Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages This information is current as of October 1, 2021. Milena Sokolowska, Li-Yuan Chen, Yueqin Liu, Asuncion Martinez-Anton, Hai-Yan Qi, Carolea Logun, Sara Alsaaty, Yong Hwan Park, Daniel L. Kastner, Jae Jin Chae and James H. Shelhamer

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 27, 2015, doi:10.4049/jimmunol.1401343 The Journal of Immunology

Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages

Milena Sokolowska,*,1 Li-Yuan Chen,*,1 Yueqin Liu,* Asuncion Martinez-Anton,* Hai-Yan Qi,* Carolea Logun,* Sara Alsaaty,* Yong Hwan Park,† Daniel L. Kastner,† Jae Jin Chae,† and James H. Shelhamer*

PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat–containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome Downloaded from activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly

activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2- mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange http://www.jimmunol.org/

protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1b secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes

patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2a by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator. The Journal of Immunology, 2015, 194: 000–000. by guest on October 1, 2021 he inflammasomes are a group of multimeric cytosolic been shown to form inflammasomes upon activation by a variety protein complexes that consist of an inflammasome sensor of stimuli, through several different activation pathways, mainly in T molecule, adaptor protein ASC, and caspase-1. Activation dendritic cells and macrophages (3). Among them, NLRP3 is best of caspase-1 by this complex leads to the processing of pro–IL-1b characterized and is connected to the pathogenesis of a variety of and pro–IL-18 to their mature active forms (1). Inflammasome- diseases (4), including inflammatory bowel disease and colon can- forming sensor molecules encompass the family of the nucleotide- cer (5), diabetes type II (6), gout (7), atherosclerosis (8), and Alz- binding domain (NBD), leucine rich repeat-containing proteins heimer disease (9). NLRP3 consists of an N-terminal pyrin domain, (NLRs), including NLRP1, NLRP3, NLRP6, NLRP7, NLRP12 a central NBD, and a C-terminal leucine-rich repeat domain (10). and NLRC4 as well as two structurally different proteins, absent Gain-of-function mutations in the NLRP3 gene, mainly clustered in in melanoma 2 and IFN-g–inducible protein 16 (2). They have the NBD, result in its activation or predisposition for activation and are associated with the cryopyrin-associated periodic fever syn- dromes (CAPS), including familial cold-induced autoinflammatory *Critical Care Medicine Department, Clinical Center, National Institutes of Health, syndrome, Muckle–Wells syndrome, and neonatal onset multisys- Bethesda, MD 20892; and †Inflammatory Disease Section, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, tem inflammatory disorder (NOMID) (11). In all three phenotypes MD 20892 the most common symptoms include periodic fever, arthralgia, 1M.S. and L.-Y.C. contributed equally to this work. rash, and conjunctivitis (12). Both genetic and nongenetic diseases Received for publication May 27, 2014. Accepted for publication March 24, 2015. in which the inflammasome axis is dysregulated point to the im- This work was supported by the National Institutes of Health Intramural Program. portance of fine-tuning and modulation of its activity to maintain Address correspondence and reprint requests to Dr. James H. Shelhamer, Critical homeostasis. Because so many exogenous and endogenous factors Care Medicine Department, Clinical Center, National Institutes of Health, 9000 are able to activate different inflammasomes, potent regulatory Rockville Pike, Bethesda, MD 20892. E-mail address: [email protected] mechanisms must exist to allow the immune system to remove any The online version of this article contains supplemental material. sources of danger without causing excessive harm to the host. Abbreviations used in this article: AA, arachidonic acid; AC, adenylate cyclase; Recently, several factors and mechanisms have been identified to alum, aluminum potassium sulfate; CAPS, cryopyrin-associated periodic fever syn- negatively regulate inflammasomes at different levels of their ac- drome; COX, cyclooxygenase; cPLA2a, cytosolic phospholipase A2 group IVA; DHA, docosahexaenoic acid; EP, PGE2 receptor subtype; Epac, exchange protein tivation, including autophagy (13), IFN type I (14), microRNAs (15), directly activated by cAMP; GPCR, G protein–coupled receptor; MDM, monocyte- docosahexaenoic acid (16), NO (17) and cAMP (18). However, the derived macrophage; NBD, nucleotide-binding domain; NLR, nucleotide-binding domain, leucine-rich repeat–containing protein; NOMID, neonatal onset multisystem full spectrum, as well as downstream events involved in the reg- inflammatory disorder; PKA, protein kinase A; siRNA, small interfering RNA. ulation of inflammasomes, has not been elucidated.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401343 2 PGE2 INHIBITS NLRP3 INFLAMMASOME

