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. Author manuscript; available in PMC: 2021 Mar 1.
Published in final edited form as: Am J Reprod Immunol. 2020 Oct 16;85(3):e13352. doi: 10.1111/aji.13352

Cytotrophoblasts suppress macrophage-mediated inflammation through a contact-dependent mechanism

Alison J Eastman 1, Erin N Vrana 2, Maria T Grimaldo 3, Amanda Jones 1, Lisa Rogers 1, Donald J Alcendor 4, David M Aronoff 1,5
PMCID: PMC7897285  NIHMSID: NIHMS1652621  PMID: 32969101

Abstract

Problem:

Gestational membrane (GM) infection provokes inflammation and can result in preterm prelabor rupture of membranes (PPROM). The choriodecidual layer of the GM includes decidual stromal cells (DSC), cytotrophoblasts (CTB) and macrophages (Mφ). Our laboratory has previously shown that DSCs suppress Mφ TNFα production through secreted prostaglandin E2. We hypothesized that CTBs would also inhibit Mφ cytokine expression through secreted mediators.

Method of Study:

THP.1 Mφ-like cells with an NFκB reporter construct or human blood monocyte-derived Mφ were co-cultured with the Jeg3 CTB cell line or primary human CTBs and challenged with Group B Streptococcus (GBS) or Toll-like receptor (TLR) agonists. Conditioned medium generated from CTB cultures was applied to Mφ cultures before infection or treatment. Alternatively, CTBs were co-incubated with, but physically separated from, Mφ and GBS or TLR-stimulated. NFκB was assessed via alkaline phosphatase assay and proinflammatory mediators were assessed by qRT-PCR and ELISA.

Results:

CTBs suppressed GBS- or TLR-stimulated Mφ NFκB activity, TNFα and MMP9 production. Direct physical contact between CTBs and Mφ was required for full immunosuppression. Immunosuppression could be overcome by increasing the ratio of Mφ to CTB.

Conclusions:

CTBs limit Mφ NFκB activation and production of TNFα and MMP9 through an as-yet unknown, cell-to-cell contact-mediated mechanism. This suppression is distinct from the PGE2-mediated Mφ TNFα suppression by DSC, suggesting that DSCs and CTBs regulate Mφ inflammation through distinct mechanisms. How Mφ integrate these signals in an intact GM will be paramount to determining causes and prevention of PPROM.

Introduction

Bacterial infection or inflammation of the gestational membranes (GM), a condition associated with acute chorioamnionitis (CAM), can trigger preterm birth (PTB), preterm prelabor rupture of membranes (PPROM), and lead to stillbirth or neonatal sepsis1–3. Nearly 25–50% of PTB is attributed to infection4,5 and, even in surviving children, CAM can leave lasting health consequences2,6.

The GMs are comprised of distinct layers of cells, originating from either the mother or the blastocyst (fetus). Within the outermost layer (facing the uterus) are the maternally-derived decidual stromal cells (DSCs), then moving inwards, the fetally-derived cytotrophoblasts (CTBs), mesenchymal cells (fibroblasts), and finally amniotic epithelial cells facing the fetus. Immune cells are also found throughout the membrane; the principal innate phagocyte of the membranes is the macrophage (Mφ)5,7–9, which may be maternal (in the decidua) or fetal (in the chorioamnion) in origin4,6,10. Immune activation within uninfected GMs during pregnancy is polarized towards supporting tolerance and alternative activation, presumably to accommodate the semiallogenic fetus11. This presents a challenge when pathogens arrive, eliciting proinflammatory responses and cytokines such as TNFα, IL-6, and IL-1β.

Our laboratory has previously shown that DSCs use prostaglandin E2 (PGE2) to suppress Mφ TNFα production in response to LPS12. Other contact-dependent mechanisms also play a role in DSC suppression of lymphocyte proliferation13. CTBs express numerous inhibitory surface proteins14–17 as well as PGE218–20, but CTB suppression of Mφ function is an incomplete picture. CTB secreted factors are known to educate immune cells at the maternal-fetal interface21–23, and it has recently been shown that soluble PD-L1 generated from CTBs can alter Mφ phenotype over a long conditioning process24.

Matrix Metalloproteinases (MMPs) are enzymes that can degrade a variety of extracellular matrix components, including collagen and gelatin, and they are associated with both term rupture of membranes and PPROM25–27. Multiple MMPs are expressed in the fetal membranes at term labor including MMP2, 3, 7, 9, and 1328, while MMP expression during preterm labor also extends to MMP3, 8 (likely macrophage-derived29), 10, and 1128. MMP9 is the principal MMP in term labor and also plays a large role in preterm pathologic labor28. MMPs can be induced by cytokines30–33, including MMP9 induction by TNFα34.

