Acetylsalicylic acid is a globally used non-steroidal anti-inflammatory drug (NSAID) with diverse pharmacological properties, although its mechanism of immune regulation during inflammation (especially at in vivo relevant doses) remains largely speculative. Given the increase in clinical perspective of Acetylsalicylic acid in various diseases and cancer prevention, this study aimed to investigate the immunomodulatory role of physiological Acetylsalicylic acid concentrations (0.005, 0.02 and 0.2 mg/ml) in a human whole blood of infection-induced inflammation. We describe a simple, highly reliable whole blood assay using an array of toll-like receptor (TLR) ligands 1–9 in order to systematically explore the immunomodulatory activity of Acetylsalicylic acid plasma concentrations in physiologically relevant conditions. Release of inflammatory cytokines and production of prostaglandin E2 (PGE2) were determined directly in plasma supernatant. Experiments demonstrate for the first time that plasma concentrations of Acetylsalicylic acid significantly increased TLR ligand-triggered IL-1β, IL-10, and IL-6 production in a dose-dependent manner. In contrast, indomethacin did not exhibit this capacity, whereas cyclooxygenase (COX)-2 selective NSAID, celecoxib, induced a similar pattern like Acetylsalicylic acid, suggesting a possible relevance of COX-2. Accordingly, we found that exogenous addition of COX downstream product, PGE2, attenuates the TLR ligand-mediated cytokine secretion by augmenting production of anti-inflammatory cytokines and inhibiting release of pro-inflammatory cytokines. Low PGE2 levels were at least involved in the enhanced IL-1β production by Acetylsalicylic acid.
Citation: Brox R, Hackstein H (2021) Physiologically relevant aspirin concentrations trigger immunostimulatory cytokine production by human leukocytes. PLoS ONE 16(8): e0254606. https://doi.org/10.1371/journal.pone.0254606
Editor: Bruno Lourenco Diaz, Universidade Federal do Rio de Janeiro, BRAZIL
Received: November 11, 2020; Accepted: June 29, 2021; Published: August 24, 2021
Copyright: © 2021 Brox, Hackstein. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Acetylsalicylic Acid (ASA) is the most common of all non-steroidal anti-inflammatory drugs (NSAIDs) worldwide. Interestingly, it has been reported that ASA, in addition to its anti-inflammatory effects, can also have marked immunomodulatory effects, e.g. on the function of critical antigen-presenting cells, which are poorly understood [1, 2]. Due to its analgesic, antipyretic, anti-thrombotic and anti-inflammatory properties, ASA is used as therapy for diverse conditions including treatment of moderate pain [3, 4], reduction of symptoms in rheumatic diseases [5, 6] and prevention of cardiovascular events [7, 8]. Moreover, several clinical studies have recently provided evidence that daily intake of low-dose aspirin may significantly prevent cancer incidence, especially in gastrointestinal tract [9–11]. Originally, the main mechanism for the pharmacological effects of ASA is the suppression of endogenous prostaglandin synthesis via inhibition of cyclooxygenase (COX) activity [12, 13]. There are two isoforms of COX identified: cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) . While the constitutively expressed COX-1 regulates homeostatic prostaglandins (PGs) to mediate “housekeeping” functions in the body, COX-2 is rapidly induced by inflammatory stimuli to release PGs at tissue site of inflammation [15, 16]. Therefore, it seems that ASA, through its well-known COX inhibitory mechanism, exhibits its immunopharmacological properties via modulation of COX-dependent production of PGs. However, there is a growing body of evidence that ASA has some COX-independent mechanisms, including inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway , induction of Nitric oxide (NO) release  and lipoxin synthesis . Besides the frequent use of low-dose ASA in antithrombotic therapy, low-dose ASA has been demonstrated by recent studies to reduce cancer incidence [20–22] and play a role in immune system and certain immunopathological conditions [19, 23, 24]. However, there is still no common agreement about the mechanism of the immunomodulatory potential of ASA. There are already some results that ASA has an immunostimulating effect after LPS stimulation but most studies administered high ASA doses that are not reached in vivo [25, 26]. Therefore, this study aimed to reinvestigate the immunomodulatory effects of ASA in the context of its easily and consistently achieved plasma concentrations after regular administration in humans and extended the investigations to multiple toll-like receptor (TLR) ligands. A randomized placebo-controlled crossover study detected after intravenous and oral administration of 500 mg ASA peak plasma concentrations of 0.05 mg/ml and 0.005 mg/ml, respectively . Furthermore, a comprehensive data collection of therapeutic blood concentrations for nearly 1000 drugs reported ASA plasma concentrations in the range of 0.02 and 0.2 mg/ml . We developed a rapid and sensitive method to assess immune-related effects of ASA, Indomethacin, and Celecoxib in human whole blood (WB) after stimulation with TLR ligands 1–9. TLRs are pattern recognition receptors on diverse cell types that play a vital role in the activation of immune response involving antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages . Stimulation of TLRs by their cognate ligands trigger the migration and production of inflammatory cytokines, upregulation of major histocompatibility complex (MHC) molecules, and co-stimulatory signals in antigen-presenting cells and can therefore be exploited as an in vitro stimulus that closely mimic the physiological immune reaction [30, 31]. Using this WB assay, we examined a variety of immunomodulatory aspects of therapeutic relevant ASA doses, including cytokines and PG release, in a highly standardized manner that requires minimal blood volumes and mimics the natural in vivo environment.
Materials and methods
Freshly drawn peripheral blood from healthy male donors aged 18–60 after obtaining their written informed consent was anticoagulated using Tri-sodium citrate monovettes (S.Monovette, Sarstedt). The study was approved by the local ethics committee of University Hospital Erlangen (346_18B, 343_18B, 357_19B). Blood samples were kept at room temperature for no longer than 2h before processing.
Stimulation of whole blood
Whole blood (WB) was diluted 1:2 with RPMI 1640 (Sigma-Aldrich) supplemented with 1% Penicillin/ Streptomycin (Sigma-Aldrich) and 2 mM L-glutamine (Gibco) and were distributed in 96-well round bottom plates (total volume 200μl/well). Samples were stimulated for 18h in 5% CO2 at 37°C with 20 μl TLR ligands from InvivoGen including Pam3CsK4 (TLR1/2), HKLM (TLR2), Poly (I:C)-HMW (TLR3), Poly (I:C)-LMW (TLR3), LPS E.coli K12 (TLR4), Flagellin-ST (TLR5), FSL-1 (TLR6/2), Imiquimod (TLR7), ssRNA40/LyoVec (TLR8), and ODN2006 (TLR9). The appropriate concentrations used in this study are depicted in Fig 1. In order to investigate immunomodulatory effects, blood samples were incubated for 6h in 5% CO2 at 37°C with acetylsalicylic acid (0.2 mg/ml/ 1.0 mM, 0.02 mg/ml/ 0.1 mM or 0.005 mg/ml/ 0.03 mM), Indomethacin (0.01 mg/ml/ 0.03 mM or 0.05 mg/ml/ 0.1 mM), Celecoxib (0.01 mg/ml/ 0.03 mM or 0.05 mg/ml/ 0.1 mM), Dexamethason (1 nM or 100 nM), PGE2 (7.5 ng/ml or 5 ng/ml) (all from Sigma Aldrich) or vehicle alone before TLR stimulation. Acetylsalicylic acid, Indomethacin and Celecoxib were dissolved in DMSO; Dexamethason and PGE2 in ethanol. After stimulation, approximately 100 μl supernatant were carefully collected from each well (without disturbing the pellet) and subsequently frozen at −20°C until use. The optimal duration of stimulation for an optimal effect on cytokine secretion was determined through prior kinetic studies.
