The Induction of Pro–IL-1Β by Lipopolysaccharide Requires

The Induction of Pro−IL-1β by Lipopolysaccharide Requires Endogenous Prostaglandin E2 Production

This information is current as Zbigniew Zaslona, Eva M. Pålsson-McDermott, Deepthi of September 29, 2021. Menon, Moritz Haneklaus, Ewelina Flis, Hannah Prendeville, Sarah E. Corcoran, Marc Peters-Golden and Luke A. J. O'Neill J Immunol published online 15 March 2017

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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 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 15, 2017, doi:10.4049/jimmunol.1602072 The Journal of Immunology

The Induction of Pro–IL-1b by Lipopolysaccharide Requires Endogenous Prostaglandin E2 Production

Zbigniew Zasłona,* Eva M. Palsson-McDermott,*˚ Deepthi Menon,* Moritz Haneklaus,* Ewelina Flis,* Hannah Prendeville,* Sarah E. Corcoran,* Marc Peters-Golden,† and Luke A. J. O’Neill*

PGE2 has been shown to increase the transcription of pro–IL-1b. However, recently it has been demonstrated that PGE2 can block the maturation of IL-1b by inhibiting the NLRP3 inﬂammasome in macrophages. These apparently conﬂicting results have led us

to reexamine the effect of PGE2 on IL-1b production. We have found that in murine bone marrow–derived macrophages, PGE2 via the cAMP/protein kinase A pathway is potently inducing IL-1b transcription, as well as boosting the ability of LPS to induce IL-1b mRNA and pro–IL-1b while inhibiting the production of TNF-a. This results in an increase in mature IL-1b production in Downloaded from macrophages treated with ATP. We also examined the effect of endogenously produced PGE2 on IL-1b production. By blocking PGE2 production with indomethacin, we made a striking ﬁnding that endogenous PGE2 is essential for LPS-induced pro–IL-1b production, suggesting a positive feedback loop. The effect of endogenous PGE2 was mediated by EP2 receptor. In primary human monocytes, where LPS alone is sufﬁcient to induce mature IL-1b, PGE2 boosted LPS-induced IL-1b production. PGE2 did not inhibit ATP-induced mature IL-1b production in monocytes. Because PGE2 mediates the pyrogenic effect of IL-1b, these effects might be especially relevant for the role of monocytes in the induction of fever. A positive feedback loop from IL-1b and back to PGE2, which itself is induced by IL-1b, is likely to be operating. Furthermore, fever might therefore occur in the absence of a septic shock http://www.jimmunol.org/

response because of the inhibiting effect of PGE2 on TNF-a production. The Journal of Immunology, 2017, 198: 000–000.

rostaglandin E2 is an important lipid mediator that reg- such as LPS induce expression and secretion of both IL-1b and ulates inflammation. The effects of PGE2 on macrophages PGE2. Human monocytes and macrophages differ in their capacity P are mostly inhibitory and typically linked to an increase in to secrete IL-1b upon LPS stimulation (8). Whereas monocytes cAMP signaling (1). PGE2 is the most abundant eicosanoid at sites need only LPS to secrete mature IL-1b, macrophages need an of inflammation and is increased in chronic as well as acute types additional signal to activate the NLRP3 inflammasome and

