Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity and are commonly used for pain relief and fever reduction. NSAIDs are used following childhood vaccinations and cancer immunotherapies; however, how NSAIDs influence the development of immunity following these therapies is unknown. We hypothesized that NSAIDs would modulate the development of an immune response to Listeria monocytogenes–based immunotherapy. Treatment of mice with the nonspecific COX inhibitor indomethacin impaired the generation of cell-mediated immunity. This phenotype was due to inhibition of the inducible COX-2 enzyme, as treatment with the COX-2–selective inhibitor celecoxib similarly inhibited the development of immunity. In contrast, loss of COX-1 activity improved immunity to L. monocytogenes. Impairments in immunity were independent of bacterial burden, dendritic cell costimulation, or innate immune cell infiltrate. Instead, we observed that PGE2 production following L. monocytogenes is critical for the formation of an Ag-specific CD8+ T cell response. Use of the alternative analgesic acetaminophen did not impair immunity. Taken together, our results suggest that COX-2 is necessary for optimal CD8+ T cell responses to L. monocytogenes, whereas COX-1 is detrimental. Use of pharmacotherapies that spare COX-2 activity and the production of PGE2 like acetaminophen will be critical for the generation of optimal antitumor responses using L. monocytogenes.

This article is featured in In This Issue, p.3665

Recent advances in understanding the immune system have led to new, powerful immunotherapy approaches against cancer, including checkpoint blockade therapies, adoptive cellular immunotherapies, and pathogen-based immunotherapies (1). One promising approach uses pathogens, such as the Gram-positive intracellular bacteria Listeria monocytogenes, that naturally trigger robust cell-mediated immune responses to program tumor-targeting Ag-specific CD8+ T cells (2). As is common with many childhood vaccines (3), immunotherapies, including L. monocytogenes immunotherapy, include nonsteroidal anti-inflammatory drugs (NSAIDs) as part of their clinical trial protocol to limit unwanted side effects of immune activation, such as fever and general malaise (48). Despite this inclusion as standard of care, how NSAIDs ultimately influence the efficacy of an adaptive immune response in the context of L. monocytogenes–stimulated immunity, or many other vaccinations and immunotherapies, remains largely unknown.

NSAIDs modulate cyclooxygenase (COX) production to limit the processing of arachidonic acid into PGs and thromboxane (9), collectively known as eicosanoids. Eicosanoids have broad impacts on both homeostatic physiology and inflammatory responses and have been implicated in both anti-inflammatory and proinflammatory signaling effects depending on the cell type that produces and/or responds to the eicosanoid, the receptor that the eicosanoid signals through, and the other inflammatory mediators present (10, 11). The eicosanoid PGE2 can inhibit T cell proliferation and cytotoxic function via signaling through EP2 and EP4 receptors (12). In the context of chronic infections such as lymphocytic choriomeningitis virus, this eicosanoid signaling has profound effects, such that PGE2 inhibition can improve the clearance of chronic infection (13). Similarly, targeted inhibition of PGE2 or the EP2/EP4 receptors improves Ag presentation, CD8+ T cell mediated immunity, and ultimately survival following influenza A infection (14). In contrast, imbalance in eicosanoid production, specifically loss of PGE2, results in impairments in Mycobacterium tuberculosis–mediated immunity, whereas administration of exogenous PGE2 improves acute survival (15). These results suggest that eicosanoids modulate immunity to pathogens and that they may modulate immunity in the context of L. monocytogenes immunotherapy.

Eicosanoids have previously been implicated in regulating acute infection by L. monocytogenes. Treatment of mice with the nonspecific COX inhibitor indomethacin increased susceptibility to acute L. monocytogenes infection, potentially through inhibition of thromboxane A2 (TXA2) synthesis (16). PGE2 can additionally limit macrophage phagocytosis of L. monocytogenes (17), further suggesting that eicosanoids modulate L. monocytogenes pathogenesis. Despite these studies on the impact of eicosanoids on acute infection with pathogenic L. monocytogenes, little is known about how eicosanoids influence adaptive immune responses to L. monocytogenes. The use of L. monocytogenes as a platform for tumor immunotherapy and the explicit treatment of patients in these trials with NSAIDs demands an understanding of how COX inhibition may influence L. monocytogenes–stimulated Ag-specific cytotoxic T cell responses (3, 7, 8). To address this question, we measured the activation of Ag-specific T cells and their function in protection from lethal challenge following L. monocytogenes immunization in the presence of various COX inhibitors and knockout mouse models. Our data suggest that COX-2, and specifically PGE2, is critical for optimal L. monocytogenes–stimulated immune responses, whereas COX-1 inhibits generation of an Ag-specific CD8+ T cell–mediated protective immune response.