PGE2 belongs to a family of bioactive lipid mediators that have NBD of NLRP3, resulting in the decrease of cAMP binding (18), a broad range of effects (19). During the acute, initial stage of the the inhibitory effect of PGE2 was observed only in high doses. inflammatory response PGE2 acts as a vasodilator and facilitates Moreover, we found that blocking cPLA2a, or COX2 resulting in tissue influx of neutrophils (20), macrophages (21), and mast cells inhibition of endogenous PGE2 production, caused an increase in (22) as well as being a regulator of nociception (23). However, NLRP3 inflammasome activation. Our results suggest that PGE2 PGE2 also has many potent immunosuppressive properties that might play an important role in maintaining homeostasis during the contribute to the resolution phase of acute inflammation (24), resolution phase of inflammation by preventing excessive inflam- facilitation of tissue regeneration (25), and the return to homeo- masome activation in infiltrating macrophages, and it might serve stasis (26). However, in the context of many immunopathologies, as a significant autocrine and paracrine regulator. those PGE2-mediated effects can lead to aggravation of the dis- ease phenotype such as chronic inflammation or cancer (27). Materials and Methods PGE2 regulates activities of both innate and adaptive immune Reagents and Abs cells. Its wide range of activities, with often opposing effects, Aluminum potassium sulfate (alum crystals), monosodium urate crystals, depends on the species, cell, and tissue types or context of action and ultrapure LPS were purchased from InvivoGen (San Diego, CA). ATP, (28). PGE2 synthesis is initiated by phospholipase A2,which nigericin sodium salt, 8-bromo-cAMP,and IBMX were obtained from Sigma- catalyzes the hydrolysis of membrane phospholipids and liberates Aldrich (St. Louis, MO); KH7 and forskolin were from Tocris Bioscience free fatty acids. Cytosolic phospholipase A group IVA (cPLA a) (Bristol, U.K.). PGE2, PF-04418948 (EP2 inhibitor), GW 627368X (EP4 2 2 inhibitor), butaprost, free acid (EP2 agonist), CAY10598 (EP4 agonist), and is selective for arachidonate in the sn-2 position of membrane NS-398 (COX2 inhibitor) were purchased from Cayman Chemical (Ann phospholipids, thus generating arachidonic acid (AA), the sub- Arbor, MI). cPLA2a inhibitor (N-[(2S,4R)-4-(biphenyl-2-ylmethyl-isobutyl- strate of cyclooxygenases (COX1 and COX2), that converts AA to amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl]-3-[4- Downloaded from PGH (29). It is then converted to downstream active prostanoid (2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl] acrylamide, HCl) was obtained 2 from Calbiochem/EMD Millipore (Gibbstown, NJ). 8-(4-Chlorophenylthio)- by the terminal synthases. In many cells of innate immunity such 2’-O-methyladenosine 39,59-cyclic monophosphate sodium salt (Epac ago- as macrophages, cPLA2a is the rate-limiting enzyme in PGE2 nist; activates Epac 1 and Epac 2) and 8-bromoadenosine 39,59-cyclic production (30). The diverse effects of PGE2 may be also monophosphothioate, Rp-isomer sodium salt (PKA inhibitor) were pur- chased from Enzo Life Sciences (Farmingdale, NY). N6-benzoyl-8- accounted for, at least in part, by the existence of four PGE2 re- ceptor subtypes (EPs), belonging to the family of G protein– piperidinoadenosine-39,59-cyclic monophosphate sodium salt (PKA agonist) http://www.jimmunol.org/ and 4-cyclopentyl-2-(2,5-dimethylbenzylsulfanyl)-6-oxo-1,6- dihydropyrimidine- coupled receptors (GPCRs), differentially expressed in cells and 5-carbonitrile (Epac inhibitor) were purchased from Axxora (Lausen, by coupling to more than one G protein, initiating various signal Switzerland). Ab against IL-1b (AF-201-NA) was purchased from R&D 2+ transduction pathways (31). Whereas EP1 mediates cytosolic Ca Systems (Minneapolis, MN). Abs against caspase-1 (2225S) and total cPLA2a mobilization (32), EP2 and EP4 couple primarily to G a, which were purchased from Cell Signaling Technology (Danvers, MA); Ab against s NLRP3 (AG-20B-0014-C100) was from Adipogen (San Diego, CA); Ab activates adenylate cyclase (AC) to convert ATP to cAMP (33, against ASC (sc-22514-R) was from Santa Cruz Biotechnology (Dallas, 34). Changes in cAMP levels are further translated into pleiotropic TX). Monoclonal HRP-conjugated b-actin Ab was obtained from GenScript intracellular effects by a panel of cAMP-binding effector proteins (Piscataway, NJ) and Cell Signaling Technology. HRP-conjugated secondary (35). The EP3 signaling pathway inhibits AC activity by coupling Abs were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). by guest on October 1, 2021 to the Gia subunit and decreasing cAMP levels (36). In macrophages, at the priming stage of NLRP3 inflammasome Primary cells and cell lines activation by TLR signaling, apart from induction of NLRP3 and Human elutriated monocytes from healthy donors were obtained by an pro–IL-1b expression, there is also an activation of cPLA2a, re- Institutional Review Board–approved protocol from the National Institutes lease of AA, and production of PGE2 and other eicosanoids (37, of Health Blood Bank (Bethesda, MD). Monocytes were resuspended in 38). Moreover, aluminum salts and silica crystals (39), hyaluronan RPMI 1640 medium with 2 mM L-glutamine and supplemented with 10% (40), as well as ATP (41) and other known activators of NLRP3 heat-inactivated FBS (Life Technologies/Thermo Scientific, Waltham, MA) overnight. The next day, 18 3 106 monocytes were seeded into a T75 tissue inflammasome further stimulate PGE2 production, although proba- culture flask in 15 ml IMDM (Life Technologies) with 10% heat-inactivated bly in an NLRP3- and caspase-1–independent manner (39). Fur- FBS and 50 ng/ml M-CSF (Life Technologies) for 7 d. Half of the medium thermore, PGE2 and other prostanoids have been shown to be was replaced after 3 d of culture. Twenty-four hours prior to experiments, primarily responsible for several immediate reactions and sudden macrophages were treated with trypsin (Lonza, Walkersville, MD) for 1 min, death in the “eicosanoid storm” after NLRC4 inflammasome ac- scraped, and plated in the IMDM medium without M-CSF into 12-well plates or 6-well plates at a density of 0.35 3 106 or 0.85 3 106,respectively. tivation by intracellular flagellin (42). In this case, this process Blood specimens from patients with CAPS were drawn after obtaining appears to be NLRC4-, casp-1–, and COX1-dependent (42). How- informed consent under a protocol approved by the National Institute of ever, the effects of exogenous or endogenous PGE2 on the NLRP3 Arthritis and Musculoskeletal and Skin Diseases/National Institute of Di- inflammasome activation have not been studied. Owing to the abetes and Digestive and Kidney Diseases Institutional Review Board. Blood specimens from healthy volunteers were obtained under a protocol growing evidence of the coexistence of potent lipid mediator sig- approved by the National Heart, Lung, and Blood Institute Institutional naling and inflammasome formation in response to infection, tissue Review Board. Blood samples were processed immediately using lymphocyte damage, or other cellular stress, we sought to analyze the effect of separation medium (Lonza) according to the manufacturer’s protocol to obtain PBMCs. PBMCs (2 3 106) were seeded, allowed to attach to a exogenous PGE2 on NLRP3 inflammasome activation. We also studied whether the endogenously produced lipid mediators might 12-well plate containing RPMI 1640 medium without serum for 20 min, immediately followed by the inflammasome activation/inhibition experi- be involved in the modulation of NLRP3 inflammasome activation. ments. THP-1 cells were obtained from the American Type Culture Col- We performed experiments in human primary monocyte-derived lection (Manassas, VA) and cultured in RPMI 1640 medium containing macrophages (MDMs) and in PBMCs from healthy donors as 10% heat-inactivated FBS and 0.05 mM 2-ME. THP-1 cells (0.5 3 106) well as in PBMCs from CAPS patients. In this study, we found that were plated into a 12-well plate in the presence of 50 nM PMA (Sigma- Aldrich) for 12 h before the experiment was performed. PGE2 decreased NLRP3 inflammasome activation, triggered by aluminum crystals, ATP, or nigericin. This effect was derived through Inflammasome activation and inhibition EP4 and through an increase in intracellular cAMP, independently Human primary MDMs were treated with/without LPS (100 ng/ml) for 4 h of protein kinase A (PKA) and exchange protein directly activated in IMDM with 10% FBS, followed by 30 min treatment with PGE2 (0.1 mM by cAMP (Epac). In patients with CAPS, with mutations in the or as indicated), EP2 agonist (butaprost, free acid; 0.5 mM), EP4 agonist The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. PGE2 inhibits alum-induced NLRP3 inflammasome activation and IL-1b and IL-18 secretion. (A–E) Human primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by 30 min treatment with PGE2 (0.1 mM) or vehicle (EtOH) (A–C) or indicated doses of PGE2 (D and E) and then stimulated with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants, lysates, and cell pellets were collected for Western blot (WB) (A and D) or ELISA (B, C, and E). (A and D) The WBs are representative of three independent experiments from three healthy donors, each showing similar results. (B) IL-1b release data represent mean 6 SEM from seven independent experiments from seven healthy donors, performed in duplicate. IL-1b data are presented as the percentage of the response after LPS/alum treatment, ranging from 61.6 to 831.6 pg/ml. (Figure legend continues) 4 PGE2 INHIBITS NLRP3 INFLAMMASOME

