17β-Estradiol Promotes TLR4-Triggered Proinflammatory Mediator Production through Direct Estrogen Receptor α Signaling in Macrophages In Vivo

Bertrand Calippe, Victorine Douin-Echinard, Laurent Delpy, Muriel Laffargue, Karine Lélu, Andrée Krust, Bernard Pipy, Francis Bayard, Jean-François Arnal, Jean-Charles Guéry and Pierre Gourdy
J Immunol July 15, 2010, 185 (2) 1169-1176; DOI: https://doi.org/10.4049/jimmunol.0902383

Abstract

17β-estradiol (E2) has been shown to promote the expression of inflammatory mediators by LPS-activated tissue resident macrophages through estrogen receptor α (ERα) signaling. However, it remained to be determined whether E2 similarly influences macrophages effector functions under inflammatory conditions in vivo, and whether this action of E2 resulted from a direct effect on macrophages. We show in this study that chronic E2 administration to ovariectomized mice significantly increased both cytokine (IL-1β, IL-6, and TNF-α) and inducible NO synthase mRNA abundance in thioglycolate (TGC)-elicited macrophages. The proinflammatory action of E2 was also evidenced at the level of released IL-1β and IL-6 by ex vivo LPS-activated macrophages. E2 concomitantly inhibited PI3K activity as well as Akt phosphorylation in TGC-elicited macrophages, suggesting that E2 promoted TLR-dependent macrophage activation by alleviating this suppressive signaling pathway. Indeed, this effect was abolished in the presence of the inhibitor wortmannin, demonstrating a key functional link between inhibition of PI3K activity and the E2 action on macrophage functions. Endogenous estrogens levels circulating in ovary-intact mice were sufficient to promote the above described actions. Finally, thanks to a CreLox strategy, targeted disruption of ERα gene in macrophages totally abolished the effect of E2 on the expression of inflammatory mediators by both resident and TGC-elicited peritoneal macrophages. In conclusion, we demonstrate that estrogens, through the activation of ERα in macrophages in vivo, enhance their ability to produce inflammatory mediators and cytokines upon subsequent TLR activation.

Besides their crucial role in reproduction, the sexual steroid hormones estrogens have been recently recognized to influence numerous immune and inflammatory responses, especially during autoimmune and infectious pathophysiological processes (1, 2). These immunomodulating actions of estrogens are thought to mainly result from their specific effects on the different cellular components of the immune system, because most of them, if not all, have been demonstrated to express estrogen receptors (ERs) (2). Among these cellular targets, macrophages may play a pivotal role in the modulation of immune responses by estrogens. Indeed, these crucial cells of the innate immune system not only contribute to the first line of defense against pathogens but also play an important role in directing adaptive immune responses (3, 4).

Recognized as a key step in innate immune processes, one of the main features of macrophages is to produce a diverse range of inflammatory mediators, including cytokines and NO, following the interaction of pathogen-associated molecular pattern molecules with their surface cognate receptors, such as TLRs. Conflicting results have been reported from studies investigating the effects of estrogens on macrophage effector functions. Whereas in vitro experiments have suggested that E2 exerts anti-inflammatory properties on monocyte/macrophage cell lines or microglial cells following their activation with LPS, the prototypic ligand of TLR4 (5–8), opposite results have been independently reported by analyzing the in vivo effects of estrogens on the same cell populations (9, 10). Indeed, in striking contrast with previous in vitro observations, long-term in vivo exposure to estrogens from endogenous or exogenous origin was first demonstrated to enhance the LPS-induced transcription of proinflammatory cytokines, namely IL-12 and TNF-α, by microglial cells through ERα-dependent mechanisms (10). Furthermore, although confirming the anti-inflammatory effect of short-term in vitro exposure to E2 on murine resident peritoneal macrophages, we recently demonstrated that chronic administration of E2 to ovariectomized female mice markedly increases the expression of numerous inflammatory cytokines and NO by these cells in response to LPS activation ex vivo (9). Noteworthy, this proinflammatory effect was mediated through ERα and resulted, at least in part, from the downmodulation of the PI3K/Akt pathway, a negative regulator of TLR4-induced activation (11–15). We therefore hypothesized that sustained ERα activation in macrophages in vivo would lead to constitutive deficiency in the PI3K signaling pathway thereby resulting in enhanced proinflammatory mediator expression upon TLR engagement (9).

However, whether estrogens similarly influence the inflammatory status of macrophages in inflamed tissues in vivo has not been explored to date. Furthermore, our previous experimental approaches did not address whether E2 exerted its proinflammatory actions through a direct effect on macrophages or thanks to indirect mechanisms involving other cellular targets (9). In the current study, we first analyzed the estrogen actions in the classical and widely used model of i.p. thioglycolate (TGC) injection in the mouse, leading to the in vivo recruitment and activation of peritoneal macrophages (16). Then, we generated and characterized a new mouse model of targeted disruption of ERα gene in macrophages to examine the effects of E2 administration on macrophage effector functions in vivo.

