR4, and TLR7, but not TLR2 or TLR9, reduced primary microglial CD36 transcription and cell surface CD36 protein and reduced Ab42 phagocytosis as well. At each step, the effects of innate immune activation on CD36 were reversed by at least 50% by an EP2 antagonist, and this partial rescue of microglia Ab42 phagocytosis was largely mediated by CD36 activity. Finally, we showed in hippocampus of wild-type mice that innate immune activation suppressed CD36 expression by an EP2-dependent mechanism. Taken together with results of others that found brain clearance of Ab peptides and behavioral improvements mediated by CD36 in mice, regulation of CD36-mediated Ab phagocytosis by suppression of EP2 signaling may provide a new approach to suppressing some aspects of Alzheimer disease pathogenesis. (Am J Pathol 2015, 185: 230e239; http://dx.doi.org/10.1016/j.ajpath.2014.09.016)

Observational studies in patients and experimental studies have reproducibly shown that nonselective COX or COX- that use a variety of model systems have concluded that 2eselective inhibition by nonsteroidal anti-inﬂammatory affected regions of the brain in Alzheimer disease (AD) drugs (NSAIDs) can suppress amyloid b (Ab) accumulation experience a proinﬂammatory, pro-oxidative state.1 Indeed, and associated behavioral changes in transgenic mouse models recent studies have highlighted several genes involved in the of AD10 and can reduce the risk of AD dementia in people by innate immune response whose variants are associated with approximately 60%.11 Together these results suggest that increased risk of AD, especially the TREM2 gene.2e4 TREM2 NSAIDs may effectively suppress some aspect of what is now encodes the triggering receptor expressed on myeloid cells-2, appreciated to be early steps in AD pathogenesis.12 These a protein expressed by myeloid lineage cells, including compelling experimental model and human data formed the microglia in brain, which can function as a receptor for rationale for multiple clinical trials. Two clinical trials targeted phagocytosis,5 modulate the innate immune response in what is now understood to be relatively advanced AD: one with brain,6,7 and diminish immune response through several NSAIDs in AD dementia and one with NSAIDs in mild signaling pathways, including suppression of Toll-like re- cognitive impairment, a diagnostic group enriched for pro- ceptor (TLR) signaling.8,9 dromal AD; both failed.13,14 A third trial targeted early-stage The prostaglandin (PG) pathway, a cascade initiated by enzyme-catalyzed synthesis of PGH2 from arachidonic acid by Supported by NIH grants ES016754, ES007032, AG005136, and the cyclooxygenases (COXs), COX-1 or COX-2, is an essential NS062684; and the Nancy and Buster Alvord Endowment. component of the innate immune response. Multiple studies Disclosures: None declared.