In this study, we examined the impact of co-culture with CTBs on Mφ activation. We hypothesized that CTBs would inhibit Mφ cytokine expression through secreted mediators, similar to DSC-mediated TNFα suppression via PGE212.

Methods

Cell lines and regents:

The human monocytic leukemia-derived cell line THP.1 expressing an NFκB reporter gene (NF-κB SEAP Reporter Monocytes, THP.1 Blue, Invivogen, San Diego, CA, USA), was maintained in RPMI 1640 with 2 mM L-glutamine, and 25 mM HEPES (Invitrogen, Carlsbad, CA) and supplemented with 10% charcoal-stripped fetal bovine serum, 1% antibiotic/antimycotic solution and 100 μg/ml Normocin (Invivogen) in T-75 flasks until use in experiments. The human immortalized Jeg3 cytotrophoblast cell line was a kind gift from Dr. Carolyn Coyne’s laboratory at the University of Pittsburgh. Jeg3 CTBs were cultured in MEM (Invitrogen) without phenol red and supplemented with 10% charcoal-stripped FBS and 1% antibiotic/antimycotic solution. Phorbol myristate acetate (PMA; Sigma (St. Louis, MO, USA)) was used at 5 ng/mL to differentiate the nonadherent THP.1 monocytes into Mφ -like cells on the day prior to co-culture with CTBs. The PMA-treated THP.1 cells were assessed for adherence to flasks as a surrogate for differentiation into Mφ. Trophoblast medium was purchased from ScienCell for use with primary CTBs and supplemented with provided FBS, trophoblast growth supplement, and antibiotic (ScienCell, Carlsbad, CA). Ficoll-Paque™ and Monocyte Isolation Kit II® were obtained from Miltenyi (Auburn, CA, USA). Cytokeratin-7 antibody clone OVTL12/30 was obtained from Invitrogen and used at 1:50. Vimentin antibody clone EPR3776 was obtained from abcam (Cambridge, MA, USA) and used at 1:100. M-CSF was obtained from Sigma-Aldrich. Cabozantanib was purchased from MedChemExpress (Monmouth Junction, NJ, USA). HLA-G neutralizing antibody (clone 87G) was obtained from Novus (Centennial, CO, USA). Tim-3 neutralizing antibody (clone F38–2E2) was obtained from BioLegend (San Diego, CA, USA). PD-L1 inhibitor 1 (also known as BMS-1) was obtained from Selleckchem (Houston, TX, USA). CD200R blocking antibodies (clone 325531) were obtained from R&D Systems (Minneapolis, MN, USA). BioRad iScript reverse transcription supermix and BioRad iQ master mix were obtained from BioRad (BioRad, Hercules, California, USA). All TaqMan probes used were obtained from Thermo-Fisher (Waltham, MA, USA): MMP1-Hs00899658_m1, MMP2- Hs01548727_m1, MMP3- Hs00968305_m1, MMP8- Hs01029057_m1, MMP9 Hs00957562_m1, MMP10- Hs00233987_m1, and GAPDH- Hs02758991_g1.

Group B Streptococcus (GBS):

GBS clinical isolate GB0011235, a capsular serotype III strain, was kindly provided by Dr. Shannon Manning at Michigan State University. It was grown overnight at 37°C to stationary phase in trypticase soy broth. Bacteria were pelleted and resuspended in PBS and washed again. GBS was resuspended in PBS and diluted to the proper inoculum (multiplicity of infection (MOI) 10) before being added to Mφ cultures. Bacteria were serially diluted and plated to confirm inoculum.

Mφ CTB co-culture experiments:

CTBs were plated on Day 0 in 96 well plates at 1 × 105 cells/well in 200 μl. On Day 1, PMA-treated (5 ng/mL overnight, the minimum concentration to promote adherence with minimal immune activation (data not shown)) THP.1 cells were added in a 1:1 mixture of antibiotic-free CTB media and Mφ media at a ratio of 1 Mφ:10 CTB for all experiments unless otherwise indicated. After 1 hour, wells were infected with GBS or treated with PAM3CSK4 or LPS (Sigma-Aldrich, Darmstadt, Germany). Infection was allowed to proceed for 1 hour and antibiotic was added to all wells to a 1x concentration. Wells were harvested after 24 hours (on Day 2) and supernatants were used for NFκB activity assay or ELISA. CTBs were plated on Day 0 in 12-well plates at 5 × 105 per well in 200 μl, and the experiment proceeded as described above. Cells were lysed with TRIzol and RNA isolated by phenol-chloroform extraction.