Materials and Methods

Mice and surgical procedure

Female C57BL/6J mice were purchased from Charles River Laboratories (L’arbresle, France). All mice were maintained under specific pathogen-free conditions, and procedures were performed in accordance with the principles and guidelines established by the National Institute of Medical Research (Institut National de la Santé et de la Recherche Médicale). Female mice were ovariectomized or sham-operated at 4 wk of age, and ovariectomized mice were given either placebo or E2 60-d time release pellets (0.1 mg, 80 μg/kg/day; Innovative Research, Sarasota, FL) implanted s.c. in the scapular region, as described previously (9). At 8 wk of age, mice received a single i.p. injection of 4% TGC (Sigma-Aldrich, St. Louis, MO) diluted in PBS. Four days later, mice were sacrificed by CO2 inhalation, peritoneal cells were collected, and uterine weight was individually recorded to assess either the efficiency of ovariectomy (uterine atrophy) or the in vivo exposure to estrogens (uterine hypertrophy).

Generation of LysM-Cre ERαL2/L2 mice

C57BL/6J LysM-Cre mice (17) were crossed with ERαflox/flox mice, which contains loxP sites in the 5′ and 3′ region of the exon 2 of the esr1 gene coding for ERα (18). The appropriate deletion was characterized in splenocytes by PCR using primers P1-P2 and P1-P3: P1 (5′-TTGCCCGATAACAATAACAT-3′), P2 (5′-ATTGTCTCTTTCTGACAC-3′), and P3 (5′-GGCATTACCACTTCTCCTGGGAGTCT-3′). P1-P2 PCR generated a 380-bp product in wild-type ERαflox/flox mice and no product in LysM-Cre+ ERαflox/flox mice. P1-P3 PCR generated 920-bp products in wild-type and 390 bp in LysM-Cre+ ERαflox/flox mice. These PCRs could also discriminate the floxed allele that exhibited an increased size because of the insertion of LoxP sites. Deletion efficiency by the Cre/loxP system was checked by Southern blot analysis on tails and various tissues of LysM-Cre+ ERαflox/flox mice. After BamHI digestion of genomic DNA, 4.4- and 8.8-kb bands represent the targeted (floxed) allele and the deleted allele, respectively (18).

TGC-elicited peritoneal macrophages preparation and cell culture conditions

TGC-elicited peritoneal cells were harvested from mice peritoneal cavities by two washes with phenol red-free serum-free X-Vivo 15 medium (Cambrex, East Rutherford, NJ) containing heparin (20 IU/ml). After centrifugation (10 min, 328 × g), cells were resuspended in X-Vivo 15 medium supplemented with 50 mg/ml gentamicin (Invitrogen Life Technologies, Carlsbad, CA), 2.5 mg/ml amphotericin B (Biochrom, Berlin, Germany), and 2 mM l-glutamine (Life Technologies, Rockville, MD). Macrophages and total cells were counted using a Neubauer slide in presence of trypan blue (Eurobio, Les Ulis, France). According to experimental design, cells were plated in either 96-well (2 × 105 macrophages/well; BD Falcon, BD Biosciences, San Jose, CA) or 12-well plates (106 macrophages/well; Costar, Corning Life Sciences, Lowell, MA). Peritoneal cells were incubated for 2 h (37°C, 5% CO2) to allow macrophage adhesion and then were washed three times to remove nonadherent cells. This protocol resulted routinely in a culture containing >95% macrophages. In some experiments, peritoneal TGC-elicited macrophages from placebo- or E2-treated mice were stimulated in vitro with LPS in presence of 1 nM E2 (Sigma-Aldrich) or 1/10,000 vehicle DMSO (endotoxin free; Sigma-Aldrich). In some experiments, macrophages were incubated with 50 nM wortmannin (United States Biological, Swampscott, MA) or vehicle DMSO 1 h before and during LPS activation, allowing the inhibition of the PI3K pathway, as described previously (9).

Quantitative real-time PCR

TGC-elicited peritoneal macrophages were plated in 12-well plates, stimulated or not with LPS (20 ng/ml) for 4 h, then total mRNA were extracted with TRIzol reagent, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA), and purified with a mammalian total RNA miniprepkit, Genelute (Sigma-Aldrich). Quantity and quality of mRNA were checked with a spectrophotometer (ND-1000; NanoDrop Technologies, Wilmington, DE). Reverse transcription was performed with 500 ng total mRNA, using High Capacity DNA Archive kit (Applied Biosystems, Foster City, CA). Twenty-five reverse-transcripted RNA were used as template for quantitative real-time PCR, set up in 96-well plates using PCR Mastermix (Power SYBR Green; Applied Biosystems). Gene expression level was quantified using ABI Prism 7900 sequence detection system (Applied Biosystems). Primers for IL-1β, TNF-α, IL-12p40, IL-6, and inducible NO synthase (iNOS) have been published previously (9, 19). Results were analyzed using the SDS program, version 2.2 (Applied Biosystems). The relative expression of target transcripts in each sample was normalized to GAPDH and compared with the expression in a stimulated or nonstimulated control according to the ΔCt method: ΔCtsample = Ctsample − CtGAPDH, ΔΔCtsample = ΔCtsample − ΔCtcontrol, relative expressionsample = 2−ΔΔCtsample.

Cytokine production by peritoneal macrophages

Macrophages were stimulated with LPS (20 ng/ml) for 24 h, and supernatants were collected and stored at −80°C until IL-6 quantification. For IL-1β, 5 mM ATP was added to culture medium during the last 3 h of stimulation to convert pro–IL-1β to IL-1β, as described previously (20, 21). IL-1β and IL-6 concentrations were determined using specific ELISAs (BD Biosciences), with a sensitivity of 15 pg/ml. Results were expressed for individual mice as cytokine amounts (nanograms) normalized to 106 input macrophages.

