Prostaglandin E2 Regulation of Macrophage Innate Immunity

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Prostaglandin E2 Regulation of Macrophage Innate Immunity Danielle Watkins Kimmel, Lisa M Rogers, David M Aronoff, and David E Cliffel Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00322 • Publication Date (Web): 10 Dec 2015 Downloaded from http://pubs.acs.org on December 22, 2015

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Chemical Research in Toxicology

Prostaglandin E2 Regulation of Macrophage Innate Immunity Danielle W. Kimmel,1 Lisa M. Rogers,2 David M. Aronoff,2 David E. Cliffel1,3,* 1. Department of Chemistry, Vanderbilt University, Nashville, TN 37235, United States 2. Department of Medicine, Division of Infectious Diseases, Vanderbilt University, Nashville, TN 37232, United States 3. Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN 37235, United States * Corresponding author at: Department of Chemistry, Vanderbilt University, VU Station B, Nashville, TN 37235-1822, United States, Fax: +1-615-343-1234, E-mail address: [email protected]

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Abstract Globally, maternal and fetal health is greatly impacted by extraplacental inflammation. Group B streptococcus (GBS), a leading cause of chorioamnionitis, is thought to take advantage of the uterine environment during pregnancy in order to cause inflammation and infection. In this study, we demonstrate the metabolic changes of murine macrophages caused by GBS exposure. GBS alone prompted a delayed increase in lactate production, highlighting its ability to redirect macrophage metabolism from aerobic to anaerobic respiration.

This production of lactate is thought to aid in the development and

propagation of GBS throughout the surrounding tissue. Additionally, this study shows that PGE2 priming was able to exacerbate lactate production, shown by the rapid and substantial lactate increases seen upon GBS exposure. These data provide a novel model to study the role of GBS exposure to macrophages with and without PGE2 priming.

Keywords Microphysiometry, group B streptococcus, pregnancy, chorioamnionitis, Prostaglandin E2

Introduction Annually, the global occurrence of inflammation of the extraplacental membranes during pregnancy (chorioamnionitis) contributes to nearly 6 million premature births, 1.6 million stillbirths, and 1.2 million neonatal sepsis episodes, creating an enormous unmet need for safe, affordable, and effective preventive approaches.1–5 Chorioamnionitis is commonly caused by bacteria that ascend from the vagina through the cervix, and into the fetal membranes that surround the amniotic fluid-bathed fetus. A major barrier to

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developing new solutions is a gap in the understanding of how bacterial pathogens invade innate immune defenses as they cross fetal membranes to cause chorioamnionitis. In the developing world, Group B Streptococcus (GBS, S. agalactiae) is a leading cause of chorioamnionitis.6 GBS is a gram-positive bacterium that colonizes healthy women asymptomatically, but can lead to pneumonia, septicemia, and meningitis in neonates and immunocompromised individuals.7–10 Pregnancy is immunologically unique because the maternal immune system must develop tolerance to the semi-allogeneic fetus. Also, the inflammatory cascades involved in active labor must be suppressed until term. A central unanswered question in maternal immunology is whether there is a cost to this immunomodulation. Studies suggest that the immune reprogramming during pregnancy increases the risk of infection and that some microbes have evolved mechanisms to exploit this altered niche.11 To reduce the burden of chorioamnionitis, it is imperative to discover potential therapeutic targets of maternal immunity that could be strengthened without compromising reproductive health. Prostaglandin (PG)E2 is a cyclooxygenase (COX)-derived lipid mediator and potent endogenous regulator of innate immunity.12 It binds to four distinct G protein-coupled Eprostanoid (EP) receptors, numbered EP1-4. PGE2 production is enhanced at sites of infection,

where

it

promotes

inflammation

through

endothelial

cell-mediated

vasodilatation and support of Th17 adaptive immune responses.13 However, PGE2 has potent anti-inflammatory and immunosuppressive properties, including the direct inhibition of neutrophil, macrophage, and epithelial cell host defense functions.14 These inhibitory effects of PGE2 primarily result from cAMP-dependent signaling processes, triggered by EP2 and/or EP4 activation.14

