Inflammasomes Inhibits the NLRP1 and NLRP3 2 the Cyclopentenone Prostaglandin 15D-PGJ

The Cyclopentenone Prostaglandin 15d-PGJ2 Inhibits the NLRP1 and NLRP3 Inflammasomes

This information is current as Nolan K. Maier, Stephen H. Leppla and Mahtab Moayeri of October 1, 2021. J Immunol 2015; 194:2776-2785; Prepublished online 13 February 2015; doi: 10.4049/jimmunol.1401611 http://www.jimmunol.org/content/194/6/2776 Downloaded from

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References This article cites 69 articles, 25 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/194/6/2776.full#ref-list-1

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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. The Journal of Immunology

The Cyclopentenone Prostaglandin 15d-PGJ2 Inhibits the NLRP1 and NLRP3 Inﬂammasomes

Nolan K. Maier, Stephen H. Leppla, and Mahtab Moayeri

Inflammasomes are cytosolic protein complexes that respond to diverse danger signals by activating caspase-1. The sensor components of the inflammasome, often proteins of the nucleotide-binding oligomerization domain–like receptor (NLR) family, 12,14 detect stress, danger stimuli, and pathogen-associated molecular patterns. We report that the eicosanoid 15-deoxy-D –PGJ2 (15d-PGJ2) and related cyclopentenone PGs inhibit caspase-1 activation by the NLR family leucine-rich repeat protein (NLRP)1 and NLRP3 inflammasomes. This inhibition was independent of the well-characterized role of 15d-PGJ2 as a peroxisome proliferator receptor-g agonist, its activation of NF erythroid 2–related factor 2, or its anti-inflammatory function as an inhibitor of

NF-kB. Instead, 15d-PGJ2 prevents the autoproteolytic activation of caspase-1 and the maturation of IL-1b through induction of a cellular state inhibitory to caspase-1 proteolytic function. The eicosanoid does not directly modify or inactivate the caspase-1 enzyme. Rather, inhibition is dependent on de novo protein synthesis. In a mouse peritonitis model of gout, using monosodium Downloaded from urate crystals to activate NLRP3, 15d-PGJ2 caused a significant inhibition of cell recruitment and associated IL-1b release. Furthermore, in a murine anthrax infection model, 15d-PGJ2 reversed anthrax lethal toxin-mediated NLRP1-dependent resistance. The findings reported in this study suggest a novel mechanism for the anti-inflammatory properties of the cyclopentenone PGs through inhibition of caspase-1 and the inflammasome. The Journal of Immunology, 2015, 194: 2776–2785.