of inflammation and infection (2). PGE2 has four receptors, caspase-1, such as ATP (8, 9). Recently in macrophages PGE2 by guest on September 29, 2021 namely EP1–4, and many of the opposing biologic effects of PGE2 through cAMP has been demonstrated to inhibit the NLRP3 can be explained by receptor expression patterns (2). Most cells inflammasome and subsequently IL-1b processing and secretion produce and respond to PGE2, which allows PGE2 to shape the (10, 11). This ability of PGE2 to boost transcription of IL-1b while tissue microenvironment, including the phenotype of long-lived inhibiting the NLRP3 inflammasome is somewhat contradictory. immune cells such as macrophages. Although PGE2 has been In the present study we have reexamined the importance of shown to be anti-inflammatory in macrophages by inhibition of exogenous PGE2 in the regulation of IL-1b transcription. We have TNF-a and in the boosting of production of IL-10 (3), it is often found that LPS treatment promotes PGE2 by inducing COX-2 and considered as a proinflammatory mediator by promotion of local inhibiting the expression of the PGE2-degrading enzyme 15- vasodilatation, inflammatory edema, and fever (2, 4). hydroxydehydrogenase (15-PGDH). LPS can further skew PGE2 In monocytes, PGE2 and cAMP have been shown to drive IL-1b signaling by decreasing EP3 and increasing EP2 receptor ex- transcription (5, 6). Previous work identified indomethacin de- pression, which will increase cAMP levels. Importantly, we rivative, which inhibited COX-1 expression, to downregulate demonstrate a critical role for endogenous PGE2 acting via EP2 in LPS-induced IL-1b production, suggesting the importance of an the production of IL-1b induced by LPS. We have found no effect endogenous prostanoid in IL-1b secretion (7). Bacterial products of PGE2 on NLRP3 and procaspase-1 expression, but a prominent positive effect of PGE2 on IL-1b secretion in monocytes, while a *School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, inhibiting the production of TNF- . Overall, therefore, PGE2 is a Trinity College Dublin, Dublin 2, Ireland; and †Division of Internal Medicine, Uni- crucial inducer of IL-1b, acting to set up a feedback loop to cause versity of Michigan, Ann Arbor, MI 48109 fever, where induction of PGE2 by IL-1b is known to be critical, ORCIDs: 0000-0003-1529-9677 (M.H.); 0000-0002-9768-0262 (M.P.-G.). while also inhibiting sepsis. Received for publication December 9, 2016. Accepted for publication February 15, 2017. Materials and Methods This work was supported by the Science Foundation Ireland (to L.A.J.O.). Cells Address correspondence and reprint requests to Prof. Luke A.J. O’Neill, Trinity Biomedical Sciences Institute, Trinity College Dublin, Inflammation Research C57BL/6 (Harlan) mice were bred under specific pathogen-free conditions, Group, 152-160 Pearse Street, Room 4.33, Dublin 2, Ireland. E-mail address: and EP2-deficient mice were bred in the University of Michigan Unit for [email protected] Laboratory Animal Medicine. Briefly, bone marrow–derived macrophages Abbreviations used in this article: BMDM, bone marrow–derived macrophage; (BMDMs) were isolated and cultured as previously described (12). BMDMs 15-PGDH, 15-hydroxydehydrogenase; PKA, protein kinase A; WT, wild-type. were differentiated in 20% L929 media until day 6, after which they were plated for experimentation. All experiments were carried out with prior Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 ethical approval from the Trinity College Dublin Animal Research Ethics

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602072 2 PGE2 INDUCES IL-1b IN MACROPHAGES