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The capacity for PGE2 to limit host inflammatory responses likely evolved to prevent inflammatory tissue damage and promote the resolution of inflammation.15–17 Notably, some microorganisms appear to use PGE2 immunomodulation to their advantage, stimulating its generation to blunt immune defense.18–23 In addition, several clinical conditions associated with an enhanced susceptibility to infection are characterized by exaggerated PGE2 production, including cancer,24,25 smoking,26 aging,25,27 HIV infection,28,29 malnutrition,30 solid organ/bone marrow transplantation31,32 and pregnancy.33,34 These facts have prompted interest in targeting PGE2 synthesis and signaling in the treatment of certain infectious diseases.12,35–38 Prostaglandins, including PGE2, have many important roles in reproduction.39 Throughout gestation, PGE2 dampens maternal immune responses against fetal tissues to contribute to maternofetal tolerance.40–43 At term, systemic and local PGE2 levels increase dramatically44,45 to induce cervical softening and uterine smooth muscle contraction that aids in delivery.46 The high concentrations of PGE2 in the gravid uterus, coupled with further stimulation of PGE2 synthesis by microbial stimulation, could create a highly immunosuppressed local environment and one that might be amenable to pharmacological targeting. Recently, it was demonstrated that PGE2 exacerbated group A Streptococcus infection in both a murine intrauterine infection model and human THP-1 macrophage-like cell experiments, allowing for an increase in bacterial dissemination.47 In addition, a stable PGE2 analogue delivered into the maternal cervix postpartum in cows increased the incidence of puerperal endometritis48 and PGE2 injections facilitated the establishment of chlamydial uterine infections in mice.18 Thus, we speculate that high PGE2 levels in the female reproductive tract during pregnancy might increase susceptibility to infection.

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These data implicate PGE2 overproduction as a causal determinant of invasive bacterial infections. However, this has not been studied in the context of chorioamnionitis, which is surprising since pregnancy is characterized by high uterine concentrations of PGE2. This work focuses on the central hypothesis that PGE2 is a critical endogenous suppressor of innate immunity within fetal membranes that is exploited by bacteria that cause chorioamnionitis. The multianalyte microphysiometer (MAMP) was utilized in these studies to investigate the role of PGE2 as a modulator of macrophage metabolic responses to infection with GBS. This instrumentation allows for simultaneous, dynamic sensing of glucose and oxygen consumption, lactate production, and extracellular acidification. Previous work with the MAMP has provided insight into atherogenic development,49 neuronal conditioning,50,51 infectious disease onset,52,53 and metabolic changes induced by toxin exposure.54 The objective of the present studies was to utilize the MAMP to describe alterations in macrophage metabolism in response to GBS infection and define the extent to which PGE2 modifies such responses.

Materials and Methods Reagents and Instrumentation. All materials were used as obtained unless otherwise noted. Lyophilized alamethicin was purchased from A.G. Scientific, Inc (San Diego, CA). RAW 264.7 cells and their culture media, DMEM, were purchased through American Type Culture Collection and cultured according to their guidelines (TIB-71, Manassas, VA). Glucose oxidase (GOx, Type IIS from Aspergillus niger), bovine serum albumin (BSA, fraction V, 96%), sodium pyruvate, and glutaraldehyde (glutaric dialdehyde, 25 wt%

