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Differential Modulation of TLR3- and TLR4-Mediated Dendritic Cell Maturation and Function by

This information is current as Leigh A. Jones, Shrook Kreem, Muhannad Shweash, of September 23, 2021. Andrew Paul, James Alexander and Craig W. Roberts J Immunol 2010; 185:4525-4534; Prepublished online 15 September 2010; doi: 10.4049/jimmunol.0901155

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Supplementary http://www.jimmunol.org/content/suppl/2010/09/13/jimmunol.090115 Material 5.DC1 http://www.jimmunol.org/ References This article cites 56 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/185/8/4525.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Modulation of TLR3- and TLR4-Mediated Dendritic Cell Maturation and Function by Progesterone

Leigh A. Jones, Shrook Kreem, Muhannad Shweash, Andrew Paul, James Alexander, and Craig W. Roberts

The role of progesterone in modulating dendritic cell (DC) function following stimulation of different TLRs is relatively unknown. We compared the ability of progesterone to modulate murine bone marrow-derived DC cytokine production (IL-6 and IL-12) and costimulatory expression (CD40, CD80, and CD86) induced by either TLR3 or TLR4 ligation and determined whether activity was via the progesterone receptor (PR) or receptor (GR) by comparative studies with the PR-specific norgestrel and the GR agonist . Progesterone was found to downregulate, albeit with different sensitivities, both TLR3- and TLR4-induced IL-6 production entirely via the GR, but IL-12p40 production via either the GR or PR. Of particular significance was that progesterone was able to significantly inhibit TLR3- but not TLR4-induced CD40 expression in bone marrow- Downloaded from derived DCs. Stimulation of the PR (with progesterone and norgestrel) by pretreatment of DCs was found to sustain IFN regulatory factor-3 phosphorylation following TLR3 ligation, but not TLR4 ligation. Overall, these studies demonstrate that progesterone can differentially regulate the signaling pathways employed by TLR3 and TLR4 to affect costimulatory molecule expression and cytokine production. The Journal of Immunology, 2010, 185: 4525–4534.

endritic cells (DCs) play a pivotal role in immunity by IL-10, and reduce secretion of inflammatory cytokines, such as http://www.jimmunol.org/ linking the innate and adaptive immune systems and are IL-12 (10, 11). The microenvironment created during pregnancy D vital in initiating protective responses to a number of differs from the normal physiological situation, particularly with pathogens (1–3). Ligation of TLRs with pathogen-associated mo- regard to circulating hormones, and levels of , lecular patterns induces maturation of DCs characterized by in- , and progesterone are all increased. This change in the creased surface expression of CD40, CD80, and CD86 accom- hormonal milieu is thought to play a major role in skewing the panied by the production of a number of cytokines. Following maternal immune response to a Th2 and T regulatory phenotype maturation and Ag uptake, DCs migrate to the lymphoid tissues (12, 13), influenced in part by actions of hormones on a number of