he inflammasome is a multiprotein complex that forms in The PGs are lipid signaling molecules derived from arachidonic http://www.jimmunol.org/ response to a variety of danger signals. Upon activation, acid that have diverse functions. Production of PGs depends on T the sensor protein, often of the nucleotide-binding oligo- the action of the cyclooxygenase (COX) enzymes of which there merization domain–like receptor (NLR) family, recruits caspase-1 are two isoforms. COX-1 is expressed constitutively and predomi- (formerly IL-cleavage enzyme). Caspase-1 undergoes autopro- nantly produces prostanoids with housekeeping functions. COX-2 teolytic activation and cleaves the zymogen forms of the proin- is induced following inflammatory stimuli and is the dominant flammatory cytokines IL-1b and IL-18 to their mature forms, source of PGs during the inflammatory response. Most PGs exert which are then secreted from the cell. Caspase-1 activation also their effects through activation of specific transmembrane G pro- leads to a rapid form of lytic cell death termed pyroptosis (1). tein–coupled receptors. These receptors activate a varied set of by guest on October 1, 2021 Inflammasome function plays a key role in the innate immune intracellular signaling pathways that lead to diverse biological response to infection (2), and the mechanisms by which pathogens activities (3). Many PGs have demonstrated proinflammatory activate and evade inflammasome detection are an active area effects, but anti-inflammatory effects have been reported for of study. However, inflammasome signaling and the associated cyclopentenone PGs. Cyclopentenone PGs contain a highly re- cytokine responses or tissue necrosis are also associated with active a,b-unsaturated carbonyl that can form covalent attach- numerous inflammatory disorders, including type 2 diabetes, ments with thiol groups on proteins, thus altering their function Alzheimer’s disease, arthritis, macular degeneration, and asthma (4). These PGs inhibit proinflammatory NF-kB activity through (1). For this reason, discovery of novel inflammasome inhibitors is covalent modification of IKK as well as direct inhibition of NF-kB of clinical importance. binding to DNA (5–7). They also upregulate and activate anti- inflammatory heat shock proteins (HSPs) such as HSP70 (8). 12,14 15-Deoxy-D –PGJ2 (15d-PGJ2) is the best studied anti- Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Dis- inflammatory PG. It is formed as a dehydration product of eases, National Institutes of Health, Bethesda, MD 20892 PGD (9) and was the first identified endogenous ligand of the Received for publication June 25, 2014. Accepted for publication January 8, 2015. 2 nuclear receptor peroxisome proliferator–activated receptor g This work was supported by the Intramural Research Program of the National Insti- tute of Allergy and Infectious Diseases. (PPARg) (10, 11). 15d-PGJ2 also activates the transcription factor NF erythroid 2–related factor 2 (NRF2) through formation of Address correspondence and reprint requests to Dr. Mahtab Moayeri, National In- stitutes of Health, Building 33, Room 1W20B, Bethesda, MD 20892. E-mail address: adducts with the NRF2 inhibitor KEAP1 (12). Both PPARg and [email protected] NRF2 are known to have anti-inflammatory roles (13, 14). It is The online version of this article contains supplemental material. through these actions that this lipid mediator can alter expression Abbreviations used in this article: BMDM, bone marrow–derived macrophage; COX, of cytokine, chemokine, and proinflammatory genes (15). A 12,14 cyclooxygenase; 15d-PGJ2, 15-deoxy-D –PGJ2; FlaTox, combination of anthrax number of studies have shown that 15d-PGJ has therapeutic po- protective Ag and fusion protein of N-terminal domain of anthrax lethal factor to 2 flagellin; FP59, fusion protein of N-terminal domain of anthrax lethal factor to the tential in animal models of inflammatory disease (for review, see ADP-ribosylating domain of Pseudomonas aeruginosa exotoxin A; HSP, heat shock Ref. 15). protein; LF, anthrax lethal factor; LFn-Fla, fusion protein of N-terminal domain of In this study, we report on a novel mechanism for the anti- anthrax lethal factor to flagellin; LT, anthrax lethal toxin (combination of anthrax protective Ag and anthrax lethal factor); MSU, monosodium urate; NLR, nucleotide- inflammatory actions of 15d-PGJ2 and related cyclopentenone binding oligomerization domain–like receptor; NLRP, nucleotide-binding oligomer- PGs. Our studies show that these PGs are potent inhibitors of the ization domain–like receptor family leucine-rich repeat protein; NRF2, NF erythroid 2–related factor 2; PA, anthrax protective Ag; PPARg, peroxisome proliferator– anthrax lethal toxin (LT) activation of the NLR family leucine-rich activated receptor g. repeat protein (NLRP)1 inflammasome and nigericin-mediated www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401611 The Journal of Immunology 2777 activation of the NLRP3 inflammasome. These PGs inhibit in- with PBS (6 ml/mouse). Infiltrating cells were counted following eryth- flammasome-mediated activation of caspase-1 and, as a conse- rocyte lysis using ACK buffer (Life Technologies). IL-1b measurements in quence, prevent maturation and release of IL-1b, both in vitro and lavage fluids were made using an ELISA kit from R&D Systems (Min- neapolis, MN). in vivo, in a manner independent of effects on NF-kB. Our find- For sensitization of anthrax spore–resistant NLRP1bS-expressing mice, ings confirm the role of these lipid mediators as anti-inflammatory 15d-PGJ2 or vehicle was administered to BALB/cJ, C57BL/6J, C57BL/ agents and support their development as therapeutic agents for the 6NTac Nlrp1bS/S, or C57BL/6NTac Nlrp1bR;R mice (100 mg, 200 ml, s.c., 3 treatment of inflammatory diseases. at 5 min and 1 h after spore infection). Animals were infected with 2–4 107 A35 spores (200 ml, s.c.) and monitored for 7 d postinfection for signs of malaise or death. Materials and Methods All animal experiments were performed in strict accordance with Reagents guidelines from the National Institutes of Health and the Animal Welfare Act, approved by the Animal Care and Use Committee of the National 15d-PGJ2, CAY10410 (9,10-dihydro–15d-PGJ2), PGD2, PGE2, PGF2a, Institute of Allergy and Infectious Diseases, National Institutes of Health. rosiglitazone, and T0070907 were purchased from Cayman Chemical (Ann Arbor, MI). PGA1, PGJ2, 4-cyclopentene-1,3-dione, 2-cyclopentenone, Cytotoxicity assays cycloheximide, actinomycin D, indomethacin, buthionine sulfoximine, and N-acetylcysteine and uric acid were from Sigma-Aldrich (St. Louis, MO). RAW264.7 cells and BALB/cJ (LT-responsive) BMDMs were grown in 96- Cyclopentanone and cyclopentene were obtained from Tokyo Chemical well plates to 90% confluence and pretreated with various drugs or vehicle Industry (Portland, OR). Structures of these prostanoids are shown in at a range of doses or times (as described in the figure legends). Cells were Table I. Nigericin, lactacystin, and ultrapure LPS were purchased from then incubated with LT or medium. Cell viability was assessed by MTT Calbiochem (San Diego, CA). (Sigma-Aldrich) as previously described (26). In selected experiments, cell death was assessed by staining cells with 5 mM propidium iodide (Sigma-