Committee. PBMCs from healthy volunteers were isolated from buffy coats could not detect any mature IL-1b in the supernatants from conditions obtained from the blood transfusion services in St. James’s Hospital (Dublin, presented in Fig. 1E (data not shown). PGE2 significantly increased Ireland). PBMCs were isolated by Ficoll/metrizoate density gradient centri- secretion of the mature form of IL-1b in BMDMs activated with ATP fugation (Lymphoprep; Nycomed, Marlow, U.K.). Monocytes were obtained by plastic adherence. to stimulate the NLRP3 inflammasome, as well as activity of caspase- 1, which cleaves pro–IL-1b (Fig. 1H, 1I). Although PGE2 increased Reagents cleaved caspase-1 in the supernatant (Fig. 1I, lane 4, fifth panel), it had RPMI 1640 culture medium and penicillin/streptomycin/amphotericin B no effect on procaspase expression in cell lysates (Fig. 1I, lane 4, a solution were purchased from Invitrogen (Carlsbad, CA). PGE2 and EP2 second panel). PGE2 inhibited induction of TNF- production in re- receptor antagonist AH6809 were purchased from Cayman Chemical (Ann sponse to LPS (Fig. 1F). PGE2 and both PKA and Epac agonists Arbor, MI); DMSO served as vehicle control. The protein kinase A boosted IL-6 production (Fig. 1G). Overall, these results indicate that (PKA)–specific cAMP analog 6-Bnz-cAMP and Epac-specific cAMP an- b alog 8-pCPT-29-O-Me-cAMP were purchased from Biolog (Bremen, PGE2 given before LPS has a positive effect on IL-1 induction by Germany). LPS was purchased from Sigma-Aldrich (St. Louis, MO). The increasing transcription and translation of IL-1b,aswellasactivating direct adenylyl cyclase activator forskolin was purchased from EMD Mil- caspase-1 cleavage and secretion of mature IL-1b. lipore (Darmstadt, Germany). Endogenous PGE is necessary for LPS-induced IL-1b RNA isolation and quantitative real-time PCR 2 production RNA extraction was performed using the RNeasy mini kit (Qiagen), and Next we explored the effect of LPS on the expression of the PGE cDNA was generated using an Applied Biosystems high-capacity cDNA 2 archive kit. The quantitative real-time PCR analysis was performed with an receptors EP1–4 and its degrading enzyme 15-PGDH. Binding of ABI 7500 Fast real-time PCR system (Applied Biosystems). Reactions PGE2 to EP2 and EP4 receptors, which are Gas coupled, stimulates Downloaded from were set up with SYBR Green PCR core reagents (Invitrogen). Data were adenylyl cyclase and increases cAMP. EP3 is Gai coupled and in- normalized to GAPDH, and mRNA expression fold change relative to 2DD hibits cAMP induction, whereas EP1 is Ga coupled and activates controls was calculated using the 2 Ct method. q phospholipase C to release diacylglycerol and IP3, which signal via Western blot protein kinase C and calcium, respectively. As shown in Fig. 2A, upon 24-h LPS stimulation expression of EP1, EP3, and EP4 was Cells were lysed in 53 sample buffer and separated by SDS-PAGE and blotted according to standard protocols. For measurement of cleaved IL-1b decreased, whereas EP2 was increased. The expression of 15- http://www.jimmunol.org/ and caspase-1 in the supernatant, proteins in the supernatant were precipi- PGDH was dramatically suppressed. These effects will enhance the tated with 1% (v/v) StrataClean resin (Agilent Technologies) and beads were effect of PGE2 on IL-1b production via increased signaling of lysed in 53 sample buffer. Proteins were visualized using the HRP substrate cAMP due to the increase in EP2 and stabilization of PGE via the WesternBright ECL spray (Advansta) on a ChemiDoc imaging system (Bio- 2 Rad). Primary Abs were: NLRP3 (1:1000, D2P5E; Cell Signaling Tech- decrease in its metabolizing enzyme. We next tested whether en- nology), b-actin (1:15,000, AC-74; Sigma-Aldrich), IL-1b (pro- and dogenously produced PGE2, which is known to be induced by LPS, cleaved; 1:1000, AF-401; R&D Systems), caspase-1 p20 (Asp297;1:1000, might be involved in the induction of IL-1b transcription by LPS. D57A2; Cell Signaling Technology), and pro–caspase-1 p45 (A-19, 1:2000, As shown in Fig. 2B, the COX1/2 inhibitor indomethacin pro- sc-622; Santa Cruz Biotechnology). HRP-conjugated secondary Abs were foundly inhibited LPS-induced IL-1b transcription. SW033291, a from Jackson ImmunoResearch Laboratories. recently developed 15-PGDH inhibitor (13), augmented IL-1b tran- by guest on September 29, 2021 ELISA scription (Fig. 2B), indicating that a positive feedback loop via PGE2 a b production may be occurring. Indomethacin also decreased LPS- TNF- , IL-6, and IL-1 concentrations in supernatants were measured by b ELISA according to the manufacturer’s instructions (R&D Systems). induced pro–IL-1 production (Fig. 2C, lane 6). The EP2 receptor antagonist AH6809 also