Macrophages exert homeostatic actions in pregnancy to protect against preterm birth and fetal inflammatory injury

Macrophages are commonly thought to contribute to the pathophysiology of preterm labor by amplifying inflammation — but a protective role has not previously been considered to our knowledge. We hypothesized that given their antiinflammatory capability in early pregnancy, macrophages exert essential roles in maintenance of late gestation and that insufficient macrophages may predispose individuals to spontaneous preterm labor and adverse neonatal outcomes. Here, we showed that women with spontaneous preterm birth had reduced CD209+CD206+ expression in alternatively activated CD45+CD14+ICAM3– macrophages and increased TNF expression in proinflammatory CD45+CD14+CD80+HLA-DR+ macrophages in the uterine decidua at the materno-fetal interface. In Cd11bDTR/DTR mice, depletion of maternal CD11b+ myeloid cells caused preterm birth, neonatal death, and postnatal growth impairment, accompanied by uterine cytokine and leukocyte changes indicative of a proinflammatory response, while adoptive transfer of WT macrophages prevented preterm birth and partially rescued neonatal loss. In a model of intra-amniotic inflammation–induced preterm birth, macrophages polarized in vitro to an M2 phenotype showed superior capacity over nonpolarized macrophages to reduce uterine and fetal inflammation, prevent preterm birth, and improve neonatal survival. We conclude that macrophages exert a critical homeostatic regulatory role in late gestation and are implicated as a determinant of susceptibility to spontaneous preterm birth and fetal inflammatory injury.

BALB/c males and examined the following morning for the presence of a vaginal plug, which indicated 0.5 days post coitum (dpc). C57BL/6, FVB/N, and C57BL/6-Tg(CAG-EGFP)131Osb/LeySop mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were bred in the animal care facility at the C.S. Mott Center for Human Growth and Development (Wayne State University, Detroit, MI, USA) and housed on a 12 h light/12 h dark cycle. Eight-to twelve-week-old C57BL/6 or FVB/N females were mated with males of proven fertility and examined daily between 8:00 and 9:00 a.m. for the presence of a vaginal plug. After observation of a vaginal plug, female mice were removed from the mating cages and housed separately. A weight gain ≥2 grams at 12.5 dpc confirmed pregnancy.
Ultrasound-guided intra-amniotic injection of LPS was performed on 16.5 dpc C57BL/6 mice, as previously described (4)(5)(6). Briefly, dam anaesthesia was induced with 2-3% isoflurane (Aerrane, Baxter Healthcare Corporation, Deerfield, IL, USA) and 1-2 L/min of oxygen, then maintained with 1.5-2% isoflurane and 1.5-2 L/min of oxygen. Dams were positioned on a heating pad and abdominal hair removed. Body temperature was monitored by rectal probe (Visual Sonics, Toronto, ON, Canada) and maintained at 37 ± 1°C, and respiratory and heart rates were monitored by electrodes embedded in the heating pad. Using an ultrasound probe fixed and mobilized with a mechanical holder, ultrasound guided intra-amniotic injection of LPS (Escherichia coli O111: B4, Sigma)(100 ng in 25 μL of sterile PBS, or 25 μL of PBS alone for controls) was performed in each amniotic sac using a 30G needle (Becton Dickinson) stabilized by a mechanical holder (VisualSonics). Following treatments, dams were either monitored by infrared camera (Sony, Tokyo, Japan) until delivery or euthanized for tissue recovery at specific time points in late gestation, as detailed below.
Preterm birth was defined as delivery within 48 h of intervention (CD11b + myeloid cell depletion or LPS administration): ≤18.0 dpc ( Figure Figure 6C), and the rate was calculated as proportion of total number of pregnant mice. Gestational length was calculated as the length of time from the presence of the vaginal plug until the appearance of the first pup in the cage bedding. Neonatal survival (% pups born) was calculated at birth, at 12-24 h after birth, and at 8 and 21 days of age. Pup weights were recorded at 12-24 h after birth, as well as 8 and 21 days of age.