AD,15 similar to the epidemiologic cohorts, but was terminated Laboratories (Logan, UT). Papain and DNase I were from because of toxicity, likely related to perturbation of prostacyclin Worthington Biochemical (Lakewood, NJ). Butaprost, 16e18 and thromboxane metabolism. Despite these disap- NS-398, 17-phenyl trinor PGE2, CAY10598, ZK118182, pointing therapeutic outcomes, some data suggest pharmaco- 17-phenyl trinor PGF2a, carbaprostacyclin, U-46619, AH dynamic effects of NSAIDs in the central nervous system of 6809, and CD36 blocking antibody (Clone JC63.1) were patients with AD.19,20 from Cayman Chemical Company (Ann Arbor, MI). We and others have sought to disentangle the potentially Dibutyryl cAMP, forskolin, tribromoethanol (Avertin), beneficial effects of NSAIDs in early AD pathogenesis from and polyinosinic-polycytidylic acid (PIC; TLR3 ligand) toxic effects by focusing on specific PG receptors distal in the were from Sigma-Aldrich (St. Louis, MO). Pam3CSK4 signaling cascade rather than proximal COX inhibition.21e23 (TLR2 ligand), loxoribine (TLR7 ligand), and CpG (TLR9 PGE2 is the principal proinflammatory PG, and increased ligand) were from Invivogen (San Diego, CA). Lipo- PGE2 concentrations in cerebrospinal fluidhavebeenfoundin polysaccharide (TLR4 ligand) was purchased from Cal- patients with AD,24 particularly early in the disease process.25 biochem (La Jolla, CA). pHrodo fluorogenic dye was from In addition, the inducible form of PGE2 synthase is increased in Invitrogen (Carlsbad, CA). b Amyloid 1-42 (Ab42)was AD brain and is a key component in Ab-mediated neurotox- from American Peptide Company (Sunnyvale, CA). icity in a mouse model of AD.26 We, therefore, have focused on PGE2 and its functionally antagonistic receptor subtypes, EP1 Animals to EP4, which are widely expressed in central nervous system cells, including microglia, astrocytes, and neurons.21 Primary C57BL/6 mice and CD36/ (B6.129S1-Cd36tm1Mfe/J) microglia from mice homozygous deficient for the EP2 mice were purchased from The Jackson Laboratory (Bar gene (PTGER2;EP2/ ) have the highly desirable dual Harbor,ME).EP2/ mice were a gift from Dr. Richard phenotype of suppressed TLR-activated microglia-mediated Breyer (Vanderbilt University, Nashville, TN). The neurotoxicity that depends on the adaptor protein DOCK2 and animals were maintained in a specific pathogen-free enhanced microglial phagocytosis of Ab that is DOCK2 environment. All procedures were approved by the e independent.27 29 These studies show that microglial EP2 is University of Washington Institutional Animal Care and necessary for lipopolysaccharide-induced microglia-mediated Use Committee. neurotoxicity in microgliaeneuron cocultures through regulation of inducible nitric oxide synthase and COX-2 and subse- Stereotactic Intracerebroventricular Injection quent neurotoxic cytokine production, including IL-1b production,27 and indicate EP2-dependent neurotoxicity in Ten-week-old male mice were anesthetized with isoflurane EP2 / microglia, with reduced paracrine neurotoxicity in inhalation and placed in a stereotactic instrument. After the response to aggregated Ab in neuronemicroglia cocultures.28 skull was exposed through skin incision, a cranial window Recently, these findings were confirmed in mice with condi- (2 to 3 mm) on the left lateral ventricle was opened by tional deletion of EP2 in myeloid lineage cells and in bone drilling with a small burr bit. Five microliter of reagent marrow transplant experiments.30,31 was injected over a period of 5 minutes by using a Microglial phagocytosis is mediated largely by a class of Hamilton microsyringe; injections were 200 ng of AH surface proteins called scavenger receptors. CD36, a type B 6809, 50 ng of PIC, AH 6809 plus PIC, or vehicle. After scavenger receptor originally identified as a receptor for long- injection, the mice were placed on an isothermal pad and chain fatty acids and oxidized low-density lipoprotein, is a continuously observed until recovery. Twenty-four hours microglial binding site for Ab,32 facilitates intracellular nucle- after injection, the mice were sacrificed by i.p. injection of ation of Ab peptides into fibrils,33 and mediates some of the tribromoethanol and perfused with ice-cold phosphate- biological effects of Ab peptides, including innate immune buffered saline, and brain tissues were rapidly harvested e activation and oxidative stress.34 36 CD36 is expressed on for analysis. microglia in AD brains.34 CD36 overexpression in human brain correlates with b-amyloid deposition but not with AD; it is Cell Culture and Exposure undetectable in healthy brains without Ab deposition.37 We are unaware of any data to link the specific PG receptors with CD36 Primary microglia and astrocytes were isolated from brains function. Here, we tested the hypothesis that EP2 suppression of of newborn mice and cultured as previously described.38,39 Ab phagocytosis is mediated, at least in part, by CD36. Medium concentrations of PGE2 were measured by ELISA exactly as described previously.38 Cells were incubated with Materials and Methods the compounds described at the indicated concentrations and times. We note that, although EP antagonist AH 6809 Materials has nearly equal affinity for EP1 and DP1 in human tissue,40 it has been shown to be selective for EP2 in mouse tissue.41 Dulbecco’s modified Eagle’s medium/F-12 medium and EP agonist butaprost was used at 20 mmol/L, based on fetal bovine serum were purchased from Hyclone previous studies that showed effects up to 100 mmol/L.42,43