Flow cytometry analysis and Abs

Before staining, total peritoneal cells (5–10 × 105) were incubated 15 min at room temperature with blocking buffer (PBS with 1% SVF, 3% normal mouse serum, 3% normal rat serum, 5 mM EDTA, and 1% NaN3) containing 5 μg/ml anti-CD16/CD32 (2.4G2; BD Biosciences) to block FcγIII/II receptors. Cells were then incubated for 30 min with mAbs diluted at the optimal concentration in FACS buffer (PBS, 5% FCS, 5 mM EDTA, and 1% NaN3): allophycocyanin- or FITC-conjugated anti-CD11b (M1/70.15; BD Biosciences), PE-conjugated anti-CD19 (1D3; BD Biosciences), and either FITC- or PE-conjugated anti-F4/80 (BM8; eBioscience, San Diego, CA) or PE-conjugated anti-TLR4 (MTS 510; eBioscience) or PE-conjugated anti-CD36 (72-1; eBioscience). Then, cells were washed three times, and data were acquired on a FACSCalibur flow cytometer (BD Biosciences). The expression levels of F4/80, MHC class II, CD36, and TLR4 on macrophages were analyzed after gating on CD11bhighCD19neg cells by using the CellQuest software (BD Biosciences).

Western blot analysis and Abs

TGC-elicited peritoneal macrophages were lysed with Laemmli buffer (25 mM Tris [pH 8], 200 mM glycine, and 0.25% SDS) supplemented with antiproteases (complete EDTA free; Roche, Basel, Switzerland), 2 mM orthovanadate (Sigma-Aldrich), 1 mM PMSF (Sigma-Aldrich), and 1 mM NaF (Sigma-Aldrich). Protein extracts were sonicated and centrifuged (16,000 × g, 4°C, 15 min) to clarify supernatant. Proteins were quantified with the bicinchoninic acid technique (Interchim, Montluçon, France). Samples were heated to 95°C at 2 min, with loading buffer (Tris [pH 8], 2% SDS, 10 mM EDTA, 0.01% Bromophenol blue, 25 mM 1,4-dithio-dl-threitol, and 250 mM 2-ME), and 20-μg proteins were separated by 10% sodium dodecyl-PAGE. Proteins were then transferred on a 0.2-μm nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) in a transfer buffer (25 mM Tris [pH 8], 200 mM glycine, and 25% ethanol). Membranes were washed and blocked with nonfat milk 5% in TBST. Abs were incubated overnight at 4°C in 5% TBST milk. Membranes were washed three times (10 min) before the addition of a secondary HRP-coupled Ab in 5% TBST milk for 1 h at room temperature. Membranes were washed four times (15 min) with TBST. Then, ECL (Pierce, Rockford, IL) was added, and the film (Kodak, Rochester, NY) was exposed and developed by Hyper Processor (Amersham Biosciences). Monoclonal rabbit anti–phospho-Akt (serine 473) and polyclonal rabbit anti-Akt, anti–phospho-ERK1/2, and anti-ERK1/2 were from Cell Signaling Technology (Beverly, MA) and were used at 1/1000. Mouse monoclonal anti-mouse β-actin (Sigma-Aldrich) was used at 1/5000. Secondary polyclonal anti-mouse or -rabbit IgG HRP-coupled Abs were used at 1/5000 (Cell Signaling Technology).

PI3K activity assay

Macrophages were plated in 12-well plates. For a negative control, wortmannin (200 nM) was added 10 min before the stimulation with LPS. Cells were washed with PBS, and lysis buffer was added (20 mM Tris-HCl [pH 8], 138 mM NaCl, 2.7 mM KCl, 5% glycerol, 1 mM MgCl2, 1 mM CaCl2, 1% Nonidet P-40, 5 mM EDTA, 1 mM NaVO3, 20 μM leupeptin, 20 μM aprotinin, and 20 mM NaF). Then, cells were scraped, and lysates were collected and centrifuged (15,000 × g, 10 min, 4°C) to clarify supernatant. Five microliters of rabbit polyclonal Ab anti-PI3Kp85 (Millipore, Billerica, MA) was added, and tubes were placed on wheel 1 h at 4°C, then 5 mg protein A-Sepharose was added, and tubes were kept on wheel for 1 h at 4°C. Then, a centrifugation was performed to pellet the protein complex, which was washed once with the lysis buffer, twice with washing buffer 1 (0.1 M Tris-HCl [pH 7.4] and 0.5 M LiCl) and twice with washing buffer 2 (20 mM HEPES [pH 7.4] and 5 mM MgCl2) to finally resuspend the pellet with 40 μl washing buffer 2. PI3K activity was determined in presence of micelles of phosphatidylserine (2 mg/ml) and of phosphoinositol (1 mg/ml), 6 mM ATP, and 100 μCi/ml [γ-32P]ATP in a final volume of 60 μl, at 37°C under agitation. Reaction was stopped with 1 N 60 μl HCl, and lipids were extracted with 200 μM methanol/CHCl3 1:1. Samples were deposed on a TLC plate preactivated with a potassium oxalate solution. The plate was developed by chromatography in CHCl3/MeOH/H2O/NH4OH 45:35:7:3.5. PI3[32P] was detected by a phosphoimager and quantified by Imagequant, and an exposed film was revealed as well.

Statistical analysis

Results were expressed as means ± SEM. Statistical analyses were performed using Statview or Microsoft Excel software. For all the experiments, mean differences between groups were evaluated using Fisher’s protected least significant difference t test, and a value of p < 0.05 was considered statistically significant.