This work was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). All protocols were reviewed and approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee.

All L. monocytogenes strains used in this study were in the 10403s background and were cultured in brain heart infusion media. The immunizing strain Lm (referred to as attenuated Lm in the figure legends) was constructed in the ΔactA and ΔinlB background as previously described (18) and expressed full length OVA and the B8R20–27 epitope. The B8R20–27 epitope and full-length OVA were engineered as a single-fusion protein behind the actA promoter and in frame with the secretion signal of the amino terminal 300 bp of the ActA gene (ActAN100) in the site-specific pPL2e vector as previously described (19).

Six- to eight-week-old C57BL/6 male and female mice were obtained from the National Cancer Institute and Charles River National Cancer Institute facility. Ptgs1−/− (COX-1−/−) mice (20) were obtained from Taconic and were back-crossed to a C57BL/6 background for six generations and maintained as heterozygote breeding pairs for use in this study. Mice were age and sex matched. Microsomal PG synthase E1 (mPGES1−/−) mice, encoded by Ptges, that lack microsomal PGE synthase, the enzyme critical for PGE2 production, have been previously described (2123). Indomethacin (Sigma) was dosed at 2 mg/kg i.p (16) at 2 h before immunization and 24 h after immunization. Celecoxib (Cayman Chemical) was milled into the standard mouse chow (Envigo) at 100 mg/kg (13) and fed ad lib for 48 h before and after immunization. PGE2 (Cayman Chemical) was diluted in PBS and administered to mice i.p. 8 (0.125 μg) and 12 (0.25 μg) h postimmunization. Acetaminophen (Infant’s Tylenol) was diluted into water at 1.1 mg/ml so that mice received ∼200 mg/kg/d as previously described for 48 h before and after immunization (24). Water consumption was measured to ensure accurate dosing.

All mice were immunized i.v. with logarithmic phase ΔactA/ΔinlB L. monocytogenes (attenuated Lm) diluted in 200 μl PBS at the doses indicated. For bacterial burden analysis, mice were sacrificed at 24 or 48 h postinfection. For challenge studies, mice immunized 30 d prior were challenged with 2LD50 (2 × 105 CFU) of virulent L. monocytogenes expressing full-length OVA, and the B8R20–27 and burdens were analyzed 68–72 h postinfection. Organs were homogenized in 0.1% Nonidet P-40 in PBS and plated on Luria–Bertani plates containing streptomycin to quantify bacterial loads.

Spleens taken at the indicated time points after immunization were made into a single-cell suspension, and RBCs were lysed in ACK buffer. Total splenocytes were counted with a Z1 Coulter counter, and a total of 1.8 × 106 splenocytes were stained for analysis. For primary T cell responses measured by intracellular cytokine staining, splenocytes were stimulated ex vivo for 5 h with B8R20–27 (TSYKFESV) or OVA257–264 (SIINFEKL) in the presence of brefeldin A (eBioscience). Stimulated cells were surface stained with anti-CD3 (clone 145-2C11) and anti-CD8α (clone 53-6.7) Abs, fixed and permeabilized using intracellular fixation and permeabilization buffer (eBioscience), then stained intracellularly for IFN-γ (clone XMG1.2), TNF-α (clone MP6-XT22), and IL-2 (clone JES6-5H4). For primary and memory tetramer analysis, cells were stained with B8R-tetramer (NIH Tetramer Facility, Atlanta, GA; contract HHSN272201300006C), followed by surface staining with anti-CD3, anti-CD8α, and anti-CD44 (clone IM7) Abs. Innate immune cells were stained with markers anti-CD3, anti-CD8α, anti-CD11c (clone N418), anti-CD86 (clone GL1), anti-CD11b (clone M1/70), anti-CD40 (clone 1C10), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8-Ly6G), and anti-NK1.1 (clone PK136). All fluorophore-conjugated Abs were obtained from eBioscience. Samples were acquired using a LSRII flow cytometer (BD Biosciences, San Jose, CA) with FACSDiva software (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Ashland, OR).