(CAY10598; 0.1 mM), 8-bromo-cAMP (5–20 mM), PKA selective ago- legend. PGE2 in cell culture supernatants was measured using an EIA kit nist (6-Bnz-8-PIP-cAMP; 20 or 50 mM), or EPAC selective agonist (8-(4- from Cayman Chemical. TNF in cell culture supernatants was measured chlorophenylthio)-29-O-methyladenosine 39,59-cyclic monophosphate using a human TNF-a ELISA kit (DTA00C) from R&D Systems. Intracel- sodium salt; 20 or 50 mM), followed by stimulation with alum (400 mg/ lular cAMP quantification was performed using an EIA kit from Enzo Life ml for 5 h), ATP (2 mM for 1 h), or nigericin (4 mMfor1h)inOpti- Sciences. Human primary MDMs seeded on a six-well plate were stimulated MEM medium (Life Technologies). EP2 inhibitor (PF-04418948; 0.5 with PGE2 (0.1 mM) or forskolin (50 mM) in the presence of IBMX (200 mM), EP4 inhibitor (GW627368X; 2 mM), PKA inhibitor (8-bromoa- mM) for 15 min, followed by 10 min cell lysis in 0.1 N HCl containing 0.1% denosine 39,59-cyclic monophosphothioate, Rp-isomer sodium salt, 50 Triton X-100 (Sigma-Aldrich). For the cAMP quantification in the experi- mM), EPAC inhibitor (4-cyclopentyl-2-(2,5-dimethylbenzylsulfanyl)-6- ments with NS398, primary MDMs were treated with LPS (100 ng/ml) with/ oxo-1,6-dihydropyrimidine-5-carbonitrile; 10 mM), or vehicle (DMSO) without NS398 (5 mM), NS398 alone, or DMSO for 4 h in complete medium were added 30 min prior treatment with PGE2 or vehicle (EtOH). KH7 (25 and then stimulated with alum for 15 min in serum-free medium followed by mM), IBMX (200 mM), or forskolin (50 mM) were added 1 h prior to 10 min cell lysis in 0.1 N HCl containing 0.1% Triton X-100 and IBMX (200 treatment with PGE2 or vehicle (EtOH). In the experiments with cPLA2a mM). Cell lysates were collected, centrifuged, and stored at 280˚C before inhibitor (2 mM) in human primary MDMs, the inhibitor or DMSO was cAMP detection according to the manufacturer’s protocol. added before and after priming or only after priming, followed by treatment with alum (400 mg/ml for 5 h) in Opti-MEM. In the experiments with COX2 Western blot inhibitor (NS398; 5 mM) in human primary MDMs, the inhibitor or DMSO Supernatants were collected and cells were lysed with RIPA buffer (Thermo was added before and after priming, followed by treatment with alum (400 Scientific, Rockford, IL) containing protease inhibitor cocktails (Roche, mg/ml for 5 h) or ATP (2 mM for 1 h) in Opti-MEM. PBMCs from CAPS Basel, Switzerland). Supernatants were precipitated with an equal volume patients or healthy volunteers were primed for 3 h with LPS (1 mg/ml) in of methanol and 1:4 volumes of chloroform (Sigma-Aldrich) and centri- RPMI 1640 with 10% FBS and followed by indicated doses of PGE2 (in fuged at room temperature at 12,000 rpm for 5 min. The upper layer was CAPS patients) or PGE2 with alum (400 mg/ml) or ATP (2 mM) (in healthy aspirated, a double volume of methanol was added, and the supernatant was volunteers) for 30 min in RPMI 1640 without serum. PMA-treated THP-1 centrifuged again. The liquid phase was discarded and the pellet was dried at Downloaded from cells were treated with cPLA2a inhibitor for 30 min, then primed with 100 50˚C for 5 min. The pellets were dissolved in 13 NuPage sample buffer ng/ml LPS and stimulated by monosodium urate (100 mg/ml for 5 h), alum (Life Technologies) and heated at 70˚C for 10 min and then subjected to (400 mg/ml for 5 h), ATP (2 mM for 1 h), or nigericin (4 mM for 45 min) in electrophoresis using the 4–12% NuPAGE Bis-Tris gels using MES buffer Opti-MEM medium. Supernatants, lysates, and cell pellets were collected for (Life Technologies). The cell lysates were placed on ice for 15 min and the following analyses. then centrifuged at 13,000 rpm for 20 min. Protein concentration of cell NLRP3, EP2, EP4, and PLA2G4A knockdown lysates was determined by a BCA kit (Pierce, Rockford, IL). Equal amounts of lysate proteins (20 mg) were resolved by 4–12% NuPAGE Bis-