NFκB assay:

Experimental culture supernatants from THP.1 Blue cells were mixed with QuantiBlue reagent (Invivogen) as per manufacturer’s instructions. Reactions were allowed to proceed for 30 minutes or until color differences were distinguishable, then plates were read on a microplate reader at 625nm. Data was normalized to untreated Mφ-alone absorbance values.

ELISA:

TNFα, IL-1β, IL-6, and CCL5 ELISAs were performed on culture supernatants using human DuoSet ELISA kits as per manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). PGE2 ELISAs were performed on culture supernatants using human PGE2 ELISA kit from Enzo (Farmingdale, NY, USA) as per manufacturer’s instructions.

Primary CTB isolation and culture:

This study was reviewed and approved by the Vanderbilt University Institutional Review Board (IRB #181998). Human placentae were obtained following informed consent from term, non-laboring Cesarean deliveries. Primary CTBs were harvested and cultured as described36. Briefly, placental explants with decidua removed were plated in Trophoblast Media with all manufacturer-supplied supplements and cultured for 14–18 days, changing media every 2 days. Outgrowth of CTB colonies was monitored and colonies were removed to sub-culture in chamber slides to confluency, then T-25 flasks, then finally to T-75 flasks where they survived for approximately 15 passages. Cell type specificity was checked by presence of cytokeratin-7 staining (CTB marker) and absence of vimentin staining (decidual stromal cell marker).

Primary monocyte-derived macrophages (MDM):

This study was reviewed and approved by the Vanderbilt University Institutional Review Board (protocol #140699). Subjects were healthy, non-pregnant adults (age 18–60). Exclusion criteria included: fever >101°F in preceding 48 h, antibiotic therapy currently, or within the prior 2 weeks, current immunosuppressive therapy or short-term steroid therapy within the prior 2 weeks, chronic viral infection, including, but not limited to, HIV infection (per self-report), and/or currently diagnosed with or undergoing treatment for cancer. Whole blood was collected into heparinized blood tubes by venous puncture.

Isolation and cultivation of MDM:

Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood by density gradient centrifugation using Ficoll-Paque™ as outlined by the manufacturer Miltenyi. Following centrifugation, monocytes were isolated from PBMCs via negative selection using the Miltenyi Monocyte Isolation Kit II® as described by the manufacturer. Monocytes were cultured in T-75 flasks at a concentration of 1 million cells/mL for seven days in 200 ng/mL M-CSF in RPMI+/+ with 20% FBS to differentiate into MDM. Flow cytometry has previously been used to confirm purity > 98% (data not shown).

Conditioned media experiments:

CTB cultures were maintained overnight in 100 μL of media in uninfected and untreated conditions in a 96-well dish; this served as CTB conditioned media (CCM). 100 μL of supernatants were frozen, then thawed or applied directly without freezing to Mφ cultures in a 96-well dish and treatment or infection was added in 100 μL for a final volume of 200 μL per well, resulting in a 1:1 dilution of the CCM. NFκB assay or TNFα ELISA was performed on the supernatants.

Tilted-plate experiments:

Attempts were made to co-culture THP.1 cells and Jeg3 CTBs either as a traditional, mixed population in a single culture dish, or as relatively separate cell populations cultured on opposite ends of the same vessel. This approach was used to assess the dependence of paracrine signaling on direct cell-cell contact versus soluble mediator-dependent communication. Thus, 12-well dishes were tilted and 500 μL of Jeg3 CTBs (5 × 106 cells total) were plated onto the bottom half of each well on Day 0, then on Day 1 the plates were tilted the opposite direction and 1 mL of Mφ cell suspension (5 × 105 cells total) was added to coat the opposite side from the CTBs with limited volume covering Jeg3 CTBs on the opposing end from the Mφ. As controls, tilted plates were seeded at the same time points with only Jeg3 or only Mφ. For comparison, Jeg3 + Mφ, Jeg3 alone, or Mφ alone wells were seeded at the same time points on plates laid flat to facilitate paracrine signaling with cell-to-cell contact. Cultures were infected with GBS or treated with LPS on Day 2. Supernatants were harvested on day 3.

qRT-PCR:

Reverse transcription was performed using BioRad iScript reverse transcription supermix according to manufacturer’s instructions. qPCR was performed using BioRad iQ master mix according to manufacturer’s instructions using the aforementioned TaqMan probes. Samples were normalized to GAPDH under the following thermal cycling conditions: 3 m at 95°C, 12 s at 95°C, and 45 s at 60°C for 40 cycles. Data are quantified and presented using the 2−ΔΔct method.