Isolation, differentiation, and adoptive transfer of bone marrow-derived macrophages
For reconstitution experiments in Cd11b DTR/DTR mice, bone marrow was collected from Cd11b WT/WT mice and treated with red blood cell lysis buffer. Cells were resuspended in complete RPMI medium (cRPMI, 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 1% L-glutamate) supplemented with 20% L929 conditioned media as a source of CSF-1 and seeded at 1 x 10 6 cells/ml, as described (7). Macrophages were collected on day 6 for adoptive transfer experiments after repeated washing with ice-cold PBS using StemPro Accutase Cell Dissociation Reagent (Life Technologies). Approximately 5-10 x 10 6 cells were resuspended in 250 μl RPMI for intravenous (i.v.) injection into each dam on both 14.5 and 16.5 dpc, prior to i.p. administration of DT, or PBS on 16.5 dpc. This number was the maximum number of cells that could feasibly be transferred by tail vein injection, and was judged sufficient to partially repopulate a CD11b DTR/DTR mouse after DT administration, based on estimates of ~3 x 10 5 CD11b + F4/80 + cells/ ml in peripheral blood, and ~1 x 10 6 CD11b + F4/80 + cells/ g of uterus (Fig. 2B).
For experiments in C57BL/6 mice, bone marrow-derived cells from C57BL/6 donors were treated with red blood cell lysis buffer and cultured in IMDM medium (Thermo Scientific) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) and 10 ng/mL of recombinant CSF1 (Cat#576402; BioLegend, San Diego, CA, USA), and incubated at 37°C with 5% CO2 for 7 days. On day 7, the culture medium was replaced with fresh IMDM medium + 10% FBS and 10 ng/mL each of recombinant IL-4 (Cat#574302, BioLegend) and IL-13 (Cat#575902, BioLegend), or fresh IMDM medium + 10% FBS and 10 ng/mL CSF1. On day 8 of culture, 14-18 hours later, macrophages were collected for adoptive transfer. Either M2-polarized BMDM or non-polarized BMDM (2 -3 x 10 6 ) were re-suspended in 100 µL sterile PBS for i.v. injection on 15.5 or 16.5 dpc, prior to the intra-amniotic administration of lipopolysaccharide (LPS), or PBS. Flow cytometry was performed to quantify expression of phenotype markers Egr-2 and Ym1/2 in both BMDM preparations (Supplementary Table 3). Some mice were administered GFP + M2-polarized BMDM to allow tracking of passively transferred macrophages recruited into gestational tissues. BMDM were recovered from C57BL/6-Tg(CAG-EGFP)131Osb/LeySop mice, cultured and administered to recipients, as described above. Leukocytes were isolated from myometrial, decidual, and placental tissues as previously described, and flow cytometry was performed to determine the numbers of GFP + F4/80 + cells in each tissue as detailed below.

Immunohistochemistry
Whole uterus (myometrium and endometrium) and placenta were collected from Cd11b DTR/DTR dams 24 h after administration of DT or PBS on 16.0 dpc. Tissue sections (5 μm thick) were cut and mounted on glass microscope slides. The sections were blocked with 15% normal mouse serum (NMS) and 15% normal rabbit serum for 30 min at 37ºC followed by overnight incubation with rat anti-mouse F4/80 in PBS (1:100 dilution), 1.5% NMS, and 1.5% normal rabbit serum (eBioscience, San Diego, CA, USA) at 4ºC in a humidified chamber. Afterwards, the tissues were incubated with biotinylated rabbit anti-rat IgG (1:200 dilution) for 40 min at room temperature (Vector Laboratories, Burlingame, CA, USA), followed by a 30 min incubation with Elite ABC reagent from the VECTASTAIN ABC Elite kit (Vector Laboratories). Horseradish peroxidase was localized with diaminobenzidine tetrachloride (DAB) (DAKO, Glostrup, Denmark), and tissues were counterstained with hematoxylin (Sigma). Negative control sections were incubated either with irrelevant primary antibody, the secondary antibody alone, or no antibody. There was no positive staining identified in the control slides. Within the labyrinth zone of the placenta, F4/80 + cells in the intervillous space containing maternal blood, identified by their characteristic structure and the presence of red blood cells, were considered macrophages of maternal origin. F4/80 + cells within the interstitial compartment, exhibiting structural features typical of villous tissue with a syncytiotrophoblast lining, were considered macrophages of fetal origin (Hofbauer cells) (8,9).