The American Journal of Pathology - ajp.amjpathol.org 231 Li et al

Quantitative Real-Time PCR presented in the text or figures were corrected for multiple comparisons when appropriate with the method of Bonferroni. Total RNA was extracted from primary microglia, astrocytes, or brain tissues by using RNeasy Mini Kit (Qiagen, Valencia, Results CA). Reverse transcription was performed with Omniscript RT Kit (Qiagen). TaqMan probes and primers (Mm01135198_m1 Microglia Express Much More CD36 than Astrocytes for CD36, fMm99999915_g1 for GAPDH) were purchased from Applied Biosystems (Carlsbad, CA). PCR were per- Microglia, and less so astrocytes, are cellular effectors of formed on an Applied Biosystems ViiA 7 Real-Time PCR innate immune response in the cerebral cortex. CD36 System by using the relative quantitative method. expression in primary wild-type (WT) cultures of microglia and astrocytes prepared from the same five mice was Flow Cytometric Analysis of Cell Surface CD36 in determined by quantitative real-time PCR with each sample Primary Microglia normalized to endogenous control GAPDH. CD36 expression in microglia was on average 143-fold greater in Microglia were preincubated with 10 mg/mL of anti-mouse microglial than in astrocytes (P < 0.0001). For this reason, CD16/CD32 antibody (BD Biosciences, San Jose, CA) for 5 subsequent experiments focused on microglia. minutes on ice to block Fc receptoremediated binding. The cells were then incubated with 5 mg/mL of phycoerythrin- Down-Regulation of CD36 Expression by Specific TLR conjugated CD36 or mouse IgA control (Santa Cruz Activation Biotechnology, Inc., Santa Cruz, CA) for 30 minutes on ice in the dark. Thereafter, microglia were washed twice with Because many endogenous activators of innate immune phosphate-buffered saline and resuspended in 4% para- response, including Ab,46e49 act through TLRs and because formaldehyde for fixation. The fluorescence intensity was one function of TREM2 is to regulate TLR signaling,8 we determined by BD FACScan flow cytometry. measured primary WT mouse microglia CD36 expression after exposure for 18 hours to specific TLR activators that 38 Ab42 Phagocytosis in Primary Microglia are known to activate microglia. Activators of TLR3, TLR4, and TLR7, but not TLR2 or TLR9, suppressed Microglia were incubated with 5 mmol/L pHrodo-labeled microglia expression of CD36 (Figure 1A). A seven-point Ab42 for 2 hours at 37 C. The nonspecific signal was time series (Figure 1B) showed no change in CD36 determined by incubation of microglia with the same con- expression after 2 hours of PIC exposure (1.03 0.05 centration of labeled Ab42 for 2 hours at 4 C. Thereafter, CD36 mRNA compared with time 0), but after 4 hours of microglia were washed three times in phosphate-buffered exposure CD36 mRNA was reduced to 0.74 0.03 and saline and fixed in 4% paraformaldehyde. The samples continued to decrease linearly to 0.16 0.03 at 18 hours were analyzed by LSRII flow cytometry (BD Biosciences) (R2 Z 0.95, P < 0.0001). A similar time course for medium with a 561-nm excitation laser. PGE2 was approximately complementary with 23 2 pg/mL at time 0 and increased to 2199 64 pg/mL at 18 CD36 Expression in CHO Cells hours (P < 0.0001). Other scavenger receptors also were altered by PIC incubation for 18 hours: microglial expres- Human CD36 was expressed in Chinese hamster ovary (CHO) sion of SR-A1 was increased 3.1- 0.7-fold (P < 0.01) and cells as described by others.44 Briefly, cells were maintained in SR-B1 was decreased 0.25- 0.06-fold (P < 0.001) Ham’s F-12 medium that contained 10% fetal bovine serum compared with vehicle-exposed cultures. and penicillin/streptomycin. Human full-length CD36 cDNA was obtained from GE Healthcare (Lafayette, CO) and cloned Regulation of CD36 Expression by EP2 into the expression vector pIRES2-EGFP at EcoRI and XbaI sites. The recombinant vector or empty vector was transfected We next surveyed specific agonists to prostanoid receptors EP, into CHO cells by Lipofectamine 2000 according to the DP, FP, IP, and TP to determine which, if any, specificproduct manufacturer’s protocol. The expression of CD36 was of COX might contribute to innate immune-mediated reduc- confirmed by quantitative PCR with Taqman expression tion in CD36 expression. Agonists of EP2 or EP4 mimicked assay. Two days after transfection, cells were collected for the effect of innate immune activation by significantly phagocytosis assay and inhibition with sulfosuccinimidyl reducing CD36 expression in primary WT mouse microglia, oleate (SSO) as described by others.45 whereas agonists of EP1 or TP had a functionally antagonistic effect; agonists of FP, IP, and DP had no significant effect on Statistical Analysis CD36 at known effective concentrations (Figure 2A).39 In contrast, a COX-2eselective inhibitor, an EP2 antagonist, and Statistical analyses were performed with GraphPad Prism 5 EP2/ microglia had progressively increasing microglial (GraphPad Inc., San Diego, CA) with a set to 0.05. All P values CD36 expression. Activation of the innate immune response

232 ajp.amjpathol.org - The American Journal of Pathology EP2-Suppressed Phagocytosis via CD36