Spleens from infected mice were flash frozen and stored at −80°C until solid phase extraction. Spleens were homogenized in 1 ml cold methanol and centrifuged to remove debris. Samples were processed as previously described (25). Briefly, samples were rapidly acidified with water pH 3.5 and loaded into conditioned solid phase C18 cartridges. Columns were washed with neutral pH water followed by hexanes and then eluted with methyl formate followed by methanol. Samples were concentrated using a nitrogen manifold and suspended in a final solution of 55:45 MeOH:H2O. Samples were analyzed on an HPLC coupled to a mass spectrometer (Q Exactive; Thermo Scientific) using a C18 Acquity BEH column (100 mm × 2.1 mm × 1.7 μm) operated in negative ionization mode. Lipid mediators were eluted with a mobile phase of 55:45:0.1 methanol:H2O:acetic acid that was shifted to 98:2:0.1 over the course of 20 min. Scanning occurred from 3.5 to 20 min and included mass-to-charge ratios (m/z) between 100 and 800. Eicosanoid standards (Cayman Chemical) were run to ensure peak specificity. Data were analyzed via MAVEN (26, 27).

Blood was obtained by cardiac puncture or retro-orbital bleed, and serum was analyzed by cytokine bead array with the mouse inflammation kit (BD Biosciences) per the manufacturer’s instructions. Samples were acquired using a LSRII flow cytometer with FACSDiva software.

Statistical analysis was performed using GraphPad Prism Software (La Jolla, CA) and analyzed with a one-way ANOVA with Bonferroni’s correction or Mann–Whitney U test unless otherwise indicated.

NSAIDs are routinely administered following vaccination to modulate eicosanoid production to prevent the unwanted effects of immune activation, such as fever and muscle soreness (3, 6, 28). Attenuated L. monocytogenes is being developed as a cancer immunotherapeutic platform to stimulate antitumor responses; yet, how eicosanoid inhibition impacts the effectiveness of L. monocytogenes immunotherapy is unknown. NSAIDs are routinely administered to patients receiving L. monocytogenes immunotherapy; therefore, it is critical to understand how eicosanoids influence L. monocytogenes–triggered immunity (7, 8). To address this question, we assessed activation of cell-mediated immunity following immunization with the attenuated ΔactA/ΔinlB strain of L. monocytogenes (attenuated Lm) in the presence or absence of the nonselective NSAID indomethacin. The ΔactA/ΔinlB strain of L. monocytogenes, currently in clinical trials as an immunotherapeutic platform, contains two genomic deletions that limit the spread of L. monocytogenes from cell to cell (ΔactA) and limit invasion of hepatocytes (ΔinlB), thus leaving L. monocytogenes to invade primarily Ag-presenting immune cells (18). CD8+ T cells comprise the majority of the adaptive immune response generated by L. monocytogenes and are the cell type responsible for controlling L. monocytogenes infection, providing long-lasting immunity, and inducing an antitumor response (29). As eicosanoids have been shown to directly impact CD8+ T cell functions (12), we hypothesized that COX inhibition through NSAID use would influence the generation of immunity to L. monocytogenes.

We dosed mice with a nonspecific COX inhibitor, indomethacin, 2 h before immunization and 24 h after immunization with 1 × 107 attenuated Lm. This dosing strategy inhibits COX activity for at least 48 h (16), the critical time of T cell priming following L. monocytogenes infection, and mimics the real-world application of COX inhibition following pathogen-based immunotherapies (8). Seven days after immunization, there were significant decreases in both Ag-specific single positive IFN-γ–producing CD8+ T cells (Fig. 1A) as well as impaired multifunctional Ag-specific T cell responses (Fig. 1B). These multifunctional cells that produce IFN-γ, TNF-α, and IL-2 simultaneously are the cell type capable of forming memory cells (3032), suggesting that administration of NSAIDs may cause defects in long-term protective immunity. To test this hypothesis, we immunized mice with a low dose of attenuated Lm (1 × 103) in the presence or absence of indomethacin, and 30 d later, challenged mice with a lethal dose of virulent L. monocytogenes. Following the challenge, mice immunized in the presence of indomethacin had significantly impaired protective immune responses, as evidenced by elevated bacterial burdens in their spleens (Fig. 1C) and livers (Fig. 1D). Taken together, these data suggest that nonspecific COX inhibition following administration of NSAIDs results in impairments in cell-mediated immune responses to L. monocytogenes–based immunotherapy.

COXs exist in two forms throughout the body. COX-1 is a constitutive enzyme largely responsible for maintenance of physiological functions throughout the body, including maintenance of the stomach lining and proper blood clotting functions (9). COX-2, in contrast, is an inducible enzyme largely expressed by immune cells that is largely responsible for the production of fever and malaise (33). Although COX-2 is thought to be the critical mediator for inflammatory signals, COX-1 has recently been implicated in inflammatory responses (34), suggesting that either COX enzyme may influence immunity.