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solution in water) were purchased from Sigma (St. Louis, MO). Lactate oxidase (LOx, stabilized) was purchased from Applied Enzyme Technology (Pontypool, UK). Experimental culture media and media supplements were obtained from the Media Core at Vanderbilt University (Nashville, TN). Gentamicin was from Life Technologies (Carlsbad, CA) and fetal bovine serum was from Hyclone (Logan, UT). Tryptic soy blood agar plates were purchased from BD Biosciences (San Jose, CA). Cytosensor® consumables were purchased from Molecular Devices Corporation (Sunnyvale, CA). PGE2 was purchased through Cayman Chemical (Ann Arbor, MI). Bacterial growth. GBS (a vaginal colonizing strain known as GB590, multilocus sequence type (ST)-19, capsular serotype (cps) III) was grown to stationary phase in an aerobic atmosphere at 37°C overnight. GBS was pelleted, washed with PBS, and serially diluted onto blood agar plates to confirm CFU/mL (colony forming units per mL). A multiple of infection (MOI) of 150:1 or 250:1 was used for all RAW cell experiments presented here. MAMP Experiments. Experiments were performed using a modified Cytosenor® Microphysiometer, previously detailed by our group.49,52–60 Briefly, a 0.1 mL/min flow rate and a flow cycle of 80 sec flow followed by 40 sec stop flow enabled the build up or depletion of analytes in the 3 μL chamber for improved detection. During the course of the experiment, extracellular acidification was obtained through use of a light addressable potentiometric sensor over the duration of each experiment.61–63 Simultaneous measurements of glucose and oxygen consumption, and lactate production were obtained through use of amperometric measurements via a Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) potentiostat and hand-cast glucose and

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lactate enzyme films, which provided measurable formation of H2O2 (+0.6 V vs. Ag/AgCl; 2 M KCl). Potentiometric measurements of the extracellular acidification were calculated using the Cytosoft program.61,62 Potentiometric measurements were calculated based on the difference in current during steady state flow and end of stop-flow. Post cell death, lactate and glucose calibrations were performed using the following concentrations of glucose and lactate, respectively: 0 mM and 0 mM, 1 mM and 0.05 mM, 3 mM and 0.1 mM, 5 mM and 0.2 mM. Oxygen calibration occurred based on literature values for dissolved oxygen.64 These calibrated values were then normalized against basal values obtained 10 min prior to the initial compound exposure of either GBS or PGE2, depending on the experiment. MAMP Experimental Protocol. RAW 264.7 cells were plated in cell cups at 2.5 x 105 cells per insert prior to each experiment. For the studies measuring the interaction of RAW 264.7 cells with GBS alone, basal metabolic rates were obtained for 60 min to allow for cellular and instrumentation equilibration, followed by a 16 min GBS exposure. After exposure, the cells were allowed a 60 min recovery time. Following recovery, cells were killed using alamethicin and calibrations were performed. For the studies looking at the interaction between RAW 264.7 cells, GBS, and PGE2, basal metabolic rates were obtained for 60 min prior to a 16 min exposure of 1 uM PGE2. This exposure time was chosen, based on previous work showing high cAMP levels 15 min post PGE2 treatment.65 The cells were allowed to recover for 16 min before being exposed to the same parameters of GBS exposure mentioned before. After GBS exposure, recovery time, alamethicin exposure, and calibrations took place as mentioned previously. MAMP data are

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presented as normalized responses (maximum peak height changes from basal metabolic rates). These data are presented as mean ± SEM. Additional statistical information is provided in the supplemental information.

Results and Discussion Macrophage Metabolic Response to GBS Exposure. Previous studies have shown a significant change in metabolic activity when murine macrophages (RAW 264.7) are exposed to lipopolysaccharide (LPS), an endotoxin found in gram-negative bacteria.53 To ensure a reliable and unique biosignature was observable using GBS instead of LPS, the metabolic responses of macrophages challenged with GBS was measured. Macrophages were exposed to live GBS for 16 min and dynamic plots are shown. Shortly after the exposure, GBS challenge had reversible, but marked responses in all measured analytes of macrophage metabolism (Figure 1). Most notably, there was a substantial increase in lactate production during GBS exposure. We did not see this effect using heat-killed GBS (data not shown). This is indicative of a metabolic switch from basal function to dependence on anaerobic respiration. Typically when exposed to an inflammatory inducing stressor, such as LPS, there is a rapid activation of the macrophage, resulting in oxidative burst to phagocytose and effectively destroy the stressor.53 The lack of metabolic hallmarks associated with oxidative burst and rapid increases in all measured analytes, suggests a mechanism by which GBS is able to advantageously direct macrophage metabolism toward anaerobic respiration in order to maximize colonization.66 As previously reported by Kling et al., GBS strains can utilize lactic acid production as a