Toxins Aldrich) in medium without phenol red. Fluorescence was measured on Downloaded from a Wallac 1420 Victor 3V (PerkinElmer, Waltham, MA) plate reader with Protective Ag (PA), lethal factor (LF), and FP59 were purified from Bacillus excitation at 530 nm and emission at 615 nm. anthracis as described previously (16, 17). FP59 is a fusion protein of the PA binding domain of LF to the ADP-ribosylation domain of Pseudomo- MEK, caspase-1, and IL-1b cleavage nas aeruginosa exotoxin A (18, 19). LFn-Fla, a toxin also delivered by PA, is a similar fusion of the first 254 aa of LF to full-length flagellin from RAW264.7 cells and BMDMs were exposed to LPS (1 mg/ml) and then Legionella pneumophila (gift of Dr. Russell Vance, University of Cal- various drugs or vehicle (at doses and timing indicated in the figure leg- ifornia at Berkeley, Berkeley, CA) (20). FlaTox is the combination of LFn- ends) prior to addition of inflammasome activators (LT, FlaTox, or niger- http://www.jimmunol.org/ Fla and PA. LT is the combination of LF and PA. Concentrations of LT icin, at indicated doses). Cells were then lysed in RIPA buffer containing correspond to the concentration of each toxin component (i.e., 1 mg/ml LT LF inhibitor PT-168541-1 (gift of Alan Johnson, Panthera Biopharma) and is 1 mg/ml PA plus 1 mg/ml LF). Concentrations of FlaTox correspond to processed for Western blotting using primary Abs against MEK1 (444942, the concentration of LFn-Fla. Concentration of PA was always twice that Calbiochem), IL-1b (AF-401-NA, R&D Systems), MEK3 (sc-959), and of LFn-Fla in FlaTox experiments (i.e., 1 mg/ml FlaTox is 2 mg/ml PA plus caspase-1 p10 (sc-514, Santa Cruz Biotechnology, Santa Cruz, CA) as 1 mg/ml LFn-Fla). previously described (26). Secondary Abs used in these studies were anti- B. anthracis spores were prepared from the nonencapsulated, toxigenic goat infrared dye (800CW) (Rockland Immunochemicals, Gilbertsville, B. anthracis Ames 35 (A35) strain (21) by growing the bacteria on spor- PA) and anti-rabbit infrared dye (800CW) (LI-COR Biosciences, Lincoln, ulation agar at 37˚C for 1 d followed by 5 d at 30˚C, and inspection by NE). Immunoblots were visualized with the Odyssey infrared imaging microscopy to verify .95% sporulation. Spores were purified from plates system (LI-COR Biosciences). by four rounds of centrifugation and sterile water washes, followed by two by guest on October 1, 2021 heat treatments at 70˚C for 30 min (to kill any vegetative bacteria). Spore In vitro caspase-1 assay quantification was performed using a Petroff Hausser counting chamber (Hausser Scientific, Horsham, PA) and verified by dilution plating. LPS (1 mg/ml for 2 h) was used to induce pro–IL-1b as a substrate for recombinant caspase-1. Approximately 107 cells were lysed per milliliter Cell culture sucrose buffer (250 mM sucrose, 10 mM HEPES [pH 7.3]) by passage through a 29-gauge needle. Lysates of LPS-treated RAW264.7 cells were RAW264.7 cells and L929 mouse fibroblast cells were grown in DMEM incubated with 1 U active recombinant mouse caspase-1 (MBL Interna- supplemented with 10% FBS, 10 mM HEPES, and 50 mg/ml gentamicin tional, Woburn, MA) per 50 ml lysate in the presence or absence of 15d- (all purchased from Life Technologies, Grand Island, NY). Selected PGJ2 or Boc-Asp(OBzl)-chloromethylketone (Anaspec, San Jose, CA) for studies used lower amounts of FBS as indicated in the figure legends. 3 h at 37˚C. In other experiments cells were first pretreated with 15d-PGJ2 Mouse bone marrow was cultured in complete DMEM (as above) sup- for 1 h prior to preparation of lysates. Caspase-1–mediated cleavage of plemented with 30% L929 cell–conditioned supernatant and grown 7– IL-1b was analyzed by Western blotting. Because 15d-PGJ2 is an inhibitor 9 d to allow time for differentiation to bone marrow–derived macrophages of the NF-kB cascade (5–7), and thus could impact LPS-mediated (BMDMs). upregulation of IL-1b, LPS priming was always performed prior to 15d- PGJ treatment in each experiment. Animal studies 2 Mice were used as source of bone marrow. BALB/cJ (harboring Nlrp1bS/S Evaluation of caspase-1 sequestration in a high m.w. complex and LT-responsive macrophages), C57BL/6J (harboring Nlrp1bR/R), and Sucrose buffer lysates of 15d-PGJ2–treated or heat-shocked (42˚C) mice deficient in NRF2 on the C57BL/6J background (N10) were pur- RAW264.7 cells (as a positive control for caspase-1 sequestration) (27) chased from The Jackson Laboratory (Bar Harbor, ME). Mice deleted for 3 S/S were centrifuged at 10,000 g for 10 min at 4˚C. Western blotting for NLRP3 have been previously described (22). C57BL/6NTac Nlrp1b caspase-1 was performed on the supernatant and pellet. congenic mice carrying the LT-responsive Nlrp1bS/S have been previously described (23). These mice are resistant to spore infection relative to their Evaluation of 15d-PGJ2 conjugation to NLRP3 congenic C57BL/6NTac Nlrp1bR/R counterparts due to LT-mediated activation of the NLRP1 inflammasome (23). COX-1 knockout mice on the C57BL/6J wild-type or NLRP3-deficient BMDMs were stimulated with C57BL/6NTac were a gift from Katrin Mayer-Barber (National Institute of LPS (1 mg/ml, 4 h) to induce upregulation of NLRP3. Cells were then Allergy and Infectious Diseases, Bethesda, MD). These mice were back- incubated with 15d-PGJ2 or biotinylated 15d-PGJ2 (Cayman Chemical) crossed (four generations) in our laboratory to the C57BL/6NTac Nlrp1bS/S (50 mM, 1 h). Sucrose buffer lysates were made in the presence of com- congenic mice carrying the LT-responsive Nlrp1bS/S allele plete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN). For the mouse uric acid–induced peritonitis studies (used as a model Approximately 200 mg cell lysate was mixed with 5 mg anti-NLRP3 Ab for the arthritic disease gout) (24), C57BL/6J mice were injected i.p. with (MAB7578, R&D Systems) and placed on a rotary shaker at 4˚C for 2 h. 125 mg 15d-PGJ2 or vehicle (10% DMSO in PBS) at 5 min prior and 4 h Protein A/G agarose slurry (20 ml, Santa Cruz Biotechnology) was added after administration of monosodium urate (MSU) crystals. MSU crystals and the incubation was continued overnight. Bound immune complexes were prepared by crystallization of uric acid as previously described (25) were washed with PBS four times prior to elution of proteins in 23 SDS and injected into mice (4 mg/250 ml PBS, i.p.). At 6 h after MSU ad- loading buffer. Biotinylated adducts were detected with IR680-conjugated ministration, mice were euthanized and peritoneal lavage was performed streptavidin (LI-COR Bioscienses) by Western blotting. 2778 CYCLOPENTENONE PGs INHIBIT THE INFLAMMASOME

