PGE2 has been shown to increase the transcription of pro–IL-1β. However, recently it has been demonstrated that PGE2 can block the maturation of IL-1β by inhibiting the NLRP3 inflammasome in macrophages. These apparently conflicting results have led us to reexamine the effect of PGE2 on IL-1β production. We have found that in murine bone marrow–derived macrophages, PGE2 via the cAMP/protein kinase A pathway is potently inducing IL-1β transcription, as well as boosting the ability of LPS to induce IL-1β mRNA and pro–IL-1β while inhibiting the production of TNF-α. This results in an increase in mature IL-1β production in macrophages treated with ATP. We also examined the effect of endogenously produced PGE2 on IL-1β production. By blocking PGE2 production with indomethacin, we made a striking finding that endogenous PGE2 is essential for LPS-induced pro–IL-1β 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-1β, PGE2 boosted LPS-induced IL-1β production. PGE2 did not inhibit ATP-induced mature IL-1β production in monocytes. Because PGE2 mediates the pyrogenic effect of IL-1β, these effects might be especially relevant for the role of monocytes in the induction of fever. A positive feedback loop from IL-1β and back to PGE2, which itself is induced by IL-1β, is likely to be operating. Furthermore, fever might therefore occur in the absence of a septic shock response because of the inhibiting effect of PGE2 on TNF-α production.

Prostaglandin E2 is an important lipid mediator that regulates inflammation. The effects of PGE2 on macrophages are mostly inhibitory and typically linked to an increase in cAMP signaling (1). PGE2 is the most abundant eicosanoid at sites of inflammation and is increased in chronic as well as acute types of inflammation and infection (2). PGE2 has four receptors, namely EP1–4, and many of the opposing biologic effects of PGE2 can be explained by receptor expression patterns (2). Most cells produce and respond to PGE2, which allows PGE2 to shape the tissue microenvironment, including the phenotype of long-lived immune cells such as macrophages. Although PGE2 has been shown to be anti-inflammatory in macrophages by inhibition of TNF-α and in the boosting of production of IL-10 (3), it is often considered as a proinflammatory mediator by promotion of local vasodilatation, inflammatory edema, and fever (2, 4).

In monocytes, PGE2 and cAMP have been shown to drive IL-1β transcription (5, 6). Previous work identified indomethacin derivative, which inhibited COX-1 expression, to downregulate LPS-induced IL-1β production, suggesting the importance of an endogenous prostanoid in IL-1β secretion (7). Bacterial products such as LPS induce expression and secretion of both IL-1β and PGE2. Human monocytes and macrophages differ in their capacity to secrete IL-1β upon LPS stimulation (8). Whereas monocytes need only LPS to secrete mature IL-1β, macrophages need an additional signal to activate the NLRP3 inflammasome and caspase-1, such as ATP (8, 9). Recently in macrophages PGE2 through cAMP has been demonstrated to inhibit the NLRP3 inflammasome and subsequently IL-1β processing and secretion (10, 11). This ability of PGE2 to boost transcription of IL-1β while inhibiting the NLRP3 inflammasome is somewhat contradictory.

In the present study we have reexamined the importance of exogenous PGE2 in the regulation of IL-1β transcription. We have found that LPS treatment promotes PGE2 by inducing COX-2 and inhibiting the expression of the PGE2-degrading enzyme 15-hydroxydehydrogenase (15-PGDH). LPS can further skew PGE2 signaling by decreasing EP3 and increasing EP2 receptor expression, which will increase cAMP levels. Importantly, we demonstrate a critical role for endogenous PGE2 acting via EP2 in the production of IL-1β induced by LPS. We have found no effect of PGE2 on NLRP3 and procaspase-1 expression, but a prominent positive effect of PGE2 on IL-1β secretion in monocytes, while inhibiting the production of TNF-α. Overall, therefore, PGE2 is a crucial inducer of IL-1β, acting to set up a feedback loop to cause fever, where induction of PGE2 by IL-1β is known to be critical, while also inhibiting sepsis.