Isolation of murine leukocytes for immunophenotyping
Maternal and fetal tissues were collected from Cd11b DTR/DTR dams 24 h after administration of DT or PBS on 16.0 dpc, and Cd11b WT/WT dams 24 h after administration of 25 ng/g of DT on 16.0 dpc, and leukocytes were isolated as previously described (10). Dams were weighed and anesthetized with avertin [tribromoethanol dissolved in amylene hydrate and distilled water] (Sigma). Peripheral blood was recovered by cardiac puncture and placed in tubes containing heparin sodium (Sigma). Mice were then euthanized by cervical dislocation, and the uterine myometrium, uterine decidua, placenta, and fetal liver were collected, rinsed in PBS, pooled (3-4 tissues, respectively), and gently dissociated in StemPro Accutase Cell Dissociation Reagent (Life Technologies) using scissors. After incubation at 37ºC for 40 min, cells were filtered using a 100 µm cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA) and washed with FACS buffer (0.1% bovine serum albumin and 0.05% sodium azide in PBS). Cell debris was removed by centrifugation in 1 mL of fetal calf serum, followed by washing with 500 μL of FACS buffer.

Immunophenotyping of murine leukocytes
Immediately after isolation, leukocyte cell pellets from the blood, myometrium, decidua, placenta, and fetal liver were re-suspended in FACS buffer and pre-incubated with Fc Block (Becton Dickinson, clone 2.4G2) for 10 min on ice and subsequently incubated with specific fluorochrome-conjugated anti-mouse antibodies shown in Supplementary Table 3. Red blood cells were lysed using RBC lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate and 0.1 mM EDTA, pH 7.2). The lineage and phenotype of viable leukocytes [4′,6diamidino-2-phenylindole (DAPI) + CD45 + cells] were analyzed using combinations of antibodies against CD45, CD11b, F4/80, CD11c, MHC class II, Ly6G, CD3, CD4, CD8, CD49b, and CD69 according to gating strategies shown in Supplementary Figures 6A, 7 and 8A. Total numbers of leukocyte subsets were determined using CountBright Absolute Counting Beads (Molecular Probes, Invitrogen, Eugene, OR, USA). The term 'cells/ml' refers to the number of cells per ml of FACS buffer, at the time of flow cytometry analysis, and is directly proportional to the number of cells/uterus. The number of cells per ml was calculated using the formula provided by CountBrightTM Absolute Counting Beads (Molecular Probes). This formula considers the number of cells, number of beads, and volume in which cells were resuspended. For tissues, cells were obtained after dissociation of ~30-50 mg of tissue resuspended in 1 ml of FACS buffer. For blood, 100 uL of blood was utilized in all experiments and white blood cells were resuspended in 1 mL of FACS buffer. The analysis and preparation of images were performed using FlowJo Software Version 10.

Gene expression analysis in murine decidual tissue
Uterine decidual tissue was collected from Cd11b DTR/DTR dams 24 h after administration of DT or PBS on 16.0 dpc. Total RNA was extracted from each sample with Trizol, following the manufacturer's instructions. RNA concentration and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and RNA integrity was evaluated using agarose gels. Complementary (c) DNA was synthesized using the RT 2 First Strand Kit (QIAGEN, Hilden, Germany). A single pooled sample was generated for each group (n = 7-8 dams/pool). The RT² Profiler Mouse Innate & Adaptive Immune Response PCR Array (Cat#PAMM-052ZA-2, QIAGEN) and RT² Profiler Mouse Inflammatory Response & Autoimmunity PCR Array (Cat#PAMM-077ZA-2, QIAGEN) were used for mRNA expression profiling and performed using RT 2 SYBR Green ROX qPCR MasterMix (QIAGEN) on a 7500 Fast Real-Time PCR System (Applied Biosystems, Life Technologies, Foster City, CA, USA) (Supplementary Table 4).
Interaction network analysis of down-regulated and up-regulated genes with protein-protein interactions was performed using STRING (Search Tool for the Retrieval of Interacting Genes) v.10.0 software (11).

Measurement of plasma progesterone
For measurement of plasma progesterone, peripheral blood was collected by cardiac puncture from Cd11b DTR/DTR dams 24 h after administration of DT or PBS on 16.0 dpc. Plasma was obtained after centrifugation at 1300 x g for 10 min at 4°C and progesterone concentrations were measured using an enzyme-linked immunoassay (Mouse/Rat Progesterone Kit, Cat#55-PROMS-E01, ALPCO Diagnostics, Salem, NH, USA), following the manufacturer's instructions.