Citrate-anticoagulated blood was stimulated with TLR-ligands for 18h. Concentration levels of cytokines [pg/ml] are presented as mean ± SD of 2 experiments, each performed in duplicate.
Measurement of cytokine production
Cytokines including TNF-α, IL-1β, IL-6, IL-10 and IFN-γ were quantified using a flow cytometry bead-based immunoassay (LEGENDplexTM human essential immune response panel, BioLegend) according to the manufacturer’s protocol and analyzed using LEGENDplex version 7.0 software (Vigene Tech). Cytokine concentrations were transformed to Log2 for TLR stimulation or expressed in percent relative to TLR agonist alone, which was defined as 100%.
Measurement of PGE2 production
PGE2 concentration was measured with a Homogenous Time Resolved Fluorescence (HTRF) kit obtained from Cisbio according to the manufacturer’s protocol. TR-FRET signal was detected by a FLUOstar Omega plate reader (BMG Labtech) with laser excitation at 337 nm and dual emission at 665 nm and 620 nm. HTRF ratios were estimated as fluorescence signal at 665 nm divided by fluorescence signal at 620 nm (acceptor/donor) and then multiplied by 104. Data were converted from HTRF ratio values to PGE2 concentration using a standard curve and then expressed in percent relative to TLR agonist alone, defined as 100%.
Cellular viability and cellular composition of WB after stimulation with TLR ligands, acetylsalicylic acid, Indomethacin, Celecoxib, PGE2 or vehicle was determined by flow cytometry (S1 Fig). WB was stained with a staining kit (Zombie Aqua Fixable Viability Kit, Biolegend) in accordance with the manufacturer’s protocol. Before staining of extracellular antigens, cells were treated with Fc receptor blocking reagent (Miltenyi Biotec). Extracellular staining was performed with monoclonal antibody for 20 minutes at 4°C in FACS buffer (PBS [Sigma-Aldrich], 2% FCS [anprotect]). Afterwards, samples were lysed with ammonium chloride solution (155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA, pH 7.4) for 10 minutes at room temperature. The lysed samples were centrifuged and washed at 300g for 5 minutes before acquisition on a CytoFLEX S (Beckman Coulter) and subsequently analyzed using FlowJo v10. Doublets, cell debris, and dead cells were excluded via forward and sideward scatter as well as Zombie AquaTM. Cell subpopulations were phenotyped with the following murine α-human monoclonal antibodies: CD14-BV605 (63D3), CD56-BV650 (5.1H11), CD16-PacBlue (3G8), CD3-AF700 (OKT3), CD19-APC/Fire (SJ25C1). All antibodies were purchased from BioLegend.
Cell populations were defined as follows: live (single cells, Zombie AquaTM), monocytes (CD14+CD16+), granulocytes (CD14-CD16+), NK cells (CD14-CD16+CD56+), T cells (CD14-CD56-CD16-CD3+), B cells (CD14-CD56-CD16-CD3-CD19+).
Data were reported as mean ± SD unless otherwise stated. Statistical analysis was performed with GraphPad Prism version 8.3.0 (GraphPad Software, San Diego, California USA). Statistical significance between groups was evaluated by two-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons. P-value less than 0.05 was considered statistically significant.
Development and validation of an in vitro whole-blood model for the evaluation of immunomodulatory agents
With the aim of investigating the immunomodulatory properties of ASA in a clinically relevant setting, we adapted a WB cytokine assay that preserves the physiological cellular interactions and environment . In this simple model for infection-induced inflammation, the cytokines secretion in citrate-anticoagulated WB cell cultures from healthy subjects were measured in response to different agonists of human TLRs 1–9. To determine the optimal concentration of TLR ligands for detecting cytokine production in WB, we first stimulated with serial dilutions of each TLR agonist and assessed the essential immune cytokines (TNF-α, IL-6, IL-10, IL-1β and IFN-γ) in the supernatant via bead-based immunoassay. After 18h incubation, a dose-dependent cytokine production was detected for all TLR ligands, such that cells in the WB culture responded differently to TLR stimulation with respect to their amount and type of cytokine secretion (Fig 1). Depending on the class of pathogen, a wide variety of cells secrete cytokines in order to coordinate the innate and adaptive immune response during host defense [31, 33]. For further experiments, we focused on the most powerful stimulants in the minimum concentration with adequate efficacy (500 ng/ml Pam3CsK4 (TLR1/2); 108 cells/ml HKLM (TLR2); 10 ng/ml LPS (TLR4); 1 μg/ml Flagellin (TLR5); and 2.5 μg/ml ssRNA40 (TLR8)) that triggered not only pro-inflammatory cytokines (IFN-γ, IL-1β and TNF-α) but also anti-inflammatory cytokines (IL-10), including those with pleiotropic activities (IL-6).
Furthermore, we validated the biological specificity of WB assay using the classical anti-inflammatory glucocorticoid Dexamethason, which mediates its anti-inflammatory properties via inhibition of intracellular signals initiated by TLRs [34–37]. WB was pre-incubated with 1 nM and 100 nM Dexamethason for 6h followed by stimulation with the various TLR ligands for 18h. From the result, 100 nM Dexamethason exhibited an almost complete inhibition of TNF-α, IL-1β, IL-6 and IFN-γ release irrespective of the TLR stimulation (Fig 2). While a similar inhibitory effect was observed for IL-10 in response to LPS and Flagellin in a weakened form (mea n = 40% and 65%, respectively), Dexamethason had no influence on IL-10 concentration after stimulation with Pam3CsK4, HKLM, and ssRNA40.
Citrate- anticoagulated blood was incubated with Dexamethason or vehicle 6h before stimulation with Pam3CsK4, HKLM, LPS, Flagellin or ssRNA40. The cytokine production by cells stimulated without Dexamethason (vehicle) was set as 100%. Data represent mean±SD of four experiments performed in duplicate. Differences were significant at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) as indicated, compared to WB incubated without Dexamethason. The concentrations of TNF-α and IFN-γ upon Pam3CsK4 stimulation were below detection limit. n.d.—not detectable.
Physiologic ASA concentrations augment TLR ligand triggered immunostimulatory cytokine production
In order to evaluate the immunomodulatory impact of low ASA concentrations, we pre-incubated WB with increasing therapeutic concentrations of ASA followed by stimulation with Pam3CsK4, HKLM, LPS, Flagellin or ssRNA40. As shown in Fig 3, ASA exhibited different effects on cytokine production depending on the TLR ligand. In the presence of ASA, Pam3CsK4 induced a concentration-dependent increase in IL-1β (Fig 3A). Similarly, a significant elevation of IL-1β was detected in the supernatant of WB cultures simulated with LPS and Flagellin (Fig 3C and 3D). At the highest concentration of 0.2 mg/ml of ASA, LPS enhanced IL-10 production (mean = 175%). For ssRNA40, we observed a moderate increase in IL-6 and IFN-γ production in cells pre-incubated with ASA (Fig 3E). Notably, the stimulatory effect on IFN-γ production declined with higher ASA concentrations. In contrast, upon stimulation with HKLM, ASA demonstrated a dose-dependent inhibition of IFN-γ up to 50% (Fig 3B). In the absence of TLR ligands, addition of ASA resulted in non-significant cytokines production.
Citrate-anticoagulated blood was incubated with ASA or vehicle for 6h before stimulation with (A) Pam3CsK4 (B) HKLM (C) LPS (D) Flagellin and (E) ssRNA40. The cytokine production by cells stimulated without ASA (vehicle) was set as 100%. Data represent mean ± SD of six experiments performed in triplicate. Differences were significant at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) as indicated, compared to WB incubated without ASA. n.d.—not detectable.