to interact with B and T cells and initiate the development of hematopoietic cells including DCs (14, 15). by guest on September 23, 2021 the adaptive response (1, 4). Depending on the cytokine milieu Glucocorticoids have been reported to influence multiple aspects created by the innate response to a particular pathogen, DCs can of DC function from initial development to T cell stimulation. orchestrate the T cell response to either a Th1 or Th2 phenotype Consequently, glucocorticoids have been shown to limit the ca- (1, 5). pacity of monocytes to develop into DCs, reduce Ag uptake, up- The induction of a Th2 response has been demonstrated to occur regulate adhesion molecule expression, downregulate maturation in the vicinity of the placenta during mammalian pregnancy, and markers, such as CD40, CD80, CD86, and MHC class II, inhibit disruption of this has been associated with abortion (6, 7). In production of proinflammatory cytokines including IL-1a, IL-1b, addition, during pregnancy, T regulatory cells specific for fetal IL-6, IL-12p70, IFN-g, and TNF-a, reduce Ag presentation via the allograft Ags are involved in expression of tolerance to the fetus MHC class II pathway, and downmodulate T cell stimulatory (8, 9). DCs have been demonstrated around the fetomaternal in- capacity (16–22). terface, and studies have shown that in this situation, their function Given the paramount importance of DCs in linking, as well as is altered to favor production of regulatory cytokines, such as driving, the innate and adaptive immune response and the well- documented ability of progesterone to modulate immune activ- ity, there have been surprisingly few studies investigating how Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strath- clyde, Glasgow, United Kingdom progesterone specifically modulates DC maturation and function. Progesterone has been shown to upregulate the differentiation of Received for publication April 16, 2009. Accepted for publication July 29, 2010. DCs as measured by increased endocytic activity from bone L.A.J. was supported by a Caledonian Research Foundation scholarship from the Carnegie Trust for the Universities of Scotland. S.K. and M.S. are supported by the marrow precursors, although following LPS stimulation, an im- Iraqi Ministry of Higher Education. mature phenotype was maintained under the influence of pro- Address correspondence and reprint requests to Dr. Craig W. Roberts, Strathclyde gesterone as demonstrated by decreased MHC class II, CD40, Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor CD54, and CD80 expression (23, 24). In contrast, a further study Street, John Arbuthnott Building, Glasgow G4 0NR, United Kingdom. E-mail ad- dress: [email protected] reported that progesterone treatment of resting murine splenic The online version of this article contains supplemental material. DCs resulted in increased expression of MHC class II and CD40 Abbreviations used in this paper: BMDC, bone marrow-derived dendritic cell; DC, and enhanced T cell stimulatory activity (25). Progesterone is a dendritic cell; GR, ; IRF, IFN regulatory factor; poly I:C, ligand for the glucocorticoid receptor (GR) as well as the pro- polyinosinic-polycytidylic acid; PR, progesterone receptor; SAPK, stress-activated gesterone receptor (PR), and we have already demonstrated that protein kinase. progesterone can differentially regulate macrophage cytokine pro- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 duction by using individual receptors (26). In the current study, we www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901155 4526 PROGESTERONE MODULATION OF DCs therefore determined, using bone marrow-derived DCs (BMDCs), and FITC-labeled anti-CD86 (IgG2a, clone GL-1; catalog number 553691). whether DC TLR-induced cytokine production and maturation All Abs were purchased from BD Pharmingen (San Diego, CA). was also GR or PR dependent. In addition, we determined whether Cytokine assays different TLR ligands that used different signaling pathways The concentration of IL-6, IL-10, IL-12p40, and IL-12p70 present in cell were equally subject to progesterone modulation. Our results in- culture supernatants was assayed by sandwich ELISA. Plates were coated dicate significant plasticity in progesterone-influenced DC activ- with capture anti-mouse mAbs IL-6, IL-10, IL-12p40, and IL-12p70 as ity, whereby the hormone cannot only use either the PR or GR or described above. Recombinant murine cytokines IL-10, IL-12p40, and IL- both to mediate effects but also can selectively modulate the ac- 12p70 were purchased from BD Pharmingen, whereas recombinant mu- tivities induced by distinct TLRs. rine IL-6 was purchased from Bender MedSystems (Vienna, Austria). The detecting Abs used were biotin-labeled rat anti-mouse mAbs (BD Phar- mingen). The conjugate used for IL-6, IL-10, and IL-12p40 detection was Materials and Methods streptavidin-alkaline phosphatase (BD Pharmingen). IL-12p70 was de- Compounds tected by addition of streptavidin-HRP (BD Pharmingen). IL-6, IL-10, and IL-12p40 ELISAs were completed by addition of p-nitrophenyl phosphate LPS from Escherichia coli 055:B5, polyinosinic-polycytidylic acid (poly (Sigma-Aldrich) in glycine buffer and absorbances measured at 450 nm I:C), progesterone, norgestrel, and dexamethasone were all obtained from using a SpectraMax 190 microtiter plate spectrophotometer (Molecular Sigma-Aldrich (Dorset, U.K.). Progesterone and norgestrel were initially Devices, Sunnyvale, CA) and SoftMax Pro 3.0 software (Molecular dissolved in 100% chloroform (BDH Laboratory Supplies, Poole, U.K.) at Devices). The substrate used for IL-12p70 detection was 1% 3, 39,5,59- a concentration of 50 mg/ml. This stock solution was further diluted with tetramethylbenzidine (Sigma-Aldrich) in sodium acetate buffer at pH 5.5 complete medium to give a 250 mM solution for experimental use. containing 0.0075% (BDH, Toronto, Ontario, Canada). Dexamethasone was initially dissolved in 100% ethanol to give a 50 mM This reaction was stopped by addition of 10% sulphuric acid. solution. This stock solution was further diluted with complete medium to Downloaded from give a 250 nM solution for experimental use. Surface marker staining Postincubation, cells were washed and stained with the Pan marker anti- DC culture and treatments for cytokine and cell viability assays CD11c and incubated with the appropriate surface marker at 4˚C for 1 h. Abs Bone marrow cells were flushed from the femurs of 8-wk-old male BALB/c used are as described above. Cells were washed, and fluorescence analysis mice. These cells were cultured to produce BMDCs using GM-CSF– was performed using the BD FACSCanto flow cytometer (BD Biosciences, enriched media obtained from 363 GM-CSF myeloma cells, which allows San Jose, CA). Positive cells were gated on obtained dot plots and data

+ http://www.jimmunol.org/ differentiation of bone marrow stem cells to immature DCs (27). BMDCs analyzed for mean fluorescence intensity of CD11c cells expressing the were grown in 75 cm3 tissue culture flasks (TPP, Trasadingen, Switzerland) chosen maturation marker using FACSDiva software (BD Biosciences). in culture medium containing RPMI 1640 (Life Technologies, Paisley, Isotype-matched negative controls were used to confirm staining specificity. U.K.), 10% heat-inactivated FCS (Harlan Sera-Lab, Loughborough, U.K.), Assessment of cell viability 10% GM-CSF–enriched medium, and 2 mM L-glutamine (GIBCO, Invi- 5 trogen, Paisley, U.K.), and incubated at 37˚C and 5% CO2 for 8 d. This BMDCs plated in triplicate at 5 3 10 cells/ml and treated with hormones/ method produces a large number of CD11c+ DCs largely free from con- stimulants as previously were tested for cell viability using alamarBlue tamination with other cell types as previously described (27). BMDCs assay (Biosource). Reagent was added at 10% of the total well volume. were then seeded at 5 3 105 cells/ml on 96-well microtiter tissue culture Wells containing medium only were used as a blank. After 48 h, well plates at 100 ml volumes and incubated under standard conditions for 48 h. contents were assayed for alamarBlue reduction by measuring the absor-