Statistical analysis:

All experiments were performed in multiple, independent biological replicates as noted in each figure. Data points from independent experiments are presented along with means and analyzed with GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA). Comparisons between two experimental groups were performed with a paired Student t test, while comparisons between three or more groups were analyzed with 2-way ANOVA with multiple comparisons. Normality was tested for using the D’Agostino & Pearson test37 and log-transformed where appropriate. Differences were considered statistically significant for P < 0.05.

Results

We first determined the behavior of Mφ in culture with and without CTBs using a THP.1 Mφ-like cell line expressing a reporter gene with an NFκB binding site encoding the gene for alkaline phosphatase, which is secreted into the media upon NFκB activation. Addition of a substrate solution results in a colorimetric assay for NFκB activation. We normalized the data to untreated Mφ activation to compare across experiments. Mφ significantly activated NFκB in response to the gram-positive GBS at a multiplicity of infection (MOI) of 10 and in response to LPS treatment (Fig. 1A). Upon direct co-culture of Mφ with the Jeg3 CTB cell line at a ratio of 10 CTBs to 1 Mφ, Mφ NFκB was significantly suppressed in response to all treatments; Mφ activation was also suppressed at baseline (Fig. 1A). Similarly, TNFα production was significantly induced in the Mφ by GBS infection and LPS stimulation, but co-culture of Mφ with CTB suppressed TNFα (Fig. 1B).

Figure 1. Cytotrophoblasts suppress macrophage NFκB activity and TNFα production in direct co-culture.

Figure 1.

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Mφ and CTBs were plated each alone or in combination, then infected with GBS strain 112 at an MOI of 10 or treated with LPS at 1 μM for 24 hours and then harvested. A) NFκB assay from THP.1 Mφ expressing an NFκB reporter construct making alkaline phosphatase under the control of the NFκB response element. B) ELISA for TNFα was performed on supernatants from THP.1 Mφ mono- and co-culture with Jeg3 CTBs. C) Blood Mφ matured from whole blood monocytes by magnetic selection and incubation over 7 days with M-CSF (monocyte derived macrophages; MDM) were incubated alone or in combination with Jeg3 CTBs or primary human CTBs and tested for TNFα concentration by ELISA. For all experiments in Figure 1, N = 9 from 3 separate, matched experiments of 3 replicates each. * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-way ANOVA with Sidak’s multiple comparisons test, log transformed where indicated on Y-axis.

To replicate these findings using primary cells, we matured CD14+ blood monocytes into Mφ (monocyte-derived macrophages; MDM) over 7 days using M-CSF and repeated the direct co-culture with Jeg3 CTBs or with primary CTBs during treatment with GBS, TLR2 agonist PAM3CSK4, and LPS. MDMs induced greater TNFα production at baseline and in response to treatment than THP.1 cells (Fig. 1C, white bars). Primary CTBs significantly suppressed MDM TNFα production at baseline while Jeg3 CTBs did not. However, Jeg3 CTBs still significantly suppressed TNFα production from MDM during GBS and LPS treatment, and primary CTBs suppressed MDM TNFα production to an even greater degree (Fig. 1C).

Comparing immune suppression from MDMs of other analytes by Jeg3 and primary CTBs, we found that only primary CTBs suppressed MDM IL-1β (Fig. S1A), while both Jeg3 and primary CTBs equally suppressed IL-6 and CCL5 production from the MDM (Fig. S1B–C).

Our laboratory has previously published that DSCs suppress Mφ TNFα production through the action of PGE2 and that this can be accomplished using uninfected decidual stromal cell-conditioned media12. We found that Jeg3 CTB made PGE2 both at baseline and during infection, although it was not induced further by infection with GBS at any MOI or treatment with LPS (Fig. S2). Conditioned medium was generated from uninfected CTB cultures (CCM) and added to Mφ cultures prior to infection with GBS. Surprisingly, CCM was unable to suppress THP.1 Mφ NFκB activity (Fig. 2A) or TNFα production (Fig. 2B). Furthermore, CCM was taken from infected CTB cultures, filtered to remove bacteria, and applied to Mφ cultures which were then infected or treated. This also did not suppress Mφ activation (data not shown).

Figure 2. Cytotrophoblast conditioned media does not suppress macrophage NFκB activity or TNFα production.

Figure 2.