Effect of indomethacin and celecoxib on TLR ligand stimulated cytokine production
Since low concentrations of ASA (0.01–0.1 mM) are demonstrated to primarily inhibit prostaglandin biosynthesis by targeting both COX-1 and COX-2 [38, 39] and at higher concentrations (> 5 mM) may exhibit an immunoregulatory effect mediated by inhibition of NF-κB , we next examined the impact of two other NSAIDs exhibiting different mechanisms of action. Indomethacin is known to inhibit COX-1 and COX-2 activity without any effect on NF-κB activation  and Celecoxib is described as a selective COX-2 inhibitor . In our WB assay, Indomethacin showed a very slight increase in few cytokines concentration compared to ASA (Fig 4). Significant higher cytokines concentration were only observed for TNF-α and IFN-γ upon stimulation by LPS and ssRNA40, respectively (Fig 4C and 4E). In contrast, similar to ASA, addition of the highest concentration of Celecoxib triggered a substantial elevation of several cytokines in response to TLR-ligands (Fig 4). Celecoxib (0.05 mg/ml) strongly upregulated the production of IL-1β by almost 100% compared to TLR stimulation alone (Fig 4A and 4C–4E). Pre-treatment with Celecoxib also elicited an increased amount of IL-6 in supernatant of WB cultures stimulated with Pam3CsK4 and LPS (mean = 220% and 160%, respectively). A considerable increase in IL-10 production by the highest dose of Celecoxib was obtained in response to LPS (mean = 208%) and Flagellin (mean = 181%). In addition, a concentration-dependent inductive effect of Celecoxib was also observed for ssRNA-stimulated IFN-γ production (Fig 4E).
Citrate-anticoagulated blood was incubated with 0.01 and 0.05 mg/ml Indomethacin, Celecoxib or vehicle 6h before stimulation with (A) Pam3CsK4 (B) HKLM (C) LPS (D) Flagellin and (E) ssRNA40. The cytokine production by cells stimulated without Indomethacin or Celecoxib (vehicle) was set as 100%. Data represent mean ± SD of six experiments performed in duplicate. Differences were significant at a p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) as indicated, compared to WB incubated without ASA. n.d.—not detectable.
ASA inhibits TLR-triggered PGE2 production in human WB in a dose-dependent manner
PGE2, the predominant eicosanoid in inflammatory response, is largely dependent on the activity of COX-2 . We examined the influence of ASA on PGE2 production in response to TLR ligands. In an initial experiment, we validated that all TLR ligands catalyzed the formation of PGE2 compared to unstimulated WB, such that HKLM was the most potent activator (Fig 5A). Pre-incubation of blood samples with different concentrations of ASA showed a dose-dependent inhibition of TLR ligand-induced PGE2 production (Fig 5B). PGE2 production was reduced by approximately 50% in all TLR ligand-stimulated cells at the highest concentration of ASA (0.2 mg/ml). A modest decrease (20%–40%) was detected at lower ASA doses in the supernatant of WB cultures incubated with Pam3CsK4, LPS, and Flagellin. In contrast, 0.0 2mg/ml and 0.005 mg/ml of ASA were insufficient to significantly suppress PGE2 production in response to HKLM and ssRNA40.
Citrate-anticoagulated blood was incubated with (A) Pam3CsK4, HKLM, LPS, Flagellin or ssRNA40 and PGE2 production was measured. Results are expressed as mean ± SD of four experiments performed in triplicate. Differences were significant at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) as indicated, compared to unstimulated WB. (B) Various concentrations of ASA or vehicle following stimulation with TLR agonists. PGE2 production is expressed in percent compared to TLR agonist alone (vehicle), which is defined as 100%. Data represent mean ± SD of four experiments performed in triplicate. (C) Various concentrations of IND, CCXB or vehicle following stimulation with TLR agonists. PGE2 production is expressed in percent compared to TLR agonist alone (vehicle), which is defined as 100%. Data represent mean ± SD of four experiments performed in duplicate. Differences were significant at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) as indicated, compared to WB incubated without ASA, IND or CCXB.
Similarly, Indomethacin and Celecoxib at both concentrations were able to significantly suppress TLR ligand-induced PGE2 production (Fig 5C). However, Indomethacin and Celecoxib showed a stronger inhibitory effect compared with ASA (45–95%).
Immunostimulatory properties of ASA are partially reversed by PGE2
To investigate whether the inhibitory effects of ASA on TLR agonist-mediated PGE2 production is responsible for the immunostimulatory cytokine production, exogenous PGE2 was added in excess to WB before stimulation. The exogenous PGE2 suppressed the release of IL-1β and IFN-γ in response to TLR ligands by 30%–70% compared to those of non-treated controls with the exception of ssRNA40 where no downregulation of IL-1β was observed (Fig 6). Strikingly, the inhibitory effect prevailed even in the presence of 0.2 mg/ml ASA and abolished the potentiating impact of ASA on IL-1β production upon stimulation with Pam3CsK4, LPS, and Flagellin (Fig 6A, 6C and 6D). Similarly, the reduced levels of IFN-γ did not change with the addition of ASA. However, addition of exogenous PGE2 resulted in a marked increase in IL-6 for all TLR ligands (mean = 190–865%), in IL-10 after stimulation with LPS (mean = 230%) and HKLM (mean = 148%), and in TNF-α after stimulation with LPS (mean = 220%), Flagellin (mean = 155%) and ssRNA40 (mean = 258%). Interestingly, co-incubation with 0.2mg/ml ASA had no effect on the elevated cytokine levels caused by PGE2. In contrast, IL-10 release was even suppressed by PGE2 when WB was stimulated with Pam3CsK4 (mean = 35%; Fig 6A) and showed no effect on Flagellin- and ssRNA40-induced IL-10 levels (Fig 6D and 6E).
PGE2 (5 ng/ml) was added to citrate-anticoagulated blood in the presence or absence of 0.2 mg/ml ASA before stimulation with TLR-ligands including (A) Pam3CsK4 (B) HKLM (C) LPS (D) Flagellin and (E) ssRNA40. Stimulated cells incubated without ASA and PGE2 (vehicle) set as 100%. Data represent mean ± SD of three experiments performed in triplicate.
In the present study, we developed a straightforward technique using human WB stimulated with different TLR ligands to investigate the immunomodulatory effects of in vivo relevant plasma concentrations of ASA. The supernatant of WB cultures offers the possibility of simultaneous and quantitative detection of multiple parameters (including cytokines and PGs) that are important players in intracellular signal transduction and intercellular communications of immune cells, without changing their relative proportions in cells . Upon stimulation with various TLR ligands, we detected typical patterns of secreted cytokines attributed to different TLR-associated signaling pathways and various types of responding cells [44–46]. The known immunosuppressive agent, Dexamethason, confirmed the functionality of the WB assay by potently inhibiting the TLR ligand-induced cytokine production. In previous studies, anti-inflammatory cytokine (IL-10) production was less inhibited by Dexamethason compared to pro-inflammatory cytokines production (TNF-α and IL-1β) [47, 48].
In comparison with previous studies, we focused on the ability of in vivo relevant ASA concentrations that are achieved after administration of therapeutic ASA doses to modulate cytokine production in human WB after TLR ligand stimulation. ASA concentrations were selected based on reported plasma levels in literature [27, 28]. Our study revealed that physiological ASA concentrations in WB significantly increase TLR-stimulated cytokine production in a dose-dependent manner. Especially, addition of 0.02 and 0.2 mg/ml ASA enhanced the production of IL-1β, IL-10, IL-6 and IFN-γ in WB culture after TLR stimulation with Pam3CsK4, LPS, Flagellin, and ssRNA40. In contrast, an inhibitory effect of ASA was only detected for HKLM-mediated IFN-γ levels. Immunostimulatory properties of ASA were reported previously, where oral administration of ASA in healthy volunteers resulted in increased IL-1β and TNF-α synthesis by PBMCs  and elevated TNF-α activity in LPS-stimulated human monocytes . An increased production of TNF-α, IFN-γ, IL-10, and IL-6 was also observed following LPS stimulation of WB and addition of higher ASA concentrations (1–5 mM) [25, 26]. In this study, we were able to detect an immunostimulatory effect on cytokine release even at therapeutic relevant ASA plasma concentrations and could show an immunostimulatory effect not only after LPS stimulation but also with various other TRL ligands, suggesting that the immunomodulatory capacity of ASA may be much broader than previously thought. Only male donors were included in the current experimental study to reduce confounding factors, as sex-related differences in cytokine production are evident following TLR7/8 stimulation of healthy human subjects [51–53]. However, as ASA is a globally used NSAID in men and women, it would be an interesting clinical question to examine the immunomodulatory effects of ASA in female donors as well.