BMDCs were then activated with either LPS (50 ng/ml or 800 ng/ml) or bance of the wells at 570 and 600 nm on a SpectroMax 190 plate reader by guest on September 23, 2021 poly I:C (50 ng/ml) in 50 ml volumes. (Molecular Devices) and the percentage reduction of alamarBlue calcu- Cells were simultaneously treated with medium alone, medium con- lated as previously described (28). taining solvent vehicle controls (either chloroform or ethanol accordingly), or hormones. Progesterone and norgestrel were added in 50 ml volumes at Western blotting 0.122–62.5 mM. Dexamethasone was added in 50 ml volumes at 0.122– Abs used in Western blotting studies, including phospho–stress-activated 62.5 nM. Cultures were incubated at 37˚C and 5% CO2 for 72 h. After this time, supernatants were collected from each triplicate well and stored at protein kinase (SAPK)/JNK (Thr183/Tyr185; catalog number 9251S), 220˚C for subsequent determination of IL-6, IL-12p40, and IL-12p70. phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204; catalog number 4377S), p44/42 MAPK (ERK1/2; catalog number 4695), phospho-IFN For cell viability assays, 20 ml alamarBlue (Biosource, Nivelle, Belgium) 396 was added to the culture 48 h before experiment termination. regulatory factor (IRF)-3 (Ser ; catalog number 4947S), IRF-3 (catalog number 4962), and phospho–NF-kB (p65; Ser536; catalog number 3031L), DC culture and treatments for maturation marker assays were purchased from Cell Signaling Technology (Beverly, MA). Abs raised against IkBa (Santa Cruz Biotechnology, Santa Cruz, CA; catalog Bone marrow cells were collected from BALB/c femurs as previously number 2607) and p65 (Santa Cruz Biotechnology; catalog number L0607) described and centrifuged at 200 3 g for 10 min at 4˚C. Cell numbers were were purchased from Insight Biotechnology, Middlesex, U.K., and anti– counted and diluted to 2.5 3 105 cells/ml preseeding at 2 ml volumes onto phospho-p38 (catalog number 44-684G) was purchased from Invitrogen. six-well plates (Nunc, Roskilde, Denmark). This procedure allows minimal All other materials used during gel electrophoresis and Western blot- disruption of DCs that could otherwise induce the nonspecific upregulation ting were of the highest commercial grade available and purchased from of maturation markers prior to experimental treatments. Following 7 d of Sigma-Aldrich. culture, BMDCs were pretreated with progesterone (62.5 mM), norgestrel (62.5 mM), or dexamethasone (62.5 nM) for 24 h preaddition of LPS (100 Analysis of protein expression and/or phosphorylation in ng/ml) or poly I:C (10 mg/ml). BMDCs were then left to incubate for 16 h BMDCs at 37˚C and 5% CO2. BMDCs (1 3 106 cells; 1 ml) prepared on 12-well plates were exposed to Abs vehicles, hormones, LPS, or poly I:C as appropriate and whole cell lysates prepared for Western blotting as described previously (29). For ELISAs, the following capture Abs were used: anti-mouse mAb IL-6 (IgG1, clone MP5-20F3; catalog number 554400), anti-mouse mAb IL-10 Data analysis, scanning densitometry, and statistical analyses (IgG1, clone JES5-2A5; catalog number 551215), anti-mouse mAb IL- 12p40 (IgG1, clone 15.6; catalog number 551219), and anti-mouse mAb Scanning densitometry was performed on Western blots using an Epson IL-12p70 (IgG2b, clone 9AG; catalog number 554658). Abs used for de- Perfection 164054 Scan jet and Epson twain 55.52 (32.32) Scanjet Picture tection were biotin-labeled rat anti-mouse anti-cytokine mAbs for IL-6 software (Epson, Long Beach, CA). All values are mean 6 SE of n =3 (IgG2a, clone MP5-32C11; catalog number 554402), IL-10 (IgM, clone experiments. Statistical significance of differences was calculated by the SXC-1; catalog number 554423), and IL-12p40 (IgG2a, clone C17.8; catalog one-way ANOVA followed by a Newman-Keuls test using GraphPad number 554476). For flow cytometry analysis, the anti-mouse Abs used were Prism Version 4.0 software (GraphPad, San Diego, CA). A p value ,0.05 PE-labeled anti-CD11c (IgG2b, clone S-HCL-3; catalog number 333149), was accepted as significant. All other sets of data were analyzed using FITC-labeled anti-CD40 (IgG2a, clone 3/23; catalog number 553790), a Mann–Whitney U test using Statview software (SAS Institute, Marlow, FITC-labeled anti-CD80 (IgG2, clone 16-10A1; catalog number 553768), U.K.) with a value of p , 0.05 taken as significant. The Journal of Immunology 4527