ON-TARGETplus SMARTpool small interfering RNA (siRNA) (Dharmacon/ Tris gels using MOPS running buffer (Life Technologies) and then trans- http://www.jimmunol.org/ Thermo Scientific, Lafayette, CO) against human NLRP3 (L-017367-00-0005 blotted to the nitrocellulose membranes using the iBlot dry blotting system or sc-45469, Santa Cruz Biotechnology), EP2 (PTGER2) (L-005712-00-0005), (Life Technologies). For ASC pyroptosome, cell pellets were cross-linked EP4 (PTGER4) (L-005714-00-0005), and cPLA2a (PLA2G4A) (L-009886- using 2 mM disuccinimidyl suberate (Thermo Scientific) for 30 min in PBS 00-005) together with ON-TARGETplus control nontargeting pool in room temperature; 63 SDS sample buffer was added and samples were (D-00181970-05) were used to perform knockdown experiments in human processed as cell lysates. The membranes were blocked with 5% nonfat primary MDMs or THP-1 cells. Human primary MDMs (0.5 3 106 cells skim milk in PBST (PBS containing 0.1% Tween 20) for 2 h and then per 100-ml cuvette) or THP-1 cells (5 3 106 per 100-ml cuvette) were incubated with primary Abs overnight at 4˚C. Subsequently, the membranes transfected with siRNA pools (1 mM or 100 nM) using a P3 Primary Cell were washed and then incubated with specific secondary Abs conjugated 4D-Nucleofector X Kit L (Amaxa, Cologne, Germany) according to the with HRP for 1 h. The blots were developed using the SuperSignal West manufacturer’s protocols for human macrophages and monocytes, respec- Femto chemiluminescent substrate kit (Thermo Scientific) and visualized tively. The silencing of gene expression was confirmed by RT-PCR. All on a ChemiDoc MP image station (Bio-Rad, Hercules, CA). by guest on October 1, 2021 experiments on transfected cells were performed after 48 h. Statistical analysis Real-time PCR Data were analyzed by one-way ANOVAwith a Holm–Sidak post hoc test, Total RNA was extracted from cells using QIAshredder columns and Kruskall–Wallis ANOVA on ranks with a Dunn post hoc test, repeated RNeasy mini kit and treated with DNase (Qiagen, Valencia, CA). Reverse measurements ANOVA, followed by a Sidak post hoc test or unpaired transcription was performed using an iScript cDNA synthesis kit (Bio-Rad, Student t test, as appropriate. Differences were considered significant when Hercules, CA). Gene expression was assessed using RT-PCR performed on p , 0.05. The data are presented as mean 6 SEM from the indicated an ABI Prism ViiA7 sequence detection system (Applied Biosystems, Foster number of independent experiments, each performed in duplicate. City, CA) using commercially available probe and primers sets (Applied Biosystems) as follows: GAPDH, Hs02758991_g1; IL-1b, Hs01555410_m1; IL-6, Hs00174131_m1; TNF, Hs99999043_m1; cPLA2a (PLA2G4A), Results Hs00233352_m1; NLRP3, Hs00918082_m1; caspase-1, Hs00354836_m1; PGE2 inhibits NLRP3-triggered inflammasome activity in EP1 (PTGER1), Hs00168752_m1; EP2, Hs04183523_m1; EP3, Hs00168755_m1; EP4, Hs00168761_m1; and iTaq universal probes supermix (Bio-Rad). Gene human macrophages expression was normalized to GAPDH transcripts and represented as a rel- To assess the effect of PGE2 on NLRP3 inflammasome activation, ative quantification compared with control. we first examined whether PGE2 could inhibit caspase-1 cleavage ELISA and IL-1b secretion. Human primary MDMs were primed with LPS followed by treatment with PGE and subsequent stimulation IL-1b and IL-18 in cell culture supernatants were measured using a human 2 IL-1 b/IL-1F2 Quantikine Kit (SLB50) from R&D Systems or a human IL- with alum to activate IL-1b maturation via NLRP3 inflamma- 18 ELISA kit from Medical and Biological Laboratories (Nagoya, Japan), some. We found that PGE2 blocked alum-induced activation of respectively, according to the manufacturers’ directions. There were sub- caspase-1 and production of mature IL-1b and IL-18 (Fig. 1A–C, stantial differences in the IL-1b and IL-18 release between the donors, as Supplemental Fig. 1). We also observed that PGE in these ex- depicted in the Supplemental Fig. 1. Therefore, to combine similar experi- 2, ments and calculate statistical significance, the values in all experiments are perimental conditions, slightly decreased the expression of pro– presented as the percentage of the response after LPS/alum treatment. The IL-1b without affecting NLRP3 or caspase-1 (Fig. 1A). The in- range of the raw values from combined experiments is included in each figure hibitory effect of PGE2 on caspase-1 cleavage, IL-1b maturation,

(C) IL-18 release data represent mean 6 SEM from five independent experiments from five healthy donors, performed in duplicate. IL-18 data are presented as the percentage of the response after LPS/alum treatment, ranging from 18.2 to 821.3 pg/ml. (E) IL-1b release data represent mean 6 SEM from six independent experiments from six healthy donors, performed in duplicate. IL-1b data are presented as the percentage of the response after LPS/alum treatment, which ranged from 11.4 to 299 pg/ml. *p , 0.05 as compared with LPS/alum treatment, as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. PGE2-induced inhibition of IL-1b production is driven through NLRP3 inflammasome. (A–E) Primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by 30 min treatment with PGE2 (0.1 mM) or vehicle (EtOH), then stimulated with ATP (2 mM for 1 h) (A–C) or nigericin (4 mM for 1 h) (D and E) in serum-free medium. Supernatants and lysates were collected for Western blot (WB) or ELISA. (A and E) The WBs are representative of three independent experiments from three healthy donors, each showing similar results. (B) IL-1b release data represent mean 6 SEM from three independent experiments from three healthy donors, performed in duplicate. Data are presented as the percentage of the response after LPS/ATP treatment, ranging from 226.6 to 5851.4 pg/ml. (C and D) Mature caspase-1 is presented in OD units after subtraction of background from three independent experiments from three healthy donors. (F and G) MDMs were transfected with NLRP3 siRNA (1 mM; L-017367-00-0005). After 48 h cells were treated with/without LPS (100 ng/ml) for 4 h in complete culture medium, followed by stimulation with alum (400 mg/ml for 5 h) (F) or ATP (2 mM for 1 h) (G) in serum-free medium. The WBs are representative of three independent experiments from three healthy donors, each showing similar results.

ASC oligomerization (Fig. 1D), and IL-1b secretion (Fig. 1E) was (43), to determine whether the suppression exerted by PGE2 dose-dependent and began with very low doses of PGE2 (0.01–0.1 specifically affected alum-dependent NLRP3 inflammasome ac- mM) in human primary MDMs. The effectiveness of these doses tivity, a phagocytosis-dependent model of NLRP3 activation (44). points to a physiologic and receptor-specific mode of PGE2-me- We found that caspase-1 cleavage and IL-1b secretion were also diated inhibition of NLRP3 activation (31). We also tested other decreased by PGE2 in an ATP-induced (Fig. 2A–C) or nigericin- activators of NLRP3 inflammasome, such as ATP and nigericin induced (Fig. 2D, 2E) NLRP3 inflammasome activation model. To 6 PGE2 INHIBITS NLRP3 INFLAMMASOME

control for NLRP3 specificity of studied stimuli in human mac- PGE2 inhibits NLRP3 inflammasome through EP4 rophages, we performed NLRP3 siRNA experiments with two PGE2 is known to exert various effects through at least four dif- different sources of siRNAs, confirming that both alum and ATP ferent receptors, that is, EP1, EP2, EP3, and EP4 (PTGER1–4), all specifically induced NLRP3-dependent caspase-1 and IL-1b belonging to the GPCR family (31). Therefore, we first analyzed cleavage (Fig. 2F, 2G). Thus, PGE2 specifically inhibited the ac- which receptors are expressed on human primary MDMs. In tivity of NLRP3 inflammasome. agreement with the literature on mouse macrophages, cell lines, and human macrophages (34), we found that EP1 basal mRNA PGE2 differentially influences the gene expression of NLRP3 expression was at the edge of the detection level; EP3 expression inflammasome complex components was very low, whereas EP2 and EP4 were abundantly expressed To evaluate the role of PGE2 on the expression of NLRP3 in- (Supplemental Fig. 2A). Furthermore, both EP2 and EP4 mRNA flammasome components, we performed a time course analysis of expression was significantly increased by LPS priming (Supplemental PGE2 on LPS-induced gene expression. Human primary MDMs Fig. 2B, 2C). To determine which receptor is responsible for the were treated with/without LPS, followed by treatment with PGE2 PGE2 effect on NLRP3 inflammasome, we used specific pharma- for 1.5, 3, and 5.5 h. We found that LPS-induced IL-1b mRNA cological agonists and antagonists of each receptor as well as expression was not significantly altered by PGE2 in either of the a pool of siRNAs to knock down each receptor. The EP4 agonist, time points, although there was a downward trend (Fig. 3A). CAY10598, but not the EP2 agonist, butaprost, blocked alum- NLRP3 mRNA expression was significantly increased by PGE2 at induced secretion of mature IL-1b and caspase-1 (Fig. 4A, 4B). the 1.5 h time point (Fig. 3B), whereas caspase-1 mRNA ex- Moreover, we found that the EP4 antagonist, GW627368X, blocked

