The establishment of pregnancy requires a finely balanced interaction between embryonal and maternal tissues that is regulated by hormones, cytokines, and other regulatory systems. Several studies in mammals have shown that inflammation is crucial at the maternal-fetal interface, both at implantation and parturition [1
]. Human placentation is characterized by extravillous trophoblast migration, invasion into the decidua, and replacement of endothelial cells in spiral arteries. This process allows spiral artery remodeling to increase blood supply to the developing fetus [3
]. Abnormal inflammation is associated with trophoblast failure to migrate and invade into blood vessels and to acquire the endothelium-like phenotype. This failure contributes to pregnancy pathologies such as preeclampsia, intrauterine growth restriction, and other pregnancy associated pathologies (reviewed in [4
The IL-36 group of cytokines comprises three pro-inflammatory agonists (IL-36α, IL-36β, and IL-36γ), and one antagonist (IL-36Ra), which are recently getting attention because of their role in inflammatory diseases like psoriasis [5
]. All members of the IL-36 family use the same receptor (IL-36R), a heterodimer composed of interleukin-1 receptor-like 2 (IL1RL2) as the ligand binding moiety and the IL-1 receptor accessory protein (IL1RAcP). This receptor induces inflammatory responses through MyD88, MAPK, NF-kB, and AP-1 pathways. IL-36Ra acts as a natural antagonist, whose binding to IL-36R does not trigger IL-1RAcP recruiting, counterbalancing the inflammatory process [9
The role of the IL-36 family in pregnancy remains largely unknown. In mice, high expression of IL-36 was reported in the uterus at estrus stage and during labor, and is highly induced by Listeria monocytogenes,
demonstrating its role in inflammation and maternal immune response to pathogens [11
]. In humans, a recent report demonstrated enhanced levels of IL-36Ra in plasma samples of pregnant compared to non-pregnant women, and decreased IL-36Ra levels in extracellular vesicles from preeclampsia versus healthy placentas [13
]. So far, these results suggest that the IL-36 system may have an important role for uterine biology and for the immunological processes required in human pregnancy. We have hypothesized that the IL-36 system regulates trophoblast functions, as specifically the communication with endothelial cells, which may be altered by pathogens and in pregnancy complications such as preeclampsia. Therefore, the aim of this work is to elucidate the potential role of IL-36 agonists in trophoblast cells and to identify their association with angiogenic factors. The expected results will contribute to a better understanding of the function of these pro-inflammatory cytokines during pregnancy including their implication in the trophoblast response to microbial components.
In the early stages of pregnancy, extravillous trophoblast cells migrate into the maternal decidua, surround the spiral arteries, access their lumen and replace their endothelial cells [3
]. This physiological process of vascular remodeling assures proper blood flow to the fetus and, hence, the materno-fetal exchange of nutrients and waste products [14
]. Disruption of this process results in aberrant placental vascularization and placental pathologies. A complex cytokine network regulates, both positively and negatively, the angiogenic pathways associated with trophoblast-endothelial interaction [20
]. Recently, members of the IL-1 superfamily (comprising the IL-36 subfamily) were found in placenta and trophoblast cells, and aberrantly expressed in preeclampsia, a pregnancy disorder often associated with defective trophoblast-driven angiogenesis [13
]. However, the functions of the IL-36 subfamily in pregnancy remains largely unknown.