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Jeg3 CTBs were cultured overnight and their supernatant was used as CTB conditioned media (CCM). THP.1 Mφ were cultured alone, with CTBs directly, or with CCM and infected with GBS strain 112 at an MOI of 10 or treated with LPS at 1 μM for 24 hours and then harvested. A) NFκB assay and B) TNFα ELISA (log transformed) run on supernatants from cultures. For all experiments in Figure 2, N = 9 from 3 separate, matched experiments with 3 replicates each. Data was log transformed where indicated on the y-axis label. * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-way ANOVA with Tukey’s multiple comparisons test.

To further determine if real-time feedback from infected CTB cultures is necessary for Mφ immune suppression, cells were co-incubated allowing either direct cell-cell contact between CTBs and Mφ or not (by physically separating the two cell types within the same culture vessel (see Methods)). While direct co-culture of Mφ and CTB in a flat plate with Mφ-CTB cell-to-cell contact significantly suppressed NFκB activity during GBS infection or LPS treatment, direct co-culture in the tilted plate did not suppress Mφ NFκB activity (Fig. 3A). Further, co-culture of Mφ and CTB in a flat plate suppressed TNFα production while direct co-culture in the tilted plate with minimal Mφ-CTB contact did not (Fig. 3B).

Figure 3. Direct contact is required for cytotrophoblast suppression of macrophage NFκB activity and TNFα production.

Figure 3.

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Jeg3 CTBs were plated in 24 well dishes tilted ~10–20 degrees to deposit cells only on half of each well in the plate on day 0. On day 1, dishes were tilted the opposite direction and THP.1 Mφ were plated on the other side. On day 2, dishes were placed flat and infected or treated as above. All experiments were compared to Jeg3 CTBs and THP.1 Mφ plated together on a flat plate following the same timeframe. A) NFκB activation in THP.1 blue cells cultured individually, in a flat plate, or in a tilted plate, was assayed by colorimetric NFκB assay and b) TNFα production was similarly assayed by ELISA. For all of Figure 3, N = 6 from 3 separate, matched experiments with 2 replicates each. *** p < 0.001 by 2-way ANOVA with Tukey’s multiple comparisons test; n.s. not significant difference.

To identify whether the immunomodulatory actions of CTBs were inducible by GBS infection or were intrinsic in uninfected cells, we infected CTBs and Mφ individually overnight with GBS on day 1, then on day 2 added infected CTBs to Mφ (which were infected or uninfected) and compared this to adding uninfected CTBs and infected with GBS overnight. We found that uninfected CTBs co-cultured with pre-stimulated Mφ suppressed Mφ NFκB activation to an equal extent as pre-stimulated CTBs did, suggesting that CTBs constitutively express the inhibitory surface molecule (Figure S3A). Furthermore, rested CTBs could suppress both rested and pre-stimulated Mφ to an equal extent (Fig. S3B).

We next tested the hypothesis that the paracrine suppression of Mφ immune responses by CTBs could be overcome by increasing the relative number of Mφ in the coculture system. To address this, we co-cultured Mφ and CTBs in ratios of 1:10, 1:5, 1:2, and 1:1 Mφ per CTB. In the 1:10 Mφ:CTB ratio, we observed significant suppression in GBS and LPS treatments (Fig. 4A). At a ratio of 1:5 Mφ:CTB, CTBs were only able to suppress NFκB activity in GBS-treated wells and not LPS (Fig. 4B). At ratios of 1:2 and 1:1 Mφ:CTB, CTB suppression of Mφ NFκB was lost (Fig. 4C–D).

Figure 4. Increasing ratios of macrophages to cytotrophoblasts is sufficient to overcome contact-mediated suppression of NFκB.

Figure 4.

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Mφ and CTBs were plated as in Figure 1, but the ratio of Mφ to CTB gradually increased from A) 1:10, to B) 1:5, to C) 1:2, to D) 1:1 with the number of CTBs staying constant and the number of Mφ increasing with the ratio. For all of Figure 4, N = 9 from 3 separate, matched experiments with 3 replicates each. * p < 0.05 by 2-way ANOVA with Sidak’s multiple comparisons test.

To determine the mechanism of Mφ-CTB cell contact-mediated suppression, we tried numerous pharmacological inhibitors and neutralizing antibodies against known suppressive Mφ or CTB surface proteins. We first investigated TAM receptor-mediated immunosuppression38,39. Treatment of cultures with the broad TAM receptor inhibitor Cabozantanib (CABO) did not rescue MDM TNFα production during co-culture with CTBs (Fig. 5A). We next investigated LILRB-HLA-G immunosuppression14,15 by treating cultures with an HLA-G neutralizing antibody. HLA-G neutralizing antibody did not rescue MDM TNFα production during co-culture with CTBs (Fig. 5B). We investigated PD-L/PD-L1-mediated immunosuppression using PD-L/PD-L1 inhibitor 1. PD-L/PD-L1 inhibition did not rescue MDM TNFα production during co-culture with CTBs (Fig. 5C). We investigated Tim3-mediated immunosuppression40 by using Tim3-neutralizing antibodies in direct MDM-CTB co-culture. Tim3 neutralizing antibody also did not rescue MDM TNFα production during co-culture with CTBs (Fig. 5D). Lastly, we tested whether CD200R inhibition by CD200R blocking antibodies could rescue the CTB-dependent immunosuppression and found that CD200R neutralizing antibody also did not rescue MDM TNFα production (Fig. 5E).