C57BL/6 (Harlan) mice were bred under specific pathogen-free conditions, and EP2-deficient mice were bred in the University of Michigan Unit for Laboratory Animal Medicine. Briefly, bone marrow–derived macrophages (BMDMs) were isolated and cultured as previously described (12). BMDMs were differentiated in 20% L929 media until day 6, after which they were plated for experimentation. All experiments were carried out with prior ethical approval from the Trinity College Dublin Animal Research Ethics Committee. PBMCs from healthy volunteers were isolated from buffy coats obtained from the blood transfusion services in St. James’s Hospital (Dublin, Ireland). PBMCs were isolated by Ficoll/metrizoate density gradient centrifugation (Lymphoprep; Nycomed, Marlow, U.K.). Monocytes were obtained by plastic adherence.

RPMI 1640 culture medium and penicillin/streptomycin/amphotericin B solution were purchased from Invitrogen (Carlsbad, CA). PGE2 and EP2 receptor antagonist AH6809 were purchased from Cayman Chemical (Ann Arbor, MI); DMSO served as vehicle control. The protein kinase A (PKA)–specific cAMP analog 6-Bnz-cAMP and Epac-specific cAMP analog 8-pCPT-2′-O-Me-cAMP were purchased from Biolog (Bremen, Germany). LPS was purchased from Sigma-Aldrich (St. Louis, MO). The direct adenylyl cyclase activator forskolin was purchased from EMD Millipore (Darmstadt, Germany).

RNA extraction was performed using the RNeasy mini kit (Qiagen), and cDNA was generated using an Applied Biosystems high-capacity cDNA archive kit. The quantitative real-time PCR analysis was performed with an ABI 7500 Fast real-time PCR system (Applied Biosystems). Reactions were set up with SYBR Green PCR core reagents (Invitrogen). Data were normalized to GAPDH, and mRNA expression fold change relative to controls was calculated using the 2−ΔΔCt method.

Cells were lysed in 5× sample buffer and separated by SDS-PAGE and blotted according to standard protocols. For measurement of cleaved IL-1β and caspase-1 in the supernatant, proteins in the supernatant were precipitated with 1% (v/v) StrataClean resin (Agilent Technologies) and beads were lysed in 5× sample buffer. Proteins were visualized using the HRP substrate WesternBright ECL spray (Advansta) on a ChemiDoc imaging system (Bio-Rad). Primary Abs were: NLRP3 (1:1000, D2P5E; Cell Signaling Technology), β-actin (1:15,000, AC-74; Sigma-Aldrich), IL-1β (pro- and cleaved; 1:1000, AF-401; R&D Systems), caspase-1 p20 (Asp297; 1:1000, D57A2; Cell Signaling Technology), and pro–caspase-1 p45 (A-19, 1:2000, sc-622; Santa Cruz Biotechnology). HRP-conjugated secondary Abs were from Jackson ImmunoResearch Laboratories.

TNF-α, IL-6, and IL-1β concentrations in supernatants were measured by ELISA according to the manufacturer’s instructions (R&D Systems).

Data are presented as mean ± SEM. Statistical significance was analyzed using the GraphPad Prism 5.0 statistical program (GraphPad Software, La Jolla, CA). Comparisons between two experimental groups were performed using the Student t test. A p value <0.05 was considered statistically significant.

We first evaluated the effect of PGE2 on IL-1β expression in murine BMDMs. Stimulation for 24 h with PGE2 alone increased IL-1β transcription in a dose-dependent manner (Fig. 1A). Forskolin, which activates adenylyl cyclase and results in an increase in intracellular cAMP, mimicked PGE2 augmentation of IL-1β and similarly decreased basal transcription of TNF-α, demonstrating opposing effects on these two proinflammatory cytokines (Fig. 1B). PGE2 increased IL-1β transcription in response to LPS, demonstrating a synergistic effect (Fig. 1C). cAMP can be sensed by PKA or exchange factor directly activated by cAMP (Epac). Specific cAMP agonists revealed the importance of PKA, but not Epac, in regulation of IL-1β transcription. The PKA agonist 6-Bnz-cAMP strongly boosted IL-1β mRNA and further enhanced LPS-induced IL-1β transcription. The Epac agonist 8-pCPT-2′-O-Me-cAMP had no effect on LPS-induced IL-1β mRNA (Fig. 1D). We next examined pro–IL-1β protein production. As shown in Fig. 1E, PGE2 (lane 2) and the PKA agonist (lane 3) increased production of pro–IL-1β and boosted this response in LPS-treated cells (lanes 5 and 6). We could not detect any mature IL-1β in the supernatants from conditions presented in Fig. 1E (data not shown). PGE2 significantly increased secretion of the mature form of IL-1β in BMDMs activated with ATP to stimulate the NLRP3 inflammasome, as well as activity of caspase-1, which cleaves pro–IL-1β (Fig. 1H, 1I). Although PGE2 increased cleaved caspase-1 in the supernatant (Fig. 1I, lane 4, fifth panel), it had no effect on procaspase expression in cell lysates (Fig. 1I, lane 4, second panel). PGE2 inhibited induction of TNF-α production in response to LPS (Fig. 1F). PGE2 and both PKA and Epac agonists boosted IL-6 production (Fig. 1G). Overall, these results indicate that PGE2 given before LPS has a positive effect on IL-1β induction by increasing transcription and translation of IL-1β, as well as activating caspase-1 cleavage and secretion of mature IL-1β.