To further investigate the influence of COX inhibition on the immunostimulatory effects of ASA, we used Indomethacin, which inhibits COX activity without any effect on NF-κB activation  and Celecoxib, which is described as a selective COX-2 inhibitor . In contrast to ASA, Indomethacin exhibited a weak effect on TLR-triggered cytokine production. A moderate stimulatory effect of Indomethacin was observed at the highest concentration for LPS-mediated TNF-α, IL-1β and IL-10 production as well as ssRNA40-stimulated IFN-γ production. Interestingly, the selective COX-2 inhibitor, Celecoxib, promoted a marked increase in several cytokines (IL-1β, IL-6, IL-10 and IFN-γ) which is comparable to the effect of ASA. These findings are in line with previously reported role of COX-2 inhibition for stimulatory effects of NSAIDs on the production of cytokines [25, 54].
Following TLR stimulation, various cell types express high levels of COX-2, which accounts for the production of large amounts of PGE2 . We therefore focused on the downstream product of COX, PGE2, to further examine the involvement of COX-2 in the immunostimulatory effects of low-dose ASA. PGE2 is an attractive key mediator in many early inflammatory events as it is able to exhibit both promotion of anti-inflammatory effects such as IL-10 production and direct suppression of multiple pro-inflammatory cytokines including IFN-γ, TNF-α, and IL-1β to limit nonspecific inflammation, depending on the context [55–58]. The biological actions of PGE2 are mediated by four distinct G protein-coupled receptors (EP1, EP2, EP3, and EP4) on the plasma membrane of target cells . We confirmed that PGE2 is generated in response to all TLR ligands and determined that COX activity is influenced by ASA as measured by the dose-dependent suppression of TLR-ligand induced PGE2 production. Indomethacin and Celecoxib also reduce the production of PGE2 to baseline levels in WB [60, 61]. Consistent with the ability of PGE2 to downregulate pro-inflammatory cytokines, addition of exogenous PGE2 to WB before TLR-stimulation suppressed the production of IFN-γ and IL-1β. In turn, we found that TLR ligand-induced IL6 concentrations were further increased after addition of exogenous PGE2. PGE2 also showed enhanced production of HKLM- and LPS-released IL-10 and increased levels of TNF-α following stimulation with LPS, Flagellin and ssRNA40. In contrast, a suppressive effect of PGE2 was observed for Pam3CsK4-induced IL-10 levels. The pleiotropic roles of PGE2 in immune regulation have been described for several immune cell types, particularly those involved in innate immunity such as macrophages, neutrophils, natural killer cells, and dendritic cells (DCs) [62–65]. For example, PGE2 strongly inhibits the production of Th1 cytokines, such as IFN-γ and IL-2, and favors type-2 responses in general . The biasing of the immune system toward Th2 and away from Th1 responses by PGE2 is further supported by the PGE2-mediated inhibition of antigen-primed DCs to produce IL-12. These DCs produce high levels of IL-10 and directly induce the differentiation of naïve T cells into Th2 cells [67–69]. In addition, NK cells secrete IFN-γ to activate macrophages during the innate immune response, which is suppressed by PGE2 . The precise mechanism of these inhibitory effects remains unclear but there is evidence that intracellular cAMP, a downstream effector molecule of PGE2 signaling through the EP2/EP4 receptors, and increased production of polarizing cytokines are involved in suppressing Th1 cell-mediated immune inflammation [71–75]. Blocking IL-1β processing and secretion involves inhibiting the NLR family pyrin domain containing 3 (NLRP3) inflammasome in human primary monocyte-derived macrophages, which is mediated through the EP4 receptor and increases intracellular cAMP [76, 77]. This is also supported by the finding that we detected no increase in IL-1β in response to PGE2 following stimulation with ssRNA40, because RNA analogs such as ssRNA40, activate IL-1β through the NLRP3 pathway [78, 79]. However, we found no inhibitory effect of PGE2 on pro-inflammatory TNF-α production in our WB assay. Various parameters could play a role in this discrepancy that highlights the artificial nature of in vitro experiments. It was previously reported that PGE2 exhibits dose-dependent effects on TNF-α release from rat macrophages: low concentrations had a stimulatory effect and high concentrations had an inhibitory effect . In addition, the temporal context could be decisive for the mode of action of PGE2, as macrophage TNF biosynthesis is inhibited by exogenously supplied PGE2 but is insensitive to endogenously produced PGE2, most likely due to a time delay in LPS- induced PGE2 biosynthesis . The induction of IL-6 by PGE2 can be explained based on activation of NF- κB [82, 83]. An increased IL-6 response to PGE2 in murine inflammatory macrophages has been suggested to be distinctively regulated than IL-10 and has been shown to be dependent on p38/MAP kinase activity . Several studies demonstrated that agents that increase cAMP levels enhance IL-10 transcription [84, 85]. This also includes PGE2, which upregulates the production of IL-10 in various cell types including macrophages , T cells , and DCs [88, 89]. In addition, investigations of the inflammatory effects of PGE2 on DC functions have shown that COX-2-mediated PGE2 accounts for the boost in IL-10 release and suppresses production of pro-inflammatory cytokines, such as IL-12p70 [88, 89]. The anti-inflammatory phenotype associated with enhanced production of IL-10 is mediated by increased intracellular cAMP via the EP2 and EP4 receptor subtypes by modulating the EP/PKA/SIK/CRTC/CREB pathway [86, 90–93]. EP2 and EP4 are Gs-coupled receptors that signal primarily through the adenylate cyclase-dependent cAMP/PKA/CREB pathway . Importantly, our results highlight that the TLR ligands investigated induced various amounts of PGE2 and similarly, adding exogenous PGE2 resulted in different effects on cytokine production depending on the TLR ligand applied. TLRs recruit a specific set of adaptor molecules, such as MyD88 and TRIF, to initiate downstream signal transduction pathways. MyD88 is used by all TLRs except TLR3 and activates the transcription factor NF-κB and mitogen-activated protein kinases (MAPK) to induce inflammatory cytokines [29, 94]. However, some TLRs utilize additional adapter proteins including TRIF, TIRAP, and TRAM to trigger different signaling pathways from different intracellular compartments [95, 96]. Investigations of TLR-mediated PGE2 production in human DCs have demonstrated that only the TLR4 and TLR7/8 ligands released PGE2, although all TLRs are expressed and functional . Differential post-transcriptional regulation was also the reason for a stronger induction of IL10 secretion via TLR4 in TLR2 and TLR4-stimulated BM derived macrophages [98, 99].