Results Cyclopentenone PGs require an a,b-unsaturated carbonyl to prevent NLRP1-dependent pyroptosis Anthrax LT contains a protease that cleaves the N terminus of rodent NLRP1 proteins, leading to caspase-1 activation (28–30). Activation of caspase-1 leads to a rapid form of cell death known as pyroptosis (for review, see Ref. 31). LT activation of the NLRP1 inflammasome in selected inbred rodent strains that harbor LT- responsive alleles leads to pyroptosis, whereas strains with resistant NLRP1 alleles can undergo apoptosis (32–35). Our earlier studies showed that heat shock inhibits the inflammasome through sequestration of procaspase-1 in a large complex (27), preventing LT-mediated pyroptosis. We tested a number of cyclopentenone PGs that induce HSPs such as HSP70 (36, 37) along with cyclopentanone PGs for the ability to inhibit the NLRP1 inflammasome- dependent pyroptosis of LT-sensitive macrophages. All the cyclopentenone PGs tested inhibited the well-characterized NLRP1- dependent LT-induced pyroptosis. Interestingly, most of the cyclopentanone PGs that were tested failed to inhibit (Fig. 1A, Table I). Downloaded from The exception was the cyclopentanone PG PGD2, which had low inhibitory activity (Fig. 1A, Table I); however, it has been shown that this PG undergoes nonenzymatic dehydration to the cyclopentenone PG PGJ2 in aqueous media (38), and many of the purported physiological effects of PGD2 are actually mediated by PGJ2 and its

downstream dehydration products such as 15d-PGJ2 (39). http://www.jimmunol.org/ The restriction of inhibitory activity to only those PGs with cyclopentenone rings suggested a requirement for an a,b-unsaturated carbonyl. We further evaluated this requirement by examining the inhibitory activity of cyclopentene, cyclopentanone, 2-cyclopentenone, and various structural analogs (Table I). Cyclopentene and cyclopentanone, which have no a,b-unsaturated carbonyls, had no FIGURE 1. Cyclopentenone PGs protect cells from LT-induced pyroptosis. RAW264.7 cells (A) or BALB/cJ BMDMs (B) were treated with effect on LT-induced pyroptosis. However, 2-cyclopentenone, which varying concentrations of PGs for 30 min in a dose range where the has one carbonyl, did demonstrate inhibitory activity. Furthermore, 4- compounds were nontoxic. Cells were then challenged with 1 mg/ml LT cyclopentene-1,3-dione, which has two a,b-unsaturated carbonyls, and viability was measured by MTT staining at 1.5–2 h after LT. All values by guest on October 1, 2021 had a lower EC50 than did 2-cyclopentenone. Finally, the 15d-PGJ2 are averages from two wells in one representative experiment. Each curve analog CAY10410, which is identical to 15d-PGJ2 except for the is representative of at least 2 and up to 10 similar experiments, depending absence of the a,b-unsaturated bond in the ring, had no inhibitory on the particular compound shown. (C) RAW264.7 cells were incubated activity. with 15d-PGJ2 (50 mM, 30 min) followed by LT (1 mg/ml) for indicated times. Western blotting of cell lysates was performed with Abs against the 15d-PGJ2 inhibited LT-induced pyroptosis of both RAW264.7 N terminus of MEK1 and MEK3. The asterisk (*) indicates a cross-reac- cells (Fig. 1A) and BMDMs (Fig. 1B) with an EC50 in the micromolar (20-40 mM) range. Reducing the amount of FBS to 1% tive protein band that acts as an internal equal loading control. Results represent three similar Western blots. or lower decreased the EC50 by up to 10-fold (Supplemental Fig. 1A). This finding is in agreement with studies showing that electrophilic cyclopentenone PGs such as 15d-PGJ are rapidly 2 IkB as well as binding of NF-kB to DNA (5–7). NF-kB signaling inactivated by FBS in cell culture medium (40). is important for priming of the inflammasome. For all studies of 15d-PGJ2 does not inhibit LT cleavage of MEKs inflammasome activation and cytokine processing, cells were primed with LPS before exposure to 15d-PGJ to avoid any NF- To determine whether 15d-PGJ2 protects against LT pyroptosis by 2 inhibiting toxin translocation to the cytosol or enzymatic activity, kB signaling inhibitory effects that could lead to differences in we examined the cleavage of the cytosolic MEK substrates of LT. priming and IL-1b levels. Using this method, no differences in LT rapidly cleaves the N terminus of multiple MEK proteins, and inflammasome priming, indicated by pro–IL-1b levels, were ob- this cleavage can be monitored by loss of an epitope (MEK1) and served in PG-treated cells in any experiments (as demonstrated by by altered mobility of the MEK3 protein (41, 42). We found that all Western blots). 15d-PGJ2 inhibited caspase-1 autoproteolysis cleavage of the MEK proteins was uninhibited by treatment of (monitored by generation of the p10 fragment) as well as maturation and secretion of IL-1b in LT-treated cells at a step subse- the cells with 15d-PGJ2, thus confirming that, in the presence of the drug, active LT translocated to the cytosol (Fig. 1C). Thus 15d- quent to NF-kB signaling (Fig. 2A). Early studies have established that MEK signaling pathways that led to NF-kB activation (41, PGJ2 does not impact the binding, endocytosis, translocation, or activity of LF. 42) are inactivated by LT, and, furthermore, inhibitors of NF-kB such as BAY11-7082, BAY11-7085, SN-50, and knockdown of 15d-PGJ2 inhibits cytokine processing of multiple p65 have previously been shown to have no effect on LT-mediated inflammasomes activation of NLRP1 (data not shown). Thus, the inhibitory effects Inflammasome activation causes rapid autoproteolysis of caspase-1, of LT on caspase-1 were independent of effects on NF-kB. In- which leads to processing and secretion of the proinflammatory terestingly, application of the drug up to 45 min after LT caused cytokine IL-1b (1). 15d-PGJ2 inhibits NF-kB phosphorylation by significant inhibition of cytokine processing, further confirming an The Journal of Immunology 2779

Table I. EC50 values of structural variants of 15d-PGJ2 15d-PGJ2 is capable of inhibiting multiple inﬂammasomes with diverse activating stimuli.

