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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC 2017 March 15.
Published in final edited form as:
PMCID: PMC4779725
NIHMSID: NIHMS752465

An M1-like macrophage polarization in decidual tissue during spontaneous preterm labor that is attenuated by rosiglitazone treatment1

Abstract

Macrophages are implicated in the local inflammatory response that accompanies spontaneous preterm labor/birth; however, their role is poorly understood. We hypothesized that decidual macrophages undergo an M1 polarization during spontaneous preterm labor and that PPARγ activation via rosiglitazone would attenuate the macrophage-mediated inflammatory response, preventing preterm birth. Herein, we show that: 1) decidual macrophages undergo an M1-like polarization during spontaneous term and preterm labor; 2) M2-like macrophages are more abundant than M1-like macrophages in decidual tissue; 3) decidual M2-like macrophages are reduced in preterm pregnancies compared to term pregnancies, regardless of the presence of labor; 4) decidual macrophages express high levels of TNF and IL12, but low levels of PPARγ, during spontaneous preterm labor; 5) decidual macrophages from women who underwent spontaneous preterm labor display plasticity by M1↔M2 polarization in vitro; 6) incubation with rosiglitazone reduces the expression of TNF and IL12 in decidual macrophages from women who underwent spontaneous preterm labor; and 7) treatment with rosiglitazone reduces the rate of LPS-induced preterm birth and improves neonatal outcomes by reducing the systemic pro-inflammatory response in B6 mice and down-regulating mRNA and protein expression of NFκB, TNF, and IL10 in decidual and myometrial macrophages. In summary, we demonstrated that decidual M1-like macrophages are associated with spontaneous preterm labor, and that PPARγ activation via rosiglitazone can attenuate the macrophage-mediated pro-inflammatory response, preventing preterm birth and improving neonatal outcomes. These findings suggest that the PPARγ pathway is a new molecular target for future preventative strategies for spontaneous preterm labor/birth.

INTRODUCTION

Preterm birth, or birth prior to 37 weeks of gestation, is the leading cause of perinatal morbidity and mortality worldwide (1). In 2013, 11.39% of all births in the United States were diagnosed as preterm (2). Preterm neonates are at an increased risk for short- and long-term morbidity, and prematurity represents a substantial burden for society and the healthcare system (3, 4). About 70% of all preterm births occur after spontaneous preterm labor (5). Therefore, it is essential to determine the mechanisms implicated in spontaneous preterm labor and to develop therapies to prevent this syndrome.

Inflammation is implicated in the physiological and pathological processes of labor for term and preterm gestations (6-9). Pathological inflammation can result from the activation of innate (10-15) or adaptive (16-19) immunity. Among innate immune cells, macrophages play a central role throughout pregnancy (20-26) and seem to participate in the mechanisms implicated in spontaneous preterm labor (27-30). Macrophages reside in human decidual tissue (27, 31), where their proportions are greater in term than in preterm gestations (32). Similarly, the proportion of murine decidual macrophages is higher in term than in mid gestation (29). Macrophage density is even greater in decidua from women who underwent term and preterm labor than in women who delivered at term without labor (27). Macrophage involvement in preterm labor was established when LPS-induced preterm birth was rescued by depletion of F4/80+ macrophages (33). Collectively, this evidence suggests a role for pro-inflammatory macrophages in the process of labor at term and preterm; yet, the functional and phenotypic properties of these innate immune cells are poorly understood.

The classical paradigm shows the existence of two distinct activated and polarized macrophage subsets: pro-inflammatory (M1) and anti-inflammatory (M2) (34-38). M1 macrophages were the first to be described and thus considered “classically” activated (34, 39). M1 macrophages are characterized by expression of IFNγ, IL12, and IL23, production of NO and reactive oxygen species, and promotion of Th1 responses (34, 36, 40-43). M2 macrophages are considered “alternatively” activated (34, 44-46) and exhibit anti-inflammatory activity through production of IL10 and up-regulation of arginase-1 (34, 36, 42, 47-50). Recently, it was demonstrated that M1- and M2 macrophages represent only the extremes of the macrophage activation spectrum (51). However, the M1/M2 macrophage paradigm provides a useful framework for selected immune-related diseases (52).

The current hypothesis states that decidual M2 macrophages are implicated in supporting maternal-fetal tolerance throughout pregnancy (25, 26, 37, 53-55). At term pregnancy, decidual macrophages seem to have a pro-inflammatory phenotype since they express pro-inflammatory cytokines during labor (31, 56, 57). Yet, whether decidual macrophages undergo an M1 polarization during spontaneous preterm labor is unknown. Herein, we hypothesized that decidual macrophages undergo an M1 polarization during spontaneous term and preterm labor, and that targeting these cells could prevent preterm birth. In search for a therapeutic drug that exerts anti-inflammatory activity in macrophages, we evaluated rosiglitazone (RSG), a selective peroxisome proliferator-activated receptor (PPAR)γ agonist (58), which has been shown to induce an M2 macrophage polarization by activating the PPARγ pathway (59). PPARγ activation suppresses gene transcription by interfering with signal transduction pathways, such as the NFκB, STAT, and AP1 pathways (60-62). PPARγ activation has been suggested as a therapeutic intervention for preventing preterm birth (63) since treatment with 15-deoxy-Δ12,14-prostaglandin J2 compound, a PPARγ agonist (64, 65), delays LPS-induced preterm birth (66). However, whether PPARγ activation reduces the pro-inflammatory phenotype of M1 macrophages in decidual tissue, preventing preterm labor/birth, has not been investigated.

The aims of this study were: 1) to determine whether decidual macrophages undergo an M1 polarization during spontaneous term and preterm labor; 2) to explore whether decidual macrophages from women who underwent spontaneous preterm labor display M1↔M2 plasticity in vitro; 3) to evaluate whether PPARγ activation in vitro through incubation with RSG reduces the expression of TNF in decidual macrophages from women who underwent spontaneous preterm labor; 4) to determine whether treatment with RSG reduces the rate of LPS-induced preterm birth and improves neonatal outcomes in B6 mice; 5) to determine whether treatment with RSG reduces the systemic pro-inflammatory response in pregnant B6 mice injected with LPS; and 6) to investigate whether treatment with RSG alters the mRNA and protein expression of NFκB, TNF, and IL10 in decidual and myometrial macrophages of pregnant B6 mice injected with LPS.