Results 2 nM; Fig. 1F), again indicating that the TLR engaged determines Progesterone uses the GR to inhibit TLR3- and TLR4-induced sensitivity to hormone treatment and activity via the GR. IL-6 production by BMDCs Progesterone uses both the GR and PR to inhibit TLR3- and The ability of BMDCs to produce IL-6 following stimulation with TLR4-induced total IL-12 production (p40) by BMDCs LPS or poly I:C was first confirmed using a simple dose-response experiment (0.012–25 mg/ml LPS/6.25–100 mg/ml poly I:C; data The ability of BMDCs to produce total IL-12 (p40) following not shown). Totals of 800 ng/ml LPS and 50 mg/ml poly I:C were stimulation with LPS or poly I:C was first confirmed using a simple identified as the most suitable stimulant concentrations for use in dose-response experiment (0.012–25 mg/ml LPS/6.25–100 mg/ml studies with hormones because they allowed both potentiation and poly I:C; data not shown). Totals of 50 ng/ml LPS and 50 mg/ml inhibition of IL-6 production to be assessed. poly I:C were identified as the most suitable stimulant concen- Once the assays had been standardized, we determined whether trations for use in studies with hormones because they allowed progesterone, norgestrel, and dexamethasone could modulate LPS- both potentiation and inhibition of IL-12 (p40) production to or poly I:C-induced IL-6 production by BMDCs. Over a number of be assessed. experiments (n = 3), progesterone caused a significant inhibition Progesterone induced a significant reduction of IL-12p40 pro- of LPS-induced IL-6 production at 62.5 mM and 31.25 mM (Fig. duction by LPS-stimulated BMDCs at all concentrations tested and 1A). Progesterone also significantly inhibited poly I:C-induced had an IC50 of 1–2 mM (Fig. 2A). Progesterone also significantly IL-6 production, but at lower concentrations .0.5 mM (IC50 of reduced IL-12p40 production from poly I:C-stimulated BMDCs at 0.5–1 nM; Fig. 1D), indicating that IL-6 production via TLR3 concentrations .1 mM and had an IC50 of 0.5–1 mM. (Fig. 2D). Downloaded from ligation was more sensitive to progesterone treatment than TLR4 Norgestrel, the PR agonist, also downmodulated LPS-induced ligation. Whereas norgestrel, the PR agonist, did not modulate IL-12p40 production at all concentrations tested and had an IC50 IL-6 production by LPS- or poly I:C-stimulated BMDCs (Fig. 1B, of 0.5–1 mM (Fig. 2B). Norgestrel also significantly inhibited 1E), the GR agonist dexamethasone demonstrated a greater ca- IL-12p40 production by poly I:C-stimulated BMDCs .31.3 mM pacity to reduce IL-6 levels compared with progesterone. Dexa- (Fig. 2E), this time indicating that engagement of different TLRs treatment using concentrations above 2 nM resulted in is subject to different hormonal sensitivity via PR. Dexamethasone http://www.jimmunol.org/ significant downregulation of LPS-induced IL-6 (IC50 of 7.8–15.7 inhibited LPS-induced IL-12p40 levels from BMDCs, with sig- nM; Fig. 1C), and at all concentrations tested, dexamethasone nificant downregulation occurring at concentrations .2 nM, dem- reduced poly I:C-induced IL-6 production by BMDCs (IC50 of 1– onstrating an IC50 of 1–2 nM (Fig. 2C). Dexamethasone also by guest on September 23, 2021

FIGURE 1. The influence of progesterone, norgestrel, and dexamethasone on IL-6 production by BMDCs stimulated with 800 ng/ml LPS or 50 mg/ml poly I:C for 72 h. Cells were cultured with LPS or poly I:C in the presence of progesterone (A, D), norgestrel (B, E), or dexamethasone (C, F). Unstimulated BMDCs failed to produce detectable levels of IL-6 (nd). Unstimulated cells treated with the solvent vehicle of 0.04% chloroform (progesterone and norgestrel) or 0.0001% ethanol (dexamethasone) produced undetectable or very low levels of IL-6 (data not shown). Stimulated control cell cultures were treated with 800 ng/ml LPS or 50 mg/ml poly I:C (closed bars). Stimulated control cell cultures treated with 800 ng/ml LPS or 50 mg/ml poly I:C and either 0.04% chloroform or 0.0001% ethanol produced levels of IL-6 that were comparable to stimulated cells treated with LPS or poly I:C alone (data not shown). Results are the mean of n = 3 treatments from one of three representative experiments. Where SEs are not evident, it is because they are too small to be visible. pp , 0.05 compared with stimulated controls using a Mann–Whitney U test. 4528 PROGESTERONE MODULATION OF DCs Downloaded from http://www.jimmunol.org/

FIGURE 2. The influence of progesterone, norgestrel, and dexamethasone on IL-12p40 production by BMDCs stimulated with 50 ng/ml LPS or 50 mg/ ml poly I:C for 72 h. Cells were cultured with LPS or poly I:C in the presence of progesterone (A, D), norgestrel (B, E), or dexamethasone (C, F). Unstimulated BMDCs produced low levels of IL-12p40 (open bars). Unstimulated cells treated with the solvent vehicle of 0.04% chloroform (progesterone and norgestrel) or 0.0001% ethanol (dexamethasone) produced undetectable or very low levels of IL-12p40 (data not shown). Stimulated control cell cultures were treated with 50 ng/ml LPS or 50 mg/ml poly I:C (closed bars). Stimulated control cell cultures treated with 50 ng/ml LPS or 50 mg/ml poly I:C by guest on September 23, 2021 and either 0.04% chloroform or 0.0001% ethanol produced levels of IL-12p40 that were comparable to stimulated cells treated with LPS or poly I:C alone (data not shown). Results are the mean of n = 3 treatments from one of three representative experiments. Where SEs are not evident, it is because they are too small to be visible. pp , 0.05 compared with stimulated controls using a Mann–Whitney U test.