15d-PGJ2 induces a cellular state inhibitory to caspase-1 Given the ability of this compound to inhibit multiple inﬂamma- somes, we hypothesized that 15d-PGJ2 could inhibit caspase-1 directly. The a,b-unsaturated carbonyl makes cyclopentenone PGs highly electrophilic and capable of conjugation to cysteine residues on various proteins, altering their functions (4). Caspase-1, a cysteine protease, contains catalytically important thiol groups that could be modiﬁed. However, using recombinant active caspase in Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

aRAW264.7 cells were treated 30 min with variable concentrations of compound. Cells were challenged with LT (1 mg/ml, 1.5 h) and cell viability was measured by MTT staining. At least two experiments represented by a single shown study were used to generate an EC50 value for each compound’s ability to inhibit LT-induced pyroptosis. effect independent of inhibition of NF-kB signaling. Inhibition of pyroptosis by 15d-PGJ2 required earlier application of the drug (Supplemental Fig. 1B, 1C). Importantly, the ability of 15d-PGJ2 to inhibit the inflammasome was not limited to NLRP1 or linked FIGURE 2. 15d-PGJ inhibits caspase-1 autoproteolysis and cytokine to pyroptosis. 15d-PGJ2 also inhibited cytokine processing and 2 secretion in response to treatment with nigericin, an ionophore maturation of multiple inflammasomes. BALB/cJ BMDMs were primed that activates the NLRP3 inflammasome (Fig. 2B). 15d-PGJ with LPS (1 mg/ml, 2 h). Cells were then exposed to 15d-PGJ2 (50 mM, 2 30min)followedby(A)LT(1mg/ml), (B) nigericin (50 mM), or (C)FlaTox demonstrated only modest inhibition of the NLR family caspase-1 (1 mg/ml) for indicated times. Western blotting of cell lysates and culture recruitment domain–containing protein 4/NLR family apoptosis supernatants was performed with Abs against the p10 fragment of caspase- inhibitory protein 5 inflammasome after stimulation with FlaTox 1 or IL-1b. The asterisk (*) indicates a cross-reactive protein band that acts (Fig. 2C). However, previous work has similarly found that FlaTox as an internal equal loading control. Results shown in these Western blots is more difficult to inhibit (43), possibly due to its ligand binding– are representative of between 4 andupto16similarexperimentsper based mechanism of activation (44, 45). These data indicate that treatment. 2780 CYCLOPENTENONE PGs INHIBIT THE INFLAMMASOME

a cell lysate assay where 15d-PGJ2 was added to lysates, we found required de novo protein translation, either to induce a cytopro- no evidence of direct caspase-1 inhibition (Fig. 3A). Interestingly, tective protein or to maintain a certain threshold level of a cas- however, lysates made from cells pretreated with 15d-PGJ2 were pase-1 inhibitory protein with normally rapid turnover (the levels inhibitory to recombinant caspase-1 (Fig. 3B). This indicated that of which diminish after inhibition of protein synthesis). Inter- a cellular state or protein that could inhibit caspase-1 enzymatic estingly, inhibition of NLRP3-mediated cytokine processing was activity was induced by 15d-PGJ2 in intact cells. not affected by cycloheximide treatment (data not shown), suggesting that the lipid mediator could have multiple inhibitory Inflammasome inhibition by 15d-PGJ is dependent on protein 2 mechanisms. translation We previously reported that both heat shock and arsenical com- Inhibition is independent of PPARg, NRF2, or COX-1 pound inhibition of inflammasome activation did not require de novo protein synthesis (27, 43). In the case of heat shock, 15d-PGJ2 is a ligand of the nuclear receptor PPARg (10, 11), procaspase-1 is trapped in a high m.w. complex that can be which is known to have anti-inflammatory roles (14). We hy- centrifuged away from the cytosolic cellular fraction (27). We pothesized that the eicosanoid could mediate its effects through this receptor. However, the PPARg agonist rosiglitazone was un- found that 15d-PGJ2, a potent inducer of heat shock proteins (8), did not induce sequestration of caspase-1 into a high m.w. com- able to inhibit LT-induced pyroptosis (Fig. 5A). Furthermore, plex (Supplemental Fig. 2). despite loss of the a,b-unsaturated bond in the ring, the 15d-PGJ2 In contrast to the response to both heat shock and arsenical analog CAY10410 retains the ability to activate PPARg (46). This compounds, the transcription inhibitor actinomycin D and the drug does not protect cells from LT-induced pyroptosis (Table I). translation inhibitor cycloheximide reversed the protective effects Similarly, these compounds were unable to prevent LT-induced Downloaded from (Fig. 5B) or nigericin-induced (data not shown) cytokine pro- of 15d-PGJ2 in a dose-dependent manner (Fig. 4A, 4B). This requirement for transcription further supported the fact that 15d- cessing. Additionally, the PPARg inhibitor T0070907 did not reverse the ability of 15d-PGJ2 to inhibit the inflammasome PGJ2 manifested its inhibitory effects on the inflammasome in a manner independent of the eicosanoid’s inhibitory effect on NF- (Fig. 5C, 5D). Taken together, these data suggest that PPARg kB transcription. Similar results were found upon an even more signaling and transcriptional processes downstream of this re- complete enzymatic inhibition of translation using FP59, a fusion ceptor are not required for the effects of 15d-PGJ2 on the http://www.jimmunol.org/ protein of an ADP-ribosylating enzyme that targets elongation inflammasome. factor 2 (Fig. 4C). Furthermore, inhibition of translation partially 15d-PGJ2 also activates the cap‘n’collar family transcription factor NRF2 (12) by binding to cysteine residues of the NRF2 restored cytokine processing in 15d-PGJ2–treated cells (Fig. 4D). repressor KEAP1. NRF2 controls the expression of a large and This evidence indicated that inflammasome inhibition by 15d-PGJ2 diverse group of cytoprotective and anti-inflammatory genes (13), which we hypothesized could be inhibitory to inflammasome function. However, this seemed unlikely as NRF2 is stabilized and activated by proteasome inhibition (47–50), and treatment of