Figure 1 Effects of TLR activators on CD36 mRNA expression. A: WT mouse primary microglia were treated with Veh or 1 mg/mL activator of TLR2 (Pam3CSK4), 20 mg/mL TLR3 (PIC), 1 mg/mL TLR4 (lipopolysaccharide), 1 mmol/L TLR7 (loxoribine), or 1 mmol/L TLR9 (CpG) for 18 hours. RNA was extracted for quantitative real-time PCR. CD36 mRNA values in each sample were normalized to endogenous control GAPDH. B: WT mouse primary microglia were treated with 20 mg/mL PIC for the indicated times. CD36 mRNA was assessed by quantitative real-time PCR, and results were normalized to GAPDH. Medium PGE2 was assayed by ELISA. Data are expressed as means SEM. n Z 3 per group. ***P < 0.001 for Bonferroni- corrected repeated pair comparisons with vehicle (A) or with value at time 0 (B). PGE2, prostaglandin E2; PIC, polyinosinic-polycytidylic acid; TLR, Toll-like receptor;Veh,vehicle;WT,wild-type. with agents speciﬁc for TLR3, TLR4, or TLR7 each reproducibly decreased microglial CD36 expression, an effect that was mostly reversed for each TLR activator with an EP2 antagonist (AH 6809) (Figure 2B); although reversal by AH 6809 was greatest in the context of TLR3 activation. A se- Figure 2 Effects of eicosanoid receptors with and without innate immune lective antagonist of EP4 is not commercially available.50,51 activation on CD36 mRNA expression by mouse primary microglia. RNA was / EP2 and EP4 are stimulatory G-protein-couple receptors, but isolated from cultures prepared from wild-type mice (dark bars) or EP2 mice e (light bar) after 18 hours of incubation. Results from quantitative real-time PCR because EP2 also can activate G-protein independent second were normalized to endogenous control GAPDH. A: Microglia exposed to vehicle 52 messenger signaling, we tested the effect of directly or 5 mmol/L agonists of EP1 (17-phenyl trinor PGE2), 20 mmol/L EP2 (butap- manipulating cAMP signaling on CD36 expression; dibutyryl rost), 2.5 mmol/L EP4 (CAY10598), 10 mmol/L DP (ZK118182), 10 mmol/L FP cAMP or forskolin decreased CD36 mRNA values by the (17-phenyl trinor PGF2a), 10 mmol/L IP (carbaprostacyclin), or 5 mmol/L TP e same magnitude as butaprost alone (Figure 2C). These data (U-46619). B: Microglia exposed to vehicle or 20 mmol/L COX-2 selective inhibitor (NS398), 20 mmol/L EP2 antagonist AH, and TLR activators (PIC for show that multiple eicosanoid products of COX exert tonic TLR3 at 20 mg/mL, LPS for TLR4 at 1 mg/mL, or loxoribine for TLR7 at 0.5 mmol/L) control over CD36 expression in mouse primary microglia and with or without 20 mmol/L AH. C: Microglia exposed to 20 mmol/L butaprost, 250 that among these EP2 or EP4 activation suppresses CD36 mmol/L db cAMP, or 10 mmol/L forskolin. Data are expressed as means SEM. expression to an amount matched by direct activation of cAMP n Z 3to12pergroup.*P < 0.05, **P < 0.01, and ***P < 0.001 for Bonferroni- y < yy < second messenger signaling. Finally, results by using an corrected repeated pair comparisons with vehicle; P 0.05 and P 0.001 for Bonferroni-corrected paired comparisons between two experimental groups. AH, antagonist show that EP2 signaling is responsible for most AH 6809; COX-2, cyclooxygenase 2; db cAMP, dibutyryl cAMP; EP2/,homo- reduced microglial CD36 expression with TLR-activated zygous EP2 deﬁcient; LPS, lipopolysaccharide; PGE2, prostaglandin E2; PIC, innate immune response. polyinosinic-polycytidylic acid; TLR, Toll-like receptor; Veh, vehicle.