IL-36 cytokines play important roles in innate and adaptive immune responses associated with inflammation in skin, kidneys, joints, brain, and lungs [21
]. Several cell types including epithelial (keratinocytes), dendritic, and T helper cells, as well as macrophages and granulocytes are important producers and responders to IL-36, and participate in pro-inflammatory diseases, such as psoriasis [7
]. Here, we report that all members of the IL-36 cytokine family are expressed constitutively in PTC as transcripts and proteins, namely IL36A
(IL-36Ra), and their receptor IL1RL2
(IL-36R). In our study, the two widely recognized trophoblast cell models JEG-3 and HTR-8/SVneo cells have similar expression of IL36A
mRNA and protein in their cell lysates. In HTR-8/SVneo cells, IL36B
transcripts were below detection limit, but both proteins were detected. It can be argued that in non-treated cells, only low levels of transcripts are available, but under exogenous treatment, mRNA expression can be induced. Differences in the expression of proteins, mRNA, and miRNAs between trophoblastic models and compared to primary cells have been cumulatively reported [24
]. Therefore, two cell models were included in this study to investigate general functions of the IL-36 system in trophoblast biology. JEG-3 cells were derived from choriocarcinoma [27
] and are often considered a model for third trimester trophoblast cells and HTR-8/SVneo cells were obtained by immortalization of isolated first trimester extravillous trophoblast cells [28
Several epidemiological and causal studies have evaluated the relation between maternal infections and pregnancy disorders. In most cases, these studies indicate an association between bacterial and viral infections and the pathogenesis of pregnancy complications [29
]. Previously, we reported a gestational age-dependent expression of IL-36 cytokines in the mouse uterus, which is strongly induced in presence of L. monocytogenes
]. To examine whether IL-36 cytokines mediate trophoblast response to local infection, we challenged PTC and trophoblastic cells with bacterial (LPS) and viral (poly I:C) components. A transient increase of IL-36 family expression was observed upon both treatments at RNA and protein levels. In our settings, in both PTC and HTR-8/SVneo cells, poly I:C was a stronger inducer of IL-36 cytokine transcripts than LPS with a peak at 12 h of stimulation, whereas, simultaneously, the induction of intracellular protein expression of IL-36 (α, β, and γ) was similar, demonstrating differences in the kinetics of protein and RNA expression. Altogether, these observations agree with other reports highlighting the IL-36 axis function in inflammatory response during bacterial, viral and fungal infections [32
]. They also add to the observation that in keratinocytes low doses of poly I:C already induce expression and release of soluble IL-36γ in a dose- and time-dependent manner [35
In our study, primary cells were more sensitive to IL-36 stimulation than HTR-8/SVneo cells, and almost no effects were observed in JEG-3 cells. Upon LPS and poly I:C administration, IL36G
had the highest expression changes in PTC. IL-36γ and IL-36R are also present in epithelial cells of the human female reproductive tract (FRT) and are induced by poly I:C [36
]. Pre-treatment with IL-36γ prior to mouse intravaginal viral challenge significantly limited vaginal viral replication and delayed disease onset, decreased disease severity, and increased mice survival [37
]. Altogether, these data suggest a role for IL-36γ in host defense against invading pathogens in placental tissues worthy to be further evaluated.
Our results show that IL-36 cytokines are induced by microbial components in trophoblast cells. Treatment with LPS and poly I:C alters trophoblast function which could impair their ability to remodel spiral arteries and contribute to the pathogenesis of pregnancy complications [38
]. Therefore, we investigated the potential effects of IL-36 on trophoblast behavior. Additional administration of recombinant IL-36γ, which is constitutively expressed by HTR-8/SVneo cells has no effect on their 2D migration. Conversely, both IL-36α and -β promoted migration, but the effect of IL-36α was significant only at low concentrations. This observation is similar to that reported for IL-1β effects on formation of tube-like structures by trophoblast cells. At low concentrations IL-1β increases and at high concentrations decreases their length [40
]. Accordingly, we assessed the effect of IL-36 (α, β, and γ) on the capacity of trophoblast to interact with endothelial cells. We found that IL-36 (α, β, and γ) induced a significant increase in the number of nodes and IL-36 (β and γ) additionally favored tube elongation in structures of trophoblastic cells when co-cultured on preformed HUVEC tubes. Node quantification and tube length are accepted parameters for the analysis of in vitro angiogenesis assays, but as they rely, respectively, on the sprouting and growing capacities of the network, which are spatially limited, results may differ among methods [41
]. In our results, both parameters pointed out to an enhancing effect of IL-36 cytokines on the interaction of trophoblast and endothelial cells. This goes in line with a report on human endothelial cells co-cultured on a monolayer of primary fibroblasts. Stimulation with IL-36α and IL-36γ results in a significant increase of tubule length and branch point number, which is partly due to the induction of VEGFA expression in fibroblast but not in endothelial cells [42
]. However, a more detailed study is needed to elucidate the molecular effects of IL-36 cytokines on trophoblasts and endothelial cells.