inhibited production of pro–IL-1b by LPS Statistical analysis (Fig. 2C, lane 4). Pretreatment with indomethacin increased TNF-a Data are presented as mean 6 SEM. Statistical significance was analyzed using mRNA (Fig. 2D), consistent with PGE2 inhibiting LPS-induced the GraphPad Prism 5.0 statistical program (GraphPad Software, La Jolla, TNF-a transcription. We next tested the importance of the EP2 re- CA). Comparisons between two experimental groups were performed using ceptor using a genetic approach. As depicted in Fig. 2E, LPS was less the Student t test. A p value ,0.05 was considered statistically significant. able to induce pro–IL-1b in EP2-deficient BMDMs (lane 5 compared with lane 6), and exogenous PGE2 was less able to boost the LPS Results response (lane 7 compared with lane 8). This confirms the importance b a PGE2 induces IL-1 while decreasing TNF- production of induced EP2 expression in LPS-treated cells for pro–IL-1b pro- We first evaluated the effect of PGE2 on IL-1b expression in murine duction. As shown in Fig. 2F, indomethacin was not able to inhibit BMDMs. Stimulation for 24 h with PGE2 alone increased IL-1b the residual, presumably PGE2-independent, pro–IL-1b induced by transcription in a dose-dependent manner (Fig. 1A). Forskolin, LPS in EP2-deficient mice (compare lane 5 to lane 7). It clearly which activates adenylyl cyclase and results in an increase in in- inhibited this response in wild-type (WT) cells (compare lane 6 to tracellular cAMP, mimicked PGE2 augmentation of IL-1b and lane 8), again pointing to the importance of endogenous PGE2 for the similarly decreased basal transcription of TNF-a, demonstrating induction of pro–IL-1b. We next examined whether molecular ma- opposing effects on these two proinflammatory cytokines (Fig. 1B). chinery responsible for the production of mature IL-1b differs be- PGE2 increased IL-1b transcription in response to LPS, demon- tween WT and EP2-deficient macrophages. Expression of NLRP3 strating a synergistic effect (Fig. 1C). cAMP can be sensed by PKA and procaspase-1 was induced by LPSinbothgenotypes(Fig.3A, or exchange factor directly activated by cAMP (Epac). Specific 3B, compare lane 1 and lane 4), but it was not affected by PGE2 or cAMP agonists revealed the importance of PKA, but not Epac, in indomethacin pretreatment (Fig. 3A, 3B, lane 5). Neither procaspase-1 regulation of IL-1b transcription. The PKA agonist 6-Bnz-cAMP nor NLRP3 protein expression was regulated by exogenous PGE2 strongly boosted IL-1b mRNA and further enhanced LPS-induced added at baseline (Fig. 3A, 3B, compare lane 1 and lane 2), before IL-1b transcription. The Epac agonist 8-pCPT-29-O-Me-cAMP had LPS induction (Fig. 3A, 3B, compare lane 4 and lane 5), and before no effect on LPS-induced IL-1b mRNA (Fig. 1D). We next exam- activation of NLRP3 with ATP (Fig. 3A, 3B, compare lane 7 and lane ined pro–IL-1b protein production. As shown in Fig. 1E, PGE2 (lane 8). Similarly, in none of the above conditions did indomethacin affect 2) and the PKA agonist (lane 3) increased production of pro–IL-1b expression of NLRP3 and procaspase-1 in cell lysates (Fig. 3A, 3B, and boosted this response in LPS-treated cells (lanes 5 and 6). We lane 6). We have tested the effect of ATP on mature IL-1b production The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. PGE2/cAMP/PKA induces IL-1b while decreasing TNF-a production. (A) BMDMs were stimulated with indicated dose of PGE2 for 24 h and mRNA was subjected to quantitative PCR analysis. (B) BMDMs were treated with 1 mM PGE2 or 20 mM forskolin for 24 h and mRNA was subjected to quantitative PCR analysis. (C) BMDMs were pretreated with 1 mM PGE2 or 20 mM forskolin or corresponding DMSO controls for 30 min and subsequently stimulated with 100 ng/ml LPS for 24 h, and mRNA was subjected to quantitative PCR analysis. (D) BMDMs were pretreated with 6-Bnz-cAMP (PKA agonist) and 8-pCPT-29-O-Me-cAMP (Epac agonist) both at 500 mM for 30 min and subsequently stimulated with 100 ng/ml LPS for 24 h, and mRNA was subjected to quantitative PCR analysis. (E) BMDMs were treated with 1 mM PGE2 or 500 mM 6-Bnz-cAMP (PKA agonist) or followed after 30 min with 100 ng/ml LPS 24 h, and cell lysates were subjected to Western blot analysis with Ab recognizing pro–IL-1b (representative blot from three independent experiments is presented). (F–H) BMDMs were treated as above, and supernatants were collected for ELISA analysis of secreted IL-1b, IL-6, or TNF-a.(I) Western blot analysis of lysates and supernatants. A representative Western blot of three experiments is presented. Data are shown from three to four independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 by multiple comparison (ANOVA). 4 PGE2 INDUCES IL-1b IN MACROPHAGES