The hypothesis of a direct correlation between cytokine release and PGE2 production by ASA remains to be confirmed by a larger sample size study . By adding exogenous PGE2 to compensate for the inhibitory effect of ASA on TLR-ligand induced PGE2 production, it was demonstrated that the potentiating effect of ASA on IL-1β formation was completely prevented. This may either be due to the supplemented amount of PGE2 or to an inhibitory effect of PGE2 on IL-1β. Previous studies have presumed that the immunostimulatory properties are caused by the loss of PGE2. A similar inhibitory effect of PGE2 was reported for the amplification of TNF-α by ASA after LPS stimulation  and for the increased production of IL-6 and TNF-α by the NSAID Indomethacin . Especially for TNF-α, it has been suggested that inhibited PGE2 production is responsible for the observed stimulatory effect [50, 62, 66]. In contrast, the upregulation of IL-10 and IL-6 by ASA in this study was probably not caused by inhibiting PGE2. Lipoxins are endogenous anti-inflammatory metabolites of the arachidonic acid pathway and ASA affects the formation of lipoxin epimers resulting in the generation of 15 epi-lipoxin A4, also known as aspirin-triggered lipoxin (ATL) [101–103]. Lipoxin A4 (LXA4) has been demonstrated to upregulate IL-10 through the Notch signaling pathway in murine BV2 microglia cells  and stimulate IL-6 generation in human monocytes . Furthermore, stable 15-epi–LXA4 analogs display potent in vivo anti-inflammatory action and induce nitric oxide production for an anti-inflammatory effect [106, 107]. Thus, in addition to inhibiting PGs, ASA also triggers the formation of lipid mediators, which can be used as targets to elucidate the immunomodulatory properties of ASA.
In addition, we emphasized the distinct effects of PGE2 on cytokine secretion to modulate various steps during the inflammatory response, which originated not only from four EP receptors, but also from various levels of expression among different tissues, differences in sensitivity, the ability to activate multiple signaling pathways, and the inflammatory stimulus used. The present study indicates that PGE2 modulates immune response via regulation of cytokine signaling, as well as cytokine production, which in turn is partly responsible for the immunostimulatory effect of ASA. In summary, we established a simple and efficient assay using human WB to monitor the immunomodulatory effects of clinically relevant ASA doses in response to various TLR ligands. We demonstrated that therapeutically achieved plasma concentrations of ASA exert a boosting effect on cytokine production following stimulation with TLR ligands such as Pam3CsK4, LPS, Flagellin, and ssRNA40. Furthermore, our results indicate a potential role of PGE2 and COX-2 in mediating the immunostimulatory effects of ASA. While the immunomodulatory effect of peak plasma concentrations of ASA is clearly demonstrated, the numerous players including the dichotomous role of PGE2 in inflammation, turnover of COX enzymes in various cell types, and different signaling pathways upon TLR stimulation, requires further investigation in order to unravel the complex mechanisms behind the immunostimulatory properties of physiologically relevant ASA concentrations. While inhibiting COX with NSAIDs is conventionally regarded as an “anti-inflammatory” strategy, an alternative possibility is that NSAIDs prevent overproduction of immunosuppressive PGE2, which may represent an “immunostimulatory” strategy.
S1 Fig. Analysis of cellular viability and cellular composition in the WB assay.
Citrate-anticoagulated blood was treated with TLR ligands, 0.2 mg/ml ASA, 0.5 mg/ml IND, 0.5 mg/ml CCXB, 10 ng/ml PGE2 or vehicle (unstimulated). The absolute number of living cells (A), the viability of cells (B), and the cellular composition within live cells (C) were analyzed by flow cytometry. Data represent three independent experiments, each performed in triplicate. Bars indicate the mean ± SD.
S1 Table. Data of cytokine release by TLR ligands 1–9.
S2 Table. Inhibitory effect of Dexamethason on TLR-ligand induced cytokine release.
S3 Table. Immunostimulatory effect of acetylsalicylic acid on TLR-ligand induced cytokine release.
S4 Table. Effect of Indomethacin and Celecoxib on TLR-ligand induced cytokine release.
S5 Table. Inhibitory effect of acetylicsalicylic acid, Indomethacin, and Celecoxib on TLR-ligand induced PGE2 production.
S6 Table. Effect of exogenous addition of PGE2 on immunomodulatory properties of acetylsalicylic acid.
We thank all voluntary blood donors for providing blood samples at the Department of Transfusion Medicine and Hemostaseology, University Hospital Erlangen and all colleagues involved for supporting this study.
- 1. Hackstein H, Morelli AE, Larregina AT, Ganster RW, Papworth GD, Logar AJ, et al. Aspirin Inhibits In Vitro Maturation and In Vivo Immunostimulatory Function of Murine Myeloid Dendritic Cells. The Journal of Immunology. 2001;166(12):7053–62. pmid:11390449
- 2. Hackstein H, Thomson AW. Dendritic cells: emerging pharmacological targets of immunosuppressive drugs. Nature reviews Immunology. 2004;4(1):24–34. pmid:14704765
- 3. Lanas A, McCarthy D, Voelker M, Brueckner A, Senn S, Baron JA. Short-Term Acetylsalicylic Acid (Aspirin) Use for Pain, Fever, or Colds—Gastrointestinal Adverse Effects. Drugs in R & D. 2011;11(3):277–88.
- 4. Limmroth V, Katsarava Z, Diener H-C. Acetylsalicylic acid in the treatment of headache. Cephalalgia. 1999;19(6):545–51. pmid:10448540
- 5. Dornan J, Reynolds WJ. Comparison of ibuprofen and acetylsalicylic acid in the treatment of rheumatoid arthritis. Can Med Assoc J. 1974;110(12):1370–2. pmid:4599671
- 6. Smolen JS. Insights into the treatment of rheumatoid arthritis: A paradigm in medicine. Journal of Autoimmunity. 2020:102425. pmid:32143989
- 7. Dai Y, Ge J. Clinical use of aspirin in treatment and prevention of cardiovascular disease. Thrombosis. 2012;2012:245037–43. pmid:22195280
- 8. Hennekens CH, Dyken ML, Fuster V. Aspirin as a Therapeutic Agent in Cardiovascular Disease. Circulation. 1997;96(8):2751–3. pmid:9355934
- 9. Ishikawa H, Wakabayashi K, Suzuki S, Mutoh M, Hirata K, Nakamura T, et al. Preventive effects of low-dose aspirin on colorectal adenoma growth in patients with familial adenomatous polyposis: double-blind, randomized clinical trial. Cancer Med. 2013;2(1):50–6. pmid:24133627
- 10. Jacobs EJ, Newton CC, Gapstur SM, Thun MJ. Daily Aspirin Use and Cancer Mortality in a Large US Cohort. Journal of the National Cancer Institute. 2012;104(16):1208–17. pmid:22888140
- 11. Bardia A, Ebbert J, Vierkant R, Limburg P, Anderson K, Wang A, et al. Association of aspirin and non-aspirin NSAIDs with cancer incidence and mortality in a large prospective cohort study. Cancer Research. 2007;67(9):3400.
- 12. Vane JR. Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirin-like Drugs. Nature New Biology. 1971;231(25):232–5. pmid:5284360
- 13. Botting RM. Vane’s discovery of the mechanism of action of aspirin changed our understanding of its clinical pharmacology. Pharmacological Reports. 2010;62(3):518–25. pmid:20631416
- 14. Xie WL, Chipman JG, Robertson DL, Erikson RL, Simmons DL. Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci U S A. 1991;88(7):2692–6. pmid:1849272
- 15. William L. Smith , David L. DeWitt a, Garavito RM. Cyclooxygenases: Structural, Cellular, and Molecular Biology. Annual Review of Biochemistry. 2000;69(1):145–82.