Cytokine secretion by myometrial and decidual macrophages
Myometrial and decidual tissue was collected from Cd11b WT/WT mice on 16.5 dpc, 17.5 dpc and 18.5 dpc, and disaggregated as described above. Single cell suspensions were then incubated with biotin-conjugated rat anti-mouse F4/80 (clone BM8, eBioscience) and streptavidin microbeads (Miltenyi Biotec) for positive selection using MS columns and a magnetic MACS separator. The purity of isolated macrophages were confirmed to be >90% by flow cytometry to detect F4/80 + cells. Cells were counted using an automatic cell counter (Cellometer Auto 2000; Nexcelom, Lawrence, MA, USA) and plated in a 24-well plate (Fisher Scientific, Waltham, MA, USA) at a density of 1×10 6 cells/mL in complete RPMI medium for culture at 37°C with 5% CO2. Macrophages were collected 6 h later and the supernatant recovered for analysis of IL-10, IL-12p70, TNF, and TGFβ concentrations using Quantikine ELISA Kits (R&D Systems, Minneapolis, MN, USA).
Gene expression in the fetal brain and lung upon treatment with M2-polarized macrophages C57BL/6 dams were administered LPS or PBS by intra-amniotic injection on 16.5 dpc, with or without adoptive transfer of M2-polarized macrophages, as described above, then 16 h later were euthanized to collect the fetal brain and lung. The tissues were stored in RNAlater Stabilization Solution (Invitrogen by Thermo Fisher Scientific, Baltics UAB, Lithuania) until RNA isolation. Total RNA was isolated using QIAshredders, RNase-Free DNase Sets, and RNeasy Mini Kits (QIAGEN), according to the manufacturer's instructions. RNA concentrations and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Scientific), and RNA integrity was evaluated with the Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE, USA). Complementary (c)DNA was synthesized using SuperScript III First-Strand Synthesis SuperMix (Invitrogen by Thermo Fisher Scientific, Carlsbad, CA, USA). Gene expression profiling was performed on the BioMark™ System for high-throughput RT-qPCR (Fluidigm, San Francisco, CA, USA) with the TaqMan® gene expression assays (Applied Biosystems, Life Technologies Corporation, Foster City, CA, USA) listed in Supplementary Table 5.    macrophages (B-F), neutrophils (G-K), dendritic cells (L-P), and T cells (CD45 + CD11b -CD3 + cells) (Q-U), in the maternal peripheral blood, uterine myometrium, uterine decidua, placenta, and fetal liver (n = 8-9 per group). Data are percentage of CD45 + leukocytes, shown as boxplots where midlines indicate medians, boxes indicate interquartile range, and whiskers indicate minimum/maximum range. Symbols are values from individual dams. Data were analyzed by ANOVA followed by post-hoc t-test. P values were considered significant when P < 0.05.       Figure 11, related to Figure 8. Effect of adoptive transfer of M2 polarized macrophages to dams on inflammatory gene expression in the brain and lung of fetuses after intra-amniotic administration of LPS. M2polarized macrophages (M2 Mɸ), or vehicle, were administered intravenously on 15.5 days post coitum (dpc) and 16.5 dpc to C57BL/6 dams, followed by intra-amniotic injection with lipopolysaccharide (LPS) on 16.5 dpc (M2+LPS). Control C57BL/6 dams were injected intra-amniotically with LPS only (LPS). Dams were euthanized 16 h after LPS injection to collect fetal brain and lung for evaluation of gene expression. (A) Expression of Il1a, Il2,Il3,Il23,Aim2,Nlrp1a,Casp11,Sele,Sell,Mki67,Vcam,Arg1, and Ym1 in the fetal brain (n = 4-10 per group). (B) Expression of Il5, Aim2, Arg1, and Ym1 in the fetal lung (n = 8-21 per group). Data are shown as boxplots where midlines indicate medians, boxes indicate interquartile range, and whiskers indicate minimum/maximum range. Symbols are values from individual fetuses. Data were analyzed by Mann-Whitney U-tests. P values were considered significant when P < 0.05. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.