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virulence factor to promote tissue necrosis, enhance bacterial adhesion, and alter the function of immunomodulating proteins.67 During exposure time, maximum peak height increases were compared against the basal metabolic rate as detailed in Table 1. We were able to confirm that GBS results in an induction of lactate production using a static in vitro culture method and a commercially available L-lactate ELISA kit (p < 0.05; data not shown). Additionally, the glucose consumption data becomes much more uncertain upon exposure in comparison to the pre-exposure data, suggesting a large variability in glucose uptake.

PGE2 Alters RAW 264.7 Metabolic Responses to GBS Exposure. Upregulation of uterine prostaglandins during pregnancy is thought to provide a unique environment that aids in the proliferation of infectious agents, however the mechanism by which this takes place is poorly understood.1,11,39,68 To better investigate how macrophages react to live GBS exposure when primed with PGE2, a dynamic metabolic biosignature was measured using the MAMP. Macrophages were exposed to PGE2 for 16 min, prior to GBS challenge. During this priming, cellular metabolism had no remarkable changes, indicating that the presence of PGE2 alone did not significantly control or contribute to macrophage metabolism (Figure 2). The cells were allowed a 16 min recovery time before being challenged with GBS for an additional 16 min. During the GBS exposure, macrophage metabolism followed the same trend as before, however when primed with PGE2 each response was exacerbated and more rapid than with GBS alone (Table 1). We did not see this effect using heat-killed GBS, data compared in Table 1. As previously reported, lactic acid production enhances adhesion of GBS in anaerobic conditions.67 This dynamic data suggests that PGE2,

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substantially amplifies the lactate production associated with GBS exposure in order to aid colonization and survival of the bacterium. During gestation, PGE2 supports maternofetal immune tolerance, whereas at term, PGE2 concentrations increase acutely because it regulates cervical softening and uterine contractions,11,40,45,46 and our data suggest PGE2 inhibits macrophage activation for oxidative burst, allowing colonization and survival of bacterial infection.

Conclusion Globally, bacterial infections during pregnancy contribute to substantial maternal and fetal complications.1–5 GBS is known to illicit chorioamnionitis, but little is understood about how the bacterium is able to advantageously proliferate in the gravid uterus. Throughout gestation, PGE2 dampens maternal immune responses against fetal tissues.40– 43,68

At term, systemic and local PGE2 levels increase dramatically44,45 to induce cervical

softening and uterine smooth muscle contraction that aids in delivery.46 These studies focused on identifying the relationship between high levels of PGE2 and the macrophage metabolic response toward GBS exposure. When perfused onto RAW 264.7 cells, live GBS prompted marked increases in lactate production and decreases in extracellular acidification. During typical macrophage oxidative burst, all measured analytes increase rapidly.49,53 The measured biosignature of GBS exposure indicates that macrophages are unable to successfully incite oxidative burst during a GBS challenge. Instead, there is a slight delay in the measured metabolic response, resulting in a shift from aerobic to anaerobic respiration. This shift allows for production of