caused significant downregulation of poly I:C-induced IL-12p40 (62.5–1000 ng/ml LPS/6.25–100 mg/ml poly I:C; data not shown). production at all concentrations tested and had an IC50 of 0.2–0.5 Totals of 100 ng/ml LPS and 10 mg/ml poly I:C were identified as nM (Fig. 2F). the most suitable stimulant concentrations for use in studies with hormones because they allowed both potentiation and inhibition of Progesterone, norgestrel, and dexamethasone have no effect on CD40, CD80, and CD86 expression to be assessed. the viability of stimulated BMDCs BMDCs were pretreated with progesterone, norgestrel, or dexa- alamarBlue, a nonspecific stable dye that is used to assess cell methasone for 24 h to investigate whether these hormones had viability and cell proliferation by measuring the reducing envi- any effect on expression of CD40, CD80, and CD86 (Fig. 3) ronment of the proliferating cell, was employed to determine following stimulation with LPS (Fig. 3) or poly I:C (Fig. 4). whether the effects of hormones on BMDC cytokine production Progesterone, norgestrel, and dexamethasone did not have an ef- were due to a specific effect on signaling pathways or a nonspecific fect on LPS-induced expression of CD40 by BMDCs (Fig. 3A). mechanism caused by cell death. Although stimulation with LPS However, progesterone had a slight but significant inhibitory effect and poly I:C significantly upregulated mitochondrial activity as on BMDC expression of CD80 and CD86 in comparison with compared with control cultures treated with medium alone, treat- LPS-stimulated control cultures (Fig. 3B,3C). Norgestrel had ment with hormones had no effect on cell viability or proliferation no significant effect on LPS-induced BMDC expression of CD40 as compared with LPS- or poly I:C-stimulated controls (data not (Fig. 3A), CD80 (Fig. 3B), or CD86 (Fig. 3C). Dexamethasone, in shown). In addition, BMDCs stimulated with LPS or poly I:C and contrast, induced a significant reduction in expression of CD86 treated with progesterone, norgestrel, or dexamethasone failed to (Fig. 3C) in comparison with LPS controls but not CD40 or CD80 produce detectable levels of IL-10 (not shown). (Fig. 3A,3B). By comparison with TLR4 ligation, progesterone demonstrated significant downregulation of poly I:C-induced The ability of progesterone to downmodulate CD40, CD80, BMDC expression of CD40, CD80, and CD86 (Fig. 4). Dexa- and CD86 expression depends on the TLR engaged methasone also inhibited poly I:C-induced BMDC’s CD40, CD80, The ability of BMDCs to express the costimulatory and CD86 (Fig. 4) expression in comparison with poly I:C-treated CD40, CD80, and CD86 following stimulation with LPS or poly controls. Norgestrel had no significant effect on poly I:C-induced I:C was first confirmed using a simple dose-response experiment expression of CD40 (Fig. 4A). In contrast to TLR4 ligation, it did The Journal of Immunology 4529

FIGURE 3. LPS-induced BMDC expression of CD40 (A), CD80 (B), and CD86 (C) preceded by preincubation with the hormones progesterone, norgestrel, and dexa- methasone. Cells were preincubated in the presence or absence of progesterone, norgestrel, and dexamethasone for 24 h followed by a 16-h stimulation with LPS. Unstimulated cells received medium alone. Unstimu- lated cells treated with the solvent vehicle of 0.04% Downloaded from chloroform (progesterone and norgestrel) or 0.001% ethanol (dexamethasone) produced levels of CD40, CD80, or CD86 that were comparable to medium con- trols (not shown). Stimulated control cells were treated with 100 ng/ml LPS. Stimulated controls receiving 100 ng/ml LPS and either 0.04% chloroform or 0.0001% http://www.jimmunol.org/ ethanol produced equivalent levels of CD40, CD80, and CD86 compared with stimulated cells receiving LPS alone. pp , 0.05 compared with stimulated controls using a Mann–Whitney U test. by guest on September 23, 2021