Figure 5. Selective blockade of several known cell contact-dependent macrophage inhibitory receptor pathways does not rescue TNFα production during cytotrophoblast co-culture.

Figure 5.

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MDMs and Jeg3 CTBs were plated as in Figure 1, but with A) TAM receptor blocker Cabozantanib, B) HLA-G neutralizing antibody C), PD-L1 inhibitor 1, D) Tim-3 neutralizing antibody, or E) CD200R neutralizing antibody added simultaneously with Mφ addition before infection or treatment. For all experiments from Figure 5, N = 6 from 3 separate, matched experiments with 2 averaged replicates each. * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-way ANOVA with Tukey’s multiple comparisons test.

To assess the impact of CTB-mediated cytokine suppression on MMP production, which is associated with preterm premature rupture of membranes28, we measured GBS, TLR2 agonist- (PAM3CSK4, a known inducer of MMP941) and TLR4 agonist (LPS)-induced production of MMP1, 2, 3, 8, 9, and 10 by qRT-PCR. We did not find expression of MMP1, 3, 8, and 10 (data not shown). There was a trend of MMP2 upregulation in Mφ-alone cultures that was suppressed when co-cultured with CTBs during GBS, PAM3CSK, and LPS stimulation, but this did not reach significance (Fig. 6A). There were no changes in MMP2 protein production by ELISA (Fig. 6B). Conversely, MMP9 mRNA and protein were significantly upregulated in Mφ-alone cultures during stimulation, but co-culture with CTBs suppressed the induction of MMP9 (Fig. 6C–D).

Figure 6. MMP9 is induced in Mφ during stimulation but this is suppressed during co-culture with CTBs.

Figure 6.

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THP.1 blue Mφ and Jeg3 CTB were plated as in Figure 1. MMP2 levels were assessed by A) qRT-PCR or B) ELISA and MMP9 levels were assessed by C) qRT-PCR or D) ELISA. (A) and (C) N = 6 from 3 separate, matched experiments with 2 replicates each, (B) and (D) N = 18 from 3 separate, matched experiments with 6 replicates each. * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-way ANOVA with Sidak’s multiple comparisons test, log transformed where indicated on Y-axis label.

Discussion

In this study, we have shown that immortalized and primary Mφ activate NFκB and make proinflammatory cytokines and MMP9 in response to GBS infection or TLR agonism, but that co-culture with immortalized and primary CTBs suppress induction of these measured outputs. Conditioned medium from CTBs and even indirect co-culture were insufficient to suppress Mφ NFκB and TNFα, suggesting that this suppression is cell-to-cell contact-dependent. Despite testing several cell contact-dependent mechanisms known to a) suppress NFκB and/or TNFα and b) be expressed at the maternal-fetal interface, the mechanism behind this cell-to-cell contact-dependent immunosuppression by CTBs remains elusive. However, CTB-mediated suppression can be overcome with a sufficiently high ratio of Mφ to CTB.

Of note, in our hands, CTBs cultured alone failed to induce secretion of any inflammatory and tissue-remodeling molecules screened (IL-1β, TNFα, CCL2, CCL5, IL-6, IL-8, MMP2, MMP9, CXCL10, IL-10, IL-4) regardless of stimulation (GBS, TLR2, TLR4). This is in contrast to reports of Jeg3 CTBs producing TNFα and IL-642. We originally selected Jeg3 cells for this study because of their reported induction of TNFα and IL-6, and we hypothesized that Jeg3 secreted factors would have a large impact on Mφ activation. This turned out not to be the case.

In this study, we used both primary human cells and immortalized cell lines. The THP.1 monocyte-like cells43 were activated overnight in a very low dose of PMA (5 ng/mL), which was sufficient to activate but minimally polarize the cells (as assessed by adherence with minimal TNFα, IL-10, and IL-12 secretion, data not shown). MDMs were cultured in M-CSF for 7 days, which is often associated with a more alternatively-activated Mφ phenotype relative to maturation with GM-CSF44. Over 24 hours of stimulation through PRRs, THP.1 cells secreted much less TNFα per milliliter than the MDM. The contact-dependent CTB immunosuppression is sufficient to inhibit both the relatively low TNFα production from the THP.1 cells and the higher TNFα production from the MDMs, suggesting that the immunosuppression may work on different polarizations and types of Mφ.