Results

Chronic E2 administration enhances the expression of inflammatory cytokine mRNAs by TGC-elicited peritoneal macrophages

To assess the influence of chronic E2 administration on peritoneal macrophages submitted to an in vivo activation process, ovariectomized female mice implanted with either placebo- or E2-releasing pellets received a single i.p. injection of TGC. As previously described (16), total peritoneal cells were collected 4 d later, allowing to determine the number of macrophages and their phenotypic characteristics using specific lineage and activation markers (4, 22). As shown in Fig. 1A, the absolute numbers of total peritoneal cell and macrophages, characterized by a double F4/80- and CD11b-positive staining, were both significantly decreased in the peritoneal collections from E2-treated mice, as compared with the placebo group. Neither the number of CD36- or TLR4-expressing macrophages nor the expression level of both molecules was affected by E2 treatment (Fig. 1B).

FIGURE 1.
FIGURE 1.

Chronic E2 treatment in vivo does not alter the expression of CD36 and TLR4 but enhances the mRNA expression of proinflammatory cytokines by TGC-elicited peritoneal macrophages. A, Flow cytometry analysis of CD11b and F4/80 expression level on TGC-elicited peritoneal cells from either placebo- or E2-treated mice. Total peritoneal cell number was determined with a Neubauer slide, and then the total number of macrophages was assessed according to CD11b and F4/80 staining. B, Flow cytometry analysis of CD36 and TLR4 surface expression on CD11b+ macrophages from either placebo- or E2-treated mice. Results are expressed as means ± SEM from two independent experiments, each including four mice. In dot plots, isotype controls are represented as thin and targets in bold. C, Cells from peritoneal cavity of placebo- or E2-treated mice, injected previously with TGC 2%, were plated. A culture of elicited peritoneal macrophages is obtained 2 h later, washing plates three times. Then cells were lysed, and the mRNA expression of cytokines was quantified by quantitative real-time PCR. Results are normalized according to GAPDH mRNA level, referring to results obtained from TGC-elicited peritoneal macrophages from placebo-treated mice group. Results are expressed as means ± SEM from four independent experiments (between 12 and 14 individual mice were considered in each group). *p < 0.05; **p < 0.005; ***p < 0.0001.

To further characterize the effect of E2 on the activation status of TGC-elicited macrophages, cytokines and iNOS mRNA abundance were determined in macrophages from either placebo- or E2-treated mice without any ex vivo stimulation. Whereas TNF-α mRNA abundance was only slightly enhanced, IL-1β, IL-6, and iNOS mRNA levels were strongly increased by E2 administration (Fig. 1C). In contrast, no change in IL-12 mRNA abundance was observed between placebo- and E2-treated mice (Fig. 1C). Altogether, these data indicate that E2 promotes constitutive expression of inflammatory mediators by TGC-elicited peritoneal macrophages.

E2 treatment inhibits the activation of the PI3K/Akt pathway in TGC-elicited macrophages

We next determined whether E2 administration in vivo influenced the capacity of TGC-elicited macrophages to produce inflammatory mediators following their activation by LPS ex vivo. As shown in Fig. 2A, E2 treatment resulted in a significant increase in IL-1β, IL-6, and iNOS mRNA abundance in LPS-activated macrophages, as observed in basal conditions. This was confirmed at the protein level by analyzing the effect of E2 on the LPS-induced production of IL-1β (+55%; p < 0.05) and IL-6 (+93%; p < 0.0001) (Fig. 2B). In contrast, the levels of IL12p40 and TNF-α gene expression were not significantly altered by E2 in LPS-activated macrophages (data not shown).

FIGURE 2.
FIGURE 2.

In vivo E2 treatment enhances the LPS-induced expression of cytokines by TGC-elicited macrophages through the modulation of PI3K activity. TGC-elicited macrophages from placebo- or E2-treated mice were stimulated by LPS (20 ng/ml) to analyze the expression of proinflammatory cytokines. A, Cells were stimulated 4 h in the presence of LPS to quantify mRNA amount of IL-1β, IL-6, and iNOS. The mRNA expression was quantified by quantitative real-time PCR. Results are normalized according to GAPDH mRNA level, referring to results obtained from TGC-elicited peritoneal macrophages stimulated by LPS from placebo-treated mice group. Results are expressed as means ± SEM from two independent experiments, eight mice per group. B, Cells were incubated with 50 nM wortmannin or 1/10,000 vehicle (DMSO) 1 h. Then they are stimulated by LPS (20 ng/ml) in the presence of wortmannin or DMSO, respectively. Culture supernatants were collected 24 h later. IL-1β and IL-6 were quantified by specific ELISA. Nine individual mice were considered in each group.*p < 0.05; **p < 0.005; ***p < 0.001.

It has been shown that the PI3K/Akt pathway negatively regulates the expression of numerous inflammatory cytokines by TLR4-stimulated APCs, including dendritic cells, monocytes, and peritoneal macrophages (9, 12, 14, 15). We therefore examined whether the inhibition of the PI3K/Akt signaling pathway by E2 leads to a higher level of proinflammatory cytokine production. To this end, TGC-elicited macrophages from placebo or E2-treated mice were incubated in presence of the pharmacological inhibitor of PI3K family wortmannin 1 h before and during LPS stimulation. PI3K activity inhibition strongly increased LPS-induced IL-1β (+85%; p < 0.05) and IL-6 (+109%; p < 0.0001) production by macrophages from placebo-treated mice (Fig. 2B), attesting that activation of the PI3K pathway inhibited the TLR4-dependent expression of proinflammatory cytokines by TGC-elicited peritoneal macrophages. In contrast, wortmannin did not enhance further proinflammatory cytokine production in macrophages from E2-treated mice (Fig. 2B), suggesting that this pathway was already inactivated in these cells in agreement with our previous work on resident peritoneal macrophages (9).