MATERIALS AND METHODS

Human subjects, clinical specimens, and definitions

Human placental basal plate and chorioamniotic membrane samples were obtained at the Perinatology Research Branch, an intramural program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U. S. Department of Health and Human Services (NICHD/NIH/DHHS), Wayne State University (Detroit, MI, USA), and the Detroit Medical Center (Detroit, MI, USA). The collection and utilization of biological materials for research purposes were approved by the Institutional Review Boards of NICHD and Wayne State University. All participating women provided written informed consent. The study groups included women who delivered at term with (TIL) or without (TNL) spontaneous labor, or preterm with (PTL) or without (PTNL) spontaneous labor. The demographic and clinical characteristics of the four groups of women are shown in Table I - III. Table I and andIIII include samples used for extracellular and intracellular immunophenotyping, and Table III includes samples used for in vitro experimentation. Labor was defined by the presence of regular uterine contractions at a frequency of at least two contractions every 10 minutes with cervical changes resulting in delivery (67). In each case, several tissue sections of the chorioamniotic membranes, umbilical cord, and placental disc were evaluated for acute or chronic chorioamnionitis, according to published criteria (68, 69), by pathologists who had been blinded to the clinical outcomes. Patients with neonates having congenital or chromosomal abnormalities were excluded.

Table I
Demographics and clinical characteristics of samples used for extracellular immunophenotyping
Table II
Demographics and clinical characteristics of decidua basalis samples used for intracellular immunophenotyping
Table thumbnail
Table III Demographics and clinical characteristics of samples used for in vitro experiments

Isolation of decidual leukocytes

Decidual leukocytes were isolated from decidual tissue of each study group (TNL, TIL, PTNL and PTL) as previously described (70). Briefly, the decidua basalis was collected from the basal plate of the placenta, and the decidua parietalis was separated from the chorioamniotic membranes. Decidual tissue was homogenized using a gentleMACS Dissociator (Miltenyi Biotec, San Diego, CA, USA) in StemPro® Accutase® Cell Dissociation Reagent (Life Technologies, Grand Island, NY, USA). Homogenized tissues were incubated for 45 min at 37°C with gentle agitation. After incubation, tissues were washed in ice-cold 1X PBS (Life Technologies) and filtered through a 100μm cell strainer (Falcon™, Corning Life Sciences, Inc., Durham, NC, USA). The resulting cell suspension was centrifuged at 300 × g for 10 min at 4°C. Decidual leukocytes were then separated by density gradient using the reagent Ficoll-Paque Plus (GE Healthcare Biosciences, Uppsala, Sweden), following the manufacturer's instructions. Cells collected from the mononuclear layer of the density gradient were washed with 1X PBS and immediately used for immunophenotyping.

Immunophenotyping of decidual macrophages

Isolated decidual mononuclear cells were incubated with 20μl of human FcR blocking reagent (Miltenyi Biotec) in 80μl of stain buffer (Cat # 554656, BD Biosciences, San Jose, CA, USA) for 10 min at 4°C. The cells were then incubated with extracellular fluorochrome-conjugated anti-human monoclonal antibodies for 30 min at 4°C in the dark (Supplementary Table I). After extracellular staining, the cells were washed with 1X PBS to remove excess antibody, re-suspended in 0.5 ml stain buffer, and acquired using the BD™ LSR II Flow Cytometer (BD Biosciences) and BD™ FACSDiva 6.0 software (BD Biosciences). For intracellular immunophenotyping, following extracellular staining, the cells were fixed and permeabilized using the BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (BD Biosciences). Next, the cells were washed with 1X BD Perm/Wash™ Buffer (BD Biosciences), re-suspended in 50μL of the same buffer, and stained with intracellular antibodies for 30 min at 4°C in the dark (Supplementary Table I). Finally, the stained cells were washed with 1X BD Perm/Wash™ Buffer, re-suspended in 0.5 ml stain buffer, and acquired using the BD™ LSR II Flow Cytometer and BD™ FACSDiva 6.0 software. The analysis and figures were performed using the FlowJo software version 10 (FlowJo, LLC, Ashland, OR, USA). ICAM3 is down-regulated in decidual macrophages compared to blood monocytes (37, 53); therefore, we characterized M1 and M2 macrophages within the ICAM3 gate, as shown in Figure 1A. Several M1 and M2 markers were included in the immunophenotyping (Supplementary Table 1). Identification of M1 (TNF, iNOS, and IL12 (71)) and M2 (IL10 (71)) mediators, as well as PPARγ (59, 72), was performed in macrophages from the decidua basalis (CD45+CD14+ cells), as shown in Figure 2A.

Figure 1
Immunophenotyping of M1- and M2-like macrophages in decidual tissues
Figure 2
Immunophenotyping of M1 and M2 macrophage mediators in decidual macrophages

Macrophage isolation from decidual tissue and in vitro polarization

Decidual leukocytes were isolated from the decidua basalis of women who underwent PTL, as previously described (70). Macrophage isolation was performed using CD14 microbeads (Miltenyi Biotec), following the manufacturer's instructions. Briefly, isolated leukocytes were labelled with CD14 microbeads and separated by positive selection using MS columns (Miltenyi Biotec) and a magnetic MACS separator (Miltenyi Biotec). Decidual macrophages (CD14+ cells) were then washed with MACS buffer [BSA 0.5% (Sigma-Aldrich, St. Louis, MO, USA), EDTA 2mM (Life Technologies), and 1X PBS] at 300 × g for 5 min. The resulting macrophage pellet was re-suspended in 1ml of RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% of penicillin/streptomycin antibiotic (Life Technologies), hereafter referred to as “supplemented RPMI,” and cells were counted using an automatic cell counter (Cellometer Auto 2000; Nexcelom, Lawrence, MA, USA). Decidual macrophages were placed into a 24-well plate (Fisher Scientific, Waltham, MA, USA) at a density of 1×106 cells/ml of supplemented RPMI medium and cultured at 37°C with 5% CO2, which was designated as Day 0. M1 macrophage polarization was induced by adding 5ng/ml of GM-CSF (R&D Systems, Minneapolis, MN, USA) on Day 0, and then 10ng/ml of LPS (Escherichia coli 0111:B4; Sigma-Aldrich) and 5ng/ml of IFNγ (Biolegend, San Diego, CA, USA) on Day 3 (37) (Figure 3A). Alternatively, M2 macrophage polarization was induced by adding 50ng/ml of M-CSF (R&D Systems) on Day 0, and then 25ng/ml of IL4 (Biolegend) and 25ng/ml of IL13 (Biolegend) on Day 3 (37). M2 macrophage polarization was also induced by adding 50ng/ml of M-CSF (R&D Systems) and 50ng/ml of IL10 (Biolegend) on Day 0 (37). Decidual macrophages were harvested for immunophenotyping or immunofluorescence before (Day 0) or after macrophage polarization (Day 6).