During the conditioned medium experiments, treatment of Mφ with CTB-conditioned medium was insufficient to suppress Mφ TNFα production and NFκB activation. Furthermore, we noted a slight increase in NFκB activation from Mφ in presence of the conditioned medium (Fig. 2A, untreated bars) that was mirrored in the tilted-plate experiments. Mφ cultured in the same culture vessel but on the opposite side from the CTBs had an elevated NFκB induction at baseline (Fig. 3A, untreated bars). However, this augmentation of NFκB in the presence of CTB-secreted factors did not correspond to an increase in TNFα production in either experiment (Fig. 2B, 3B) under either unstimulated or stimulated conditions. It is unclear whether the baseline NFκB increase corresponded to increases in other secreted cytokines at baseline or functional differences in the Mφ activation.

MMPs are induced through multiple mechanisms32,45. Among these, MMP9 is known to be upregulated in THP.1 cells by TLR2 stimulation via the NFκB signaling pathway41. Our data is consistent with these studies: NFκB was suppressed in THP.1 cells and this suppression was mirrored by MMP9 during GBS and TLR stimulation while in direct co-culture with CTBs. While MMPs are made by invasive trophoblasts46, we expect that the MMP9 in our system is derived from Mφ due to its absence in CTB-only cultures (data not shown). We show here that MMP9 is suppressed in Mφ during co-culture with CTBs; however, it is unclear whether the MMP9 suppression is regulated by the same mechanism governing TNFα suppression or whether MMP9 suppression follows TNFα suppression and is not induced because TNFα is not induced. The lack of expression of many other MMPs known to be found in gestational membranes28 may be due to the in vitro nature of the experiments, and other MMPs may be subject to CTB-mediated suppression in vivo.

A remaining question is, why is CTB-conditioned media, which contains PGE2 (Fig. S2), unable to suppress TNFα as it does during co-culture between Mφ and DSCs12? It is possible that there are changes to prostaglandin receptor expression on the Mφ47, or changes to other necessary pathways’ gene expression when Mφ are co-cultured with CTB versus DSC. In preliminary experiments employing transwell tri-culture of DSC, CTB, and Mφ, TNFα is inhibited, but it is unclear whether it is through the DSC-mediated PGE2 production, the unknown CTB contact-mediated mechanism, a novel mechanism, or combination.

Recent publications have found that soluble PD-L124 and exogenous Leukemia Inhibitory Factor (LIF)48 at the maternal-fetal interface can alter Mφ inflammatory properties, including TNFα secretion, and affect Mφ-CTB interactions. Zhang et al. found that 6–7 days of incubation with CTB-conditioned medium in the presence of PD-1 blocking antibodies changed the phenotype of Mφ from M2 to M1, but it is unclear the minimum length of time necessary to achieve M1 suppression. Hamelin-Morrissette et al. studied the impact of Mφ activation with and without LIF on CTB invasion in transwell co-culture studies using HTR8/SVneo CTBs and human blood monocyte-derived Mφ, and found that pre-treatment of IFNγ-activated Mφ with LIF abolished their inhibition of CTB invasion. Our data here suggests that there is a contact-dependent mechanism at play which, in less than 24 hours, dramatically suppresses Mφ TNFα. This may be a useful strategy in the chorion to educate Mφ that have migrated from other parts of the tissue. In combination with our laboratory’s previous work showing DSC suppression of Mφ TNFα through secreted PGE212, multiple mechanisms are at play in a complete model of the maternal-fetal interface to suppress Mφ proinflammatory responses.

There are several limitations to this study. Of greatest significance is the absence of a defined mechanism behind the contact-dependent immunosuppression. Potential candidates for cell surface receptors implicated in the immunosuppression were known immunosuppressive surface receptors expressed on Mφ that act on the NFκB and/or TNFα pathways, and whose ligands are known to be expressed on CTBs. None of the screened surface receptors were individually responsible for suppression of Mφ TNFα production, but the possibility remains for the involvement of numerous redundant immunosuppressive pathways- multiple mechanisms, both those screened here and unscreened, may be at play.