To test this assumption, TGC-elicited macrophages from either placebo- or E2-treated mice were harvested for in vitro PI3K activity measurement, and for the phosphorylation status of Akt, a direct target of PI3K activity through the generation of phosphatidylinositol-(3,4,5)-triphosphate and activation of PDK1 at the plasma membrane. Fig. 3A and 3B shows that E2 treatment in vivo led to a significant constitutive inhibition of both PI3K activity and of Akt phosphorylation at the serine 473 site in TGC-elicited macrophages. This effect was maintained following TLR4 activation as shown by the reduced Akt phosphorylation in TGC-elicited macrophages from E2-treated mice activated with LPS (Fig. 3B). Interestingly, E2 administration simultaneously led to an increased phosphorylation state of ERK1/2 in LPS-activated macrophages (Fig. 3C). Furthermore, wortmannin strongly enhanced the constitutive and LPS-induced phosphorylation of ERK1/2 (Fig. 3C), thus demonstrating that the PI3K/Akt pathway acts as a negative regulator of MAPK phosphorylation in TGC-elicited macrophages in perfect agreement with previous observations from transgenic mice with reduced PI3K activity because of the invalidation of the p85α regulatory subunit (14). Altogether, these data establish that in vivo exposure of macrophages to estrogens downregulates the PI3K/Akt pathway, which contributes to the increased production of proinflammatory cytokines by macrophages in response to TLR4 activation.

FIGURE 3.
FIGURE 3.

In vivo E2 treatment alters the PI3K/Akt pathway in TGC-elicited macrophages. A, The p85 PI3K subunit was immunoprecipitated from TGC-elicited macrophages of placebo- or E2-treated mice to analyze PI3K activity. In the negative control, cells were preincubated with 200 nM wortmannin for 10 min, and then cells were stimulated by LPS (20 ng/ml) for 10 min. PI3K activity was quantified as the capacity of immunoprecipitated complex to convert phosphatidylinositol into PI3[32P]. Cell lysates and immunoprecipitated complexes were analyzed by Western blot to control the amounts of p85 (α,β) before (IB) and after (IP➢IB) immunoprecipitation. Results were normalized to the activity in placebo group and expressed as means ± SEM from three independent experiments. *p < 0.05. B, TGC-elicited macrophages were stimulated by LPS (20 ng/ml) for 10 or 30 min to analyze Akt phosphorylation (serine 473 site) by Western blot. C, TGC-elicited macrophages were stimulated by LPS (20 ng/ml) 30 min to analyze Akt (serine 473 site) and ERK1/2 phosphorylation by Western blot. Macrophages were preincubated with wortmannin (50 nM) or 0.1% DMSO for 2 h to explore the regulation of MAPK activation by the PI3K/Akt pathway. Data are representative of at least three separate experiments each including three to five individual mice.

Endogenous estrogens are sufficient to mediate a proinflammatory effect on TGC-elicited peritoneal macrophages

The experiments demonstrating the proinflammatory effect of in vivo exposure to estrogens on both resident and TGC-elicited peritoneal macrophages have been conducted with stable plasma E2 concentrations in the range of those observed during estrus or early pregnancy in the mouse. Thus, we then wondered whether endogenous estrogens influenced the activation status of TGC-elicited macrophages by comparing the properties of cells from ovariectomized and sham-operated intact female mice. No significant difference was observed in the number of total peritoneal cells and macrophages between ovariectomized or sham-operated mice (data not shown). The expression level of surface markers by macrophages was similar in both groups. As shown in Fig. 4A, unstimulated macrophages from sham-operated mice were characterized by a higher expression of IL-1β (+40%; p < 0.05), IL-6 (+283%; p < 0.001) and iNOS (+112%; p < 0.001) mRNA, and in a lesser extent of TNF-α mRNA (+36.9%; p = 0.035; data not shown). Likewise, endogenous estrogens led to a significant decrease in Akt phosphorylation in TGC-elicited macrophages without any activation ex vivo (data not shown).

FIGURE 4.
FIGURE 4.

Endogenous estrogens exert a proinflammatory effect on TGC-elicited peritoneal macrophages. TGC-elicited macrophages from OVX or sham-operated mice were stimulated by LPS (20 ng/ml) to analyze the expression of proinflammatory cytokines. A, Cells were stimulated 4 h in presence of LPS to quantify the mRNA amount of IL-1β, IL-6, and iNOS. The mRNA expression was quantified by quantitative real-time PCR. Results are normalized according to GAPDH mRNA level, referring to results obtained from TGC-elicited peritoneal macrophages from OVX mice group. Results are expressed as means ± SEM from two independent experiments, eight mice per group. B, Cells were incubated with 50 nM wortmannin or 1/10,000 vehicle (DMSO) 1 h. Then they are stimulated by LPS (20 ng/ml) in the presence of wortmannin or DMSO, respectively. Culture supernatants were collected 24 h later. IL-1β and IL-6 were quantified by specific ELISA. Nine individual mice were considered in each group.*p < 0.05; ***p < 0.001. OVX, ovariectomized.