pression was unchanged (Fig. 3C). TNF and IL-6 usually serve as PGE2-induced inhibition of NLRP3 inflammasome activation, Downloaded from controls for specific inflammasome and nonpriming effecting whereas the EP2 antagonist, PF-04418948, had no effect (Fig. 4C, mechanisms. However, both of these cytokines have been reported 4D). Both inhibitors applied simultaneously had a similar effect to to be regulated by PGE2 and cAMP in opposite directions in EP4 antagonist alone (Fig. 4C, 4D). The function of EP2 and EP4 macrophages and through mechanisms not affecting intracellular agonists and antagonists was controlled by measuring TNF canonical TLR signaling (45, 46). Interestingly, we found that LPS- (Supplemental Fig. 3A, 3B), which inhibition was reported to be induced IL-6 mRNA expression was not changed by the addition of driven through both of these receptors in human macrophages (47). http://www.jimmunol.org/ PGE2 after LPS stimulation (Fig. 3D), as opposed to TNF-a,in Knockdown of EP2 or EP4 was performed by electroporation with which mRNA expression was significantly decreased at each time the specific pools of siRNAs. The knockdown efficiency for EP2 point studied (Fig. 3E). Taken together, the lack of the effect of and EP4 was 97 and 65%, respectively. When both siRNAs were PGE2 on the transcription of genes encoding proteins involved in applied together, then EP2 was reduced by 93% and EP4 was re- the NLRP3 inflammasome complex formation, as well as its in- duced by 50% as assessed by RT-PCR (Fig. 4E). Similar to the hibitory effect on mature caspase-1, ASC oligomerization, and antagonists results, EP4 knockdown, as opposed to EP2 knock- constitutively expressed IL-18, as shown in the previous paragraph, down, reduced PGE2-induced inhibition of NLRP3 inflammasome suggests that PGE2 acts on NLRP3 inflammasome assembly. activation (Fig. 4F, 4G). Simultaneous knockdown did not have an by guest on October 1, 2021

FIGURE 3. PGE2 does not decrease gene expression of proteins involved in NLRP3 inflammasome complex formation. (A–E) Primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by treatment with PGE2 (0.1 mM) or vehicle (EtOH) for indicated time in serum-free medium. At each time point, cells were lysed and total RNA was extracted. Gene expression was assessed using RT-PCR, normalized to GAPDH transcripts and represented as a relative quantification (RQ) compared with vehicle-treated cells from the first time point. Data represent mean 6 SEM from four independent experiments from four healthy donors, each performed in duplicate. *p , 0.05 as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. PGE2 inhibits NLRP3 inflammasome through EP4. (A and B) Primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by 30 min treatment with EP2 agonist (butaprost, free acid, 0.5 mM), EP4 agonist (CAY10598, 0.1 mM), PGE2 (0.1 mM), or vehicle (EtOH) and then stimulated with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for Western blot (WB) (A)or IL-1b ELISA (B). IL-1b release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 111.5 to 139.9 pg/ml. (Figure legend continues) 8 PGE2 INHIBITS NLRP3 INFLAMMASOME

effect on PGE2-induced inhibition of NLRP3 inflammasome acti- specific inhibitor (8-bromoadenosine 39,59-cyclic monophosphothioate) vation, probably because of the low efficiency of the knockdown of did not block PGE2-mediated NLRP3 inflammasome inhibition EP4 (Fig. 4E–G). Collectively, these data suggest that PGE2 (Fig. 6C, 6D). Similarly, Epac selective agonist (8-(4-chlor- decreases NLRP3 inflammasome activity through EP4. ophenylthio)-29-O-methyladenosine 39,59-cyclic monophosphate) did not inhibit caspase-1 cleavage and production of mature IL-1b cAMP, independent of PKA and Epac, is involved in (Fig. 6E, 6F), and Epac selective inhibitor (4-cyclopentyl-2-(2,5- PGE -mediated NLRP3 inflammasome inhibition 2 dimethylbenzylsulfanyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile) The most widely described signaling pathway through EP4 in did not reverse PGE2-mediated NLRP3 inflammasome inhibition macrophages is coupling with Gsa subunit and activation of AC (Fig. 6G, 6H). At the same time, these agonists and antagonists (34). AC converts ATP to cAMP, which is an important second had an effect on TNF production (Supplemental Fig. 3C–F), as messenger. Intracellular cAMP levels are further controlled by previously reported (52, 53), proving that they were effective the actions of phosphodiesterase, which converts cAMP to in- and, more importantly, suggesting separate pathways of cAMP- active 59-AMP, by degrading the phosphodiester bond (48). dependent NLRP3 inflammasome and TNF inhibition. Collec- Signaling through EP4 increases intracellular cAMP levels (34). tively, these data suggest that PGE2-mediated NLRP3 inflamma- It was previously demonstrated that cAMP inhibits NLRP3 some inhibition is exerted through cAMP, but not through PKA or inflammasome through direct interaction with NLRP3 protein in Epac. mouse bone marrow–derived macrophages and in human PBMCs (18). To determine whether this is the major mechanism of the PGE2-mediated NLRP3 inflammasome inhibition in CAPS patients requires higher doses observed effect of PGE2-induced NLRP3 inhibition in human primary MDMs, we applied several approaches that involved the As mentioned above, it has been previously reported that cAMP Downloaded from key molecules in the regulation of intracellular cAMP levels. We binds directly to the NBD (NACHT) of NLRP3 protein, and this used KH7, an AC inhibitor that decreases basal or activated cAMP binding is impaired in cases of the most common mutations of levels (49); forskolin, a direct AC activator that increases intra- NLRP3 gene, present in patients with CAPS (18). Therefore, to cellular cAMP levels (50); and IBMX, a phosphodiesterase in- further analyze whether PGE2-mediated NLRP3 inhibition is hibitor that increases cAMP levels by blocking its degradation (51). cAMP-NBD specific, we studied the effect of PGE2 on IL-1b se- First, we confirmed that in human primary MDMs, 15 min treat- cretion in PBMCs from mutation-positive CAPS patients. PBMCs http://www.jimmunol.org/ ment with PGE2 stimulated cAMP production (Fig. 5C). Next, we of these patients are especially prone to NLRP3 inflammasome found that blocking AC with KH7 in the presence or absence of activation and abundant secretion of mature IL-1b in response to alum significantly reduced PGE2-mediated NLRP3 inflammasome LPS priming only, without other NLRP3 stimuli (54). Monocytes inhibition (Fig. 5A, 5B) and IL-1b secretion (Fig. 5B). Forskolin from healthy subjects are also reported to respond to LPS signal treatment significantly decreased NLRP3 activation (Fig. 5A) and only, probably by intrinsic release of ATP, but the second signal IL-1b secretion (Fig. 5B), which was further decreased by adding makes their response much more robust (55). We found here that in PGE2, suggesting additive PGE2-mediated mechanisms of AC monocytes from healthy subjects PGE2 decreased alum or ATP- activation. Not surprisingly, prolonged IBMX treatment completely dependent mature IL-1b secretion in similar doses as in monocyte- by guest on October 1, 2021 abrogated NLRP3 activation (Fig. 5A) and IL-1b secretion (Fig. 5B). derived macrophages (0.1–10 mM) (Fig. 7A). However, in patients To further confirm cAMP-mediated NLRP3 inflammasome inhi- with familial cold autoinflammatory syndrome or Muckle–Wells bition, we used 8-bromo-cAMP, a cell-permeable analog of cAMP. syndrome with known mutations in NLRP3 (Fig. 7B), these doses We found that 8-bromo-cAMP in a dose-dependent manner sup- were less effective and the PGE2 inhibitory effect was visible in pressed activation of caspase-1 and production of mature IL-1b doses 10- to 100-fold higher than in healthy subjects (Fig. 7B). (Fig. 5D) and in the dose of 20 mM significantly decreased IL-1b These results suggest that PGE2-mediated NLRP3 inhibition is secretion (Fig. 5E). In macrophages, cAMP might act through diminished in patients carrying an NLRP3 mutation in the NBD. ubiquitously expressed intracellular cAMP receptors, the classic a protein kinase A (PKA), and exchange protein directly activated by cPLA2 and COX2 are involved in endogenous NLRP3 cAMP (Epac), or it might directly bind to the NLRP3 protein (18). inflammasome regulation To study whether the PGE2-dependent cAMP-mediated effect on Having found that exogenous PGE2 inhibits NLRP3 inflamma- NLRP3 is derived through PKA or Epac, we used their specific some activation in human primary MDMs, we wondered whether agonists and antagonists. We found that PKA selective agonist endogenous PGE2 production might serve as a regulatory auto- (6-Bnz-8-PIP-cAMP) did not mimic PGE2-dependent NLRP3 inflam- crine or paracrine mechanism against excessive NLRP3 activation. masome inhibition (Fig. 6A, 6B). Also, we observed that PKA- Along with others, we have previously shown that LPS induces