FIGURE 2. Endogenous PGE2 is necessary for LPS-induced IL-1b production. (A) BMDMs were treated for 24 h with 100 ng/ml LPS, and mRNA was subjected to quantitative PCR analysis. (B) BMDMs were pretreated with 10 mM indomethacin or 1 mg/ml SW033291 (15-PGDH inhibitor) for 1 h and subsequently stimulated with 100 ng/ml LPS for 24 h, and mRNA was subjected to quantitative PCR analysis. (C) BMDMs were pretreated with 10 mM indomethacin or 10 mM EP2 antagonist (AH6809) for 60 min followed by stimulation with 100 ng/ml LPS for 24 h, and cell lysates were subjected to Western

blot analysis with Ab recognizing Downloaded from pro–IL-1b (representative blot from three independent experiments is presented). (D) BMDMs were pretreated with 10 mM indomethacin for 1 h and subsequently stimulated with 100 ng/ml LPS for 24 h, and mRNA was subjected to quantitative PCR http://www.jimmunol.org/ analysis. (A)–(D) represent three to six independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 compared with controls (ANOVA). (E and F) BMDMs from WT and EP22/2 mice were pretreated with

1 mMPGE2 or with 10 mMindo- methacin for 60 min followed by stimulation with 100 ng/ml LPS for by guest on September 29, 2021 24 h, and cell lysates were subjected to Western analysis analysis with Ab recognizing pro–IL-1b (representative blot from three mice per group is presented).

in LPS primed from WT and EP2-deficient BMDMs. As shown in cretion in monocytes following 24 h treatment with LPS (Fig. 5A, Fig. 3C, ATP was less able to induce secreted IL-1b in EP2-deficient right side), although this effect was not evident at the shorter time BMDMs. This difference between two genotypes was greatly enhanced point of 3 h (Fig. 5A, left side). As shown in Fig. 5C, expression of b in BMDMs pretreated with exogenous PGE2.ThatPGE2 pretreatment NLRP3 followed the same trend as pro–IL-1 ; that is, it was in- increased IL-1b secretion in EP2-deficient mice suggests that there is duced by PGE2 and PKA agonist in LPS-treated monocytes (fourth probably some compensatory effect of EP4 signaling, which, simi- panel, compare lanes 6 and 7 to lane 5), whereas procaspase-1 was larlytoEP2,isaPGE2 receptor that increases cAMP levels. These not affected by PGE2 or cAMP treatment (fifth panel). Examination results further implicate the importance of EP2 for IL-1b production of supernatant revealed that in monocytes treated with LPS alone and with LPS followed by ATP, pretreatment with PGE signifi- PGE boosts LPS-induced IL-1b in human monocytes 2 2 cantly increased secreted pro–IL-1b (top panel, compare lane 6 to We next explored whether our findings held true in human mono- lane 5), whereas the effect on secretion of mature IL-1b and cleaved cytes obtained from PBMCs. PGE2 alone did not cause a significant caspase-1 was not evident (compare lane 5 to lane 6 and lane 9 to increase in IL-1b mRNA (data not shown), but it significantly lane 10). Interestingly, the PKA agonist only minimally increased boosted LPS-induced IL-1b mRNA (Fig. 4A), whereas TNF-a secretion of pro–IL-1b, but it drastically increased secretion of mRNA was decreased (Fig. 4B). LPS increased EP2 expression in mature IL-1b (second panel) and cleaved caspase-1 (third panel) in monocytes (Fig. 4C). Similar to BMDMs, indomethacin decreased monocytes treated by LPS followed by ATP (compare lane 9 and LPS-induced pro–IL-1b production (Fig. 4D, lane 4) and increased lane 11). When human monocytes were treated with PGE2 after LPS LPS-induced TNF-a (Fig. 4E). and before ATP to specifically affect the NLRP3 inflammasome, we Monocytes do not need NLRP3 inflammasome activation to se- did not observe an inhibition of IL-1b secretion (Fig. 5B), in contrast crete mature IL-1b, because LPS can also activate NLRP3 via a to what has been demonstrated previously for macrophages. Taken pathway involving RIPK1/FADD/CASP8 (8). We therefore tested together, results obtained from human monocytes suggest that PGE2 whether PGE2 would boost this response. Pretreatment of human and PKA are boosting LPS-induced IL-1b production, with PGE2 monocytes with PGE2 or the PKA agonist upregulate IL-1b se- having a net positive effect on the IL-1b system. The Journal of Immunology 5