Following ex vivo stimulation with LPS, the respective production of IL-1β and IL-6 was significantly enhanced by endogenous estrogens, as assessed at both mRNA and protein levels (Fig. 4). Furthermore, supporting the involvement of the PI3K/Akt pathway in this effect, IL-1β and IL-6 productions were no longer enhanced by endogenous estrogens when cells were incubated in presence of wortmannin (Fig. 4B). Altogether, these observations extent to endogenous levels the ability of estrogens to influence the activation status of TGC-elicited macrophages.

LysM-Cre efficiently mediates depletion of floxed ERα in macrophages

Although the proinflammatory action of E2 on peritoneal macrophages has been demonstrated to be mediated by ERα, whether estrogens exert this effect by targeting this nuclear receptor in macrophages remained to be determined. To this end, we developed a new mouse model characterized by the specific abolition of ERα expression in monocytes/macrophages and neutrophils using the Cre/loxP technology. ERαflox/flox mice, carrying the ERα gene in which the second coding exon is flanked by loxP sites, were crossed with mice expressing the Cre recombinase under the control of the LysM promoter (Fig. 5A). Southern blot analysis showed a Cre-mediated recombination of the floxed ERα gene in tissues particularly rich in macrophages, namely the lung and the spleen (Fig. 5B). Moreover, deletion of ERα gene was almost complete in purified TGC-elicited macrophages from LysM-Cre+ mice (Fig. 5B).

FIGURE 5.
FIGURE 5.

Evidence for efficient deletion of ERα-floxed allele in macrophages from LysM-Cre+ERαflox/flox mice. A, Restriction map of the floxed ERαL2/L2 targeting construct and the Cre-generated deletion allele. The Cre-mediated deletion event eliminates both the exon 2 of ERα and the additional BamHI site inserted together with the 5′ LoxP site. BamHI-digested DNA was hybridized with a specific DNA probe located downstream from the exon 2 as indicated; 8.8- and 4.4-kb bands represent the deleted allele (KO) and the targeted allele (Floxed), respectively. B, Southern blot analysis of genomic tail DNA and DNA obtained from various tissues of LysM-Cre+ERαflox/flox mice as indicated. C, PECs were used as a source of macrophages and were obtained from mice injected i.p. 4 d before with 1 ml 3% TGC. PECs were incubated on tissue culture plates, and adherent cells were recovered and stained with CD11b and F4/80 mAbs for flow cytometry analysis. D, RNA was isolated from purified macrophages and reverse transcribed. Real-time PCR analysis of ERα transcripts was then performed and normalized to hypoxanthine phosphoribosyltransferase transcripts abundance analyzed in parallel. KO, knockout; PECs, peritoneal exudate cells.

To definitely validate ERα deletion in macrophages from LysM-Cre+ERαflox/flox mice, TGC-elicited macrophages were harvested and ERα mRNA abundance was determined by quantitative RT-PCR. These cell preparations obtained after a 2 h adhesion step contained 87–89% TGC-elicited macrophages highly expressing CD11b and F4/80 (Fig. 5C). The LysM-Cre genotype did not alter the proportion of macrophages, but ERα expression was dramatically reduced (−85%) in macrophages from LysM-Cre+ as compared with wild-type ERαflox/flox mice (Fig. 5D).

ERα deficiency in macrophages abolishes the proinflammatory effect of E2

We then investigated whether the cell-specific abrogation of ERα expression in myeloid cells altered the action of E2 on macrophage inflammatory responses. As expected, in control ERαflox/flox mice, chronic E2 administration significantly reduced the number of TGC-elicited macrophages in the peritoneal cavity (−50%; p < 0.001), and increased the abundance of cytokines and iNOS mRNA in these cells, without further ex vivo stimulation (IL-6 and iNOS) or following LPS activation: IL-1β (+147%; p < 0.0001), IL-6 (+127%; p < 0.05) and iNOS (+533%; p < 0.005) (Fig. 6A). In striking contrast, these actions of E2 on macrophage number (−6%, NS) and functions (Fig. 6A) were totally abolished in LysM-Cre+ERαflox/flox mice. As expected, the results obtained at the protein level (Fig. 6B) paralleled those at the mRNA level (Fig. 6A), further emphasizing the crucial role of ERα signaling in monocytes/macrophages in the proinflammatory effect of estrogens.

FIGURE 6.
FIGURE 6.

Proinflammatory effect of E2 is abolished in TGC-elicited macrophages from LysM-Cre+ERαflox/flox mice. TGC-elicited peritoneal macrophages from OVX mice treated with placebo or E2 were stimulated by LPS to analyze the expression of proinflammatory cytokines and iNOS expression by quantitative real-time PCR or ELISA. A, TGC-elicited macrophages were stimulated 4 h in presence of LPS (20 ng/ml) to quantify the mRNA amount of IL-1β, IL-6, and iNOS. Results are normalized according to GAPDH mRNA level, referring to results obtained from TGC-elicited peritoneal macrophages from placebo-treated LysM-Cre-ERαflox/flox mice group. Results are expressed as means ± SEM from an experiment; four to five mice per group. B, TGC-elicited peritoneal macrophages were stimulated 24 h in presence of LPS (20 ng/ml) to quantify IL-1β and IL-6 in supernatants by specific ELISA. Results are expressed as means ± SEM from an experiment; four to five mice per group. *p < 0.05; **p < 0.005; ***p < 0.001.