Members of the VEGF family, VEGFA and PGF are critical for embryonic angiogenesis [43
]. During pregnancy, VEGFA and PGF are expressed in villous and extravillous trophoblast cells, villous vascular endothelium, and decidual natural killer cells, and are altered in pregnancies with adverse outcomes [44
]. In this study, IL-36 agonists (α, β, and γ) induced VEGFA
mRNA in a dose- and cell type-dependent manner in trophoblastic cells. In contrast, no changes were observed in VEGFA
mRNA after IL-36 (α, β, and γ) stimulation in HUVECs (Supplementary Figure S4
) although these cells constitutively express IL-36R. Abnormal vascular growth and impaired endothelial function are often associated with pregnancy disorders such as preeclampsia. However, data on VEGF
expression in normal pregnancy and preeclampsia are still controversial as their mRNA levels have been reported to be decreased, increased or unchanged in preeclamptic placental tissue [45
]. Here, we report induction of VEGFA and PGF mRNA transcript and protein expression in trophoblastic cells by IL-36 and its correlation with promotion of tube formation at low doses.
miRNAs have been established as major regulators of gene expression. Several miRNA species have been suggested to play a role in placental vascular formation by targeting VEGFA
and other factors (reviewed in [51
]). The miRNA profile in trophoblast cells changes with the gestational age and in presence of Leukemia Inhibitory Factor (LIF), a predominant cytokine present in the placenta during early pregnancy [19
]. Here, we are reporting a general induction of miRNAs associated with angiogenesis or pregnancy pathologies in trophoblast cells upon IL-36 administration. We identified miR-146a-3p as strongly induced in PTC by IL-36 cytokines. miR-146a was also identified as the most upregulated miRNA in hepatocellular carcinoma (HCC) tissue and cells in vitro. This miRNA enhances endothelial cell activities associated with angiogenesis [52
]. Likewise, miR-141-5p and miR-141-3p were induced by IL-36 agonists, in HTR-8/SVneo cells by more than 10-fold. Previously, we have reported upregulation of miR-141-3p in placenta tissue from pregnancies complicated with preeclampsia. This miRNA is exported from trophoblastic cells via extracellular vesicles (EVs) and changes proliferation in target immune cells [53
]. In a model of ovarian cancer, miR-141-3p-containing EVs induce the expression of VEGFR-2 in endothelial cells promoting migration and angiogenesis [54
]. These observations advocate for a dysregulation of miR-141-3p in malignancies, which can result in aberrant angiogenesis and cell-to-cell communication with immune cells. The implication of the IL-36 axis on these changes in the first stages of pregnancy remains to be elucidated.
Altogether, the results of this study indicate that IL-36 cytokines may play a role in the immune response of trophoblast cells to local infection and/or inflammation. They act as positive regulators of trophoblast migration and angiogenic potential. Their effects seem to be cell type specific and depend on their local concentration.
4. Materials and Methods
The Placenta Lab strictly applies quality management and is certified after DIN EN ISO 9001.
4.1. Isolation of PTC
PTC were isolated from third trimester placentas as described before [53
]. In brief, placental villi were cut into small pieces, washed in sterile PBS with 1% penicillin/streptomycin, and then enzymatically digested at 37 °C in three cycles of 12 min with digestion enzyme solution containing 0.1 mg/mL of DNase type IV, 0.5 mg/mL of Collagenase type IV and 1 mg/mL of Protease type IV (all from Sigma-Aldrich Taufkirchen, Germany) in Dulbecco’s Modified Eagle’s Medium (DMEM) serum free medium (Gibco Thermo Fischer, Carlsbad, CA, USA). Enzymatic activity was stopped by adding equal amount of DMEM supplemented with 10% fetal bovine serum (FBS). Cell suspension was filtered through 100 μm cell strainers, centrifuged for 20 min at 700× g
and resuspended in supplemented DMEM. A PercollTM
(Merck KGaA, Darmstadt, Germany) gradient (60% and 25%) was used to separate cells. After a spin at 750× g
for 30 min without brake, the cell layer between 25 and 60% PercollTM
was collected. Trophoblast solution was washed twice with supplemented DMEM medium and centrifuged at 700× g
for 5 min. Contaminating erythrocytes were eliminated by incubation with 4 mL of 1X RBC lysis buffer (BioLegend, Koblenz, Germany) at room temperature and protected from light for 10 min. After RBC lysis, the cell suspension was centrifuged at 350× g
for 5 min and resuspended in 2 mL Hanks’ Balanced Salt Solution (HBSS). Leucocytes and fibroblasts were depleted by negative isolation using Dynabeads®
(Invitrogen Life Technologies, Darmstadt, Germany) magnetic beads coated with anti-CD45 (BioLegend) and anti-CD82 (Dako Denmark A/S, Glostrup, Denmark) antibodies. The obtained supernatant containing the unbound trophoblast cells was centrifuged at 350× g
for 5 min. The trophoblast pellet was resuspended in supplemented DMEM and isolated trophoblast cells were seeded in a well plate and cultured at 37 °C in 5% CO2
for further experiments.