macrophages with the proteasome inhibitor lactacystin does not by guest on October 1, 2021 prevent nigericin-based NLRP3-dependent cytokine processing (data not shown and Ref. 35). We tested the effects of 15d-PGJ2 in BMDMs lacking NRF2 and found that the drug was capable of inhibiting nigericin-induced cytokine processing, indicating that the effects of this eicosanoid on the inflammasome did not involve this transcription factor (Fig. 5E). Recent studies have linked inflammasome activation with release of a variety of lipid mediators described as an “eicosanoid storm” (20). We hypothesized that modulation of this prostanoid release by exogenous 15d-PGJ2 could alter inflammasome function. However, 15d-PGJ2 inhibited LT-mediated pyroptosis and cytokine processing independent of COX-1, a central enzyme in the PG biosynthetic pathway (Fig. 6). Additionally, the cyclooxygenase inhibitor indomethacin did not reverse 15d-PGJ2– mediated protection from pyroptosis (Supplemental Fig. 3A).

Glutathione modulation does not alter inhibition Cyclopentenone PGs form conjugates with cellular glutathione that

FIGURE 3. 15d-PGJ2 does not inhibit caspase-1 enzymatic activity di- can alter the physiological effectiveness of the PG (51). Increasing rectly but induces a cellular state inhibitory to caspase-1. (A) RAW264.7 the levels of cellular glutathione using the glutathione precursor cells were primed with LPS (1 mg/ml, 2 h). Sucrose lysates were incubated N-acetylcysteine or decreasing cellular levels of glutathione using with recombinant active caspase-1 (1 U/50 ml, 3 h, 37˚C) in the presence the glutathione synthesis inhibitor buthionine sulfoximine had no or absence of 15d-PGJ2 at indicated concentrations or positive control effect on the protection of cells from LT-induced pyroptosis by caspase-1 inhibitor Boc-Asp(OBzl)-chloromethylketone (400 mM). (B) 15d-PGJ (Supplemental Fig. 3B, 3C). RAW264.7 cells were primed with LPS (1 mg/ml, 2 h) followed by 2 Cyclopentenone PGs are also potent inducers of intracellular treatment with 15d-PGJ2 (50 mM, 1 h). As above, sucrose lysates were incubated with recombinant active caspase-1. The asterisk (*) indicates reactive oxygen species (52), and oxidative stress is a potential a cross-reactive protein band that acts as an internal equal loading mechanism of inﬂammasome regulation (53). However, the failure control. Western blot results are representative of at least three similar of N-acetylcysteine, a potent antioxidant, to alter 15d-PGJ2 pro- experiments. tection suggests that induction of reactive oxygen species does not The Journal of Immunology 2781 Downloaded from

FIGURE 4. 15d-PGJ2–mediated inhibition of the NLRP1 inﬂammasome requires protein synthesis. (A) BALB/cJ BMDMs or (B) RAW264.7 cells were incubated with variable concentrations of cycloheximide or actinomycin D for 1 h. Cells were then exposed to 15d-PGJ2 (50 mM, 30 min) followed by LT (1 mg/ml, 1.5–2 h). (C) RAW264.7 cells were treated with 10 ng/ml PA with or without 100 ng/ml FP59 for 2 h. PA receptor saturation in this cell type occurs at doses 20-fold higher than that used here (69). Thus, sufﬁcient receptor remained on the cell surface to mediate LT intoxication. Unbound toxin was then washed from the cells, and the cells were incubated with 15d-PGJ2 and LT as above. Cell viability was measured by MTT staining. Each point was assayed in triplicate. Shown results are for a representative of two identical experiments. In (C), error bars represent SEM. (D) BALB/cJ BMDMs were http://www.jimmunol.org/ treated with LPS (1 mg/ml, 3 h). Cells were then allowed to rest in LPS-free media for 1 h before cycloheximide treatment (2 mg/ml, 1 h). 15d-PGJ2 (50 mM, 30 min) followed by LT (1 mg/ml, 75 min) were then applied. Cell lysates were analyzed for IL-1b maturation by Western blot. The asterisk (*) indicates a cross-reactive protein band that acts as an internal equal loading control. Western blot results are representative of three similar experiments.

play a role in the effects of 15d-PGJ2 on the inflammasome. and did not find any evidence that 15d-PGJ2 directly bound to Furthermore, alterations of oxidative state by 15d-PGJ2 are usu- NLRP3 (data not shown), suggesting that a novel inhibitory ally coupled with direct modification of proteins. We used a bio- mechanism is operating that does not involve covalent adduct tin-labeled 15d-PGJ2 analog coupled with immunoprecipitation formation to NLRP3. by guest on October 1, 2021

FIGURE 5. 15d-PGJ2 inﬂammasome inhibition is independent of PPARg and NRF2. (A) RAW264.7 cells were incubated with variable concentrations of PPARg agonist rosiglitazone for 1.5 h. Cells were exposed to LT (1 mg/ml, 2 h) and cell viability was assessed by MTT staining. (B) BALB/cJ BMDMs were primed with LPS (1 mg/ml, 2 h) followed by 15d-PGJ2, CAY10410, or rosiglitazone (50 mM, 30 min) and LT (1 mg/ml, 75 min). IL-1b in cell lysates and supernatants was assessed by Western blot. (C) RAW264.7 cells were treated with variable concentrations of PPARg antagonist T0070907 for 1 h before treatment with 15d-PGJ2 (50 mM, 30 min). Following LT treatment (1 mg/ml, 2 h), cell viability was assessed by MTT staining. (D) RAW264.7 cells were primed with LPS (1 mg/ml, 2 h) followed by treatment with T0070907 (30 mM, 1 h). Cells were then incubated with 15d-PGJ2 (50 mM, 30 min) followed by LT (1 mg/ml, 1.5 h). (E) 2/2 C57BL6/J wild-type and NRF2 BMDMs were primed with LPS (1 mg/ml, 2 h) followed by 15d-PGJ2 (50 mM, 30 min) and then with nigericin (50 mM, 30 min). Cell lysates and culture supernatants were analyzed for IL-1b maturation by Western blot. In (A)and(C), each condition was assayed in duplicate. Curves are representative of at least two similar experiments. The asterisk (*) indicates a cross-reactive protein band that acts as an internal equal loading control. 2782 CYCLOPENTENONE PGs INHIBIT THE INFLAMMASOME

zation to spore infection and a susceptibility comparable to mice harboring the Nlrp1b R/R allele (Fig. 7C, 7D). Because the effects of LT on susceptibility to spore are uniquely independent of NF- kB signaling, the 15d-PGJ2 effects in this model, unlike the MSU cell recruitment model, are likely to be on a downstream inﬂam- masome activation event required for IL-1b release.