Treatment with the nonselective COX inhibitor indomethacin (35) inhibited L. monocytogenes–stimulated immunity. To understand whether COX-1 or COX-2 differentially affects the generation of immune responses to L. monocytogenes, we used both genetic and pharmacologic approaches to isolate the role of each COX. COX-1 selective inhibitors exist against purified enzymes (36); however, in vivo, these inhibitors are nonselective (37). Therefore, we used COX-1−/− mice (20) to isolate the role of COX-1. In contrast to treatment with indomethacin, immunization of COX-1−/− mice resulted in improved Ag-specific CD8+ Tcell responses 7 d after immunization, both in the effector subset (Fig. 2A) as well as the multifunctional CD8+ T cell subset (Fig. 2B). Similar effects were seen with the use of a B8R-specific tetramer (Supplemental Fig. 1A), suggesting an improved T cell priming and overall CD8+ T cell effector response, as opposed to just an increased functional response. Additionally, similar increases in T cell activation were observed in response to the SIINFEKL peptide of OVA, suggesting that this increased response is not Ag specific (Supplemental Fig. 1B, 1C). Consistent with increased T cell priming in the absence of COX-1, COX-1−/−-immunized mice were more protected from a secondary lethal challenge of virulent L. monocytogenes, harboring lower bacterial burdens in their spleens (Fig. 2C), whereas burdens in the liver were similar to COX-1–sufficient mice (Fig. 2D). These results are in direct contrast to the inhibitory effect of indomethacin and suggest that COX-1 activity is inhibitory to the development of L. monocytogenes–stimulated immunity.

Given our unexpected observations that COX-1 activity is detrimental to immunity generated by L. monocytogenes, we hypothesized that inhibition of COX-2 was the basis of the inhibitory effects of indomethacin. To assess the role of COX-2, we used a COX-2 specific inhibitor, celecoxib, that has previously been shown to specifically impair COX-2 at the dose used (13). Mice given celecoxib had impaired Ag-specific effector and multifunctional CD8+ T cell responses (Fig. 2A, 2B, black diamonds), similar to the impairments seen in mice dosed with indomethacin. Mice treated with celecoxib also had decreased B8R-specific tetramer responses (Supplemental Fig. 1A) and OVA-specific responses (Supplemental Fig. 1B, 1C). Consistent with decreased CD8+ T cell activation, inhibition of COX-2 also impaired long-term protective immunity, as celecoxib-treated animals had increased burdens following lethal challenge (Fig. 2C, 2D, black diamonds), suggesting that inhibition of COX-2 impairs immune responses to L. monocytogenes.

Our findings suggest that COX-1 is detrimental to the immune response generated to L. monocytogenes, whereas COX-2 is critical to forming an effective immune response. Most NSAIDs, like indomethacin, are nonselective (35); therefore, we asked which effect would be dominant by treating COX-1−/− mice with the COX-2–specific inhibitor. Similar to what we observed with the nonselective COX inhibitor, indomethacin, COX-1−/− mice treated with celecoxib had impaired effector and multifunctional Ag-specific responses (Fig. 2A, 2B, gray diamonds), B8R-tetramer responses (Supplemental Fig. 1A), and OVA-specific responses (Supplemental Fig. 1B, 1C), although not quite to the level of COX-2 inhibition alone. Consistent with the poor effector and multifunctional T cell responses following immunization, we also observed impairments in clearance of the secondary challenge (Fig. 2C, 2D, gray diamonds). Taken together, our results suggest that COX-1 activity negatively impacts the development of immunity to L. monocytogenes, whereas COX-2 activity is critical. Further, nonspecific NSAIDs administered following immunization with L. monocytogenes–based immunotherapeutics likely inhibit optimal T cell responses, suggesting that the use of COX-1 specific inhibitors or non-NSAID analgesics may improve the immune response to L. monocytogenes in clinical trials.

Consistent with the way that NSAIDs are traditionally used following vaccination, our experimental approach examines the effect of COX during the first 48 h after immunization with L. monocytogenes, which is the critical period when the majority of T cell priming occurs (38). We hypothesized that altering the eicosanoid milieu at this critical time point could influence the three signals needed for T cell priming, namely Ag presentation, costimulation, or inflammation (39), particularly as eicosanoids can increase dendritic cell (DC) maturation states (40). We did not observe any differences in viable bacterial burdens in the spleens or livers of mice following COX modulation throughout the first 48 h after immunization (Supplemental Fig. 1D–G). We also examined the total number of DCs, both conventional CD11c+ cells and the critical cross-presenting CD8α+ CD11c+ cells, and their maturation state with both CD86 and CD40 expression. We observed slight differences in the number of DCs and their costimulatory expression, but these differences did not correlate with either T cell responses or protective immunity (Supplemental Fig. 1H–K), suggesting that modulation of COX was not significantly altering either of the first two steps of T cell priming.