FIGURE 3. Endogenous PGE2 boosts mature IL-1b secretion. (A) BMDMs from WT and (B) EP22/2 mice were pretreated with 1 mM

PGE2 or with 10 mM indomethacin for 60 min followed by stimulation with 100 ng/ml LPS for 24 h and ATP for 1 h. Cell lysates were subjected to Western blot analysis with Ab recognizing procaspase-1 or NLRP3 (representative blot from three mice per group is presented). (C) BMDMs from WT and EP22/2 mice were treated as above and supernatant was collected for ELISA analysis of secreted IL-1b. Data are Downloaded from shown from one experiment with three mice per group. *p , 0.05, ***p , 0.001 (Student t test). http://www.jimmunol.org/

b Discussion pathway. The mechanism whereby PGE2 boosts IL-1 transcription is b In this study, we have found that PGE2 boosts IL-1b production in likely to involve CREB, which has been shown to drive IL-1 tran- a murine BMDMs and human monocytes while inhibiting TNF-a.PGE2 scription (6). Evidence also exists for PGE2 stabilizing HIF1- increases IL-1b production by upregulating transcription of IL-1b (14, 15), which has also been shown to drive IL-1b (12). In general, the mRNA, leading to an increase in pro–IL-1b via the cAMP–PKA effect of PGE2 on the macrophage transcriptional machinery is very by guest on September 29, 2021

FIGURE 4. PGE2 induces IL-1b transcription in human monocytes. Monocytes were isolated from PBMCs from buffy coats by the plastic adherence method. (A–C) Human

monocytes were treated with 1 mM PGE2 or 100 ng/ml LPS, and mRNA was subjected to quantitative PCR analysis. (D) Human monocytes were pretreated with 10 mM indomethacin for 60 min followed by stimulation with 100 ng/ml LPS for 24 h, and cell lysates were subjected to Western blot analysis with Ab recognizing pro–IL-1b.(E) Human monocytes were pretreated with 10 mM indomethacin for 1 h and subsequently stimulated with 100 ng/ml LPS for 24 h, and mRNA was subjected to quantitative PCR analysis. Data are presented from three to four independent experiments. *p , 0.05, **p , 0.01 (Student t test). 6 PGE2 INDUCES IL-1b IN MACROPHAGES Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. PGE2 boosts LPS-induced IL-1b production in human monocytes. Monocytes were isolated from PBMCs from buffy coats by the plastic adherence method. (A) Human monocytes were treated with 1 mM PGE2 or 500 mM 6-Bnz-cAMP (PKA agonist) for 30 min and subsequently followed by 100 ng/ml LPS for 3 or 24 h, after which supernatants were collected for ELISA analysis. (B) Human monocytes were stimulated with 100 ng/ml LPS for