FIGURE 1.

PGE2/cAMP/PKA induces IL-1β while decreasing TNF-α 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 μM PGE2 or 20 μM forskolin for 24 h and mRNA was subjected to quantitative PCR analysis. (C) BMDMs were pretreated with 1 μM PGE2 or 20 μM 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-2′-O-Me-cAMP (Epac agonist) both at 500 μM 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 μM PGE2 or 500 μM 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-1β (representative blot from three independent experiments is presented). (FH) BMDMs were treated as above, and supernatants were collected for ELISA analysis of secreted IL-1β, IL-6, or TNF-α. (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).

FIGURE 1.

PGE2/cAMP/PKA induces IL-1β while decreasing TNF-α 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 μM PGE2 or 20 μM forskolin for 24 h and mRNA was subjected to quantitative PCR analysis. (C) BMDMs were pretreated with 1 μM PGE2 or 20 μM 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-2′-O-Me-cAMP (Epac agonist) both at 500 μM 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 μM PGE2 or 500 μM 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-1β (representative blot from three independent experiments is presented). (FH) BMDMs were treated as above, and supernatants were collected for ELISA analysis of secreted IL-1β, IL-6, or TNF-α. (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).

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Next we explored the effect of LPS on the expression of the PGE2 receptors EP1–4 and its degrading enzyme 15-PGDH. Binding of PGE2 to EP2 and EP4 receptors, which are Gαs coupled, stimulates adenylyl cyclase and increases cAMP. EP3 is Gαi coupled and inhibits cAMP induction, whereas EP1 is Gαq coupled and activates phospholipase C to release diacylglycerol and IP3, which signal via protein kinase C and calcium, respectively. As shown in Fig. 2A, upon 24-h LPS stimulation expression of EP1, EP3, and EP4 was decreased, whereas EP2 was increased. The expression of 15-PGDH was dramatically suppressed. These effects will enhance the effect of PGE2 on IL-1β production via increased signaling of cAMP due to the increase in EP2 and stabilization of PGE2 via the decrease in its metabolizing enzyme. We next tested whether endogenously produced PGE2, which is known to be induced by LPS, might be involved in the induction of IL-1β transcription by LPS. As shown in Fig. 2B, the COX1/2 inhibitor indomethacin profoundly inhibited LPS-induced IL-1β transcription. SW033291, a recently developed 15-PGDH inhibitor (13), augmented IL-1β transcription (Fig. 2B), indicating that a positive feedback loop via PGE2 production may be occurring. Indomethacin also decreased LPS-induced pro–IL-1β production (Fig. 2C, lane 6). The EP2 receptor antagonist AH6809 also inhibited production of pro–IL-1β by LPS (Fig. 2C, lane 4). Pretreatment with indomethacin increased TNF-α mRNA (Fig. 2D), consistent with PGE2 inhibiting LPS-induced TNF-α transcription. We next tested the importance of the EP2 receptor using a genetic approach. As depicted in Fig. 2E, LPS was less able to induce pro–IL-1β in EP2-deficient BMDMs (lane 5 compared with lane 6), and exogenous PGE2 was less able to boost the LPS response (lane 7 compared with lane 8). This confirms the importance of induced EP2 expression in LPS-treated cells for pro–IL-1β production. As shown in Fig. 2F, indomethacin was not able to inhibit the residual, presumably PGE2-independent, pro–IL-1β induced by LPS in EP2-deficient mice (compare lane 5 to lane 7). It clearly inhibited this response in wild-type (WT) cells (compare lane 6 to lane 8), again pointing to the importance of endogenous PGE2 for the induction of pro–IL-1β. We next examined whether molecular machinery responsible for the production of mature IL-1β differs between WT and EP2-deficient macrophages. Expression of NLRP3 and procaspase-1 was induced by LPS in both genotypes (Fig. 3A, 3B, compare lane 1 and lane 4), but it was not affected by PGE2 or indomethacin pretreatment (Fig. 3A, 3B, lane 5). Neither procaspase-1 nor NLRP3 protein expression was regulated by exogenous PGE2 added at baseline (Fig. 3A, 3B, compare lane 1 and lane 2), before LPS induction (Fig. 3A, 3B, compare lane 4 and lane 5), and before activation of NLRP3 with ATP (Fig. 3A, 3B, compare lane 7 and lane 8). Similarly, in none of the above conditions did indomethacin affect expression of NLRP3 and procaspase-1 in cell lysates (Fig. 3A, 3B, lane 6). We have tested the effect of ATP on mature IL-1β production in LPS primed from WT and EP2-deficient BMDMs. As shown in Fig. 3C, ATP was less able to induce secreted IL-1β in EP2-deficient BMDMs. This difference between two genotypes was greatly enhanced in BMDMs pretreated with exogenous PGE2. That PGE2 pretreatment increased IL-1β secretion in EP2-deficient mice suggests that there is probably some compensatory effect of EP4 signaling, which, similarly to EP2, is a PGE2 receptor that increases cAMP levels. These results further implicate the importance of EP2 for IL-1β production