*p , 0.05 as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test (B). (C and D) Primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete culture medium, then 30 min with EP2 inhibitor (PF-04418948, 0.5 mM), EP4 inhibitor (GW627368X, 2 mM), both inhibitors, or vehicle (DMSO), followed by 30 min treatment with PGE2 (0.1 mM) or vehicle (EtOH) and stimulation with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for WB (C) or IL-1b ELISA (D). IL-1b release data represent mean 6 SEM from five in- dependent experiments from five healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 36.6 to 372.6 pg/ml (D). (E–G) MDMs were transfected with EP2 siRNA (1 mM), EP4 siRNA (1 mM), or both. After 48 h cells were treated with/without LPS (100 ng/ml) for 4 h in complete culture medium, followed by 30 min treatment with PGE2 (0.1 mM) or vehicle (EtOH), and then stimulated with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for RT-PCR (E), WB (F), or IL-1b ELISA (G). Gene expression was normalized to GAPDH transcripts and represented as a relative quantification (RQ) compared with vehicle-treated control siRNA transfected cells. Data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. *p , 0.05 as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test (E). IL-1b release data represent mean 6 SEM from three in- dependent experiments from three healthy donors, performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 34.6 to 491.5 pg/ml (G). *p , 0.05 as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test (D and G). The WBs are representative of three independent experiments from three healthy donors, each showing similar results (A, C, and F). The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. cAMP is involved in PGE2-mediated NLRP3 inhibition. (A and B) Primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by treatment for 1 h with KH7 (25 mM), IBMX (200 mM), or forskolin (50 mM) and followed by PGE2 (0.1 mM) or vehicle (EtOH) for 30 min and stimulated with alum for 5 h (400 mg/ml) in serum-free medium. Supernatants and lysates were collected for Western blot (WB) (A) or IL-1b ELISA (B). IL-1b release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in du- plicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 76.7 to 562.8 pg/ml. #p , 0.05 as compared with LPS/alum treatment, *p , 0.05 as assessed by one-way ANOVA, followed by a Holm–Sidak post hoc test (B). (C) Primary MDMs were treated with IBMX

(200 mM) with either vehicle (EtOH), PGE2 (0.1 mM), or forskolin (50 mM) for 15 min. cAMP measured in cell lysates represents mean 6 SEM from three independent experiments from three healthy donors, each performed in triplicate. *p , 0.05 as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test. (D and E) MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by 30 min treatment with indicated doses of 8-bromo-cAMP or vehicle (Tris) and then stimulated with alum for 5 h (400 mg/ml) in serum-free medium. Supernatants and lysates were collected for WB (D) or IL-1b ELISA (E). IL-1b release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 70.2 to 1100.9 pg/ml. *p , 0.05 as compared with LPS/alum treatment, as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test (E). The WBs are rep- resentative of three independent experiments from three healthy donors, each showing similar results (A and D). 10 PGE2 INHIBITS NLRP3 INFLAMMASOME Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. PGE2-derived NLRP3 inflammasome inhibition does not require PKA or EPAC. (A and B) Primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by 30 min treatment with PKA selective agonist (6-Bnz-8-PIP-cAMP, 20 or 50 mM), PGE2 (0.1 mM), or vehicle and then stimulated with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for Western blot (WB) (A)orIL-1b ELISA (B). IL-1b release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 44.7 to 103 pg/ml. *p , 0.05 as compared with LPS/ alum, as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test (B). (C and D) Primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete culture medium and then 30 min with PKA inhibitor (8-bromoadenosine 39,59-cyclic monophosphothioate, Rp-isomer sodium salt, 50 mM) or vehicle (DMSO), followed by 30 min treatment with PGE2 (0.1 mM) or vehicle (EtOH) and stimulation with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for WB (C) or IL-1b ELISA (D). IL-1b release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 43.9 to 167.7 pg/ml. *p , 0.05 as compared with LPS/alum, as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test (D). (E and F) Primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by 30 min treatment with EPAC selective agonist (8-(4-chlorophenylthio)-29-O-methyladenosine 39,59-cyclic monophosphate sodium salt, 20 or 50 mM), PGE2 (0.1 mM), or vehicle and then stimulated with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for WB (E) or IL-1b ELISA (F). IL-1b release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 82.9 to 245.6 pg/ml. *p , 0.05 as compared with LPS/alum, as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test (F). (G and H) Primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete culture medium and then 30 min with EPAC inhibitor (4-cyclopentyl- 2-(2,5-dimethylbenzylsulfanyl)-6-oxo-1,6-dihydropyrimidine-5-carbon- itrile, 10 mM) or vehicle (DMSO), followed by 30 min treatment with PGE2 (0.1 mM) or vehicle (EtOH) and stimulation with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for WB (G) or IL-1b ELISA (H). IL-1b release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 206.2 to 226.4 pg/ml. *p , 0.05 as compared with LPS/alum, as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test (H).