3h,followedby1mMPGE2 or DMSO for 30 min, washed, and followed by 1 h with 5 mM ATP, after which supernatants were collected for ELISA analysis. Data are presented from three to four independent experiments. *p , 0.05 (ANOVA). (C) Human monocytes were treated with 1 mM PGE2 or 500 mM 6-Bnz-cAMP (PKA agonist) or 8-pCPT-29-O-Me-cAMP (Epac agonist) for 30 min and subsequently followed by 100 ng/ml LPS for 24 h and 60 min of ATP, after which cell lysates and concentrated supernatants were subjected to Western blot analysis. Data show a representative Western blot from two experiments. (D) Schematic representation of the effect of PGE2 on IL-1b production. LPS induces COX-2 and inhibits expression of the PGE2 metabolizing enzyme 15-PGDH. This results in an increase in PGE2. LPS increases expression of EP2 receptor, and PGE2/EP2 signaling boosts pro–IL-1b production. This results in mature IL-1b production via NLRP3 activation in macrophages by ATP or in monocytes by activation of Toll/IL-1R domain–containing adaptor inducing IFN-b (TRIF)–RIPK1–caspase-8. IL-1b will further boost PGE2 via a feed-forward loop. complex and affects a large group of cytokines, including induction of systemically, which include induction of fever and the acute phase re- IL-10 by the PKA–SIK–CRTC3 pathway (16) and inhibition of TNF-a sponse, as well as limiting the damaging effects of TNF-a,aknown by inhibition of NF-kB (17). IL-6 was demonstrated to be boosted by inducer of sepsis. Additionally, LPS has been shown to induce higher PGE2 (18), and we further implicated PKA and Epac agonist in that amounts of PGE2 in monocytes compared with macrophages, process. Furthermore, induction of IL-1b by LPS requires endoge- strengthening the positive feedback loop in monocytes (20). LPS further nously produced PGE2. This is likely to drive a positive feedback loop promotes IL-1b transcription by stabilizing PGE2 by negative regula- induced by LPS, because IL-1b itself is a potent inducer of PGE2 (19). tion of its degrading enzyme 15-PGDH, and by promoting expression Upon infection, bacterial components such as LPS induce PGE2 as of EP2, which activates adenylyl cyclase and cAMP signaling. Inhibi- well as proinﬂammatory cytokines, including IL-1b and TNF-a.PGE2 tion of 15-PGDH has been demonstrated in vivo in LPS-induced fever in a cAMP-dependent manner inhibits TNF-a and boosts IL-1b.This (21) and sepsis (22). We interpret the PGE2–IL-1b positive feedback observation has the potential to promote the beneﬁcial effects of IL-1b loop as favorable for host defense during infection. For example, in The Journal of Immunology 7