Eicosanoids are known to influence chemoattractant signals as well as the production of cytokines from innate immune cells, which can ultimately modulate T cell responses (4044). Thus, we analyzed the innate immune response consisting of both cellular infiltrate and cytokine production at 24 and 48 h after infection. We did not observe any changes in immune cell recruitment, including monocytes, inflammatory monocytes, neutrophils, or NK cells, into the spleen following COX modulation (Supplemental Fig. 2A–D). We next assessed a variety of proinflammatory cytokines in the serum following various COX manipulations and saw no significant differences in levels of MCP-1, IL-12p70, IL-6, or TNF-α (Supplemental Fig. 2E–H). We observed a significant elevation in IFN-γ at 24 postinfection when either COX-1 or COX-2 were inhibited, which persisted only in the COX-2 inhibition group at 48 h postinfection (Supplemental Fig. 2I). We also observed significantly lower levels of the anti-inflammatory cytokine IL-10 when COX-2 was inhibited (Supplemental Fig. 2J). These results suggest that, similar to previous reports, COX-2 promotes an immunoregulatory environment.

To determine what eicosanoids are essential for optimal L. monocytogenes–triggered immunity, we first assessed what eicosanoids are present. Many eicosanoids act locally, so we assessed the eicosanoid milieu in the spleen using the highly specific and sensitive method of liquid chromatography–mass spectrometry over the first 48 h postimmunization (25). We examined PGE2, an eicosanoid with pleotropic effects on the immune system, PGD2, an eicosanoid with largely immunosuppressive effects, thromboxane B2 (TXB2; the stable breakdown product of TXA2), and leukotriene B4 (LTB4), a major by-product of lipoxygenase activity, the other arm of arachidonic acid breakdown. We observed a trend for increased PGE2 by 8 h after immunization that became significant by 12 h after immunization, resulting in an ∼6–8-fold increase in PGE2 levels over uninfected mice (Fig. 3A). PGE2 quickly declined to baseline levels by 24 h postimmunization and remained there throughout 48 h postimmunization. Additionally, we observed significant increases in PGD2 at 8 and 12 h postimmunization of 2–3 fold over uninfected mice, although this was not consistent in every animal (Fig. 3B). Throughout the first 48 h after immunization, we did not observe any changes in TXB2 (Fig. 3C) or LTB4 (Fig. 3D), suggesting that PGs are specifically induced downstream of COX function following L. monocytogenes immunization. Taken together, these results suggest that L. monocytogenes immunization transiently, but specifically, increases the levels of PGE2 and PGD2, with a more pronounced increase in PGE2.

PGE2 is one of the major prostanoids produced downstream of COX-2 stimulation (45). Given that we found that COX-2 is critical for immunity to L. monocytogenes and that PGE2 is induced following L. monocytogenes immunization, we hypothesized that PGE2 is critical for generation of an immune response to L. monocytogenes. To test this hypothesis, we immunized mice deficient in mPGES1−/− that lack the major enzyme responsible for PGE2 synthesis in the mouse (21). Seven days after immunization, we observed impaired Ag-specific IFN-γ production (Fig. 4A) and a significant impairment for multifunctional CD8+ T cell responses (Fig. 4B) similar to what we observed following treatment with the COX-2–specific inhibitor celecoxib. Similarly, mPGES1−/− mice had a trend for impaired B8R-specific tetramer responses (Supplemental Fig. 3A), consistent with a decreased effector CD8+ T cell response, and decreased OVA-specific responses, indicating an Ag nonspecific response (Supplemental Fig. 3B, 3C). PGE2-dependency is conserved independent of immunizing dose as mPGES1−/− mice immunized with attenuated Lm at a low dose (1 × 103 cfu) similarly had impaired IFN-γ B8R-specific response (Supplemental Fig. 3E) and multifunctional B8R-specific CD8+ T cell response (Supplemental Fig. 3F). mPGES1−/− mice immunized with a low dose of attenuated Lm also had impaired B8R-specific tetramer (Supplemental Fig. 3D) and OVA-specific responses (Supplemental Fig. 3G, 3H), suggesting that the observed effects occur independent of immunizing dose and Ag. Consistent with these defects in primary T cell activation, mPGES1−/− mice were unable to clear a secondary challenge of virulent L. monocytogenes and harbored increased bacterial burdens in their spleens and livers when compared with wild-type mice (Fig. 4C, 4D). mPGES1−/− mice were not innately more sensitive to L. monocytogenes infection as naïve mPGES1−/− harbored similar burdens to mPGES1+/+ mice in both their spleens (Fig. 4C) and livers (Fig. 4D). Finally, deficits in effector T cell function could result in impaired memory responses and ultimately contribute to loss of protective immunity. To assess memory responses, we examined B8R-specific CD44+ CD8+ T cells present 30 d postimmunization with a high dose (1 × 107 cfu) of attenuated Lm. mPGES1−/− mice strongly trended toward having impaired B8R-specific memory responses at 30 d postimmunization when compared with wild-type mice (Supplemental Fig. 3I), suggesting that deficits in effector T cell function are maintained throughout the memory period. Consistent with the elevated inflammatory cytokines and diminished anti-inflammatory cytokines observed in COX-2 inhibited mice, IL6 and TNF-α were elevated in mPGES1−/− mice, whereas IL-10 was diminished (Supplemental Fig. 4C, 4D, 4F). These data suggest a role for PGE2 in preventing hyperinflammation to promote T cell priming following L. monocytogenes immunization, consistent with previous reports that PGE2 promotes an immunosuppressive environment (40). Taken together, these results suggest that PGE2 is critical for generation of cell-mediated immunity to L. monocytogenes.