The American Journal of Pathology - ajp.amjpathol.org 233 Li et al

CD36 expression is transcriptionally regulated, but we effects on Ab42 phagocytosis. We quantified phagocytosis of nevertheless sought to validate the changes observed in Ab42 labeled with a pH-sensitive dye (pHrodo) and under the microglia CD36 mRNA expression with changes in CD36 same conditions that affected CD36 mRNA and cell surface protein. Flow cytometric analysis of primary WT mouse protein values (shown in Figures 2 and 3). Butaprost microglia labeled with phycoerythrin-conjugated anti-CD36 significantly decreased Ab42 phagocytosis in WT microglia, antibody; 24 hours of exposure to butaprost, an EP2 agonist, validating previous experiments from our laboratory that / significantly reduced CD36 cell surface protein to values used EP2 microglia with synthetic Ab42 and human that were proportionate to mRNA reduction (Figure 3). brain-derived Ab peptides (Figure 4A).28,30,53 TLR3 acti- Conversely, exposure to AH 6809 increased CD36 cell vation from PIC exposure significantly reduced Ab42 surface protein values. These results are consistent with the phagocytosis by WT microglia, and this immune suppression known transcriptional regulation of CD36 protein and was significantly but partially reversed by AH 6809, paral- indicate that changes in microglia cell surface CD36 protein leling the effect of PIC on CD36 expression and indicating follow the same pattern as CD36 mRNA in response to an that EP2 activation accounted for approximately one-half of agonist or antagonist of EP2. PIC-suppressed Ab42 phagocytosis. CD36 inhibitor SSO reversed this rescue, consistent with CD36-dependent phagocytosis. Because SSO may have actions in addition Inhibition of CD36-Dependent Phagocytosis by EP2 to inhibiting CD36, we sought to validate this finding with Activation three different experiments. In the first, we used AH 6809 (coincubated with control IgA) to achieve increased Ab / 42 Our next series of experiments used primary WT and CD36 phagocytosis (P < 0.01) that paralleled increased CD36 mouse microglia to investigate whether CD36 mediated EP2 expression (Figure 4B); this prophagocytic effect of AH 6809 was completely blocked by a CD36 neutralizing antibody. Consistent with this, CD36 blocking antibodies did not further inhibit butaprost-treated cells. In the second set of experiments, we compared phagocytosis in microglia prepared from WT and CD36/ mice (Figure 4C). CD36/ microglia had reduced Ab42 phagocytosis compared with WT microglia (P < 0.001). Importantly, increased Ab42 phagocytosis seen with exposure to AH 6809 was ablated in CD36/ microglia, whereas butaprost had no significant effect on Ab42 phagocytosis in these cells. In combination, results from WT microglia with the use of SSO or a CD36- neutralizing antibody and from CD36/ microglia support the conclusion that the effects of EP2 activation on Ab42 phagocytosis are largely mediated by CD36. Finally, we transfected CHO cells with human CD36 as described by 44 others. Vector-transfected CHO cell uptake of Ab42 was defined as 100%. Expression of human CD36 significantly increased uptake of Ab42 by CHO cells to 182% 6% of vehicle-transfected control (P < 0.001), similar to what has been reported previously by others.44 Also validating the results of others,45 exposure to SSO by using the same method as described above for mouse primary microglia completely inhibited Ab42 uptake by human CD36- Figure 3 Effects of TLR3 activator and prostaglandin EP2 signaling on expressing CHO cells to 102% 9% of vehicle-transfected CD36 protein. WT microglia were treated with 20 mmol/L butaprost, 20 control; Ab42 uptake by vehicle-transfected CHO cells was mmol/L AH, 20 mg/mL PIC, or a combination of PIC and AH for 24 hours. not significantly altered by SSO (94% 8% of vector- CD36 protein on cell surface was assessed by staining with PE-labeled anti- transfected cells without SSO). CD36 antibody or isotype-matched PE-IgA (isotype control). A: Represen- tative histogram from flow cytometric assay of CD36 immunofluorescence. B: Geometric means of fluorescence intensity was normalized to vehicle. Reduced CD36 Expression by PIC Largely Depends on Data are expressed as geometric means of fluorescence intensity SEMs. EP2 in Mouse Brain n 3 per group. **P < 0.01 and ***P < 0.001 for Bonferroni-corrected y < yy < repeated paired comparisons with vehicle; P 0.05 and P 0.001 for Finally, we investigated whether EP2 regulated CD36 Bonferroni-corrected paired comparisons between two experimental groups. AH, AH 6809; Buta, butaprost; CT, control; EP2, E2 receptor sub- expression in a relevant region of WT mouse brain. Hip- type 2; PE, phycoerythrin; PIC, polyinosinic-polycytidylic acid; TLR, Toll- pocampal CD36 mRNA values were quantified 24 hours like receptor; WT, wild-type. after 50 ng intracerebroventricular injection of PIC into the

234 ajp.amjpathol.org - The American Journal of Pathology EP2-Suppressed Phagocytosis via CD36 left lateral ventricle with or without two different doses of posttests were not significant for comparison of PIC with the EP2 antagonist AH 6809 (200 or 50 ng) (Figure 5). Our PIC plus 200 ng of AH 6809. Similar results were achieved results showed that direct exposure to TLR3 activator PIC when using 50 ng (0.86 0.21) rather than 200 ng of AH significantly reduced ipsilateral hippocampal CD36 6809 (0.81 0.22). expression to one-half of vehicle (0.51 0.09; P < 0.05 by Bonferroni-corrected posttest) and that this was reversed by simultaneous exposure to AH 6809. Indeed, two-way Discussion analysis of variance had P < 0.01 for an interaction term between PIC and AH 6809, and Bonferroni-corrected Considerable data from observational studies of human brain and experimental models support the hypothesis that microglial innate immune activation is a feature and possible contributor to AD pathogenesis, a hypothesis recently rein- forced by genetic association of AD dementia with several genes that regulate innate immunity, especially TREM2.Re- sults from these studies are further buttressed by compelling epidemiologic and experimental model data that support inhibition of the PG cascade as an effective therapeutic approach early in the pathogenesis of AD before clinical expression or behavioral impairments in transgenic mouse models. Enthu- siasm over these observational and experimental data are tempered by failed trials that used NSAIDs to suppress innate immunity in subjects with mild cognitive impairment or AD dementia and the termination of an AD prevention trial because of toxicity. Although the clinical trial results clearly are disappointing, they do not necessarily indict the innate immunity hypothesis for AD but rather may reflect limitations of intervening too late in the course of AD and/or therapeutic shortcomings of proximal inhibition of the PG cascade in people. Together, these results cautiously animate pursuit of components distal in the PG cascade as a potentially more effective means of suppressing microglial innate immunity while averting unwanted side effects. Previously, with the use of primary mouse microglia, we have shown that suppression of EP2 signaling leads to a therapeutically desirable dual phenotype of reduced innate