cPLA2a activation, AA release, and downstream PGE2 production primaryMDMs,andcPLA2a siRNA-transfected THP-1 cells (37, 38). Moreover, ATP or crystals can lead to cPLA2a-depen- were used. We found that inhibition of cPLA2a enhanced NLRP3 dent PGE2 production (39, 41). Therefore, we studied whether inflammasome activation in THP-1 cells (Fig. 8A). Similarly, blockade of AA release and PGE2 production would influence knockdown of cPLA2a in THP-1 cells resulted in an increase of NLRP3 activation. PMA-induced THP-1 macrophages, human NLRP3 inflammasome activation (Fig. 8B). We also studied pri- The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 7. PGE2-induced NLRP3 inflammasome inhibition requires higher doses in CAPS patients. (A) PBMCs from four healthy subjects were treated with LPS (1 mg/ml) for 3 h in complete medium, followed by treatment with the indicated doses of PGE2 or vehicle (EtOH) and ATP (2 mM) or alum (400 mg/ml) for 30 min in serum-free medium. Supernatants and lysates were collected for Western blot (WB). (B) PBMCs from five CAPS patients with designated mutations in NLRP3 were treated with LPS (1 mg/ml) for 3 h in complete medium, followed by treatment with indicated doses of PGE2 or vehicle (EtOH) for 30 min in serum-free medium. Supernatants and lysates were collected for WB. 12 PGE2 INHIBITS NLRP3 INFLAMMASOME Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 8. cPLA2a is involved in endogenous NLRP3 inflammasome regulation. (A) THP-1 cells were treated with cPLA2 inhibitor (pyrrolidone derivative, 2 mM, for 30 min) or vehicle (DMSO) and then primed with LPS (100 ng/ml) for 4 h in serum-free medium, followed by treatment with nigericin (4 mM for 45 min), ATP (1 mM, for 1 h), monosodium urate crystals (MSU, 100 mg/ml, for 5 h) or alum (200 mg/ml, for 5 h). Supernatants and lysates were collected for Western blot (WB). The WBs are representative of two independent experiments, each showing (Figure legend continues) The Journal of Immunology 13 Downloaded from FIGURE 9. COX2 is involved in endogenous NLRP3 inflammasome regulation. (A–D) Primary MDMs were treated with/without LPS (100 ng/ml) with NS398, a specific COX2 inhibitor (5 mM), or DMSO for 4 h in complete medium, followed by subsequent treatment with NS398 (5 mM) or DMSO for 30 min and then stimulated with ATP (2 mM for 1 h) (A and C) or alum (400 mg/ml for 5 h) (B and D) in serum-free medium. Supernatants and lysates were collected for PGE2 ELISA (A and B) or Western blot (WB) (C and D). The WBs are representative of three independent experiments from three healthy donors, each showing similar results. PGE2 release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. *p , .05 as indicated, #p , .05 as compared to LPS, &p , .05 as compared to LPS/ATP or LPS/alum, as assessed by Kruskal– E

Wallis ANOVA on ranks, followed by Dunn’s post hoc test. ( ) Primary MDMs were treated with LPS (100 ng/ml) with/without NS398 (5 mM), NS398 http://www.jimmunol.org/ alone, or DMSO for 4 h in complete medium and then stimulated with alum for 15 min in serum-free medium. cAMP measured in cell lysates represents the mean 6 SEM from three independent experiments from three healthy donors, each performed in triplicate. *p , 0.05 as assessed by Kruskal–Wallis ANOVA on ranks, followed by a Dunn post hoc test.

mary human MDMs, which were treated with cPLA2 inhibitor PGE2 production, are involved in the regulation of NLRP3 before the priming step or after priming to block cPLA2a-de- inflammasome activation. pendent signals during inflammasome activation. We first con- firmed that PGE production was induced by LPS treatment and Discussion

2 by guest on October 1, 2021 further increased by alum (Fig. 8C, 8F). PGE2 production induced Monocytes and macrophages are among the first cells involved in the by LPS alone was blocked in the presence of cPLA2 inhibitor acute phase of inflammation. They infiltrate the tissue in response to added before priming (Fig. 8C) and was significantly decreased chemotactic agents and respond to the invading pathogen or tissue when added before alum treatment (Fig. 8F). Under the same trauma by several mechanisms, including inflammasome activation. experimental conditions we found that cPLA2a inhibitor added The inflammatory response should subside once it has carried out its before or after priming increased NLRP3 inflammasome activa- function to maintain homeostasis. Activated NLRP3 inflammasome tion in human primary MDMs (Fig. 8D, 8E, 8G, 8H). To more needs to be counterbalanced by the existence of local mediators or selectively block only PGE2 production, we also inhibited COX2 mechanisms that quickly quiet the response and lead to the resolution by using its selective inhibitor NS398. First, we confirmed that of acute inflammation. In the present study, we have found that PGE2 NS398 blocked LPS/ATP-induced (Fig. 9A) as well as LPS/alum- inhibited NLRP3 inflammasome activation in human primary induced PGE2 (Fig. 9B) production. Similar to cPLA2 inhibition, MDMs. We have demonstrated that this effect depends on EP4 and inhibition of COX2 led to an increase of NLRP3 inflammasome an increase in intracellular cAMP, but not on PKA or Epac- activation (Fig. 9C, 9D). Additionally, we also noted that treat- mediated pathways. We also found that PGE2 decreased IL-1b ment with NS398 decreased intracellular cAMP (Fig. 9E). Col- secretion in patients with CAPS, but in higher doses than in healthy lectively, these data suggest that cPLA2a and COX2, through subjects. Moreover, we have demonstrated that this mechanism is