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lactate, which eventually aids in the proliferation of GBS infection.67 The static culture ELISA data confirmed the substantial increases of lactate production upon GBS exposure. In our dynamic model, PGE2 primed macrophages to rapidly increase lactate production. The measured increase in primed macrophages occurred immediately upon GBS exposure, unlike the slight delay seen with GBS exposure without PGE2 priming. This suggests that the high concentrations of PGE2 seen in the gravid uterus might create an environment capable of allowing GBS to promote anaerobic respiration from the macrophage. These data provide evidence of GBS infection enhancement by a physiological PGE2 environment, suggesting a mechanism by which GBS advantageously thrives during pregnancy. Future studies should focus on a variety of GBS strains, to better understand how the differences in each strain of GBS contribute to infection. This information will enable better diagnostics and therapeutics to ensure rapid detection and resolution of a potentially life threatening GBS infection. Additionally, this model should be replicated with primary macrophages to fully correlate to a clinical setting. Eventually, primary macrophages will be utilized in a lab-on-a-chip device to simplify dynamic measurements.

Supporting Information Available Table of p-values for simplified statistical testing of RAW 264.7 macrophage metabolic response comparisons. This material is available free of charge via the Internet at http://pubs.acs.org.

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Funding Information This research was funded in part by the Defense Threat Reduction Agency (DRTA) HDTRA 1-09-0013 and the Global Alliance to Prevent Prematurity and Stillbirth (GAPPS). We also would like to thank Shellie Richards for editorial assistance.

Abbreviations Group B streptococcus – GBS Prostaglandin E2 – PGE2 Cyclooxygenase – COX E-prostanoid – EP Multianalyte microphysiometer – MAMP Glucose oxidase – GOx Bovine serum albumin – BSA Lactate oxidase – LOx Multiple of infection – MOI Vanderbilt Institute for Integrative Biosystems Research and Education – VIIBRE Lipopolysaccharide – LPS

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Table Live GBS Exposure

Glucose (%) Lactate (%) -27 ± 18 76 ± 55

Oxygen (%) -15 ± 4

Acidification (%) -37 ± 6

During Exposure Baseline 0.24 2.4 1.5 4.6 Uncertainty Dead GBS During 15 ± 5 18 ± 6 22 ± 8 13 ± 3 Exposure Exposure Baseline 3.3 2.1 3.4 0.8 Uncertainty Live GBS During 34 ± 13 540 ± 290 -42 ± 11 -62 ± 8 Exposure Exposure after PGE2 Baseline 3.7 1.4 3.7 3.7 Priming Uncertainty Table 1. RAW 264.7 macrophage metabolic response comparisons. The mean peak height change during exposure, as well as the standard errors, are reported for each analyte measured. The insignificance seen in lactate is due to biological variation in the samples upon exposure to GBS. While the exact values are not be statistically significant, the overall trend observed is indicative of a metabolic change during macrophage exposure to live GBS, indicating importance.

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Figures

Figure 1. Live GBS exposure to RAW 264.7 macrophages. The black bar along the x-axis indicates GBS exposure time (16 min). Glucose (black) and oxygen (blue) consumption, lactate production (green), and extracellular acidification (red) are shown. Decreasing metabolic analytes suggest an inhibition of oxidative burst induction, allowing for bacterial survival. Lactate production increased during GBS exposure (MOI = 250:1), suggesting a mechanism by which this strain directs macrophage metabolism to anaerobic respiration.

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Figure 2. RAW 264.7 macrophage metabolic response to priming with PGE2. Red bar along x-axis is indicating PGE2 exposure (16 min) and subsequent live GBS exposure (16 min; indicated by black bar along x-axis; MOI = 150:1). Glucose (black) and oxygen (blue) consumption, lactate production (green), and extracellular acidification (red) are shown. Rapid and significant increases of lactate production suggest PGE2 aids in GBS survival by forcing aerobic respiration and increasing lactate production while simultaneously decreasing oxygen consumption, effectively prohibiting oxidative burst.

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figure1 254x190mm (72 x 72 DPI)



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figure 2 254x190mm (72 x 72 DPI)


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Prostaglandin E2 Regulation of Macrophage Innate Immunity

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