significantly downregulate expression of CD80 and CD86 (Fig. treated with the same hormones and exposed to poly I:C or LPS for 4B,4C). Plots showing the mean fluorescence intensities for Figs. 5–120 min (data not shown). 3 and 4 are shown in Supplemental Fig. 1. It was observed that both poly I:C and LPS were also able to activate, in parallel to the pathways detailed above, the IRF-3 Ligation of the PR sustains IRF-3 phosphorylation following cascade, as measured by Western blotting using antisera specific TLR3 ligation for the phosphorylation of Ser396 in the C terminus of IRF-3. TLR3- and TLR4-stimulated cellular responses are well recognized Pretreatment of cells with progesterone or norgestrel for 1 h as being mediated via recruitment of a number of signaling cas- prior to addition of agonist prolonged poly I:C-stimulated IRF-3 cades: classical p42/44 MAPK, p38 MAPK, JNK, NF-kB, and phosphorylation in BMDCs significantly at 2 h postexposure IRF-3/7 pathways. To examine further the effects of hor- to poly I:C (Fig. 5). Pretreatment of cells with dexamethasone, mones on TLR3- and TLR4-mediated signaling events that may in contrast, had minimal effect upon poly I:C-stimulated IRF-3 precede cytokine expression, experiments were constructed to mea- phosphorylation over a similar time course but inhibited agonist- sure the expression and/or phosphorylation of key protein com- stimulated IRF-3 phosphorylation at later time points (4 and 8 h; ponents of these cascades following exposure of cells to LPS or Fig. 5). Pretreatment (1 h) with progesterone, norgestrel, or dexa- poly I:C over an acute time frame of 0–8 h. Although both LPS and methasone did not influence LPS-stimulated IRF-3 phosphoryla- poly I:C were effective activators (5–120 min) of the p42/44 tion in BMDCs, again, as assessed by Western blotting (Fig. 6). MAPK, p38 MAPK, and JNK phosphorylation and parallel IkBa degradation and p65 RelA phosphorylation in BMDCs, cells pre- treated (1 h) with the steroid hormones progesterone, norgestrel, Discussion and dexamethasone did not influence phosphorylation of any of the It is well documented that much of the immunomodulation occurring MAPK/SAPKs as assessed by Western blotting (data not shown). during pregnancy is due to alterations in sex hormone levels, Furthermore, no modulation of p65 phosphorylation and IkBa de- particularly progesterone (6, 30). Given the critical role that DCs gradation within the NF-kB cascade were observed in cells pre- play in orchestrating immune responses and their significant pre- 4530 PROGESTERONE MODULATION OF DCs

FIGURE 4. Poly I:C-induced BMDC expression of CD40 (A), CD80 (B), and CD86 (C) preceded by preincubation with the hormones progesterone, nor- gestrel, and dexamethasone. Cells were preincubated in the presence or absence of progesterone, norgestrel and dexamethasone for 24 h followed by a 16-h stimulation with poly I:C. Unstimulated cells received

medium alone. Unstimulated cells treated with the Downloaded from solvent vehicle of 0.04% chloroform (progesterone and norgestrel) or 0.001% ethanol (dexamethasone) pro- duced levels of CD40, CD80, or CD86 that were comparable to medium controls (not shown). Stimu- lated control cells were treated with 10 mg/ml poly I:C. Stimulated controls receiving 10 mg/ml poly I:C and either 0.04% chloroform or 0.0001% ethanol produced http://www.jimmunol.org/ equivalent levels of CD40, CD80, and CD86 compared with stimulated cells receiving poly I:C alone. pp , 0.05 compared with stimulated controls using a Mann– Whitney U test. by guest on September 23, 2021

sence at the fetal–maternal interface (11, 31), the current study dependent (34). In addition, the TRIF-related adaptor molecule investigated the role of progesterone in modulating DC function. As TRAM is an essential part of and restricted to the TLR4 MyD88- a result, a number of key findings have arisen from our inves- independent pathway (35). Recent studies have demonstrated that tigations: first, that the ability of progesterone to downregulate not only does TLR3 mediate a more potent antiviral response than BMDC inflammatory cytokine production and costimulatory mol- TLR4 (33), but also that TLR3 contributes critically to the de- ecule expression depends in large part on what TLR is engaged; bilitating effects of a damaging host inflammatory response as a second, progesterone downmodulates some BMDC TLR-induced result of influenza A (36), and, despite a higher viral inflammatory mediators via the GR alone and others via the PR production in the lungs, mice deficient in TLR3 had a significant and the GR; and third, PR agonists sustain IRF-3 phosphorylation survival advantage. Consequently, with regard to the well being following TLR3 ligation but not TLR4 ligation. of the fetus, there could well be an evolutionary advantage in In the current study, we demonstrate that TLR3- but not TLR4- the apparent enhanced sensitivity of TLR3-induced inflammatory induced CD40 expression can be downmodulated by progesterone. processes to downmodulation by progesterone. In addition, the induction of IL-6 via TLR3 ligation is an order of It has been reported that women who use the progesterone magnitude more sensitive to inhibition by progesterone than IL-6 contraceptive depot medroxyprogesterone acetate are at greater produced via TLR4 ligation. That DC TLR-induced activity can be risk of contracting the HIV and show increased incidence of HSV differentially sensitive to progesterone depending on the TLR reactivation and viral shedding (37, 38). A recent study has sug- engaged is likely a result of the subtly different signaling pathways gested that progesterone could be limiting the antiviral response associated with TLR4 and TLR3 (32). Thus, although both TLRs by inhibiting TLR7- and TLR9-induced IFN-a production by can use MyD88-independent pathways, TLR3-induced NF-kB plasmacytoid DCs (39). TLR3 is also known to be a vital com- activation is entirely TRIF dependent (33), and, unlike other ponent of antiviral responses by binding dsRNA and ssRNA mol- TLRs that use IL-1R–associated kinase to activate NF-kB, TLR3- ecules to induce not only IFNs but also proinflammatory cyto- induced NF-kB activation is receptor interacting protein kinase kines, which have a key role to play in antiviral defense (32, 40, The Journal of Immunology 4531 Downloaded from http://www.jimmunol.org/