similar results. (B) THP-1 cells were transfected with cPLA2a siRNA or control siRNA (1 mM). After 48 h transfected cells were primed with LPS (100 ng/ml) for 4 h in serum-free medium, followed by stimulation with alum (200 mg/ml for 5 h). Supernatants and lysates were collected for WB. The WBs are representative of two independent experiments, each showing similar results. (C–E) Human primary MDMs were treated with/without LPS (100 ng/ml) with cPLA2 inhibitor (2 mM) or DMSO for 4 h in complete medium, followed by subsequent treatment with cPLA2 inhibitor or DMSO for 30 min, and then stimulated with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for PGE2 ELISA (C), WB (D), or IL-1b ELISA (E). PGE2 (C) and IL-1b (E) release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. IL-1b data are presented as the percentage of the response after LPS/alum treatment, which ranged from 409.4 to 647.6 pg/ml (E). (F–H) Human primary MDMs were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by treatment with cPLA2 inhibitor or DMSO for 30 min, and then stimulated with alum (400 mg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for PGE2 ELISA (F), WB (G), or IL-1b ELISA (H). PGE2 (F) and IL-1b (H) release data represent mean 6 SEM from three independent experiments from three healthy donors, each performed in duplicate. IL-1b data are presented as the percentage of the response after LPS/alum treatment, which ranged from 261.2 to 516.7 pg/ml (E). The WBs are representative of three independent experiments from three healthy donors, each showing similar results (D and G). *p , 0.05 as assessed by Kruskal– Wallis ANOVA on ranks, followed by a Dunn post hoc test. a.p., after priming; b.p., before priming. 14 PGE2 INHIBITS NLRP3 INFLAMMASOME involved in an autocrine or paracrine loop, possibly controlling the decrease NLRP1 and NLRP3 activation. b-arrestin-2 was shown extent of NLRP3 inflammasome activation. to bind to full-length NLRP3 and its C-terminal leucine-rich re- Our results are consistent with the recent findings that cAMP is peat domain and NBD (NACHT) as well as to NLRP1 in an directly involved in the endogenous control of NLRP3 inflam- HEK293T overexpression system, but in Arrb22/2 knockout mice masome activation (18). Calcium sensing receptor, belonging to the reversal of DHA or eicosapentaenoic acid–mediated inhibition the GPCR family, in response to the extracellular Ca2+ increase was incomplete. However, DHA via this axis is able to suppress activates phospholipase C and inhibits AC. As a result there is an high fat diet–induced NLRP3 inflammasome activation and the increase in intracellular Ca2+ and decrease in cAMP, both of prevention of NLRP3 inflammasome-dependent insulin resistance which can independently activate NLRP3 inflammasome (18). In in vivo (16). We have recently determined that DHA also through this study, we also provided three lines of evidence showing that GRP120 activates cPLA2a and induces PGE2 production in RAW PGE2-mediated inhibition of NLRP3 inflammasome occurred by 264.7 cells and human primary macrophages (59). This suggests an increase in intracellular cAMP. First, KH7, an AC inhibitor, that the proposed PGE2-mediated inhibition of NLRP3 by increase reversed PGE2-mediated NLRP3 inflammasome inhibition. Sec- in cAMP might be an additional mechanism of omega-3–derived ond, forskolin itself as an AC activator decreased NLRP3 acti- effects. vation, and this effect was enhanced by cotreatment with PGE2. The role of PGE2 on IL-1b production in monocytes and mac- Moreover, IMBX, a phosphodiesterase inhibitor, totally blocked rophages has been previously suggested; however, several contra- alum-induced NLRP3 activation. Finally, 8-bromo-cAMP, a cell- dictory results have been reported and the mechanisms involved permeable analog of cAMP, in a dose-dependent manner mim- have not been described. Early studies suggested that PGE2 inhibits icked PGE2-mediated NLRP3 inflammasome inhibition. Another IL-1b secretion in macrophages (60–62) and monocytes (63, 64), recent study in which the authors used relatively low concen- not at the level of gene transcription but by a posttranscriptional Downloaded from trations of forskolin (up to 20 mM) reports extracellular Ca2+ mechanism, which was not well defined (63). Others reported that sensing by both calcium sensing receptor and GPRC6A in NLRP3 in monocytes, PGE2 enhances IL-1b mRNA expression and pro- activation; however, cAMP involvement was not demonstrated tein production via cAMP (65–67) and PKA-CREB–dependent (56). We have shown that even when forskolin was used in transcriptional events (68). With these differences in mind, we a concentration of 50 mM, there was still an additive effect of studied the effect of PGE2 on inflammasome activation. We found http://www.jimmunol.org/ PGE2, suggesting that AC might require stronger activation to that PGE2 added after LPS priming decreased NLRP3 inflamma- elicit its full cAMP-producing potential. Interestingly, it has been some activation via a cAMP-dependent mechanism as assessed by demonstrated that this cAMP effect is not dependent on PKA, the decrease in mature IL-1b and mature caspase-1 release and IL- a major cAMP binding protein, but is driven by direct binding of 18 release, as well as a decrease in ASC oligomerization. This cAMP to the NBD of NLRP3 protein (18). We have confirmed might account for previous observations that were noted before the these findings and extended them, showing that cAMP-mediated development of the inflammasome concept. Nonetheless, having inhibition of NLRP3 is also not dependent on Epac, the second controlled for IL-1b mRNA and protein expression, we also noted major intracellular cAMP binding protein. Similarly, we have also a nonsignificant decrease at the mRNA level, although reflected by found that in patients with CAPS, carrying mutations in the NBD the slight decreases in total pro–IL-1b levels in the cells, pointing by guest on October 1, 2021 of NLRP3 gene that result in the impairment of cAMP binding out the complexity of PGE2 in the control of IL-1b release. (18), the PGE2-mediated NLRP3 inflammasome inhibition was Our findings that cPLA2a or COX2 inhibition resulting in the less efficient and required 10- to 100-fold higher doses of PGE2 to decrease of PGE2 led to an increase in NLRP3 inflammasome elicit similar effect as in healthy subjects. This suggests that activation and IL-1b secretion in macrophages are consistent with patients with CAPS might be more resistant to signals leading to the several early works studying effects of various nonsteroidal resolution of inflammation, resulting in prolonged inflammation. anti-inflammatory drugs on IL-1b production. Indomethacin (61– They might also generate more PGE2 as a derivative of an ex- 64, 69) and piroxicam (69) were reported to increase IL-1b pro- cessive need to extinguish NLRP3 inflammasome and increased duction. Alternatively, it was reported that inhibition of cPLA2 or IL-1b signaling giving the positive feedback for activation of COX2 leads to a decrease in mature IL-1b secretion (55, 70, 71). cPLA2a and expression of COX2 and mPGES1 (57). The pleio- The differences with our findings might result from the dose and tropic and detrimental effects of prolonged, excessive PGE2 sig- type of inhibitors used in those studies, as well as the type of cells, naling in various tissues might lead to secondary pathologies (27). their redox state, and EP expression. Our results of pharmaco- Indeed, this has been demonstrated in patients with NOMID, the logical inhibition with a low dose of a highly specific COX2 in- most severe phenotype among CAPS variants, who suffer from hibitor, cPLA2a inhibitor, and cPLA2a knockdown by siRNA, in severe arthropathy and develop bulky masses in their long bones conjunction with the observations of the exogenous PGE2 effects (58). There is an increase in PGE2, cAMP, and PKA activity in on NLRP3 inflammasome activation, suggest that this axis might NOMID tumor cells, leading to abnormal Wnt signaling and play a regulatory role in human macrophages. proliferation, whereas inhibition of both PGE2 and caspase-1 leads In summary, our results suggest that PGE2 might serve as a local to a decrease in NOMID cell proliferation (58). inhibitor of NLRP3 inflammasome in monocytes and macro- PGE2 acts through four different receptors designated EP1, phages at the site of acute inflammation. In general, this mecha- EP2, EP3 and EP4, with expression variations occurring in dif- nism might serve as a switch to resolution of inflammation and ferent cells and tissue types. Using multiple pharmacological and return to homeostasis, but once dysregulated, it might lead to molecular approaches, we have determined that PGE2 acted spe- detrimental effects and pathology. cifically through EP4 to decrease NLRP3 inflammasome activa- tion in human primary MDMs. Recently, two other GPCRs have Acknowledgments been shown to be involved in the negative regulation of NLRP1 We are grateful to Drs. Chong-Shan Shi and Jehad Edwan for input and and NLRP3 inflammasomes. Yan et al. (16) have determined that helpful discussions. omega-3 fatty acids such as eicosapentaenoic acid and docosa- hexaenoic acid (DHA) acting through GPR120 and GPR40 and at Disclosures least partially by their downstream scaffolding protein b-arrestin-2 The authors have no financial conflicts of interest. The Journal of Immunology 15

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