Figure 3
In vitro M1↔M2 polarization of decidual macrophages from women who underwent spontaneous preterm labor

Confocal Microscopy

Decidual macrophages were placed into a 4-well Lab-Tek® chamber slide (Thermo Fisher Scientific, Rochester, NY, USA), fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and washed with 1X PBS. Next, macrophages were permeabilized with 0.25% Triton X-100 (Promega Corporation, Madison, WI, USA) for 5 min, and the non-specific antibody interactions were blocked using Dako® Protein Block Serum-Free Solution (Cat # X0909, Dako Corp., Carpinteria, CA, USA) for 30 min at room temperature. Chamber slides were then incubated with the following antibodies: mouse anti-human CD68 biotin-conjugated (Clone # 815CU17, eBioscience, San Diego, CA, USA), mouse anti-human CD80 (Clone # 2A2, Abcam, Cambridge, MA, USA,), and rabbit anti-human CD209 (Abcam) at 4°C overnight. Following incubation, chamber slides were washed with 1X PBS containing 0.1% Tween® 20 (Sigma-Aldrich). Secondary goat anti-rabbit IgG-Alexa Fluor 647 (Invitrogen-Molecular Probes, Eugene, OR, USA), goat anti-mouse IgG-Alexa Fluor 555 (Invitrogen-Molecular Probes), and streptavidin conjugated with Alexa Fluor 488 (Invitrogen-Molecular Probes) were added, and chamber slides were incubated for 1h at room temperature. Chamber slides were then washed and mounted with the ProLong® Gold Antifade Mountant with DAPI (Life Technologies). Immunofluorescence was visualized using a Zeiss LSM 780 laser scanning confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) at the Microscopy, Imaging, and Cytometry Resources Core at the Wayne State University School of Medicine (http://micr.med.wayne.edu/). Immunofluorescence signals for Alexa Fluor 647, Alexa Fluor 555, and Alexa Fluor 488 were excited with a 633nm HeNe laser, a 550nm HeNe laser, and a 488nm line of a multiline argon laser, respectively. The DAPI signal was excited with a 405nm diode laser.

In vitro effect of rosiglitazone in human decidual macrophages

Decidual mononuclear cells were isolated from the decidua basalis of women who underwent PTL, as previously described (70). Decidual mononuclear cells were washed with supplemented RPMI medium, re-suspended in 3ml of the same medium, and counted using an automatic cell counter. Next, decidual mononuclear cells were placed into a 24-well plate at a density of ~2×106 cells/ml of supplemented RPMI medium and cultured at 37°C with 5% CO2 for 1 hour in order to allow the macrophages to attach to the plate. Following incubation, the culture medium was gently aspirated, fresh supplemented RPMI medium containing 5ng/ml GM-CSF was added, and attached macrophages were cultured at 37°C with 5% CO2 for 3 days. Macrophages were then treated with 20μM of RSG (Selleckchem, Houston, TX, USA) dissolved in DMSO (1:1000; Sigma-Aldrich) or with DMSO alone as a vehicle control in the presence of 10μg/ml of Brefeldin A (BD Biosciences). After 6 hours of treatment, the cells were harvested for TNF, IL12 and IL10 expression analysis by flow cytometry. The percentage of cytokine expression in RSG-incubated macrophages was calculated in comparison to cytokine expression in DMSO-incubated macrophages, which was considered as 100%.

Mice

B6 (C57BL/6J) mice were bred in the animal care facility at the C. S. Mott Center for Human Growth and Development at Wayne State University, Detroit, Michigan, USA, and housed under a circadian cycle (light:dark=12:12 hours). Females 8-12 weeks old were mated with male mice of proven fertility. Female mice were examined daily between 8:00 a.m. and 9:00 a.m. for the presence of a vaginal plug, which denoted 0.5 days post coitum (dpc). Upon observation of vaginal plugs, female mice were then separated from the males and housed in different cages. Procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Wayne State University (Protocol No. A09-08-12).

LPS-induced preterm birth in mice treated with rosiglitazone

Pregnant mice were categorized into four different groups and injected intraperitoneally on 16.5 dpc with: 1) 10μg of LPS (Escherichia coli O111:B4; Sigma-Aldrich) in 200μL of 1X PBS (n=10); 2) 200μL of 1X PBS as a control; 3) 10μg of LPS in 200μL of 1X PBS followed by 10mg/kg of RSG 6 hours after the initial injection (n=10); and 4) 10mg/kg of RSG as a control (Figure 5A). RSG was dissolved in 1:10 DMSO; therefore, pregnant mice were also intraperitoneally injected on 16.5 dpc with 1:10 DMSO as a control (n=6). Pregnancy parameters including gestational age and the rates of preterm birth and pup mortality were recorded via video camera (Sony Corporation, Tokyo, Japan). Preterm birth was defined as delivery before 18.0 dpc, and its rate was defined as the percentage of dams delivering preterm among all births. The rate of pup mortality was defined as the percentage of pups born dead among the total litter size. All DMSO control mice delivered at term (19.5 ± 0.5 dpc); therefore, the gestational age is shown instead of the rate of preterm birth. Gestational age was calculated from the presence of the vaginal plug (0.5 dpc) until the observation of the first pup born.

Figure 5
Rosiglitazone treatment reduces the rate of LPS-induced preterm birth and improves neonatal outcomes

Fetal and placental weights

A second cohort of pregnant mice was injected intraperitoneally on 16.5 dpc with LPS, PBS, LPS + RSG or RSG (n=8 each), as described previously. Two hours after injection, mice were euthanized, and peripheral blood was collected by cardiac puncture and placed into a 1.5 safe-lock Eppendorf tube (Fisher Scientific) (Figure 7A). Animal dissection and tissue collection (myometrial and decidual tissues) were performed as previously described (73). The pup and placenta weights were determined using a weight scale (DIA-20, American Weight Scales, Norcross, GA, USA).