- 16. Funk CD. Prostaglandins and Leukotrienes: Advances in Eicosanoid Biology. Science. 2001;294(5548):1871–5. pmid:11729303
- 17. Kopp E, Ghosh S. Inhibition of NF-κB by Sodium Salicylate and Aspirin. Science. 1994;265(5174):956–9. pmid:8052854
- 18. Taubert D, Berkels R, Grosser N, Schröder H, Gründemann D, Schömig E. Aspirin induces nitric oxide release from vascular endothelium: a novel mechanism of action. British journal of pharmacology. 2004;143(1):159–65. pmid:15289285
- 19. Morris T, Stables M, Hobbs A, de Souza P, Colville-Nash P, Warner T, et al. Effects of Low-Dose Aspirin on Acute Inflammatory Responses in Humans. The Journal of Immunology. 2009;183(3):2089–96. pmid:19597002
- 20. Downer MK, Allard CB, Preston MA, Wilson KM, Kenfield SA, Chan JM, et al. Aspirin Use and Lethal Prostate Cancer in the Health Professionals Follow-up Study. European Urology Oncology. 2019;2(2):126–34. pmid:31017087
- 21. Kehm RD, Hopper JL, John EM, Phillips K-A, MacInnis RJ, Dite GS, et al. Regular use of aspirin and other non-steroidal anti-inflammatory drugs and breast cancer risk for women at familial or genetic risk: a cohort study. Breast Cancer Research. 2019;21(1):52. pmid:30999962
- 22. Rodríguez-Miguel A, García-Rodríguez LA, Gil M, Montoya H, Rodríguez-Martín S, de Abajo FJ. Clopidogrel and Low-Dose Aspirin, Alone or Together, Reduce Risk of Colorectal Cancer. Clinical Gastroenterology and Hepatology. 2019;17(10):2024–33.e2. pmid:30580092
- 23. Schroecksnadel K, Winkler C, Wirleitner B, Schennach H, Fuchs D. Aspirin down-regulates tryptophan degradation in stimulated human peripheral blood mononuclear cells in vitro. Clinical & Experimental Immunology. 2005;140(1):41–5. pmid:15762873
- 24. Buckland M, Lombardi G. Aspirin and the induction of tolerance by dendritic cells. Handbook of experimental pharmacology. 2009(188):197–213. pmid:19031027
- 25. Härtel C, Puttkamer J, Gallner F, Strunk T, Schultz C. Dose-dependent Immunomodulatory Effects of Acetylsalicylic Acid and Indomethacin in Human Whole Blood: Potential Role of Cyclooxygenase-2 Inhibition. Scandinavian journal of immunology. 2004;60:412–20. pmid:15379866
- 26. Osnes LTN, Haug KBF, Joø GB, Westvik ÅB, Øvstebø R, Kierulf P. Aspirin Potentiates LPS-induced Fibrin Formation (FPA) and TNF-α-synthesis in Whole Blood. Thromb Haemost. 2000;83(06):868–73.
- 27. Nagelschmitz J, Blunck M, Kraetzschmar J, Ludwig M, Wensing G, Hohlfeld T. Pharmacokinetics and pharmacodynamics of acetylsalicylic acid after intravenous and oral administration to healthy volunteers. Clin Pharmacol. 2014;6:51–9. pmid:24672263
- 28. Schulz M, Iwersen-Bergmann S, Andresen H, Schmoldt A. Therapeutic and toxic blood concentrations of nearly 1,000 drugs and other xenobiotics. Crit Care. 2012;16(4):R136. pmid:22835221
- 29. Chang ZL. Important aspects of Toll-like receptors, ligands and their signaling pathways. Inflammation Research. 2010;59(10):791–808. pmid:20593217
- 30. Buchta CM, Bishop GA. Toll-like receptors and B cells: functions and mechanisms. Immunologic Research. 2014;59(1):12–22. pmid:24847763
- 31. Hood JD, Warshakoon HJ, Kimbrell MR, Shukla NM, Malladi SS, Wang X, et al. Immunoprofiling toll-like receptor ligands: Comparison of immunostimulatory and proinflammatory profiles in ex vivo human blood models. Human vaccines. 2010;6(4):322–35. pmid:20372068
- 32. Langezaal I, Coecke S, Hartung T. Whole blood cytokine response as a measure of immunotoxicity. Toxicology in Vitro. 2001;15(4):313–8. pmid:11566555
Muñoz-Carrillo JL, Contreras-Cordero J, Gutiérrez-Coronado O, Villalobos-Gutiérrez P, Ramos-Gracia L, Hernández-Reyes V. Cytokine Profiling Plays a Crucial Role in Activating Immune System to Clear Infectious Pathogens: IntechOpen: London, UK; 2018. 30 p. https://doi.org/10.1016/j.drudis.2018.01.023 pmid:29317340
- 34. Moynagh PN. Toll-like receptor signalling pathways as key targets for mediating the anti-inflammatory and immunosuppressive effects of glucocorticoids. Journal of Endocrinology. 2003;179(2):139–44. pmid:14596665
- 35. Creed TJ, Lee RW, Newcomb PV, di Mambro AJ, Raju M, Dayan CM. The Effects of Cytokines on Suppression of Lymphocyte Proliferation by Dexamethasone. The Journal of Immunology. 2009;183(1):164–71. pmid:19542427
- 36. Barnes PJ. Anti-inflammatory Actions of Glucocorticoids: Molecular Mechanisms. Clinical Science. 1998;94(6):557–72. pmid:9854452
- 37. Speer EM, Dowling DJ, Ozog LS, Xu J, Yang J, Kennady G, et al. Pentoxifylline inhibits TLR- and inflammasome-mediated in vitro inflammatory cytokine production in human blood with greater efficacy and potency in newborns. Pediatric Research. 2017;81:806. pmid:28072760
- 38. Flower RJ. Drugs Which Inhibit Prostaglandin Biosynthesis. Pharmacological Reviews. 1974;26(1):33–67. pmid:4208101
- 39. Wu KK. Aspirin and Salicylate. Circulation. 2000;102(17):2022–3. pmid:11044413
- 40. Yin M-J, Yamamoto Y, Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of IκB kinase-β. Nature. 1998;396(6706):77–80. pmid:9817203
- 41. FitzGerald GA, Patrono C. The Coxibs, Selective Inhibitors of Cyclooxygenase-2. New England Journal of Medicine. 2001;345(6):433–42.
- 42. Kalinski P. Regulation of immune responses by prostaglandin E2. Journal of immunology 2012;188(1):21–8. pmid:22187483
- 43. Reddy M, Eirikis E, Davis C, Davis HM, Prabhakar U. Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function. Journal of Immunological Methods. 2004;293(1):127–42.