Figure 4 Regulation of mouse primary microglia phagocytosis of Ab42 by prostaglandin EP2 receptor signaling. After exposure to agents,

phagocytosis was determined by incubation with pHrodo-labeled Ab42 for 2 hours at 37C. Fluorescence intensities were determined by ﬂow cytometry, and geometric means of ﬂuorescence intensity were normalized to vehicle- exposed WT cells. A: WT microglia were pretreated with 20 mmol/L EP2 agonist butaprost, 20 mg/mL TLR3 activator PIC, or 20 mg/mL PIC plus 20 mmol/L EP2 antagonist AH 6809 for 24 hours and then incubated with

pHrodo-labeled Ab42. Cultures treated with PIC þ AH 6809 were also incubated with 500 mmol/L CD36 inhibitor, SSO, for an additional hour before phagocytosis assay. B: WT microglia were treated with 20 mmol/L AH 6809 or 20 mmol/L agonist butaprost for 24 hours, followed by incubation with 10 mg/mL anti-CD36 IgA or mouse control IgA for 1 hour, and then phagocytosis was determined. C: Primary microglia from WT or CD36/ mice were treated with 20 mmol/L EP2 antagonist AH 6809 or 20 mmol/L agonist butaprost for 24 hours, and then phagocytosis was determined. Data are expressed as means SEM. n Z 3 per group (A and C); n Z 3or4 per group (B). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 for Bonferroni-corrected repeated paired comparisons with vehicle; yP < 0.05 and yyP < 0.01 for Bonferroni-corrected paired comparisons

between two experimental groups. Ab42, b-amyloid 1-42; EP2, E2 receptor subtype 2; PIC, polyinosinic-polycytidylic acid; SSO, sulfosuccinimidyl oleate; TLR, Toll-like receptor; WT, wild-type.

The American Journal of Pathology - ajp.amjpathol.org 235 Li et al

Vehicle Current data support reduced clearance as the dominant PIC mechanism for Ab accumulation in the most common forms 54 1.00 of AD (reviewed in Wildsmith et al ). Although Ab may be transported out of brain, it also may be cleared by microglial 0.75 phagocytosis. Indeed, several lines of evidence point to Ab ** phagocytosis as a potentially important process in AD. 0.50 These include the increased genetic risk of AD with TREM2 variants, reduced expression of the phagocytosis-related 0.25 protein beclin 1 in diseased brain regions and isolated 55,56 CD36 (fold mRNA of Veh) 0.00 microglia from patients with AD, and reduced CD36 No AH 6809 Plus AH 6809 expression on leukocytes in patients with AD dementia or 57 Figure 5 Modulation of CD36 expression in mouse hippocampus by PIC mild cognitive impairment. It is important to note that, and prostaglandin E2 receptor subtype 2 antagonist AH 6809. Wild-type although we have focused on its phagocytic function, the mice received intracerebroventricular injection into the left lateral CD36/Ab complex is pleiotropic and interacts with several ventricle with vehicle, 50 ng PIC, 200 ng AH 6809, or 50 ng PIC plus 200 ng intracellular molecules, such as p130Cas, Pyk2, and Pax- AH 6809. Ipsilateral hippocampus was isolated after 24 hours, mRNA values illin, and initiates signaling pathways that ultimately lead to of CD36 were determined by quantitative real-time PCR, and results were 58 normalized to endogenous control GAPDH. Data are expressed as F-actin polymerization and cytoskeleton reorganization. means SEM. n Z 6 mice per group. Two-way analysis of variance (df Our results from mouse microglia confirmed those from 1,1,1, 20) had **P < 0.01 for PIC and interaction between PIC and AH other cell types that have shown decreased expression of 6809 but was insignificant for AH 6809 alone. Bonferroni-corrected post- CD36 after TLR3 or TLR4 activation.59,60 We expanded tests were significant only for the pair with no AH 6809 (**P < 0.01) but fi this survey to include TLR7, activation of which by an were insigni cant for the pair with AH 6809. PIC, polyinosinic-polycytidylic 