Discussion In this study, we show that cyclopentenone PGs inhibit activation of caspase-1 and maturation of IL-1b by the NLRP1 and NLRP3 inflammasomes. This inhibition is shown to be independent of the drugs’ inhibitory effects on NF-kB signaling and transcription and not due to interference with inflammasome-activating stimuli or direct modification of caspase-1. Inflammasome inhibition oc- curred instead through induction of a cellular state inhibitory to casapse-1 enzymatic activity. This cellular state was dependent on de novo protein translation but independent of the PPARg or NRF2 signaling and transcription pathways often associated with the actions of these PGs. Our results suggest that a novel mechanism and transcription pathway are responsible for the anti- Downloaded from inflammatory effects of these PGs. There is substantial evidence that the reactive a,b-unsaturated carbonyl group is necessary for many of the biological activities of cyclopentenone PGs (4). The unsaturated carbonyl imparts a reactive electrophilic character to carbon 9 of the molecule. http://www.jimmunol.org/ FIGURE 6. 15d-PGJ2 inflammasome inhibition is independent of This allows the compound to form adducts with cellular thiols COX-1. (A) BMDMs from Cox12/2 (on the C57BL/6NTac Nlrp1bS/S such as glutathione or cysteine residues on proteins (4). The background) mice were primed with LPS (1 mg/ml, 2 h). Cells were then a,b-unsaturated carbonyl appears to be necessary for inflamma- incubated with 15d-PGJ2 (50 mM, 30 min) followed by LT (1 mg/ml, 75 some inhibition, as PGs lacking the cyclopentenone ring were min). Cell lysates were analyzed for IL-1b maturation by Western blot. (B) inactive. Similarly, 2-cyclopentenone, but not the related com- 2/2 COX-1 BMDMs were exposed to varying concentrations of 15d-PGJ2 pounds cyclopentene and cyclopentanone, was able to inhibit the for 30 min. Cells were challenged with 1 mg/ml LT and viability was inflammasome. This requirement for a chemically reactive center measured by MTT staining at 1.5 h after LT. Each condition was assayed in suggests that the mechanism of inflammasome inhibition is triplicate. Curves are representative of two or more similar experiments. through conjugation to exposed thiol residues on a key inflam- The asterisk (*) indicates a cross-reactive protein band that acts as an by guest on October 1, 2021 internal equal loading control. masome regulatory component. However, we found no evidence of direct inactivation of caspase-1 or NLRP3. The finding that de novo protein synthesis is required for the compound’s effects 15d-PGJ2 inhibits the inflammasomes in mice suggests that direct modification of the inflammasome is not To investigate the effects of 15d-PGJ2 in vivo, we used two models. likely, but we cannot rule out the possibility of 15d-PGJ2–medi- The MSU-induced peritonitis model used to replicate the in- ated modification of an unknown yet important inflammasome flammation associated with gout requires NLRP3 activation and component. Our finding that inhibition of NLRP3 was not de- caspase-1–dependent release of IL-1b for cell recruitment (24). pendent on protein synthesis suggests that the mechanism of 15d- We used this model in mice treated with vehicle or 15d-PGJ2 to PGJ2 inhibition differs across inflammasomes. This may be due to assess the relative cell numbers recruited to the peritoneum and the varied biochemical mechanisms of inflammasome activation. associated levels of secreted IL-1b. MSU crystals induced a robust NLRP1, for example, is activated in rodent macrophages through recruitment of cells to the peritoneum that was significantly direct proteolytic cleavage by LT (28–30). NLR family caspase-1 inhibited by the PG treatment (Fig. 7A). Similarly, the IL-1b re- recruitment domain–containing protein 4, in contrast, acts as an lease associated with injection of MSU was significantly lower in adapter for a number of NAIP proteins that directly bind to fla- mice treated with 15d-PGJ2 (Fig. 7B). To test the effects of the gellin and flagellin-like proteins (44, 45), and NLRP3 responds drug on the NLRP1 inflammasome in vivo, we took advantage to a wide range of danger signals through an as yet unresolved of the finding that inbred mouse strains with the LT-responsive mechanism (1). Nlrp1bS/S allele (such as BALB/cJ) are highly resistant to an- PPARg and NRF2 are the primary transcriptional pathways thrax spore infection compared with strains (such as C57BL/6 activated by 15d-PGJ2 (15), and we hypothesized that one of them mice) that express the NLRP1bR variant (23). Similarly, C57BL/ may play a role in the ability of this PG to inhibit the inflam- 6JNTac congenic mice, which are identical at .99% of loci but masome. 15d-PGJ2 was the first identified endogenous ligand of differ at the Nlrp1b locus, have striking different susceptibility to PPARg, and many of the biological effects of this PG result from spore infection that is linked to LT-induced, NLRP1b-mediated PPARg activation (10, 11). However, the compound also induces caspase-1–dependent control of IL-1b responses and neutrophil PPARg-independent reactions. In fact, prior studies have demon- recruitment (23). The mice in which LT can cleave and activate strated that 15d-PGJ2 has anti-inflammatory effects on macro- NLRP1b harbor myeloid cells that undergo pyroptosis in con- phages deficient in PPARg (54), and we found that inflammasome junction with an unprimed IL-1b response that leads to resistance. inhibition is also independent of PPARg. A recent study demon- We found that inhibition of this activation of NLRP1b by treat- strated that NRF2-deficient BMDMs are impaired in inflamma- ment of spore-resistant BALB/cJ or C57BL/6NTac Nlrp1bS/S some function (55). Although we also found a slight reduction of congenic mice with 15d-PGJ2 led to a rapid and striking sensiti- cytokine processing in NRF2-deficient BMDMs relative to wild- The Journal of Immunology 2783