FIGURE 2.

Endogenous PGE2 is necessary for LPS-induced IL-1β 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 μM indomethacin or 1 μg/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 μM indomethacin or 10 μM 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 pro–IL-1β (representative blot from three independent experiments is presented). (D) BMDMs were pretreated with 10 μM indomethacin for 1 h and subsequently stimulated with 100 ng/ml LPS for 24 h, and mRNA was subjected to quantitative PCR 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 EP2−/− mice were pretreated with 1 μM PGE2 or with 10 μM indomethacin for 60 min followed by stimulation with 100 ng/ml LPS for 24 h, and cell lysates were subjected to Western analysis analysis with Ab recognizing pro–IL-1β (representative blot from three mice per group is presented).

FIGURE 2.

Endogenous PGE2 is necessary for LPS-induced IL-1β 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 μM indomethacin or 1 μg/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 μM indomethacin or 10 μM 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 pro–IL-1β (representative blot from three independent experiments is presented). (D) BMDMs were pretreated with 10 μM indomethacin for 1 h and subsequently stimulated with 100 ng/ml LPS for 24 h, and mRNA was subjected to quantitative PCR 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 EP2−/− mice were pretreated with 1 μM PGE2 or with 10 μM indomethacin for 60 min followed by stimulation with 100 ng/ml LPS for 24 h, and cell lysates were subjected to Western analysis analysis with Ab recognizing pro–IL-1β (representative blot from three mice per group is presented).

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FIGURE 3.

Endogenous PGE2 boosts mature IL-1β secretion. (A) BMDMs from WT and (B) EP2−/− mice were pretreated with 1 μM PGE2 or with 10 μM 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 EP2−/− mice were treated as above and supernatant was collected for ELISA analysis of secreted IL-1β. Data are shown from one experiment with three mice per group. *p < 0.05, ***p < 0.001 (Student t test).

FIGURE 3.

Endogenous PGE2 boosts mature IL-1β secretion. (A) BMDMs from WT and (B) EP2−/− mice were pretreated with 1 μM PGE2 or with 10 μM 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 EP2−/− mice were treated as above and supernatant was collected for ELISA analysis of secreted IL-1β. Data are shown from one experiment with three mice per group. *p < 0.05, ***p < 0.001 (Student t test).

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We next explored whether our findings held true in human monocytes obtained from PBMCs. PGE2 alone did not cause a significant increase in IL-1β mRNA (data not shown), but it significantly boosted LPS-induced IL-1β mRNA (Fig. 4A), whereas TNF-α mRNA was decreased (Fig. 4B). LPS increased EP2 expression in monocytes (Fig. 4C). Similar to BMDMs, indomethacin decreased LPS-induced pro–IL-1β production (Fig. 4D, lane 4) and increased LPS-induced TNF-α (Fig. 4E).

FIGURE 4.