Figure 7
Rosiglitazone treatment reduces the mRNA expression of Nfκb1, Tnf, and Il10 in decidual and myometrial macrophages

Chemokine/cytokine serum concentrations

Peripheral blood samples were centrifuged at 491 × g for 10 min at 4°C, and serum was separated and stored at −20°C until analysis. The Milliplex MAP Mouse Cytokine/Chemokine Kit (MCYTOMAG-70K-PX32, EMD Millipore, Billerica, MA, USA) was used to measure the concentrations of G-CSF, GM-CSF, IFNγ, IL1α, IL1β, IL2, IL3, IL4, IL5, IL6, IL7, IL9, IL10, IL12p40, IL12p70, IL13, IL15, IL17, CCL11, CXCL10, CXCL1, LIF, CXCL5, CCL2, M-CSF, CXCL9, CCL3, CCL4, CXCL2, CCL5, and TNF in the serum samples, according to the manufacturer's instructions. Plates were read using the Luminex 100 System (Luminex Corporation, Austin, TX, USA), and analyte concentrations were calculated using the xPONENT3.1 software (Luminex). The sensitivities of the assays were: 1.7pg/ml (G-CSF), 1.9pg/ml (GM-CSF), 1.1pg/ml (IFNγ), 10.3pg/ml (IL1α), 5.4pg/ml (IL1β), 1.0pg/ml (IL2), 1.0pg/ml (IL3), 0.4pg/ml (IL4), 1.0pg/ml (IL5), 1.1pg/ml (IL6), 1.4pg/ml (IL7), 17.3pg/ml (IL9), 2.0pg/ml (IL10), 3.9pg/ml (IL12p40), 4.8pg/ml (IL12p70), 7.8pg/ml (IL13), 7.4pg/ml (IL15), 0.5pg/ml (IL17), 1.8pg/ml (CCL11), 0.8pg/ml (CXCL10), 2.3pg/ml (CXCL1), 1.0pg/ml (LIF), 22.1pg/ml (CXCL5), 6.7pg/ml (CCL2), 3.5pg/ml (M-CSF), 2.4pg/ml (CXCL9), 7.7pg/ml (CCL3), 11.9pg/ml (CCL4), 30.6pg/ml (CXCL2), 2.7pg/ml (CCL5), and 2.3pg/ml (TNF). Inter-assay and intra-assay coefficients of variation were below 15% and 4.9%, respectively.

Macrophage isolation from murine myometrial and decidual tissues

Immediately after collection, myometrial and decidual tissues were mechanically disaggregated in the StemPro® Accutase® Cell Dissociation Reagent using scissors for approximately 1-2 min, as previously described (73). Samples were then incubated at 37°C for 35 min with gentle shaking (MaxQ™ 4450 Benchtop Orbital Shaker, Thermo Fisher Scientific, Marietta, OH, USA). The cell suspensions were filtered using a 100μm cell strainer (Fisher Scientific, Hanover Park, IL, USA) and washed with staining buffer [Bovine-serum albumin 0.1% (Sigma Aldrich), sodium azide 0.05% (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA), 1X PBS (Fisher Scientific Bioreagents)]. The resulting cell pellet was re-suspended in 96μl of staining buffer, and 4μl of anti-mouse F4/80 biotin-conjugated (clone BM8, eBioscience) were added. The cell suspension was then incubated for 15 min at 4°C. After incubation, the cells were washed with 2ml of staining buffer and centrifuged at 1250 × g for 7 min at 4°C. The cell pellet was re-suspended in 90μl of staining buffer, and 10μl of streptavidin microbeads (Miltenyi Biotec) were added. This cell suspension was incubated at 4°C for 15 min. Following incubation, the cells were washed with 2ml of staining buffer, and the cell pellet was re-suspended in 500μL of MACS buffer. F4/80+ cells (macrophages) were separated by positive selection using MS columns and a magnetic MACS separator. Macrophages were then washed with MACS buffer at 1250 × g for 7 min at 4°C, and an aliquot was taken to confirm purity by flow cytometry using an anti-mouse F4/80-PE (Clone BM8, eBioscience; Figure 7A). The cell pellet was re-suspended in 400μL of RNeasy Lysis Buffer (Qiagen, Germantown, MD, USA) and placed into a 1.5mL safe-lock Eppendorf tube (Fisher Scientific), which was stored at −80°C until RNA isolation.

RNA isolation, cDNA synthesis, and qRT-PCR

Total RNA was isolated from myometrial and decidual macrophages using the RNeasy mini kit (Qiagen), following the manufacturer's instructions. RNA concentrations and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and RNA integrity was evaluated with the 2100 Bioanalyzer system (Agilent Technologies, Wilmington, DE, USA) using the Agilent RNA 6000 Pico Kit (Agilent). cDNA was synthesized by using iScript Reverse Transcription Supermix for RT-qPCR kits (Bio-Rad Laboratories, Hercules, CA, USA) on the Applied Biosystems GeneAmp PCR System 9700 (Life Technologies), following the manufacturer's instructions. cDNA was amplified using the SsoAdvanced PreAmp Supermix (Catalog #1725160; Bio-Rad Laboratories) on the Applied Biosystems GeneAmp PCR System 9700. mRNA expression of Nfκb1, Tnf, Il10, and housekeeping genes (Actb, Gapdh, and Tbp) was determined by qPCR using the LuminoCtÒ SYBR Green qPCR ReadyMix (Sigma Aldrich) on the CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories), according to the manufacturer's instructions. Primers are described in Supplementary Table II.

Immunofluorescence

Myometrial and decidual tissues from mice injected with LPS, PBS, LPS + RSG or RSG (n=5 each) were immediately frozen in Tissue-Tek® O.C.T Compound (Sakura Finetek USA Inc., Torrance, CA, USA). Ten-μm-thick cryosections were cut and placed on Fisherbrand Superfrost Plus microscope slides (Thermo Scientific). After fixation with 4% paraformaldehyde (Electron Microscopy Sciences), the slides were washed with 1X PBS containing 0.1% Tween® 20, and permeabilized with 1X PBS containing 0.25% Triton® X-100 for 10 min. Non-specific antibody interaction was blocked using serum (KPL, Gaithersburg, MD, USA) at room temperature for 1 hour. Slides were then incubated with the following rabbit anti-mouse antibodies: 10μg/ml IL10 (Cat # ab9969, Abcam), 33μg/ml TNF (Cat # NBP1-19532, Novus Biologicals, Littleton, CO, USA) or 2μg/ml NFκB1 (Cat # NBP1-77395, Novus Biologicals) at 4°C overnight. After washing with PBS containing 0.1% Tween® 20, the slides were incubated with 2μg/ml goat anti-rabbit secondary antibody conjugated to Alexa Fluor 594 (Cat # A11072, Invitrogen Molecular Probes) at room temperature for 1 hour, washed again with 1X PBS containing 0.1% Tween® 20, and incubated with 2μg/ml rat anti-mouse F4/80 antibody directly conjugated to FITC (Cat # ab60343, Abcam) at room temperature for two hours. After mounting with ProLong® diamond antifade mountant with DAPI (Life Technologies), immunofluorescence was visualized using an Olympus BX 60 fluorescence microscope (Olympus Corporation, Tokyo, Japan) at 1000X magnification. The pictures were taken using an Olympus DP71 camera and DP Controller Software (Olympus Corporation). The images were merged using ImageJ 1.44p (National Institute of Health, USA).