- 44. Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdörfer B, Giese T, et al. Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol. 2002;168(9):4531–7. pmid:11970999
- 45. Barr TA, Brown S, Ryan G, Zhao J, Gray D. TLR-mediated stimulation of APC: Distinct cytokine responses of B cells and dendritic cells. European Journal of Immunology. 2007;37(11):3040–53. pmid:17918201
- 46. Satoh T, Akira S. Toll-Like Receptor Signaling and Its Inducible Proteins. Microbiology Spectrum. 2016;4(6). pmid:28084212
- 47. Franchimont D, Louis E, Dewe W, Martens H, Vrindts-Gevaert Y, De Groote D, et al. Effects of dexamethasone on the profile of cytokine secretion in human whole blood cell cultures. Regulatory Peptides. 1998;73(1):59–65. pmid:9537674
- 48. Smits HH, Grünberg K, Derijk RH, Sterk PJ, Hiemstra PS. Cytokine release and its modulation by dexamethasone in whole blood following exercise. Clin Exp Immunol. 1998;111(2):463–8. pmid:9486420
- 49. Endres S, Whitaker RE, Ghorbani R, Meydani SN, Dinarello CA. Oral aspirin and ibuprofen increase cytokine-induced synthesis of IL-1 beta and of tumour necrosis factor-alpha ex vivo. Immunology. 1996;87(2):264–70. pmid:8698389
- 50. Osterud B, Olsen JO, Wilsgård L. Increased lipopolysaccharide-induced tissue factor activity and tumour necrosis factor production in monocytes after intake of aspirin: possible role of prostaglandin E2. Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis. 1992;3(3):309–13. pmid:1643209
- 51. Berghöfer B, Frommer T, Haley G, Fink L, Bein G, Hackstein H. TLR7 Ligands Induce Higher IFN-α Production in Females. The Journal of Immunology. 2006;177(4):2088–96. pmid:16887967
- 52. Khan N, Summers CW, Helbert MR, Arkwright PD. Effects of age, gender, and immunosuppressive agents on in vivo toll-like receptor pathway responses. Human Immunology. 2010;71(4):372–6. pmid:20096740
- 53. Torcia MG, Nencioni L, Clemente AM, Civitelli L, Celestino I, Limongi D, et al. Sex Differences in the Response to Viral Infections: TLR8 and TLR9 Ligand Stimulation Induce Higher IL10 Production in Males. PLOS ONE. 2012;7(6):e39853. pmid:22768144
- 54. Sironi M, Gadina M, Kankova M, Riganti F, Mantovani A, Zandalasini M, et al. Differential sensitivity of in vivo TNF and IL-6 production to modulation by anti-inflammatory drugs in mice. International journal of immunopharmacology. 1992;14(6):1045–50. pmid:1428359
- 55. Phipps RP, Stein SH, Roper RL. A new view of prostaglandin E regulation of the immune response. Immunology Today. 1991;12(10):349–52. pmid:1958288
- 56. Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arteriosclerosis, thrombosis, and vascular biology. 2011;31(5):986–1000. pmid:21508345
- 57. Takayama K, García-Cardeña G, Sukhova GK, Comander J, Gimbrone MA, Libby P. Prostaglandin E2 Suppresses Chemokine Production in Human Macrophages through the EP4 Receptor. Journal of Biological Chemistry. 2002;277(46):44147–54. pmid:12215436
- 58. He W, Pelletier J-P, Martel-Pelletier J, Laufer S, Di Battista JA. Synthesis of interleukin 1beta, tumor necrosis factor-alpha, and interstitial collagenase (MMP-1) is eicosanoid dependent in human osteoarthritis synovial membrane explants: interactions with antiinflammatory cytokines. The Journal of Rheumatology. 2002;29(3):546–53. pmid:11908571
- 59. Hata AN, Breyer RM. Pharmacology and signaling of prostaglandin receptors: Multiple roles in inflammation and immune modulation. Pharmacology & Therapeutics. 2004;103(2):147–66. pmid:15369681
- 60. Bour AMJJ, Westendorp RGJ, Laterveer JC, Bollen ELEM, Remarque EJ. Interaction of indomethacin with cytokine production in whole blood. Potential mechanism for a brain-protective effect. Experimental Gerontology. 2000;35(8):1017–24. pmid:11121687
- 61. Koeberle A, Northoff H, Werz O. Curcumin blocks prostaglandin E2 biosynthesis through direct inhibition of the microsomal prostaglandin E2 synthase-1. Molecular cancer therapeutics. 2009;8(8):2348–55. pmid:19671757
- 62. Spengler RN, Spengler ML, Strieter RM, Remick DG, Larrick JW, Kunkel SL. Modulation of tumor necrosis factor-alpha gene expression. Desensitization of prostaglandin E2-induced suppression. J Immunol. 1989;142(12):4346–50. pmid:2723432
- 63. Agard M, Asakrah S, Morici L. PGE2 suppression of innate immunity during mucosal bacterial infection. Frontiers in Cellular and Infection Microbiology. 2013;3(45). pmid:23971009
- 64. Rodríguez M, Domingo E, Municio C, Alvarez Y, Hugo E, Fernández N, et al. Polarization of the Innate Immune Response by Prostaglandin E2: A Puzzle of Receptors and Signals. Molecular Pharmacology. 2014;85(1):187–97. pmid:24170779
- 65. Harris SG, Padilla J, Koumas L, Ray D, Phipps RP. Prostaglandins as modulators of immunity. Trends in Immunology. 2002;23(3):144–50. pmid:11864843
- 66. Hilkens CM, Snijders A, Snijdewint FG, Wierenga EA, Kapsenberg ML. Modulation of T-cell cytokine secretion by accessory cell-derived products. The European respiratory journal Supplement. 1996;22:90s–4s.
Kaliński P, Hilkens CMU, Snijders A, Snijdewint FGM, Kapsenberg ML. Dendritic Cells, Obtained from Peripheral Blood Precursors in the Presence of PGE2, Promote Th2 Responses. In: Ricciardi-Castagnoli P, editor. Dendritic Cells in Fundamental and Clinical Immunology: Volume 3. Boston, MA: Springer US; 1997. p. 363–7.
- 68. Kaliński P, Schuitemaker JH, Hilkens CM, Kapsenberg ML. Prostaglandin E2 induces the final maturation of IL-12-deficient CD1a+CD83+ dendritic cells: the levels of IL-12 are determined during the final dendritic cell maturation and are resistant to further modulation. J Immunol. 1998;161(6):2804–9. pmid:9743339
- 69. Fabricius D, Neubauer M, Mandel B, Schütz C, Viardot A, Vollmer A, et al. Prostaglandin E2 Inhibits IFN-α Secretion and Th1 Costimulation by Human Plasmacytoid Dendritic Cells via E-Prostanoid 2 and E-Prostanoid 4 Receptor Engagement. The Journal of Immunology. 2010;184(2):677–84. pmid:20018632
- 70. Mailliard RB, Alber SM, Shen H, Watkins SC, Kirkwood JM, Herberman RB, et al. IL-18-induced CD83+CCR7+ NK helper cells. The Journal of experimental medicine. 2005;202(7):941–53. pmid:16203865
- 71. Aloisi F, Simone RD, Columba-Cabezas S, Levi G. Opposite effects of interferon-γ and prostaglandin E2 on tumor necrosis factor and interleukin-10 production in microglia: A regulatory loop controlling microglia pro- and anti-inflammatory activities. Journal of Neuroscience Research. 1999;56(6):571–80. pmid:10374812
- 72. Wall EA, Zavzavadjian JR, Chang MS, Randhawa B, Zhu X, Hsueh RC, et al. Suppression of LPS-Induced TNF-α Production in Macrophages by cAMP Is Mediated by PKA-AKAP95-p105. Science Signaling. 2009;2(75):ra28–ra. pmid:19531803
- 73. Yao C, Hirata T, Soontrapa K, Ma X, Takemori H, Narumiya S. Prostaglandin E₂ promotes Th1 differentiation via synergistic amplification of IL-12 signalling by cAMP and PI3-kinase. Nat Commun. 2013;4:1685. pmid:23575689
- 74. Maseda D, Johnson EM, Nyhoff LE, Baron B, Kojima F, Wilhelm AJ, et al. mPGES1-Dependent Prostaglandin E2 (PGE2) Controls Antigen-Specific Th17 and Th1 Responses by Regulating T Autocrine and Paracrine PGE2 Production. The Journal of Immunology. 2018;200(2):725–36. pmid:29237778
- 75. Koga K, Takaesu G, Yoshida R, Nakaya M, Kobayashi T, Kinjyo I, et al. Cyclic Adenosine Monophosphate Suppresses the Transcription of Proinflammatory Cytokines via the Phosphorylated c-Fos Protein. Immunity. 2009;30(3):372–83. pmid:19285436
- 76. Mortimer L, Moreau F, MacDonald JA, Chadee K. NLRP3 inflammasome inhibition is disrupted in a group of auto-inflammatory disease CAPS mutations. Nat Immunol. 2016;17(10):1176–86. pmid:27548431
- 77. Sokolowska M, Chen LY, Liu Y, Martinez-Anton A, Qi HY, Logun C, et al. Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages. J Immunol. 2015;194(11):5472–87. pmid:25917098
- 78. Rawat P, Teodorof-Diedrich C, Spector SA. Human immunodeficiency virus Type-1 single-stranded RNA activates the NLRP3 inflammasome and impairs autophagic clearance of damaged mitochondria in human microglia. Glia. 2019;67(5):802–24. pmid:30582668
- 79. Allen IC, Scull MA, Moore CB, Holl EK, McElvania-TeKippe E, Taxman DJ, et al. The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA. Immunity. 2009;30(4):556–65. pmid:19362020
- 80. Renz H, Gong JH, Schmidt A, Nain M, Gemsa D. Release of tumor necrosis factor-alpha from macrophages. Enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides. The Journal of Immunology. 1988;141(7):2388–93. pmid:2844899
- 81. Tang T, Scambler TE, Smallie T, Cunliffe HE, Ross EA, Rosner DR, et al. Macrophage responses to lipopolysaccharide are modulated by a feedback loop involving prostaglandin E2, dual specificity phosphatase 1 and tristetraprolin. Scientific Reports. 2017;7(1):4350. pmid:28659609
- 82. Hinson RM, Williams JA, Shacter E. Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: possible role of cyclooxygenase-2. Proc Natl Acad Sci U S A. 1996;93(10):4885–90. pmid:8643498
- 83. Williams JA, Pontzer CH, Shacter E. Regulation of Macrophage Interleukin-6 (IL-6) and IL-10 Expression by Prostaglandin E2: The Role of p38 Mitogen-Activated Protein Kinase. Journal of Interferon & Cytokine Research. 2000;20(3):291–8.