Given that PGE2 is critical for generating immunity to L. monocytogenes and PGE2 is one of the major eicosanoid products produced downstream of COX-2 (45), we next asked whether PGE2 is sufficient for rescuing immunity during COX-2 inhibition. Celecoxib-treated mice were immunized with 1 × 107 CFU attenuated Lm and subsequently treated with 0.125 μg PGE2 at 8 h postimmunization followed by an additional 0.25 μg PGE2 at 12 h postimmunization, and this dosing was based on the amount of PGE2 present following immunization with attenuated Lm in a wild-type mouse (Fig. 3A). Treatment with PGE2 led to a significant rescue to wild-type levels of both B8R-specific IFN-γ production as well as multifunctional T cell responses at 7 d postimmunization (Fig. 4E, 4F). Consistent with a rescue of T cell responses in celecoxib-treated mice, tetramer staining revealed a trend toward restoration of the number of B8R-specific CD8+ T cells when PGE2 was supplemented following celecoxib treatment (Supplemental Fig. 4G), and treatment with PGE2 also significantly improved OVA-specific responses, suggesting that the observed results were independent of Ag (Supplemental Fig. 4H, 4I). These results suggest that PGE2 downstream of COX-2 is both necessary and sufficient for generating cell-mediated immunity to L. monocytogenes.

Commercially available NSAIDs are nonselective in vivo (35) and thus likely limit the production of the critical molecule, PGE2. We therefore asked whether a non-NSAID analgesic capable of limiting fever and malaise, such as acetaminophen, would be a better alternative to NSAIDs during L. monocytogenes immunotherapy. We administered acetaminophen orally in the drinking water at a concentration (200 mg/kg) that is routinely used to provide pain relief for mice (24, 46) and found that it did not impair the generation of Ag-specific effector (Fig. 5A) or multifunctional (Fig. 5B) CD8+ T cell responses following L. monocytogenes immunization. Consistent with the lack of effect on the primary CD8+ T cell response, immunized acetaminophen-treated mice had bacterial burdens similar to untreated immunized mice (Fig. 5C, 5D), further suggesting that acetaminophen does not negatively impact the cell-mediated immune response to L. monocytogenes. Taken together, our results suggest that the use of non-NSAIDs such as acetaminophen may be a suitable alternative to mitigate the unwanted side effect of L. monocytogenes immunization while promoting the immunogenicity of this immunotherapy.

NSAIDs are routinely given with childhood vaccinations, as well as cancer immunotherapies (3, 8); however, how they influence the generation of cell-mediated immunity remains unknown. In this article, we show that eicosanoids, particularly PGE2 downstream of COX-2, play a crucial role in the immune response generated to the intracellular pathogen and immunotherapeutic platform, L. monocytogenes. Surprisingly, we observed that the two forms of the COX enzyme that convert arachidonic acid into PGs and thromboxane have inverse effects on the immune response to L. monocytogenes: normal levels of COX-1 activity associated with L. monocytogenes immunization are detrimental to the development of cell-mediated immunity, whereas L. monocytogenes induction of COX-2 activity is critical for generating robust Ag-specific CD8+ T cell–dependent immunity to L. monocytogenes. Our results suggest that it would be beneficial to use analgesics that spare COX-2 such as acetaminophen for purposes of L. monocytogenes immunotherapy and that more studies should be done to understand the effects of NSAIDs in the context of other pathogen-based vaccines and immunotherapies.