Statistical analysis

Flow cytometry and qRT-PCR data analysis were performed in R (http://www.R-project.org/), and for all other data analyses, IBM SPSS Version 19.0 (IBM Corporation, Armonk, NY, USA) was used. For flow cytometry and qRT-PCR data analysis, the statistical significance of group comparisons was assessed using the Mann-Whitney U test. For qRT-PCR data, gene expression levels were computed as negative ΔCt values determined using two reference genes (Gapdh and Actb or Tbp) averaged within each sample. For in vitro experimentation with RSG, the statistical significance of group comparisons was assessed using the T test. For the rate of pup mortality, the statistical significance of group comparisons was assessed using the Mann-Whitney U test. For fetal and placental weights, the statistical significance of group comparisons was assessed using T-tests. For cytokine concentrations, multiple comparisons were performed using analysis of variance followed with Sidak post-hoc tests. For human demographic data, the group comparisons were performed using the Chi-square test for proportions as well as the Mann-Whitney U and Kruskal-Wallis tests for non-normally distributed continuous variables. A p-value < 0.05 was used to determine statistical significance.

RESULTS

Decidual macrophages undergo an M1-like polarization during spontaneous term and preterm labor

First, we determined whether decidual macrophages from spontaneous term and preterm labor cases were undergoing an M1 polarization. Although several M1 and M2 markers were included in the immunophenotyping, only a few markers were differentially expressed in decidual macrophages among the study groups. This is not surprising since the in vivo phenotype of decidual macrophages does not fit the conventional in vitro M1/M2 paradigm (23). CD80 (an M1 marker), CD163 and CD209 (M2 markers) were differentially expressed in decidual macrophages among the study groups; therefore, we termed these cells “M1-like” (CD45+CD14+ICAM3CD80+ cells) and “M2-like” (CD45+CD14+ICAM3CD163+CD209+ cells) macrophages (Figure 2A). In the decidua basalis, the proportion of M1-like macrophages was greater in women who underwent spontaneous term or preterm labor than in women who delivered at term or preterm without labor, respectively (TIL vs. TNL, PTL vs. PTNL; Figure 1B). In the decidua basalis, the proportion of M2-like macrophages was lower in preterm deliveries with and without labor than in term deliveries without labor (PTNL or PTL vs. TNL; Figure 1C). In the decidua parietalis, the proportion of M1-like macrophages was greater in women who underwent spontaneous term labor than in women who delivered at term without labor (TIL vs. TNL; Figure 1D). The proportion of decidua parietalis M1-like macrophages was also higher in spontaneous preterm labor than in preterm deliveries without labor; yet, this increase did not reach statistical significance (PTL vs. PTNL; Figure 1D). In both the decidua basalis and decidua parietalis, M1-like macrophages were greater in spontaneous preterm labor than in term without labor (PTL vs. TNL; Figure 1B & 1D). In the decidua parietalis, the proportion of M1-like macrophages was higher in women who delivered preterm than in those who delivered at term without labor (PTNL vs. TNL; Figure 1D). In contrast to the decidua basalis, the proportion of M2-like macrophages in the decidua parietalis did not vary among groups (Figure 1E). These results demonstrate that decidual macrophages undergo an M1-like polarization during spontaneous term and preterm labor.

Regardless of the gestational age or presence of labor, we found that the proportion of decidual M2-like macrophages was higher than the proportion of decidual M1-like macrophages (Figure 1C & 1E vs. Figure 1B & 1D), and that the decidua basalis had a higher proportion of M2-like macrophages than the decidua parietalis (Supplementary Figure 1A). Only in spontaneous term labor was the proportion of M1-like macrophages higher in the decidua basalis than in the decidua parietalis (Supplementary Figure 1B).

No differences were found in the proportions of HLA-DR+ (M1-like) or CD206+ (M2-like) decidual macrophages among the study groups (Supplementary Figure 2A & 2B).

Decidual macrophages express high levels of TNF and IL12, but low levels of PPARγ, during spontaneous preterm labor

Next, we investigated whether decidual M1-like and M2-like macrophages expressed pro-inflammatory or anti-inflammatory cytokines, respectively. Since both M1-like and M2-like expressed the pro-inflammatory cytokines TNF and IL12 (data not shown) and the M1-like population was low in number, we decided to report the expression of M1 (TNF, iNOS and IL12 (71)) and M2 (IL10 (71)) mediators, as well as PPARγ (59, 72), in the total decidual macrophage population (Figure 2A). Decidual macrophages from women who underwent spontaneous preterm labor expressed greater levels of TNF and IL12 than those from women who underwent spontaneous term labor (PTL vs. TIL; Figure 2B & 2D). Decidual macrophages from women who underwent spontaneous term labor expressed lower levels of TNF than those from women who delivered at term without labor (TIL vs. TNL; Figure 2B). Decidual macrophages from women who underwent spontaneous preterm labor expressed higher levels of IL12 than those from women who delivered preterm without labor (PTL vs. PTNL; Figure 2D). In contrast to M1 mediators, decidual macrophages from women who underwent spontaneous preterm labor expressed lower levels of PPARγ than those from women who delivered preterm without labor (PTL vs. PTNL; Figure 2E). Interestingly, preterm non-labor cases expressed higher levels of PPARγ when compared to term non-labor cases (PTNL vs. TNL; Figure 2E). The expression of iNOS and IL10 did not vary among the groups of women (Figure 2C & 2F). These results confirm that decidual macrophages have a pro-inflammatory phenotype during spontaneous preterm labor as they express high levels of TNF and IL12. The data also show that decidual macrophages down-regulate the expression of PPARγ during spontaneous preterm labor.

Decidual macrophages from women who underwent spontaneous preterm labor display plasticity by M1↔M2 polarization in vitro

Decidual leukocytes include both M1-like and M2-like macrophages. This is consistent with the fact that M1 and M2 polarization factors such as M-CSF, IL4, IL13, IFNγ and GM-CSF are present at the maternal-fetal interface throughout pregnancy (74-77). Therefore, we examined whether this heterogeneous population could be polarized in vitro by these mediators to become a more homogeneous M1-like or M2-like population as proof of macrophage plasticity. The potentially therapeutic ability to shift macrophages from a pro-inflammatory to an anti-inflammatory state was a major concept of our study. Decidual macrophages were cultured with GM-CSF followed by IFNγ and LPS to induce an M1 polarization or with M-CSF followed by IL13 and IL4 to induce an M2 polarization (37). Consistent with the previous experiment, we found that untreated decidual tissue from women who underwent spontaneous preterm labor had low proportions of M1-like macrophages (CD80+ cells, ~15%; Figure 3A) and high proportions of M2-like macrophages (CD209+ cells; ~27%; Figure 3B). Culture with GM-CSF followed by IFNγ and LPS stimulation increased the proportion of M1-like macrophages by 1.5 fold, but reduced the proportion of M2-like macrophages by 1.4 fold, compared to untreated macrophages (Figure 3A vs. 3B). Conversely, culture with M-CSF followed by IL13 and IL4 stimulation reduced the proportion of M1-like macrophages by 3.9 fold, but increased the proportion of M2-like macrophages by 1.4 fold, compared to untreated macrophages (Figure 3A vs. 3B). The immunostaining results confirmed our flow cytometry findings, showing that M1-like macrophages (CD80+ cells, red signal) were apparent when GM-CSF, LPS, and IFNγ were added to the culture, while M2-like macrophages (CD209+ cells, pink signal) were evident when M-CSF, IL4, and IL13 were added to the culture (Figure 3C).