- 84. Kim S-H, Serezani CH, Okunishi K, Zaslona Z, Aronoff DM, Peters-Golden M. Distinct Protein Kinase A Anchoring Proteins Direct Prostaglandin E2 Modulation of Toll-like Receptor Signaling in Alveolar Macrophages*. Journal of Biological Chemistry. 2011;286(11):8875–83. pmid:21247892
- 85. Eigler A, Siegmund B, Emmerich U, Baumann KH, Hartmann G, Endres S. Anti-inflammatory activities of cAMP-elevating agents: enhancement of IL-10 synthesis and concurrent suppression of TNF production. Journal of Leukocyte Biology. 1998;63(1):101–7. pmid:9469479
- 86. MacKenzie KF, Clark K, Naqvi S, McGuire VA, Nöehren G, Kristariyanto Y, et al. PGE2 Induces Macrophage IL-10 Production and a Regulatory-like Phenotype via a Protein Kinase A–SIK–CRTC3 Pathway. The Journal of Immunology. 2012:1202462.
- 87. Demeure CE, Yang LP, Desjardins C, Raynauld P, Delespesse G. Prostaglandin E2 primes naive T cells for the production of anti-inflammatory cytokines. Eur J Immunol. 1997;27(12):3526–31. pmid:9464843
- 88. Harizi H, Juzan M, Pitard V, Moreau JF, Gualde N. Cyclooxygenase-2-issued prostaglandin e(2) enhances the production of endogenous IL-10, which down-regulates dendritic cell functions. J Immunol. 2002;168(5):2255–63. pmid:11859113
- 89. Józefowski S, Bobek M, Marcinkiewicz J. Exogenous but not endogenous prostanoids regulate cytokine secretion from murine bone marrow dendritic cells: EP2, DP, and IP but not EP1, EP3, and FP prostanoid receptors are involved. International Immunopharmacology. 2003;3(6):865–78. pmid:12781703
- 90. Cheon H, Rho YH, Choi SJ, Lee YH, Song GG, Sohn J, et al. Prostaglandin E2 Augments IL-10 Signaling and Function. The Journal of Immunology. 2006;177(2):1092–100. pmid:16818766
- 91. Linke B, Schreiber Y, Picard-Willems B, Slattery P, Nüsing RM, Harder S, et al. Activated Platelets Induce an Anti-Inflammatory Response of Monocytes/Macrophages through Cross-Regulation of PGE2 and Cytokines. Mediators Inflamm. 2017;2017:1463216. pmid:28592915
- 92. Clark K, MacKenzie KF, Petkevicius K, Kristariyanto Y, Zhang J, Choi HG, et al. Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages. Proceedings of the National Academy of Sciences. 2012;109(42):16986–91. pmid:23033494
- 93. Platzer C, Meisel C, Vogt K, Platzer M, Volk H-D. Up-regulation of monocytic IL-10 by tumor necrosis factor-α and cAMP elevating drugs. International Immunology. 1995;7(4):517–23. pmid:7547677
- 94. Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol. 2014;5:461-. pmid:24478774
- 95. El-Zayat SR, Sibaii H, Mannaa FA. Toll-like receptors activation, signaling, and targeting: an overview. Bulletin of the National Research Centre. 2019;43(1):187.
- 96. Leifer CA, Medvedev AE. Molecular mechanisms of regulation of Toll-like receptor signaling. Journal of Leukocyte Biology. 2016;100(5):927–41. pmid:27343013
- 97. Salvi V, Vaira X, Gianello V, Vermi W, Bugatti M, Sozzani S, et al. TLR Signalling Pathways Diverge in Their Ability to Induce PGE2. Mediators Inflamm. 2016;2016:10. pmid:27630451
- 98. Teixeira-Coelho M, Guedes J, Ferreirinha P, Howes A, Pedrosa J, Rodrigues F, et al. Differential post-transcriptional regulation of IL-10 by TLR2 and TLR4-activated macrophages. Eur J Immunol. 2014;44(3):856–66. pmid:24227629
- 99. van Riet E, Everts B, Retra K, Phylipsen M, van Hellemond JJ, Tielens AGM, et al. Combined TLR2 and TLR4 ligation in the context of bacterial or helminth extracts in human monocyte derived dendritic cells: molecular correlates for Th1/Th2 polarization. BMC Immunol. 2009;10(1):9. pmid:19193240
- 100. Bujang MA, Baharum N. Sample Size Guideline for Correlation Analysis. World Journal of Social Science Research. 2016;3:37.
- 101. Chandrasekharan JA, Sharma-Walia N. Lipoxins: nature’s way to resolve inflammation. J Inflamm Res. 2015;8:181–92. pmid:26457057
- 102. Clària J, Serhan CN. Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions. Proc Natl Acad Sci U S A. 1995;92(21):9475–9. pmid:7568157
- 103. Chiang N, Hurwitz S, Ridker PM, Serhan CN. Aspirin has a gender-dependent impact on antiinflammatory 15-epi-lipoxin A4 formation: a randomized human trial. Arterioscler Thromb Vasc Biol. 2006;26(2):e14–7. pmid:16293793
- 104. Wu J, Ding D-h, Li Q-q, Wang X-y, Sun Y-y, Li L-J. Lipoxin A4 Regulates Lipopolysaccharide-Induced BV2 Microglial Activation and Differentiation via the Notch Signaling Pathway. Frontiers in Cellular Neuroscience. 2019;13(19). pmid:30778288
- 105. Andersson P, Serhan CN, Petasis NA, Palmblad J. Interactions between lipoxin A4, the stable analogue 16-phenoxy-lipoxin A4 and leukotriene B4 in cytokine generation by human monocytes. Scand J Immunol. 2004;60(3):249–56. pmid:15320881
- 106. Serhan CN. Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution. Prostaglandins, Leukotrienes and Essential Fatty Acids. 2005;73(3):141–62. pmid:16005201
- 107. Paul-Clark MJ, van Cao T, Moradi-Bidhendi N, Cooper D, Gilroy DW. 15-epi-lipoxin A4–mediated Induction of Nitric Oxide Explains How Aspirin Inhibits Acute Inflammation. Journal of Experimental Medicine. 2004;200(1):69–78. pmid:15238606