COX-1 is a constitutive enzyme with homeostatic functions but whose role in inflammation and immunity has often been overlooked in favor of its physiologic functions. We and others have begun to elucidate its critical role in modulating immune responses (34). In our model, we see that COX-1 is detrimental and impairs development of CD8+ T cell–mediated immunity. Other groups have shown detrimental effects of COX-1 following inflammasome activation (47) and some forms of arthritis (48). What downstream product mediating these detrimental effects in our system is unclear. COX-1 has been shown to preferentially, although not exclusively, couple with the enzymes responsible for TXA2 synthesis and PGD2 (49, 50). TXA2 has potent vasoconstrictive properties that can negatively modulate DC–T cell interactions (51). TXA2 produced from neutrophils can also limit T cell migration into the lymph nodes and ultimately a recall response, suggesting that the overall role of TXA2 is immunosuppressive (43). We were unable to observe any changes in TXB2 synthesis (the stable breakdown product of TXA2) over the course of L. monocytogenes infection; however, it is possible that examining TXA2 production from a certain cell type, as opposed to the entire spleen, may elucidate differences. Additionally, use of TXA2-specific inhibitors (43) or thromboxane receptor–deficient mice (51) may illuminate roles for TXA2 downstream of COX-1 following L. monocytogenes immunization. Although we were unable to observe changes in TXB2, we did observe increases in PGD2 expression following L. monocytogenes immunization to 2–3-fold levels over mock-infected controls. PGD2 has largely been implicated in allergic responses as its production largely comes from mast cells and can result in the hallmark symptoms of an asthma exacerbation, including bronchoconstriction and airway eosinophil accumulation (52, 53). PGD2 can also be produced by DCs and Th2 cells where it can limit Ag presentation and DC migration (54) to decrease T cell activation. Whether this minimally transient increase in PGD2 is dependent on COX-1 and can explain our observed increases in immunity in COX-1−/− mice remains to be determined.

In contrast to COX-1, COX-2 preferentially couples with the enzymes responsible for prostacyclin and PGE2 expression (45). Consistent with our observations of impaired immunity following COX-2 inhibition, we similarly observed impairments in immunity when PGE2 was absent, suggesting that our phenotypes due to COX-2 inhibition are dependent on PGE2. PGE2 has been implicated in many immunosuppressive as well as inflammatory effects, affecting both the innate and adaptive immune responses. PGE2 can promote regulatory T cell formation (55), modulate T cell cytokine production toward a Th2 profile (56), and promote immunosuppressive cytokine production from DCs (40). In contrast, PGE2, when found in an already inflammatory environment, can enhance DC costimulatory expression, migration, and lymph node homing allowing for more efficient T cell priming (57, 58). The pleiotropic effects of PGE2 are in part determined by the four receptors through which it signals. EP2 and EP4 signal through the G-protein–coupled receptor Gs to result in activation of adenylate cyclase (59, 60), whereas EP3 signals through Gi to inhibit adenylate cyclase (61), and EP1 works through calcium release (62). Each receptor has different affinities for PGE2, leading to another level of regulation: EP3 and EP4 are high-affinity receptors that are rapidly desensitized following ligand binding,whereas EP1 and EP2 are low-affinity receptors that respond to prolonged PGE2 signaling (63). We observe only a transient increase in PGE2 levels throughout the first 48 h after immunization, suggesting that a high-affinity receptor may be responsible for our effects. The EP4 receptor is abundantly expressed in the spleen and can promote anti-inflammatory cytokine responses (64). Consistent with this, we observe increased proinflammatory cytokine production and decreased IL-10 production when we inhibit COX-2 or PGE2. Recent work with L. monocytogenes suggests that, although inflammation is critical for generation of effective CD8+ T cell immunity (65), excessive amounts of inflammation or increased sensitivity to inflammatory mediators such as TNF-α, IFN-γ, or type I IFNs actually impairs CD8+ T cell immunity (66, 67). We suggest that the role of COX-2 and PGE2 in our system may be immunoregulatory to limit excessive innate immune responses and inflammation that have previously been shown to be detrimental in the context of L. monocytogenes immunization (6668).