PGE2 induces IL-1β transcription in human monocytes. Monocytes were isolated from PBMCs from buffy coats by the plastic adherence method. (AC) Human monocytes were treated with 1 μM PGE2 or 100 ng/ml LPS, and mRNA was subjected to quantitative PCR analysis. (D) Human monocytes were pretreated with 10 μM 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-1β. (E) Human monocytes were pretreated with 10 μM 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).

FIGURE 4.

PGE2 induces IL-1β transcription in human monocytes. Monocytes were isolated from PBMCs from buffy coats by the plastic adherence method. (AC) Human monocytes were treated with 1 μM PGE2 or 100 ng/ml LPS, and mRNA was subjected to quantitative PCR analysis. (D) Human monocytes were pretreated with 10 μM 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-1β. (E) Human monocytes were pretreated with 10 μM 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).

Close modal

Monocytes do not need NLRP3 inflammasome activation to secrete mature IL-1β, because LPS can also activate NLRP3 via a pathway involving RIPK1/FADD/CASP8 (8). We therefore tested whether PGE2 would boost this response. Pretreatment of human monocytes with PGE2 or the PKA agonist upregulate IL-1β secretion in monocytes following 24 h treatment with LPS (Fig. 5A, right side), although this effect was not evident at the shorter time point of 3 h (Fig. 5A, left side). As shown in Fig. 5C, expression of NLRP3 followed the same trend as pro–IL-1β; that is, it was induced by PGE2 and PKA agonist in LPS-treated monocytes (fourth panel, compare lanes 6 and 7 to lane 5), whereas procaspase-1 was not affected by PGE2 or cAMP treatment (fifth panel). Examination of supernatant revealed that in monocytes treated with LPS alone and with LPS followed by ATP, pretreatment with PGE2 significantly increased secreted pro–IL-1β (top panel, compare lane 6 to lane 5), whereas the effect on secretion of mature IL-1β and cleaved caspase-1 was not evident (compare lane 5 to lane 6 and lane 9 to lane 10). Interestingly, the PKA agonist only minimally increased secretion of pro–IL-1β, but it drastically increased secretion of mature IL-1β (second panel) and cleaved caspase-1 (third panel) in monocytes treated by LPS followed by ATP (compare lane 9 and lane 11). When human monocytes were treated with PGE2 after LPS and before ATP to specifically affect the NLRP3 inflammasome, we did not observe an inhibition of IL-1β secretion (Fig. 5B), in contrast to what has been demonstrated previously for macrophages. Taken together, results obtained from human monocytes suggest that PGE2 and PKA are boosting LPS-induced IL-1β production, with PGE2 having a net positive effect on the IL-1β system.

FIGURE 5.

PGE2 boosts LPS-induced IL-1β production in human monocytes. Monocytes were isolated from PBMCs from buffy coats by the plastic adherence method. (A) Human monocytes were treated with 1 μM PGE2 or 500 μM 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 3 h, followed by 1 μM PGE2 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 μM PGE2 or 500 μM 6-Bnz-cAMP (PKA agonist) or 8-pCPT-2′-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-1β 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-1β production. This results in mature IL-1β production via NLRP3 activation in macrophages by ATP or in monocytes by activation of Toll/IL-1R domain–containing adaptor inducing IFN-β (TRIF)–RIPK1–caspase-8. IL-1β will further boost PGE2 via a feed-forward loop.

FIGURE 5.

PGE2 boosts LPS-induced IL-1β production in human monocytes. Monocytes were isolated from PBMCs from buffy coats by the plastic adherence method. (A) Human monocytes were treated with 1 μM PGE2 or 500 μM 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 3 h, followed by 1 μM PGE2 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 μM PGE2 or 500 μM 6-Bnz-cAMP (PKA agonist) or 8-pCPT-2′-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-1β 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-1β production. This results in mature IL-1β production via NLRP3 activation in macrophages by ATP or in monocytes by activation of Toll/IL-1R domain–containing adaptor inducing IFN-β (TRIF)–RIPK1–caspase-8. IL-1β will further boost PGE2 via a feed-forward loop.