type BMDMs, this difference is far less pronounced than previously reported. In striking contrast, treatment with 15d-PGJ2 po- tently inhibits inflammasome function in a manner independent of NRF2. The inducible cyclooxygenase COX-2 is thought to have a dual role in inflammation, both establishing the initial inflammatory response and leading to its resolution at later times. COX-2 is rapidly induced during inflammation and contributes to acute inflammation through production of proinflammatory signaling molecules (56). However, COX-2 induction during late stages of inflammation leads to production of anti-inflammatory PGs such as 15d-PGJ2 (57). These PGs along with additional lipid mediators called lipoxins, resolvins, and protectins thus constitute important endogenous molecules in the resolution of inflammation (58). In vivo concentrations of 15d-PGJ2 are thought to be in the nanomolar range (59). Thus, one may question the physiological relevance of the micromolar concentrations necessary for inflammasome inhibition in vitro. However, it has been demonstrated that upon dilution into culture media containing FBS, most (97–99%) of the PG is inactivated and does not enter cells (40). Downloaded from Thus, the levels actually available for the cells in vitro are likely at physiological concentrations. Additionally, it can be postulated that systemic measurements of free PG in vivo are an underesti- mation of true levels found within localized pockets of inflammation or in the cell vicinity, given the high reactivity of these eicosanoids with serum and intracellular proteins (60). http://www.jimmunol.org/ Exogenous administration of 15d-PGJ2 also has efficacy as an anti-inflammatory treatment (15). In the present study we show that 15d-PGJ2 can inhibit MSU-mediated neutrophil recruitment in an NLRP3-dependent inflammatory peritonitis model. Owing to the reactivity and resulting instability of cyclopentenone PGs, most experimental studies rely on administration of relatively high doses of 15d-PGJ2. Thus, alternative delivery mechanisms for the PG may increase its therapeutic potential. Loading of poly- by guest on October 1, 2021 glycolide nanocapsules with the PG increased serum availability and improved the anti-inflammatory activity of 15d-PGJ2 in a peritonitis model (61). Retroviral overexpression of the PG synthase responsible for 15d-PGJ2 production has also been suc- cessful in a murine air-pouch model of acute inflammation, as well as in bleomycin-induced lung injury and scleroderma models (62– 64). Synthetic derivatives of 15d-PGJ2 with similar biological activities have also been reported (65, 66). Administration as an anti-inflammatory treatment in animal models of inflammation, including ischemic brain injury, LPS-

panels. (C and D) Anthrax spore–resistant NLRP1bS/S-expressing BALB/cJ (n = 5) and C57BL/6NTac Nlrp1bS/S (n = 5) mice were treated with 15d-

PGJ2 (100 mg, 200 ml, s.c., at 5 min and 1 h after spore infection) or with vehicle and infected with 2 3 107 A35 spores (C)or43 107 A35 spores (D). Spore-sensitive NLRP1bR/R-expresssing C57BL/6J (n = 5) and C57BL/6NTac Nlrp1bR;R (n = 4) mice infected with the same spore dose served as controls. For (C), the p value comparing vehicle-treated, spore-

infected BALB/cJ mice to the 15d-PGJ2–treated infected BALB/cJ group or infected C57BL/6J mice is ,0.007. There is no significant difference FIGURE 7. 15d-PGJ2 inhibition of the inflammasome in mice. (A and B) between the infection susceptibility for drug-treated BALB/cJ mice and A well-established mouse monosodium urate induced peritonitis model genetically susceptible C57BL/6J mice infected with B. anthracis spores. was used to assess impact of 15d-PGJ2 on the NLRP3 inflammasome For (D), the p value comparing vehicle-treated, spore-infected C57BL/ S/S in vivo. C57BL/6J mice (n = 9/group) were injected i.p. with 125 mg 15d- 6NTac Nlrp1b to 15d-PGJ2–treated infected group is 0.0065. The p S/S PGJ2 or vehicle (10% DMSO in PBS) at 5 min prior and 4 h after ad- value comparing vehicle-treated, spore-infected C57BL/6NTac Nlrp1b ministration of MSU crystals. Control mice were injected with vehicle (n = mice to similarly infected C57BL/6NTac Nlrp1bR;R mice is 0.00429. The 4) or drug (n = 7) alone. MSU crystals were injected into mice (4 mg/250 ml susceptibility curve for the drug-treated and spore-infected C57BL/6NTac PBS, i.p.) and after 6 h infiltrating cells were counted (A) and IL-1b levels Nlrp1bS/S mice is not significantly different from the spore-infected ge- assessed (B). The p values (unpaired t test) comparing the MSU (plus netically susceptible C57BL/6NTac Nlrp1bR/R mice. The log-rank test was vehicle) groups to MSU (plus 15d-PGJ2) groups are ,0.0001 in both used for assessment of all p values in (C) and (D). 2784 CYCLOPENTENONE PGs INHIBIT THE INFLAMMASOME induced fever, spinal cord injury, chronic obstructive pulmonary 13. Baird, L., and A. T. Dinkova-Kostova. 2011. The cytoprotective role of the Keap1–Nrf2 pathway. Arch. Toxicol. 85: 241–272. disease, colitis, myocarditis, pancreatitis, and arthritis, has been 14. Clark, R. B. 2002. The role of PPARs in inflammation and immunity. J. Leukoc. reported (15). 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