FIGURE 5. The effect of progesterone, norgestrel, and dexamethasone on poly I:C-stimulated IRF-3 phosphorylation in BMDCs. BMDCs were treated with vehicle, progesterone (62.5 mM), norgestrel (62.5 mM), or dexamethasone (62.5 nM) for 1 h pre-exposure to 25 mg/ml poly I:C for the indicated times. Whole-cell extracts were prepared and IRF-3 phosphorylation and expression assessed by Western blotting using specific Abs as detailed in the Materials and Methods section. Results in A are representative of three independent experiments, and associated data for experiments examining pretreatment with

progesterone, norgestrel, and dexamethasone were quantified by scanning densitometry and are depicted in B–D, respectively (n = 3). by guest on September 23, 2021

41). We believe that our data demonstrate a further mechanism to differentially use these receptors to cause downregulation of through which progesterone could be functioning to limit the IL-6 and IL-12 production. Whereas progesterone mediated a antiviral response by reducing TLR3-induced inflammatory re- reduction in TLR4- and TLR3-induced IL-6 production exclu- sponses. sively through the GR, inhibition of IL-12 production was medi- The release of proinflammatory cytokines, such as IL-6 and ated through the GR and PR. Although it has been shown that IL-12, as induced by infectious disease is incompatible with glucocorticoids and progesterone can induce upregulation of the successful pregnancy, and, consequently, we examined the role of anti-inflammatory cytokine IL-10 in human DCs (46), our studies progesterone on DC release of these cytokines (6). Progesterone (results not shown) as well as one using rat-derived DCs treated reduced the production of IL-6 and IL-12 by murine BMDCs with progesterone did not show an upregulation of IL-10 (47). The following stimulation with either LPS or poly I:C. Estrogen has timing of LPS treatment relative to the seeding of DC cultures previously been shown to reduce cytokine and chemokine pro- in preparation of stimulation experiments is critical in defining duction following TLR3 stimulation in a human cell line (42). secretion patterns of IL-12 and IL-10 that likely explain the dif- However, to our knowledge, this is the first report to demonstrate ferences between such studies (48). Nonetheless, we show that the role of progesterone in modulating TLR3-induced pathways progesterone can reduce IL-12 levels in a manner independent of IL-6 and IL-12 induction. Previous reports have described of IL-10. a reduction in IL-12 levels following progesterone treatment of Our studies provide further evidence of immunomodulatory ac- lymphocytes from pregnant mice and murine splenic-derived tivity through the PR by using the PR-specific agonist norgestrel. DCs (25, 43). However, the observed reduction in IL-6 production In addition, the action of hormones had no effect on cell metab- is in contrast to studies carried out by Huck and colleagues (23), olism in the current study as shown by a lack of any alteration to who failed to show any significant effects of progesterone on pro- alamarBlue reduction in treated cells. Therefore, progesterone is duction of this cytokine. Indeed, Yang et al. (25) reported increased directly modulating signaling pathways, leading to the production levels of intracellular IL-6 in murine DCs following progesterone of IL-6 and IL-12 by differential usage of the GR and PR. Im- treatment, although this study differed from ours in that cells were munomodulatory actions through the GR have been well docu- left unstimulated and were derived from spleen and not bone mar- mented. These include its ability to interact with the transcription row. However, with regard to a successful pregnancy, it is the reg- factor NF-kB (49). The PR has also been reported to physically ulation of excessive inflammatory responses that is crucial. interact with NF-kB, linked to cytokine production (49, 50). Progesterone functions by binding to both the GR and PR However, it would seem unlikely that the PR is inhibiting IL-12 (44–46). Using the receptor-specific agonists dexamethasone and production in this manner, as it would then be expected to induce norgestrel, we demonstrate in this study the ability of progesterone downregulation of IL-6. It has been suggested that steroid hor- 4532 PROGESTERONE MODULATION OF DCs Downloaded from http://www.jimmunol.org/