4.2. Cell Culture
JEG-3 and HUVEC cell lines were purchased from the Leibniz Institute—German Collection of Microorganisms and Cell Cultures DSMZ (Braunschweig, Germany). The immortalized cell line HTR-8/SVneo was provided by Dr. Charles H. Graham, (Queen’s University, Kingston, ON, Canada). Cell cultures were performed at 1 × 106 cells in a 75 cm2 flask and maintained under standard conditions (37 °C, 5% CO2 and humid atmosphere) in DMEM, Ham’s F-12 Nutrient Mix or RPMI 1640 Medium (Gibco Thermo Fischer) for PTC, JEG-3 and HTR-8/SVneo cells, respectively. All media were supplemented with 10% FBS and 1% penicillin-streptomycin antibiotic solution. HUVEC culture was maintained in Endothelial Cell Growth Medium (ECGM) supplemented with 10% FBS and PromoCell Supplement Mix (Promo Cell GmbH, Heidelberg, Germany).
4.3. Cell Stimulation
Trophoblastic cell lines (4 × 105 HTR-8/SVneo and JEG-3 per well) were seeded in 6-well culture plates and allowed to attach overnight. Cells were stimulated with 100 ng/mL LPS, 25 μg/mL poly I:C (both from Sigma-Aldrich) or medium alone as control for 6, 12 and 24 h. Based on the results from cell lines, PTC were incubated for 12 h with the same concentrations of LPS and poly I:C.
For the assessment of angiogenic factors and miRNAs, 2 × 105 HTR-8/SVneo, PTC or HUVEC cells were cultured in 12-well plates and then treated for 24 h with 20 or 50 ng/mL of IL-36 (α, β, or γ; ImmunoTools GmbH, Friesoythe, Germany).
After stimulation, cells were harvested for RNA isolation or Western blotting.
4.4. RNA Isolation and qPCR
RNA was isolated from cells before and after stimulation using TRIzol reagent (Invitrogen, Darmstadt, Germany). Total RNA concentration was determined in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Samples with A260/A280 ratio >1.8 were stored at −80 °C until being processed. Expression of IL-36 members and IL-6 was determined by reverse transcription using High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was performed using TaqMan assays (IL36A, Assay ID: Hs00205367_m1; IL36B, Assay ID: Hs00758166_m1; IL36G, Assay ID: Hs00219742_m1; IL36RN, Assay ID: Hs00202179_m1; IL1RL2, Assay ID: Hs00187259_m1; IL6, Assay ID: Hs00174131_m1; VEGFA, Assay ID: Hs00903127_m1; PGF, Assay ID: Hs01119259_m1; and GAPDH, Assay ID: Hs02758991_g1) and TaqMan Universal PCR Master Mix reagents (Applied Biosystems). qPCR was run on a Mx3005P qPCR System (Applied Biosystems). Expression of IL-36 members, IL-6, VEGFA and PGF were normalized using the 2−ΔCt method relative to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Expression level of seven miRNAs (has-miR-132-3p, Assay ID: 000457; has-miR-141-3p, Assay ID: 000463; has-miR-141-5p, Assay ID: 002145; has-miR-146a-3p, assay ID: 000468; has-miR-193b-3p, Assay ID: 002367; has-miR-210-3p, Assay ID: 000512; has-miR-378a-5p, Assay ID: 000567) was tested by applying individual TaqMan miRNA Assays (Applied Biosystems) according to the protocol provided by the supplier. Reverse transcription was performed with miRNA specific stem-loop RT primers and TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Real time PCR was performed using specific TaqMan Assays and TaqMan Universal PCR Master Mix. All reactions were run in duplicates including no-template controls in 96-well plates on Mx3005P qPCR System (Applied Biosystems). Fold changes were calculated by the formula 2−ΔΔCt using RNU48 as housekeeping control and normalized to non-treated cells.