Recently, it was demonstrated that first trimester-derived M2 decidual macrophages are induced by IL10 rather than by IL4/IL13 (37). We, therefore, incubated decidual macrophages from women who had undergone spontaneous preterm labor with M-CSF and IL10. Consistent with the aforementioned study (37), we found that IL10 induced the expression of M2 markers, CD163 and 209 (Figure 3D).

Collectively, these results demonstrate that decidual macrophages from women undergoing spontaneous preterm labor display plasticity by an M1↔M2 polarization in vitro, and that IL10 is a central mediator in this process.

Rosiglitazone incubation reduces the expression of M1 cytokines in decidual macrophages from women who underwent spontaneous preterm labor

Up to this point, our data have shown that decidual macrophages display plasticity in vitro and that their PPARγ expression is down-regulated during spontaneous preterm labor. These findings led us to hypothesize that a down-regulation of PPARγ expression/activity is linked to the premature onset of labor, and that activation of this pathway could reduce the expression of M1 mediators associated with spontaneous preterm labor. Therefore, we first evaluated whether activation of the PPARγ pathway through incubation with RSG would reduce the expression of TNF and IL12, M1 cytokines (71), or upregulate IL10, an M2 cytokine (71), in decidual macrophages. The expression of cytokines by decidual macrophages from women who underwent spontaneous preterm labor was evaluated by flow cytometry after incubation with RSG or DMSO (vehicle). Incubation with RSG reduced the expression of TNF (Figure 4A & 4B; ~2-fold decrease) and IL12 (Figure 4C; ~2-fold decrease; non-significant) in decidual macrophages from women who underwent spontaneous preterm labor. However, RSG did not have an effect on IL10 expression (Figure 4D). These results demonstrate that in vitro PPARγ activation via RSG reduces the expression of M1 cytokines in decidual macrophages from women who underwent spontaneous preterm labor.

Figure 4
Incubation of decidual macrophages with rosiglitazone

Rosiglitazone treatment reduces the rates of LPS-induced preterm birth and pup mortality

In order to study the in vivo effect of RSG, we determined whether this PPARγ agonist could rescue LPS-induced preterm birth (Figure 5A). As expected, LPS injection induced 80% of preterm births (78) (Figure 5B). When pregnant mice were injected with LPS followed by treatment with RSG, a 30% reduction in the rate of preterm birth was observed (LPS vs. LPS + RSG; Figure 5B). No preterm births were observed in mice injected with PBS or RSG alone (Figure 5B). The rate of pup mortality in mice injected with LPS was 100% (Figure 5C); yet, this was reduced by 41% after the treatment with RSG (LPS vs. LPS + RSG; Figure 5C). Since RSG was dissolved in DMSO, it was essential to investigate whether DMSO alone had adverse effects in pregnancy. All mice injected with DMSO delivered at term, as in mice injected with PBS (19.5 ± 0.5 days; Figure 5D). Also, mice injected with DMSO had a low rate of pup mortality, similar to those injected with PBS (8-13%; Figure 5E). Next, we examined the effects of RSG treatment on fetal and placental weights. LPS injection reduced fetal weight (LPS vs. PBS; Figure 5F), but body mass was restored following treatment with RSG (LPS+RSG vs. LPS; Figure 5F). The administration of RSG alone did not alter fetal weight (RSG vs. PBS; Figure 5F). LPS injection reduced placental weight (LPS vs. PBS; Figure 5G), and this was not restored by treatment with RSG (LPS + RSG vs. RSG; Figure 5G). The administration of RSG alone did not alter placental weight (RSG vs. PBS; Figure 5G). These data demonstrate that treating mice with RSG reduces the rate of LPS-induced preterm birth, as well as improves adverse neonatal outcomes.

Rosiglitazone treatment attenuates the systemic pro-inflammatory response induced by LPS

Preterm labor/birth is associated with a systemic pro-inflammatory response (8, 18, 79-87). Therefore, we determined whether treatment with RSG reduced the elevated systemic concentrations of cytokines/chemokines induced by LPS. As expected, LPS increased the systemic concentration of several cytokines and chemokines when compared with the PBS control group (LPS vs. PBS; Figure 6A & 6B and Supplementary Figure 3). Treatment with RSG attenuated the LPS-induced pro-inflammatory response by reducing the concentrations of several cytokines and chemokines including TNF, IL1β, IL3, IL4, IL9, IL10, IL12 (p40 and p70), IL13, IL15, GM-CSF, CCL2, CCL3, CCL4, CXCL2, CXCL5, and CXCL10 (LPS+RSG vs. LPS, Figure 6A & 6B). In addition, treatment with RSG increased the systemic concentrations of IL5 and CXCL9 in mice injected with LPS (LPS+RSG vs. LPS, Figure 6A & 6B). However, treatment with RSG did not reduce the systemic concentration of LPS-induced IFNγ, IL1α, IL2, IL6, LIF, G-CSF, M-CSF, CCL5, and CCL11 (Supplementary Figure 3). The administration of RSG alone did not alter the basal concentrations of cytokines/chemokines (RSG vs. PBS, Figure 6A & 6B and Supplementary Figure 3). These results demonstrate that treatment with RSG attenuates the LPS-induced pro-inflammatory response in the mother, which provides insight into the systemic immune mechanisms whereby this PPARγ agonist prevents LPS-induced preterm birth.