Mφ, as motile cells, may travel through the GMs and will be subject to immunoregulation by the cell types to which it is in contact and to which it is within distance of paracrine signaling. How the Mφ integrates these signals from multiple layers of the membrane is of great importance, particularly given what may be conflicting signals: co-culture of DSC with Mφ increases many proinflammatory cytokines upon stimulation, while co-culture of CTB (the adjacent layer in the GM) suppresses many of the same proinflammatory cytokines, such as IL-1β and CCL5 (not shown). The amnion layer of the GM, frequently employed in immunomodulatory studies49, adds another layer of complexity to Mφ integration of signals. More work modeling all layers of the GM and their interactions with immune cells is necessary to form a complete understanding of immune responses to pathogens in the GM.

Supplementary Material

Figure S1

Supplemental Figure 1. Primary cytotrophoblasts inhibit macrophage proinflammatory cytokine production to an equal or greater degree than immortalized cytotrophoblasts. Blood Mφ matured from whole blood monocytes (MDM) by magnetic selection and incubation over 7 days with M-CSF were incubated in alone or in combination with Jeg3 CTBs or primary human CTBs and tested for A) IL-1β, B) IL-6, or C) CCL5 concentration by ELISA. N = 6 from 3 experiments of 2 replicates each. * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-way ANOVA with Tukey’s multiple comparisons test.

Figure S2

Supplemental Figure 2. Jeg3 cytotrophoblasts make PGE2. Supernatants from CTB monoculture were infected with GBS at increasing MOIs or treated with LPS and ELISAs performed for PGE2. No significant differences were detected by student’s t-test.

Figure S3

Supplemental Figure 3. Jeg3 cells constitutively express mechanism for Mφ TNFα immunosuppression. THP.1 blue cells were activated with PMA overnight on day 0, and infected overnight with GBS or rested on day 1. Jeg3 cells were plated on day 0 and on day 1 were infected overnight with GBS or rested. On day 2, the THP.1 blue cells were added to the Jeg3 cultures at a ratio of 1:10 and infected with GBS or left untreated. NFκB activity was assayed by alkaline phosphatase QuantiBlue assay. A) Mφ were co-cultured with pre-activated CTBs or rested CTBs. *** p < 0.001 by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. B) CTBs were co-cultured with pre-activated Mφ or rested Mφ and compared to their respective pre-activated Mφ or rested Mφ alone. *** p < 0.001 by 2-way ANOVA with Sidak’s multiple comparisons test. For all of Figure S3, N = 12 from three separate, matched experiments with 4 replicates each.

Acknowledgments

This work was funded by the NIH (1R01AI134036-01, T32AI095202-02) and the March of Dimes (#6‐FY17‐295). The authors declare we have no conflict of interest. We would like to thank Carolyn Coyne for the gift of Jeg3 cells and Nadja for assistance in focus.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

Supplemental Figure 1. Primary cytotrophoblasts inhibit macrophage proinflammatory cytokine production to an equal or greater degree than immortalized cytotrophoblasts. Blood Mφ matured from whole blood monocytes (MDM) by magnetic selection and incubation over 7 days with M-CSF were incubated in alone or in combination with Jeg3 CTBs or primary human CTBs and tested for A) IL-1β, B) IL-6, or C) CCL5 concentration by ELISA. N = 6 from 3 experiments of 2 replicates each. * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-way ANOVA with Tukey’s multiple comparisons test.

NIHMS1652621-supplement-Figure_S1.jpg (376KB, jpg)
Figure S2

Supplemental Figure 2. Jeg3 cytotrophoblasts make PGE2. Supernatants from CTB monoculture were infected with GBS at increasing MOIs or treated with LPS and ELISAs performed for PGE2. No significant differences were detected by student’s t-test.

NIHMS1652621-supplement-Figure_S2.jpg (104KB, jpg)
Figure S3

Supplemental Figure 3. Jeg3 cells constitutively express mechanism for Mφ TNFα immunosuppression. THP.1 blue cells were activated with PMA overnight on day 0, and infected overnight with GBS or rested on day 1. Jeg3 cells were plated on day 0 and on day 1 were infected overnight with GBS or rested. On day 2, the THP.1 blue cells were added to the Jeg3 cultures at a ratio of 1:10 and infected with GBS or left untreated. NFκB activity was assayed by alkaline phosphatase QuantiBlue assay. A) Mφ were co-cultured with pre-activated CTBs or rested CTBs. *** p < 0.001 by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. B) CTBs were co-cultured with pre-activated Mφ or rested Mφ and compared to their respective pre-activated Mφ or rested Mφ alone. *** p < 0.001 by 2-way ANOVA with Sidak’s multiple comparisons test. For all of Figure S3, N = 12 from three separate, matched experiments with 4 replicates each.

NIHMS1652621-supplement-Figure_S3.jpg (250.5KB, jpg)

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