FIGURE 6. The effect of progesterone, norgestrel, and dexamethasone on LPS-stimulated IRF-3 phosphorylation in BMDCs. BMDCs were treated with vehicle, progesterone (62.5 mM), norgestrel (62.5 mM), or dexamethasone (62.5 nM) for 1 h pre-exposure to 1 mg/ml LPS for the indicated times. Whole- cell extracts were prepared and IRF-3 phosphorylation and expression assessed by Western blotting using specific Abs as detailed in the Materials and Methods section. Results in A are representative of three independent experiments, and associated data for experiments examining pretreatment with progesterone, norgestrel, and dexamethasone were quantified by scanning densitometry and are depicted in B–D, respectively (n = 3). pp , 0.05, sta- tistically significant versus agonist alone. by guest on September 23, 2021 mone receptors can differentially modulate genes at the promoter detailed reduced expression of IL-12 p35 in response to viral in- level, which could explain our observations (50, 51). Alternatively, fection due to constitutive activation of IRF-3 and its function as divergences upstream of NF-kB in the individual signaling path- a repressor of transcription via binding to bp 2172 to 2122 of the ways leading to the synthesis of IL-6 and IL-12 may be targeted p35 promoter (53). Whether this regulatory function is apparent in by norgestrel to result in the specific inhibition of IL-12. BMDCs and can be correlated with IRF-3 protein phosphorylation Related to these points, we sought to characterize further the status remains to be determined. molecular mechanisms that underlie PR-mediated inhibition/sup- The mechanism by which poly I:C-stimulated IRF-3 phos- pression of LPS- and poly I:C-stimulated cytokine production. phorylation is sustained following progesterone and norgestrel In this study, as a first step toward pursuing the potential role of pretreatment also remains to be characterized thoroughly. This signaling cascades in these processes, we examined the modulation may be a consequence of PR/GR-stimulated elevation of cellular of key signaling molecules recognized to be sensitive to TLR3/4 kinase activity; potentially via the inducible isoform of IkB kinase activation (52). PG, norgestrel, and dexamethasone collectively and/or TRAF-associated NF-kB activator-binding kinase 1 as the did not influence the phosphorylation status of the MAPKs and the recognized upstream kinase regulators of this transcription fac- activation of the NF-kB cascade, commonly engaged by this re- tor (52). Alternatively, there may be a decrease in phosphatase- ceptor superfamily. This suggested these pathways were not in- catalyzed dephosphorylation of IRF-3. Again, relevant candidate fluenced by pretreatment. It must be noted, phosphatases remain to be identified. GR-mediated immunosup- however, that in this study, experiments were limited to exami- pression has also been reported to be reliant on a glucocorticoid nation of the signaling cascades at the level of the MAPK/SAPKs receptor-interacting protein 1–IRF-3 interaction (54) in the for- and the p65/IkBa complex directly. Further investigation is re- mation of a protein complex capable of repressing, although the quired to determine whether agonists at the GR and PR influence influence of IRF-3 phosphorylation status upon this interaction has events further downstream in the cascade—in particular, nuclear not been detailed. Overall, from our studies to date, it appears that functions. these effects are specific to TLR3-mediated responses and as such It was noted, however, that progesterone and norgestrel pre- may represent a mechanism by which progesterone and norgestrel treatment led to sustained IRF-3 phosphorylation that was specific mediate modulation of IRF-3 phosphorylation and therefore may to the poly I:C–TLR3 response; this was not apparent for LPS– potentially influence transcription factor activation, downstream TLR4 signaling. Numerous studies have described that elevated cytokine production, and costimulatory molecule expression. IRF-3 activation leads to enhanced antiviral responses, including In addition to the vital role that DCs play in initiating the innate gross expression of IFNs (53). Therefore, sustained phosphoryla- immune response by production of cytokines and chemokines, tion may imply an expected elevation of cytokine production. these cells are critically important in triggering an effective adap- More recently, however, experiments in macrophages (54) have tive immune response. During the maturation process, DCs up- The Journal of Immunology 4533 regulate the expression of cell surface molecules, such as CD40, 19. Matyszak, M. K., S. Citterio, M. Rescigno, and P. Ricciardi-Castagnoli. 2000. Differential effects of during different stages of dendritic cell CD80, and CD86, that are important for interaction with B and maturation. Eur. J. Immunol. 30: 1233–1242. T cells and subsequent development of the adaptive immune re- 20. Pan, J., D. Ju, Q. Wang, M. Zhang, D. Xia, L. Zhang, H. Yu, and X. Cao. 2001. sponse (1, 4). Expression of these maturation markers is signifi- Dexamethasone inhibits the antigen presentation of dendritic cells in MHC class II pathway. Immunol. Lett. 76: 153–161. cantly upregulated following treatment of BMDCs with the TLR3 21. Piemonti, L., P. Monti, P. Allavena, M. Sironi, L. Soldini, B. E. Leone, C. Socci, ligand poly I:C and the TLR4 ligand LPS. Of particular note in and V. Di Carlo. 1999. Glucocorticoids affect human dendritic cell differentia- addition to the different sensitivities of these maturation factors to tion and maturation. J. Immunol. 162: 6473–6481. 22. Tuckermann, J. P., A. Kleiman, K. G. McPherson, and H. M. Reichardt. 2005. progesterone depending on the particular TLR engaged was that Molecular mechanisms of glucocorticoids in the control of inflammation progesterone, but not dexamethasone or norgestrel, significantly and lymphocyte apoptosis. Crit. Rev. Clin. Lab. Sci. 42: 71–104. inhibited TLR4-induced CD80, whereas progesterone inhibited 23. Huck, B., T. Steck, M. Habersack, J. Dietl, and U. Ka¨mmerer. 2005. Pregnancy associated hormones modulate the cytokine production but not the phenotype of TLR3-induced CD40 and CD80 to a significantly greater level PBMC-derived human dendritic cells. Eur. J. Obstet. Gynecol. Reprod. Biol. than ligation of either PR or GR alone. This would indicate that 122: 85–94. progesterone can downregulate these molecules either by PR- or 24. Liang, J., L. Sun, Q. Wang, and Y. Hou. 2006. Progesterone regulates mouse dendritic cells differentiation and maturation. Int. Immunopharmacol. 6: 830–838. GR-independent mechanisms. A non-PR and GR mechanism could 25. Yang, L., X. Li, J. Zhao, and Y. Hou. 2006. Progesterone is involved in the mat- result from progesterone binding to membrane-associated recep- uration of murine spleen CD11c-positive dendritic cells. 71: 922–929. 26. Jones, L. A., J. P. Anthony, F. L. Henriquez, R. E. Lyons, M. B. Nickdel, tors resulting in rapid, nongenomic effects on intracellular signal- K. C. Carter, J. Alexander, and C. W. 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