Figure 6
Rosiglitazone treatment attenuates the systemic inflammatory response induced by LPS

Rosiglitazone treatment attenuates macrophage activation in myometrial and decidual macrophages induced by LPS

PPARγ activation suppresses gene transcription by interfering with signal transduction pathways, such as the NFκB, STAT, and AP1 pathways (60-62), and induces an M2 macrophage polarization (59). Therefore, we investigated whether PPARγ activation in vivo through treatment with RSG alters the NFκB pathway, and the expression of M1 and M2 cytokines in decidual and myometrial macrophages from pregnant mice injected with LPS (Figure 7A). Decidual and myometrial macrophages (F4/80+ cells) were isolated, and their purity was confirmed by flow cytometry (88%-97%; Figure 7A). mRNA expression of Nfκb1 (a pathway regulated by PPARγ activation (60)), Tnf (an M1 cytokine (71)), and Il10 (an M2 cytokine (71)) were determined in isolated macrophages. LPS injection up-regulated the expression of Nfκb1, Tnf, and Il10 in decidual and myometrial macrophages (LPS vs. PBS; Figure 7B-7G); however, the mRNA abundance of these genes was down-regulated after treatment with RSG (LPS+RSG vs. LPS; Figure 7B-7G). In decidual macrophages, the LPS-induced expression of IL10 was partially reduced after treatment with RSG (LPS+RSG vs. LPS; Figure 7D). The administration of RSG alone did not alter the expression of any of these genes (RSG vs. PBS; Figure 7B-7G).

The protein expression of NFκB, TNF and IL10 was also determined via immunofluorescence in myometrial and decidual macrophages. Consistent with our mRNA data, LPS-induced expression of NFκB, TNF and IL10 in myometrial macrophages was reduced upon treatment with rosiglitazone (Figure 8). Similar results were found in decidual macrophages (data not shown).

Figure 8
Rosiglitazone treatment reduces the protein expression of NFκB, TNF, and IL10 in myometrial macrophages

Collectively, these results demonstrate that treatment with RSG down-regulates the LPS-induced mRNA and protein expression of NFκB, TNF, and IL10 in decidual and myometrial macrophages, which provides insight into the local immune mechanisms whereby this PPARγ agonist prevents LPS-induced preterm birth.

DISCUSSION

Herein, we demonstrated that 1) decidual macrophages undergo an M1-like polarization during spontaneous term and preterm labor; 2) M2-like macrophages are more abundant than M1-like macrophages in decidual tissue; 3) decidual M2-like macrophages are more abundant in term than in preterm gestations, yet their proportions do not change with the onset of labor; 4) decidual macrophages express high levels of TNF and IL12, but low levels of PPARγ, during spontaneous preterm labor; 5) decidual macrophages from women who underwent spontaneous preterm labor display plasticity by M1↔M2 polarization in vitro; 6) incubation with RSG reduces the expression of TNF and IL12, M1 cytokines, in decidual macrophages from women who underwent spontaneous preterm labor; 7) treatment with RSG reduces the rates of LPS-induced preterm birth and pup mortality, and restores body mass in fetuses of pregnant mice injected with LPS; 8) treatment with RSG reduces the systemic pro-inflammatory response in pregnant mice injected with LPS; and 9) treatment with RSG down-regulates the mRNA and protein expression of NFκB, TNF, and IL10 in decidual and myometrial macrophages from pregnant mice injected with LPS. Collectively, these results demonstrate that M1-like macrophages expressing TNF and IL12 are abundant in decidual tissue from women who underwent spontaneous preterm labor and that in vitro treatment with RSG attenuates this pro-inflammatory response. The study herein also demonstrates that in vivo PPARγ activation via RSG reduces the LPS-induced systemic and local inflammatory response mediated, in part, by decidual and myometrial macrophages, preventing preterm birth and improving neonatal outcomes.

Macrophages are classically activated (40) or become M1-polarized (88, 89) through stimulation by IFNγ alone or together with TNF or TLR ligands such as LPS. Once activated, M1 macrophages exhibit their function by producing NO and pro-inflammatory cytokines such as IL12, IL23, and TNF (34, 42, 48, 71, 88, 90). Pro-inflammatory macrophages are present in reproductive tissues and at the maternal-fetal interface (decidual tissue) immediately before and during spontaneous term and preterm labor (27, 29, 31, 32, 91). In the study herein, we demonstrated that a proportion of decidual macrophages undergo an M1-like polarization during spontaneous preterm labor. We also found that M2-like macrophages express TNF and IL12, which is consistent with the fact that M2b and M2d macrophages can release pro-inflammatory cytokines (92). These decidual M1-like and M2-like macrophages showed an increased expression of TNF and IL12 when compared to those who underwent spontaneous term labor. Therefore, pro-inflammatory macrophages are increased in both spontaneous term and preterm labor, yet these innate cells are more activated in women who delivered preterm. Together, these findings demonstrate that decidual pro-inflammatory macrophages are implicated in the inflammatory response at the maternal-fetal interface that is associated with spontaneous preterm labor.

In the study herein, a high proportion of decidual macrophages at term and preterm gestations display an M2-like phenotype. This is consistent with previous studies demonstrating that immunoregulatory macrophages are present in decidual tissue throughout pregnancy (25, 26, 37, 53-55). M2-like or alternatively activated macrophages exhibit an immunoregulatory phenotype that functions in homeostasis, dampening inflammation, and promoting tissue remodeling and tumor progression (34, 36, 50, 93, 94). These macrophages are characterized by increased phagocytic activity and expression of scavenging mannose and galactose receptors, as well as a high production of ornithine and polyamines through the arginase pathway (36, 50). M2 macrophage polarization can be achieved upon stimulation with IL4 and IL13 or with IL10 (34, 36, 44, 93). M2 macrophages typically express high levels of IL10 (47, 48), a cytokine that dampens inflammation (95), and also suppress M1 cytokines such as IL12 (89, 96). The aforementioned studies led to the current hypothesis that decidual M2-like macrophages have an immunoregulatory role and participate in maternal-fetal tolerance during early pregnancy (25, 26, 37, 53). However, the roles of M2-like macrophages in late pregnancy and the process of labor were unknown. Herein, we demonstrated that the proportions of decidual M2-like macrophages did not vary with the onset of labor; however, these cells express pro-inflammatory mediators and were reduced in preterm deliveries. These findings suggest that decidual M2-like macrophages are abundant at term pregnancy where they contribute to the pro-inflammatory process of labor, and may have an immunoregulatory role prior to term gestation.

Regardless of the gestational age and presence of labor, decidual M2-like macrophages were more abundant in the decidua basalis than in the decidua parietalis. A previous study demonstrated at term and mid pregnancies that macrophages from the decidua basalis express higher levels of CD105, CD209, and CD206 than macrophages from the decidua parietalis (22). CD105 is an accessory receptor for TGFβ (97-99), CD209 is a receptor for ICAM3 that facilitates APC function (100), and CD206 is the macrophage mannose receptor that facilitates phagocytosis (101). CD105 is implicated in wounding and inflammation (102-104) , and the expression of CD209 and CD206 in CD11chi decidual macrophages has been associated with APC function (23). Taken together, these results suggest that besides having an immunoregulatory role, M2-like macrophages in the decidua basalis may have an APC function and facilitate T-cell tolerance during late gestation. Recently, we provided evidence to support this hypothesis by demonstrating that macrophage activation occurs simultaneously to T-cell activation at the maternal-fetal interface in a preterm birth model induced by iNKT cell activation (105).