4.5. Western Blotting Analysis
HTR-8/SVneo, JEG-3, PTC and HUVEC cell pellets were lysed using RIPA lysis buffer (1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM NaCl and 50 mM Tris–HCl) containing protease inhibitors. Total protein concentrations were assessed using the Pierce™ Micro BCA™ Protein-Assay (Thermo Scientific). Protein extracts were loaded on a 12% precast gel SERVAGel™ (SERVA Electrophoresis GmbH, Heidelberg Germany), and resolved proteins were transferred to a nitrocellulose membrane (Hybond-P; GE Healthcare, Freiburg, Germany). Non-specific binding sites were blocked by incubation with TBS-T containing 5% (w
) non-fat dried milk for 1 h at room temperature. Membranes were immunoblotted with specific primary antibodies overnight at 4 °C, followed by 1 h incubation at room temperature with the respective HRP-conjugated secondary antibody. The following primary monoclonal antibodies (goat) were purchased from R&D Systems (Minneapolis, MI, USA) and diluted 1:500, anti-IL-36α (Cat.Nr: AF-1078), anti-IL-36β (Cat.Nr: AF-10998), anti-IL-36γ (Cat.Nr: AF-2320), anti-VEGF (Cat.Nr: AF-293), anti-PGF (Cat.Nr: AF-264). Anti-goat-HRP was diluted 1:5000 (Santa Cruz Biotechnology, Heidelberg, Germany, Cat.Nr: sc-2768) for their detection. Rabbit- anti-IL-36R (Abcam, Cat.Nr: ab180894), -anti-human GAPDH (Cell Signaling, Cat.Nr 2118S) and -anti-β-actin (Cell Signaling, Cat.Nr 4970L) were applied at a 1:500 dilution and detected with anti-rabbit-HRP diluted 1:5000 (Cell Signaling, Cat.Nr 7076P2). To show equal loading, membranes were treated twice with stripping buffer (0.2 M Glycine, 0.1% (w
) SDS and 1% Tween20, pH 2.2) for 10 min at room temperature, washed with PBS and TBS-T, and blocked with TBS-T containing 5% (w
) non-fat dried milk for 1 h at room temperature before being re-probed with anti-GAPDH (Figure 1
) or anti-β-actin (Figure 2
) antibodies. Blots were developed using an enhanced chemiluminescence (ECL) detection kit (Millipore, Schwalbach, Germany). Bands were detected by a MF-ChemiBis 3.2 gel documentation system with Totallab TL100 software version 2006 (Biostep GmbH, Jahnsdorf, Germany).
4.6. “Wound Healing” Assay (Cell Migration)
For the migration assay, ibidi™ culture inserts (Ibidi GmbH, Cat.Nr. 80209, Gräfelfing, Germany) were used. Briefly, the inserts consist of two chambers separated by a 0.5 mm divider (forming the “wound”), each chamber with a growth area of 0.22 cm2. The inserts were set into wells of a 24-well plate by using sterile tweezers and 70,000 cells were transferred to each chamber. After 4 h, cells were adhered, and the culture inserts were gently removed. The wells were filled with 1 mL of medium supplemented with 0, 20 or 50 ng/mL of IL-36 (α, β, or γ) and further incubated for 24 h. The rate of “wound” closure was monitored at regular intervals of 1 h using the JuLI™ Stage automated cell imaging system (NanoEnTek, Seoul, Korea). The JuLI™ Stage software offers image capturing and time-lapse recording with multiposition scanning.
4.7. Tube Formation Assay
Wells of an angiogenesis 96 wells μ–plate (“ibiTreat”, Ibidi, Cat.Nr: 89646) were coated with 10 μL growth factor reduced Matrigel® (Corning, Wiesbaden, Germany, Cat.Nr: 356230) (37 °C for 30 min), filled with 50 μL endothelial cell growth medium (ECGM; PromoCell, C-22010) per well and incubated overnight. 6500 HUVEC cells were stained with 10 nM CellTracker™ Green for 30 min at 37 °C (Sigma-Aldrich, Cat.Nr: C2925), added to each well and incubated at 37 °C for at least 4 h to allow tube formation. Medium was removed and co-culture was started by adding 6500 HTR-8/SVneo cells previously stained with CellTracker™ Orange (10 nM, 30 min; Sigma-Aldrich, Cat.Nr: C2927). After 20 h of co-culture, changes in the tube formation were observed and pictures were taken using an Olympus IX-81 inverted system microscope. Tube formation assay was analyzed with the Angiogenesis Analyzer plugin developed by Carpentier, implemented in the software ImageJ. Tube formation and stability were quantified as mean number of unions between three or more tubular structures (nodes) from stimulated and non-stimulated co-cultures. The tube length (μm) was additionally analyzed using the “Tube Formation FastTrack AI Image Analysis” (MetaVi Labs Inc., Bottrop, Germany).
4.8. Statistical Analysis
Experiments were repeated independently at least 3 times. Unpaired two-tailed Student’s t-test with Welch´s correction and one-way ANOVA with Bonferroni multiple comparison test were performed for comparisons as indicated in the figure legends using Prism software version 6 (GraphPad, San Diego, CA, USA). A p value < 0.05 was considered significant.