As i.p. injection of TGC induces a sterile peritonitis which involves a coordinate and time-dependent recruitment of inflammatory leukocytes, including neutrophils and inflammatory monocytes/macrophages, the eventual involvement of ERα-expressing neutrophils in the proinflammatory action of E2 on TGC-elicited macrophages cannot be ruled out. Thus, we finally examined the influence of E2 on resident macrophages from LysM-Cre+ERαflox/flox. Indeed, in absence of TGC-induced inflammatory process, flow cytometry analysis did not identify any neutrophils in the peritoneal cavity, irrespective of E2 treatment (data not shown). As expected, chronic E2 administration in vivo significantly enhanced the production of IL-1β (+138%; p < 0.005) and IL-6 (+71%; p < 0.05) by resident macrophages from control ERαflox/flox mice following a 24 h activation with LPS ex vivo (Fig. 7). By contrast, this effect of E2 was no longer observed in LysM-Cre+ERαflox/flox mice, demonstrating that estrogens exert their proinflammatory effects through the direct activation of ERα signaling in macrophages.

FIGURE 7.
FIGURE 7.

Proinflammatory effect of E2 is abolished in resident peritoneal macrophages from LysM-Cre+ERαflox/flox mice. Resident peritoneal macrophages from OVX mice treated with placebo or E2 were stimulated 21 h in presence of LPS (20 ng/ml) to quantify IL-1β and IL-6 in supernatants by specific ELISA. Results are expressed as means ± SEM from two independent experiments, five to seven mice per group. *p < 0.05; **p < 0.005. nd, nondetectable.

Discussion

In line with the recently identified proinflammatory effect of in vivo E2 treatment on murine resident peritoneal macrophages and microglial cells (9, 10), the current study demonstrates that chronic exposure to estrogens enhances the production of various proinflammatory mediators by TGC elicited (i.e., “inflamed” peritoneal macrophages), contrasting with the anti-inflammatory effect of a short-term exposition to the hormone in vitro, previously reported in the same cell types (6–8). Altogether, these observations clearly demonstrate that the experimental results obtained with a short-term E2 treatment in vitro are not predictive of the effect of a long-term exposure to estrogens in vivo on macrophage functions.

Whereas the proinflammatory effect of chronic E2 on resident peritoneal macrophages could be evidenced only after ex vivo stimulation (9), we now recognized the proinflammatory effect of E2 in TGC-elicited macrophages without any further ex vivo stimulation, definitively demonstrating that this sex hormone altered the ongoing in vivo inflammatory process as well. Indeed, IL-1β, IL-6, and iNOS mRNA contents and TNF-α, to a lesser extent, were significantly increased in macrophages by in vivo chronic E2, giving evidence of their activated status. Thus, these observations strongly suggest that the proinflammatory influence of chronic exposure to estrogens on macrophages, first demonstrated following their activation with LPS ex vivo, applies also to in vivo inflammatory situations.

Furthermore, we found that in vivo exposure to E2 significantly alters the signaling events occurring in TGC-activated macrophages, whereas no influence was observed on TLR4 surface expression. The molecular events induced by TLR4-mediated stimulation are now well identified, and the current study reinforces the evidence that the inhibition of the PI3K/Akt upregulates the expression of inflammatory cytokines in response to LPS, as previously reported in cells of the monocyte/macrophage lineage (9, 11, 12, 14, 15). Indeed, we showed that PI3K activity and the phosphorylation status of the downstream target Akt were markedly reduced in TGC-elicited macrophages from E2-treated mice, demonstrating a specific effect of estrogens on this crucial signaling pathway. Moreover, we definitively demonstrate that the inhibitory effect of E2 treatment on the PI3K/Akt pathway contributes in a large part to the increased expression of IL-1β and IL-6 in response to LPS, in line with previous results in resident peritoneal macrophages (9).

Interestingly, previous experimental studies revealed that, besides the enhancing effect on inflammatory mediator expression, inhibition of the PI3K/Akt pathway resulted in the LPS-induced activation of MAPKs and various transcription factors including NF-κB in monocytic cells (11–14). We previously demonstrated that the transcriptional activity of nuclear NF-κB p65 was significantly enhanced in peritoneal resident macrophages from E2-treated mice as a possible consequence of the reduced PI3K activity in these cells (9). However, Luyendyk et al. (14) recently proposed that the PI3K/Akt pathway negatively regulates LPS induction of inflammatory cytokines in TGC-induced peritoneal macrophages mainly by inhibiting MAPK activation. According to this hypothesis, E2 concomitantly inhibited the PI3K/Akt pathway and enhanced ERK1/2 phosphorylation in our experimental settings, suggesting that this latter signaling event contributes to their action on gene expression in TGC-elicited macrophages.

Noteworthy, E2 only exerted a weak effect on TNF-α and did not influence IL-12 gene expression in the present work, although both cytokines have been previously demonstrated to be controlled by the PI3K/Akt pathway in monocyte/macrophages (13–15). However, these data are consistent with our previous findings in resident peritoneal macrophages because E2, but also PI3K inhibition, exerted a lesser effect on TNF-α than on IL-1β, IL-6, and iNOS expression in this model (9). Similarly, genetic invalidation of the PI3K p85α regulatory subunit was reported to exert a more pronounced enhancing effect on IL-6 than on TNF-α production by TGC-elicited macrophages (14). Moreover, this trend may also result from discrepancies in the experimental settings between our work and previous ones, especially regarding cell types and the LPS concentrations used for macrophage activation ex vivo: 20 ng/ml in the current study, initially determined as optimal to study the action of E2 on IL-1β and IL-6 (9), versus at least 1 μg/ml (12–15).