Our data also showed that decidual macrophages expressed reduced levels of PPARγ in spontaneous preterm labor when compared to preterm non-labor controls. PPARγ is a hormone nuclear receptor (106, 107) that binds lipid metabolites including eicosanoids, polyunsaturated fatty acids, and oxidized phospholipids as well as synthetic thiazolidinediones (e.g., RSG and GW1929) (108). PPARγ is mainly expressed by adipose tissue and immune cells (109) including monocytes/macrophages (60, 61, 110, 111). Macrophages from immune cell-specific PPARγ null mice (PPARγ fl/fl; MMTV-Cre+) down-regulate M2 markers (IL13 and Arg1) and up-regulate M1 markers (CCL1 and CCL17) (112). Indeed, in macrophage-specific PPARγ knockout mice, the alternative macrophage activation is impaired (72). This suggests that decidual macrophages down-regulated PPARγ in order to prompt an M1-like phenotype in spontaneous preterm labor.

PPARγ activation up-regulates the mRNA and protein expression of PPARγ (113, 114) and suppresses gene transcription by interfering with signal transduction pathways, such as the NFκB, STAT, and AP1 pathways (60-62). Herein, we found that treatment with RSG, a selective PPARγ agonist (58), down-regulated the expression of TNF and IL12 in decidual macrophages from women who underwent spontaneous preterm labor. These results led us to suggest that PPARγ activation in vivo could prevent spontaneous preterm labor/birth.

PPARγ is expressed in the fetal membranes and placenta (115, 116) as well as in reproductive tissues (117-119). PPARγ has an essential role in placental development as its depletion interferes with terminal differentiation of the trophoblast and placental vascularization, causing fetal death (120). In later stages of gestation, the PPARγ pathway is linked to the inflammatory process of parturition in both term and preterm stages (116, 119, 121-127). Defective PPARγ signaling is associated with pregnancy complications (63, 128) including gestational diabetes mellitus (129), intrauterine growth restriction (130, 131), preeclampsia (132-135), and preterm birth (124, 136). Conversely, PPARγ activation via 15-deoxy-Δ-12,14-Prostaglandin J2 delays LPS-induced preterm birth and reduces neonatal mortality by promoting the resolution of inflammation (66). However, the administration of 15-deoxy-Δ-12,14-Prostaglandin J2 alone has negative effects on gestational length, causing late preterm birth (66). In the study herein, PPARγ activation via RSG reduced the rates of LPS-induced preterm birth and pup mortality, and the administration of RSG alone did not have any noticeable adverse effects on the mother or offspring. In order to explore the effect of RSG in preventing preterm birth, we analyzed the systemic and local inflammatory response induced by LPS. Treatment with RSG reduced the systemic concentrations of several inflammatory cytokines in pregnant mice injected with LPS. The systemic inhibitory activity of RSG for inflammatory cytokines was previously demonstrated in non-pregnant mice (137-139) and rats (140, 141) injected with inflammatory stimuli, as well as in patients with diabetic and nondiabetic coronary artery disease (142) or obesity (143). Locally, treatment with RSG reduced the mRNA and protein expression of NFκB, TNF, and IL10 by myometrial and decidual macrophages from mice injected with LPS. The suppressive role of PPARγ activation in macrophages has been previously demonstrated in the NFκB pathway (60, 61, 144), and in the expression/secretion of TNF (111, 144-146) and IL10 (147, 148). Taken together, these data demonstrate that RSG prevents LPS-induced preterm birth by attenuating the systemic immune response in the mother and suppressing the local pro-inflammatory response mediated, at least in part, by decidual and myometrial macrophages.

Herein, we also demonstrated that treatment with RSG increased the serum concentration of LPS-induced IL5 and CXCL9. Recently, in a model of iNKT-cell activation-induced preterm birth, we also demonstrated that RSG triggers a systemic anti-inflammatory response in the mother by increasing the serum concentration of IL10, IL12p40, IL3 and IL5 (105). IL5 is a Th2 cytokine that is implicated in eosinophilic responses and B-cell proliferation (149), and CXCL9 is involved in T-cell trafficking (150) in chronic inflammatory processes (151, 152). Previous reports, however, have demonstrated that treatment with RSG reduces bronchoalveolar lavage fluid or serum concentrations of IL5 in rodent models of asthma (153, 154) and attenuates tissue eosinophilia induced by this cytokine (155). In the same fashion, treatment with RSG inhibits the release of CXCL9 induced by the IFNs (α,γ,β) in primary cultures of human thyroid follicular cells (156) and by IFNγ and TNF in primary cultures of thyrocytes, retrobulbar fibroblasts, and retrobulbar preadipocytes obtained from Graves' ophthalmopathy patients (157). These disparities may be due to the fact that our studies were performed in pregnant mice and the serum sampling was done shortly after administration of the inflammatory stimulus. Further research is needed in order to investigate whether increased serum concentrations of IL5 and CXCL9 contribute to the effectiveness of RSG in preventing LPS-induced preterm birth.

In summary, the study herein demonstrated that an M1-like macrophage polarization at the maternal-fetal interface is associated with spontaneous preterm labor, and that PPARγ activation via RSG can attenuate the LPS-induced systemic and local pro-inflammatory responses mediated by macrophages, preventing preterm birth and improving neonatal outcomes. These findings suggest that the PPAR-γ pathway is a new molecular target for future preventative strategies for spontaneous preterm labor/birth.

Supplementary Material

ACKNOWLEDGEMENTS

We gratefully acknowledge Mary Olive, Ronald Unkel, Marcia Arenas-Hernandez, Dr. Zhong Dong, Lorri McLuckie, Yang Jiang and, and Po Hung Chiang for their contributions to the execution of this study. We thank the physicians and nurses from the Center for Advanced Obstetrical Care and Research (Wayne State University & Detroit Medical Center), and the research assistants from the Perinatology Research Branch (NICHD, NIH) Clinical Laboratory, for their help in collecting human samples. We also thank staff members of the Perinatology Research Branch Histology and Pathology Units for their examination of the pathological sections, and Maureen McGerty (Wayne State University) for her critical readings of the manuscript.

Non-standard abbreviations

dpc
days post coitum
PPAR
peroxisome proliferator-activated receptor
PTL
preterm with labor
PTNL
preterm without tabor
RSG
rosiglitazone
TIL
term with labor
TNL
term without labor

Footnotes

1This research was supported by the Perinatology Research Branch, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U. S. Department of Health and Human Services (NICHD/NIH/DHHS), and by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health.

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