61 acid; Veh, vehicle. miRNA can propagate neurodegeneration. Our data also showed that the innate immune-mediated decrease in CD36 was mostly, but not fully, reversed by an EP2 antagonist, immune-mediated neurotoxicity and enhanced Ab phago- suggesting that some other proinflammatory factor(s) also cytosis, including neuritic plaque material.28 Subsequently, inhibit microglial CD36 expression. One possibility is tumor we and others have validated these findings in macrophages necrosis factor-a,62,63 although this is complicated because and in vivo in mouse brain by using homozygous deficient tumor necrosis factor-a secretion depends in part on EP2.38 mice, conditional deletion mice, and bone marrow transplant Our data also suggest EP4 as another possibility. However, mice. Although we have made some progress in under- we were limited in pursuing this further because of lack of a standing the molecular mechanisms of EP2 regulation of selective EP4 antagonist. In contrast to innate immune microglia-mediated neurotoxicity, the mechanisms by activation effects on CD36 expression, a variety of anti- which EP2 signaling regulates Ab42 phagocytosis remained inflammatory mediators increase CD36 expression, unclear. Here, we showed that scavenger receptor CD36, a including IL-4, IL-13, and peroxisome proliferator-activated 64e67 key regulator of microglia Ab42 phagocytosis, is closely receptor g agonist. Blocking CD36 with an antibody regulated by EP2 signaling in primary WT mouse microglia reduces cytokine and nitric oxide when microglia are both without further activation or in the context of TLR incubated with PrP protein.68 Although difficult to classify activators. We focused our work on primary microglia broadly as either proinflammatory or anti-inflammatory, we because they express the majority of CD36 among found that an agonist of EP1 or TP also increased CD36 phagocytosis-competent cells in the central nervous system. expression in microglia. Functional antagonism is observed Primary WT microglial expression of CD36 was increased commonly among eicosanoid receptors.21 Our results point by an EP2 antagonist and decreased by an EP2 agonist; the to a dominant effect of EP2-mediated suppression of CD36 latter was associated with proportionately reduced CD36 expression in the context of microglial innate immune cell surface protein and Ab42 phagocytosis. We showed that activation, perhaps related to its higher binding affinity for 21 specific TLR activators led to decreased microglia CD36 PGE2 than for EP1. transcription and that this translated into reduced cell sur- Our in vivo experiments showed that CD36 expression in face CD36 protein and reduced Ab42 phagocytosis. At each WT mouse hippocampus followed the same mechanisms as step in this process, we showed that the effects of innate microglia. Specifically, activation of innate immune immune activation on CD36 could be reversed by at least response with PIC significantly decreased CD36 expression, half by suppressing EP2 signaling and that the effects of although the magnitude was somewhat less than that EP2 activation on Ab42 phagocytosis are largely mediated observed in primary microglia. The reasons for this differ- by CD36. Together, our results from primary microglia ence in magnitude of effect by PIC in vivo versus in vitro are cultures indicated tonic control of CD36 expression by EP2 not clear from our experiments but may be related to the signaling, and that most innate immune-mediated suppres- influence of other cell types and clearance of the inflam- sion of Ab42 phagocytosis depended on EP2, and that this magen. Regardless, our results with a highly selective EP2 effect was CD36 dependent. activator of TLR3 are consistent with those from AD