It is well established that macrophages, as most immune cells, express ERα, and we recently demonstrated that the activation of this nuclear receptor in vivo mediates the proinflammatory effect of estrogens on resident peritoneal macrophages (9). One important remaining question was whether E2 exerted its proinflammatory actions through a direct effect on macrophages or by indirect mechanisms involving other cellular targets. Indeed, in addition to resident macrophages, peritoneal exsudates contain numerous immune cells, such as lymphocytes or NK cells, which could represent the pivotal target of E2 for the modulation of macrophage activation (23, 24). Furthermore, the peritoneal inflammation induced by TGC results in the sequential influx of inflammatory cells including neutrophil and eosinophil polymorphonuclear cells, although inflammatory macrophages, also called TGC-elicited macrophages, represent the main inflammatory population 72 h after TGC injection (25). To definitely determine whether E2 directly influences macrophages and their functions in vivo, we generated a mouse model allowing a specific invalidation of the ERα gene in granulocytes through a CreLox strategy under the control of the LysM promoter (17, 26). Noteworthy, this cell-specific gene inactivation totally abolished the effect of E2 on the expression of inflammatory mediators by both resident and TGC-elicited peritoneal macrophages, strongly suggesting that estrogens directly target ERα-expressing macrophages. Indeed, it seems unlikely that the proinflammatory effect of E2 on resident macrophages results from the targeting of neutrophils, because these cells are not recruited in the peritoneal cavity apart from inflammatory situations.

Besides the enhancing effect of E2 on macrophage inflammatory status, we found that the hormonal treatment significantly decreased the number of TGC-elicited peritoneal macrophages observed 4 d after peritonitis induction. This latter observation suggest that, in parallel to their proinflammatory effect at the cellular level, estrogens could prevent the recruitment of immune cells in the site of inflammation, as previously proposed in various pathophysiological situations, such as atherosclerosis or autoimmune diseases (1, 27, 28). Furthermore, our results in LysM-Cre+ERαflox/flox mice indicate that the decreasing effect of E2 treatment on the number of TGC macrophages, which could result from changes in macrophage recruitment and/or survival in the inflamed peritoneal cavity, requires ERα expression in myeloid cells. However, whether the modulating action of E2 on macrophages occurs during monocyte/macrophage differentiation, or specifically during the TGC-induced in vivo inflammatory process, remains to be characterized.

In our experimental settings, the chronic s.c. administration of E2 leads to stable plasma hormonal concentrations averaging 0.3 nM, a level reached during the peak of the estrus cycle. Interestingly, the comparison of sham-operated mice to ovariectomized mice demonstrates that, whereas no change in the number of peritoneal macrophages is observed, endogenous estrogens significantly promote the expression of cytokines by TGC-elicited macrophages. Furthermore, as described above for E2 treatment, endogenous estrogens increased the expression of both IL-1β and IL-6 induced by TLR4 activation of TGC-elicited macrophages ex vivo, and this effect was totally abolished in presence of wortmannin. Accordingly, we found an inhibition of Akt phosphorylation status in TGC elicited from sham-operated mice, demonstrating that endogenous estrogens also downmodulate the PI3K pathway. Thus, the physiological cyclic changes of endogenous estrogens levels are sufficient to influence macrophage functions, whereas higher E2 concentrations in the range of those observed during estrus or early gestation (29) are required to prevent the immune cell recruitment in the peritoneum in response to TGC.

In vivo estrogens have been recently recognized to enhance the capacity of murine microglial cells (10) and dendritic cells (30, 31) to produce Th1 polarizing cytokines. Thus, it is tempting to speculate that these specific effects of estrogens on professional APCs largely contribute to enhance the effector functions of Ag-specific CD4+ T cells (24) and invariant NK T lymphocytes (23), characterized by a strong increase in IFN-γ production in these lymphocyte subpopulations. The present study provides further evidence that both physiological endogenous estrogens levels as well as exogenous E2 promote the inflammatory status of macrophages and thus the ability to mount inflammatory and immune responses. The characterization of these various actions of estrogens should allow deciphering gender impact as well as the influence of puberty, pregnancy, or menopause and its associated hormonal therapy on various pathophysiological situations where the inflammatory innate immune system plays a role.

Acknowledgments

We thank F. Lenfant and H. Laurell for helpful discussion. We also thank the animal facilities staff and Y. Barreira (Service de Zootechnie, Institut Fédératif de Recherche 150) and J.J. Maoret (Molecular Biology Platform, Institut Fédératif de Recherche 150) for technical assistance.

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by a grant from the Agence Nationale de la Recherche (Grant 06-Physio-010), the Institut National de la Santé et de la Recherche Médicale, the European Vascular Genomics Network Grant 503254, the Fondation de France, the Université de Toulouse, the Association pour la Recherche sur la Sclérose en Plaques, and the Conseil Régional Midi-Pyrénées. P.G. was supported by a grant (Contrat Interface) from the Institut National de la Santé et de la Recherche Médicale.

  • Abbreviations used in this paper:

    E2
    17β-estradiol
    ER
    estrogen receptor
    iNOS
    inducible NO synthase
    KO
    knockout
    nd
    nondetectable
    OVX
    ovariectomized
    PECs
    peritoneal exudate cells
    TGC
    thioglycolate.

  • Received July 22, 2009.
  • Accepted May 6, 2010.

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