In addition to promoting anti-inflammatory cytokines, PGE2 can limit type I IFN production during infections, such as influenza and M. tuberculosis (14, 15). Whether PGE2 is critical or detrimental in these infections is ultimately determined by the role for type I IFN; in influenza infection, type I IFNs are critical for controlling viral replication and mounting antiviral responses. The addition of PGE2 is detrimental in this system as type I IFN levels are decreased. In contrast, in an M. tuberculosis infection, type I IFN promotes bacterial pathogenesis (69) and limits immunity (15), and PGE2 is critical for immunity by limiting the detrimental effects of type I IFN. Similar to M. tuberculosis, type I IFN production is detrimental to the host both acutely (70, 71) and for generation of an adaptive immune response (67) following L. monocytogenes immunization, suggesting that the critical role for PGE2 in generating cell-mediated immune response may be through control of type I IFN. Whether type I IFNs are altered following COX modulation in a PGE2-dependent manner and how they ultimately influence the immune response will be critical to address in the future.

Interestingly, we observe tissue-specific effects for both COX-1 and COX-2; lack of COX-1 improves protective immunity in the spleen, whereas lack of COX-2 impairs protective immunity in the liver. COX-2 has been implicated in multiple liver-specific pathologies, including hepatocellular carcinoma, fibrosis, nonalcoholic steatohepatitis, and nonalcoholic fatty liver disease (72). Hepatocytes contain receptors for many of the COX-2–specific stimulators, including TNF-α, IL-1, and LPS, and liver-specific Kuppfer and stellate cells have specifically been shown to upregulate COX-2 expression following inflammatory stimuli (72, 73). Similarly, following LPS administration, COX-2 pathway activity increases in the liver (74), whereas COX-1 pathway activity has been shown to increase in the spleen and not the liver (75), similar to the phenotypes we see. Although the mechanisms governing these distinct tissue-specific differences remain unclear, it will be critical in the future to elucidate the tissue-specific differences to ensure the most therapeutic outcome pending the tissue affected.

Almost all commercially available NSAIDS exhibit nonspecific COX inhibition in vivo (35); thus, using pharmacotherapies that are able to limit the unwanted side effects of L. monocytogenes immunotherapy while preserving the critical role of PGE2 will be critical. In contrast to NSAIDs, such as indomethacin and celecoxib, acetaminophen is a common non-NSAID pharmacotherapy for pain relief and fever reduction that lacks a well-defined mechanism of action (76). Acetaminophen metabolites have been shown to modulate endogenous cannabinoid receptor activity centrally to provide pain relief (77), suggesting that it acts centrally to exhibit its effects. Additionally, similar to NSAIDs, acetaminophen can inhibit COX enzymes, although its inhibition is specific to CNS COX activity, whereas it lacks an effect against peripheral COX activity (78). These contrasting sites of action, centrally in the case of acetaminophen and peripherally in the case of NSAIDs, likely explain why in our system acetaminophen administration does not impair the development of CD8+ T cell immunity, whereas it still maintains effective antipyretic activity.

We and others have previously shown that the inflammatory environment in which T cell priming occurs following L. monocytogenes immunization modulates the magnitude of the response (65, 66, 68). In this article, we present evidence that, in addition to the traditional cytokine mediators of inflammation, lipid mediators play significant roles in the generation of cell-mediated immunity. Understanding how COX therapy modulates inflammation and which inflammatory mediators are detrimental will be critical as COX inhibitors are routinely given with vaccinations and immunotherapies and are specifically given during L. monocytogenes immunotherapy (7, 8). We suggest that development of novel, specific COX-1 inhibitors; therapies that spare COX-2 and PGE2 production; or use of non-COX acting pharmacotherapies such as acetaminophen that limit alterations in peripheral inflammation will be critical for optimal immune responses to L. monocytogenes as well as other platforms that require the generation of cell-mediated immunity.

We thank Dr. Charles Serhan for technical assistance and protocols for eicosanoid extraction. We also thank the NIH Tetramer Core Facility for provision of MHC-I B8R tetramers.

This work was supported by the following grants: National Institutes of Health R01CA188034 (to J.-D.S.) and F30 CA210912 (to E.T.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

COX

cyclooxygenase

DC

dendritic cell

LTB4

leukotriene B4

mPGES1

microsomal PG synthase E1

NIH

National Institutes of Health

NSAID

nonsteroidal anti-inflammatory drug

TXA2

thromboxane A2

TXB2

thromboxane B2.

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The authors have no financial conflicts of interest.

Supplementary data