236 ajp.amjpathol.org - The American Journal of Pathology EP2-Suppressed Phagocytosis via CD36 transgenic mice that also observed reduced CD36 expres- Collinge J, Pocock J, Lashley T, Williams J, Lambert JC, Amouyel P, sion in the brain,63 a finding confounded by the multiple Goate A, Rademakers R, Morgan K, Powell J, St George-Hyslop P, actions of Ab peptides. Most importantly, coadministration Singleton A, Hardy J; Alzheimer Genetic Analysis Group: TREM2 variants in Alzheimer’s disease. N Engl J Med 2013, 368:117e127 of AH 6809 largely reversed the effect of PIC, so much so 4. Jonsson T, Stefansson H, Steinberg S, Jonsdottir I, Jonsson PV, that there was no significant difference between hippo- Snaedal J, Bjornsson S, Huttenlocher J, Levey AI, Lah JJ, Rujescu D, campal CD36 mRNA values in the PIC alone group versus Hampel H, Giegling I, Andreassen OA, Engedal K, Ulstein I, the PIC plus AH 6809 group. Djurovic S, Ibrahim-Verbaas C, Hofman A, Ikram MA, van We did not attempt to duplicate behavioral experiments in Duijn CM, Thorsteinsdottir U, Kong A, Stefansson K: Variant of TREM2 associated with the risk of Alzheimer’s disease. N Engl J mice because several other laboratories already have Med 2013, 368:107e116 investigated CD36 in the context of mouse behavior and 5. Hsieh CL, Koike M, Spusta SC, Niemi EC, Yenari M, Nakamura MC, models of immune activation or AD. Indeed, CD36- Seaman WE: A role for TREM2 ligands in the phagocytosis of apoptotic deficient mice show significant learning impairment as neuronal cells by microglia. J Neurochem 2009, 109:1144e1156 assessed by radial arm maze.69 In contrast, increased CD36 6. Forabosco P, Ramasamy A, Trabzuni D, Walker R, Smith C, Bras J, Levine AP, Hardy J, Pocock JM, Guerreiro R, Weale ME, Ryten M: expression by peroxisome proliferator-activated receptor g/ Insights into TREM2 biology by network analysis of human brain retinoid X receptor a agonists increased microglial Ab gene expression data. Neurobiol Aging 2013, 34:2699e2714 phagocytosis and improved cognitive performance in an AD 7. Sieber MW, Jaenisch N, Brehm M, Guenther M, Linnartz-Gerlach B, transgenic mouse.67 Similarly, increased CD36 expression Neumann H, Witte OW, Frahm C: Attenuated inflammatory response after brain microinjection of IL-4/IL-13 into another AD in triggering receptor expressed on myeloid cells 2 (TREM2) knock- out mice following stroke. PLoS One 2013, 8:e52982 transgenic mouse was associated with increased Ab clear- 8. Ito H, Hamerman JA: TREM-2, triggering receptor expressed on 66 ance and improved cognitive performance. myeloid cell-2, negatively regulates TLR responses in dendritic cells. Eur J Immunol 2012, 42:176e185 9. Sun M, Zhu M, Chen K, Nie X, Deng Q, Hazlett LD, Wu Y, Li M, Conclusions Wu M, Huang X: TREM-2 promotes host resistance against Pseu- domonas aeruginosa infection by suppressing corneal inflammation In summary, our data are consistent with an innate immune via a PI3K/Akt signaling pathway. Invest Ophthalmol Vis Sci 2013, hypothesis for AD in which age, inherited deficits, or 54:3451e3462 b 10. Lim GP, Yang F, Chu T, Chen P, Beech W, Teter B, Tran T, environmental factors impair the clearance of A peptides Ubeda O, Ashe KH, Frautschy SA, Cole GM: Ibuprofen suppresses either by reduced transport and/or by reduced phagocytosis. plaque pathology and inflammation in a mouse model for Alz- This promotes accumulation of Ab peptides and other debris heimer’s disease. J Neurosci 2000, 20:5709e5714 that function as endogenous ligands of TLRs and other 11. Szekely CA, Zandi PP: Non-steroidal anti-inflammatory drugs and pattern recognition receptors that activate an innate immune Alzheimer’s disease: the epidemiological evidence. CNS Neurol Disord Drug Targets 2010, 9:132e139 response; this in turn further lowers phagocytic activity. Our 12. Sperling RA, Aisen PS, Beckett LA, Bennett DA, Craft S, Fagan AM, results are consistent with others in supporting a key role for Iwatsubo T, Jack CR Jr, Kaye J, Montine TJ, Park DC, Reiman EM, CD36 in Ab phagocytosis by microglia and highlight EP2 Rowe CC, Siemers E, Stern Y, Yaffe K, Carrillo MC, Thies B, antagonists as an effective means to suppress this toxic Morrison-Bogorad M, Wagster MV, Phelps CH: Toward defining the reinforcing cycle. Combined with results of others, our data preclinical stages of Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on suggest that EP2 antagonists have the potential as a thera- diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement peutic approach to suppressing key aspects of AD 2011, 7:280e292 pathogenesis. 13. Aisen PS, Schafer KA, Grundman M, Pfeiffer E, Sano M, Davis KL, Farlow MR, Jin S, Thomas RG, Thal LJ; Alzheimer’s Disease Cooperative Study: Effects of rofecoxib or naproxen vs placebo on Acknowledgments Alzheimer disease progression: a randomized controlled trial. JAMA 2003, 289:2819e2826 We thank Samantha Rice for assistance with animal hus- 14. Thal LJ, Ferris SH, Kirby L, Block GA, Lines CR, Yuen E, Assaid C, bandry and Aimee Schantz and Carol Arnold for managerial Nessly ML, Norman BA, Baranak CC, Reines SA; Rofecoxib Protocol / 078 study group: A randomized, double-blind, study of rofecoxib in support. EP2 mice were a gift from Dr. Richard Breyer patients with mild cognitive impairment. Neuropsychopharmacology (Vanderbilt University, Nashville, TN). 2005, 30:1204e1215 15. ADAPT Research Group, Martin BK, Szekely C, Brandt J, Piantadosi S, Breitner JC, Craft S, Evans D, Green R, Mullan M: Cognitive function References over time in the Alzheimer’s Disease Anti-inflammatory Prevention Trial (ADAPT): results of a randomized, controlled trial of naproxen and 1. Galasko D, Montine TJ: Biomarkers of oxidative damage and celecoxib. Arch Neurol 2008, 65:896e905 inflammation in Alzheimer’s disease. Biomark Med 2010, 4:27e36 16. Hoozemans JJ, Veerhuis R, Rozemuller JM, Eikelenboom P: 2. Lambert JC, Ibrahim-Verbaas CA, Harold D, Naj AC, Sims R, Soothing the inflamed brain: effect of non-steroidal anti-inflammatory Bellenguez C, et al: Meta-analysis of 74,046 individuals identifies 11 drugs on Alzheimer’s disease pathology. CNS Neurol Disord Drug new susceptibility loci for Alzheimer’s disease. 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