defense against Mycobacterium tuberculosis,bothIL-1b and PGE2 are 1 beta gene is necessary for induction in U937 and THP-1 monocytic cell lines. Mol. Cell. Biol. 13: 6678–6689. critical regulators of the infection and necessary for the host to mount a 7. Poyet, P., F. Doualla-Bell, D. Le´vesque, N. Ritchot, J. M. Guay, F. Marceau, and proper defense (23, 24). R. C. Gaudreault. 1998. Down-regulation of interleukin-1b production and PGE2 In macrophages, mature IL-1b is produced in response to NLRP3 accumulation by an indomethacin-phenylalanine derivative in human monocytes. Life Sci. 62: 2241–2247. inflammasome activation. When PGE2 was added after LPS and 8.Netea,M.G.,C.A.Nold-Petry,M.F.Nold,L.A.Joosten,B.Opitz,J.H.van before activation of NLRP3 it was demonstrated to significantly der Meer, F. L. van de Veerdonk, G. Ferwerda, B. Heinhuis, I. Devesa, et al. inhibit the NLRP3 inflammasome and IL-1b production (10). 2009. 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Shelhamer. 2015. ever, PGE2/cAMP/PKA signaling in the regulation of NLRP3- b Prostaglandin E2 inhibits NLRP3 inflammasome activation through EP4 re- dependent IL-1 production is not always straightforward because ceptor and intracellular cyclic AMP in human macrophages. J. Immunol. 194: in cryopyrin-associated periodic syndrome patients who have an 5472–5487. activating mutation in NLRP3, inhibition by PGE is ablated (25). In 11. Mortimer, L., F. Moreau, J. A. MacDonald, and K. Chadee. 2016. NLRP3 2 inflammasome inhibition is disrupted in a group of auto-inflammatory disease another study adenosine acting via cAMP/PKA/CREB was dem- CAPS mutations. Nat. Immunol. 17: 1176–1186. onstrated to sustain inflammasome activation (26). Inflammasome 12.Palsson-McDermott,E.M.,A.M.Curtis,G.Goel,M.A.Lauterbach, activation has been shown to induce high amounts of PGE (27). F. J. Sheedy, L. E. Gleeson, M. W. van den Bosch, S. R. Quinn, R. Domingo- Downloaded from 2 Fernandez, D. G. Johnston, et al. 2015. Pyruvate kinase M2 regulates Hif-1a Therefore, endogenous PGE2 might act back on the inflammasome activity and IL-1b induction and is a critical determinant of the Warburg effect and regulate IL-1b production. Our experiments with indomethacin in LPS-activated macrophages. [Published erratum appears in 2015 Cell b Metab. 21: 347.] Cell Metab. 21: 65–80. pretreatment have demonstrated that for LPS-induced IL-1 tran- 13. Zhang, Y., A. Desai, S. Y. Yang, K. B. Bae, M. I. Antczak, S. P. Fink, S. Tiwari, scription, COX-1/-2 products and EP2 signaling are essential. J. E. Willis, N. S. Williams, D. M. Dawson, et al. 2015. Tissue regeneration. In our study, the results we obtained in human monocytes are es- Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration. Science 348: aaa2340. pecially noteworthy. NLRP3 is activated in these cells by TLR4 14. Speth, J. M., J. Hoggatt, P. Singh, and L. M. Pelus. 2014. Pharmacologic increase http://www.jimmunol.org/ signaling acting via RIPK1/FADD and caspase-8 (9). This process is in HIF1a enhances hematopoietic stem and progenitor homing and engraftment. Blood 123: 203–207. not inhibited by PGE2, because in response to PGE2, induction of 15. Liu, X. H., A. Kirschenbaum, M. Lu, S. Yao, A. Dosoretz, J. F. Holland, and mature IL-1b is boosted. In human monocytes, a PKA agonist but not A. C. Levine. 2002. Prostaglandin E2 induces hypoxia-inducible factor-1a sta- PGE2 drastically increased secretion of cleaved caspase-1 and mature bilization and nuclear localization in a human prostate cancer cell line. J. Biol. IL-1b. This could be explained by the higher variability of EP re- Chem. 277: 50081–50086. 16. MacKenzie, K. F., K. Clark, S. Naqvi, V. A. McGuire, G. Noëhren, ceptor expression on monocytes from donors and the more potent Y. Kristariyanto, M. van den Bosch, M. Mudaliar, P. C. McCarthy, M. J. Pattison, effect of the artificial and stable PKA agonist compared with PGE2. et al. 2013. PGE2 induces macrophage IL-10 production and a regulatory-like The function of this process may be to promote IL-1b production by phenotype via a protein kinase A–SIK–CRTC3 pathway. J. Immunol. 190: 565– 577. by guest on September 29, 2021 monocytes, which could be important for the induction of fever. This 17. Kim, S. H., C. H. Serezani, K. Okunishi, Z. Zaslona, D. M. Aronoff, and may explain why nonsteroidal anti-inflammatory drugs are especially M. Peters-Golden. 2011. Distinct protein kinase A anchoring proteins direct effective antipyretics because they would block both the production of prostaglandin E2 modulation of Toll-like receptor signaling in alveolar macrophages. J. Biol. Chem. 286: 8875–8883. PGE2 for pyrogenicity and also the positive feedback loop driving 18. Williams, J. A., C. H. Pontzer, and E. Shacter. 2000. Regulation of macrophage IL-1b production, which will drive further PGE2 production. We have interleukin-6 (IL-6) and IL-10 expression by prostaglandin E2: the role of p38 depicted the link between PGE and IL-1b production in Fig. 5D. To mitogen-activated protein kinase. J. Interferon Cytokine Res. 20: 291–298. 2 19. O’Neill, L. A. J., M. L. Barrett, and G. P. Lewis. 1987. Induction of cyclo- our knowledge, our study identifies for the first time the critical in- oxygenase by interleukin-1 in rheumatoid synovial cells. FEBS Lett. 212: terplay between PGE2 and IL-1b as a likely determinant for IL-1b 35–39. production during infection and inflammation. 20. Endo, Y., K. Blinova, T. Romantseva, H. Golding, and M. Zaitseva. 2014. Dif- ferences in PGE2 production between primary human monocytes and differentiated macrophages: role of IL-1b and TRIF/IRF3. PLoS One 9: e98517. Disclosures 21. Ivanov, A. I., A. C. Scheck, and A. A. Romanovsky. 2003. 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