Close modal

In this study, we have found that PGE2 boosts IL-1β production in murine BMDMs and human monocytes while inhibiting TNF-α. PGE2 increases IL-1β production by upregulating transcription of IL-1β mRNA, leading to an increase in pro–IL-1β via the cAMP–PKA pathway. The mechanism whereby PGE2 boosts IL-1β transcription is likely to involve CREB, which has been shown to drive IL-1β transcription (6). Evidence also exists for PGE2 stabilizing HIF1-α (14, 15), which has also been shown to drive IL-1β (12). In general, the effect of PGE2 on the macrophage transcriptional machinery is very complex and affects a large group of cytokines, including induction of IL-10 by the PKA–SIK–CRTC3 pathway (16) and inhibition of TNF-α by inhibition of NF-κB (17). IL-6 was demonstrated to be boosted by PGE2 (18), and we further implicated PKA and Epac agonist in that process. Furthermore, induction of IL-1β by LPS requires endogenously produced PGE2. This is likely to drive a positive feedback loop induced by LPS, because IL-1β itself is a potent inducer of PGE2 (19).

Upon infection, bacterial components such as LPS induce PGE2 as well as proinflammatory cytokines, including IL-1β and TNF-α. PGE2 in a cAMP-dependent manner inhibits TNF-α and boosts IL-1β. This observation has the potential to promote the beneficial effects of IL-1β systemically, which include induction of fever and the acute phase response, as well as limiting the damaging effects of TNF-α, a known inducer of sepsis. Additionally, LPS has been shown to induce higher amounts of PGE2 in monocytes compared with macrophages, strengthening the positive feedback loop in monocytes (20). LPS further promotes IL-1β transcription by stabilizing PGE2 by negative regulation of its degrading enzyme 15-PGDH, and by promoting expression of EP2, which activates adenylyl cyclase and cAMP signaling. Inhibition of 15-PGDH has been demonstrated in vivo in LPS-induced fever (21) and sepsis (22). We interpret the PGE2–IL-1β positive feedback loop as favorable for host defense during infection. For example, in defense against Mycobacterium tuberculosis, both IL-1β and PGE2 are critical regulators of the infection and necessary for the host to mount a proper defense (23, 24).

In macrophages, mature IL-1β is produced in response to NLRP3 inflammasome activation. When PGE2 was added after LPS and before activation of NLRP3 it was demonstrated to significantly inhibit the NLRP3 inflammasome and IL-1β production (10). However, this inhibitory response was rapid and short-lived (11). In our study in murine BMDMs when PGE2 is added before LPS, the ability of ATP to increase IL-1β is potentiated, as evidenced by increased cleaved caspase-1 and mature IL-1β in supernatants. This increase was in part caused by accumulation of pro–IL-1β. However, PGE2/cAMP/PKA signaling in the regulation of NLRP3-dependent IL-1β production is not always straightforward because in cryopyrin-associated periodic syndrome patients who have an activating mutation in NLRP3, inhibition by PGE2 is ablated (25). In another study adenosine acting via cAMP/PKA/CREB was demonstrated to sustain inflammasome activation (26). Inflammasome activation has been shown to induce high amounts of PGE2 (27). Therefore, endogenous PGE2 might act back on the inflammasome and regulate IL-1β production. Our experiments with indomethacin pretreatment have demonstrated that for LPS-induced IL-1β transcription, COX-1/-2 products and EP2 signaling are essential.

In our study, the results we obtained in human monocytes are especially noteworthy. NLRP3 is activated in these cells by TLR4 signaling acting via RIPK1/FADD and caspase-8 (9). This process is not inhibited by PGE2, because in response to PGE2, induction of mature IL-1β is boosted. In human monocytes, a PKA agonist but not PGE2 drastically increased secretion of cleaved caspase-1 and mature IL-1β. This could be explained by the higher variability of EP receptor expression on monocytes from donors and the more potent effect of the artificial and stable PKA agonist compared with PGE2. The function of this process may be to promote IL-1β production by monocytes, which could be important for the induction of fever. This may explain why nonsteroidal anti-inflammatory drugs are especially effective antipyretics because they would block both the production of PGE2 for pyrogenicity and also the positive feedback loop driving IL-1β production, which will drive further PGE2 production. We have depicted the link between PGE2 and IL-1β production in Fig. 5D. To our knowledge, our study identifies for the first time the critical interplay between PGE2 and IL-1β as a likely determinant for IL-1β production during infection and inflammation.

This work was supported by the Science Foundation Ireland (to L.A.J.O.).

Abbreviations used in this article:

BMDM

bone marrow–derived macrophage

15-PGDH

15-hydroxydehydrogenase

PKA

protein kinase A

WT

wild-type.

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The authors have no financial conflicts of interest.