Dominance of pro-inflammatory cytokines over anti-inflammatory ones in placental bed of creta cases Abdel-Hamid AA, Mesbah Y, Soliman MF, Firgany AE,

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ORIGINAL ARTICLE

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Dominance of pro-inflammatory cytokines over anti-inflammatory ones in placental bed of creta cases

Ahmed A. M. Abdel-Hamid¹, Yaser Mesbah², Mona F.M. Soliman², Alaa El-Din L. Firgany³
¹ Department of Anatomy, Taibah College of Medicine, Taibah University, Almadina Almonawara, Medina, Kingdom of Saudi Arabia; Department of Medical Histology and Cell Biology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
² Department of Obstetrics and Gynecology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
³ Department of Medical Histology and Cell Biology, Faculty of Medicine, Mansoura University, Mansoura, Egypt; Department of Basic Medical Sciences, Unaizah College of Medicine, Qassim University, Unaizah, Kingdom of Saudi Arabia

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Date of Submission	08-Sep-2021
Date of Decision	04-Oct-2021
Date of Acceptance	07-Oct-2021
Date of Web Publication	14-Nov-2022

Abstract

Objective: The objective of the study is to investigate changes occurring in key inflammatory cytokines at molecular level (including genetic and protein) in placental bed of placenta creta compared to that of normal placenta and their correlation to interstitial extravillous trophoblasts (EVT) number. Subjects and Methods: Case–control study including placentas of patients with invasive placentation (creta placentas, n = 19) compared with those of normal placentation (n = 19). Besides routine histology and immunocytochemistry detection (cytokeratin-7 [CK-7]), addition to biochemical evaluation of expression of various cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL6, IL-1RA, IL-8, IL-10, and IL-13 was carried out. Results: Routine histological examination of placentas of creta cases revealed CK-7⁺ extravillous trophoblasts (EVT) penetrating deeply the myometrium with various histopathological arrangements and trophoblastic vascular invasion of the deep myometrial blood vessels. A significant increase (P < 0.05) in the mRNA expression of TNF-α, IL-1 β, and IL6 with an insignificant decrease in placental bed IL-1RA, IL-8, IL-10, and IL-13 was observed in creta cases compared to the control ones. A corresponding significant increase was detected in the protein levels of TNF-α, IL-1 β, and IL-6 as well as an insignificant decrease in placental bed IL-1RA, IL-8, IL-10, and IL-13 in creta cases compared to the normal ones. Moreover, we displayed a significant positive correlation (P < 0.05) between interstitial EVT number and mRNA expression of almost all pro-inflammatory cytokines with negative but insignificant correlation with anti-inflammatory cytokines in creta cases. Conclusion: The upregulated pro-inflammatory cytokines and the correlation of their expression with the increased interstitial EVT provide a supporting evidence of their potentially more relevant role in the development of placenta creta than the anti-inflammatory ones.

Keywords: Extravillous trophoblasts, invasive placentation, inflammatory cytokines

How to cite this URL:
Abdel-Hamid AA, Mesbah Y, Soliman MF, Firgany AE. Dominance of pro-inflammatory cytokines over anti-inflammatory ones in placental bed of creta cases. J Microsc Ultrastruct [Epub ahead of print] [cited 2023 Apr 1]. Available from: https://www.jmau.org/preprintarticle.asp?id=361125

Highlights

Vascular invasion of cytokeratin-7⁺ extravascular trophoblast (EVT) with various histopathological patterns was observed in placental bed of creta cases
A significantly upregulated expression of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1 beta [IL-1β], IL-6) was detected in placental bed of creta cases compared to control ones
An insignificantly downregulated expression of anti-inflammatory cytokines (IL-1RA, IL-8, IL-10, and IL-13) was detected in placental bed of creta cases compared to control placentas
A significant positive correlation was detected between EVT number and mRNA expression most of the pro-inflammatory cytokines in creta cases.

Introduction

Placenta accreta spectrum (PAS) is one disorder of abnormal placentation associated with increased maternal morbidity. Although it has been classically described after placental manual elimination, endometritis, or curettage of endometrium, scarring in the uterus particularly resulted form delivery by cesarean section is the main cause of PAS nowadays.^[1] The diagnosis of PAS is essentially made by the absence of decidua and invasion of chorionic villi to myometrial fibers on histopathological examination of the placenta. The exact mechanism of pathogenesis of PAS is complex that has not clearly identified; it involves angiogenesis, proliferation, and inflammation.^[2]

Cytokines and their receptors are normally expressed by cells of placenta.^[3],[4] Balanced cytokine level has a crucial role in the maintenance of normal placentation as their relative local concentrations can influence trophoblastic invasion.^[5] Tumor necrosis factor-alpha (TNF-α) is a powerful pro-inflammatory cytokine localized to proliferating extra-villous trophoblasts (EVT) in the placental bed and in decidual cells.^[6] Interleukin-1 beta (IL-1β) is another pro-inflammatory cytokine that has naturally occurring receptor antagonist (IL-1Ra).^[7],[8]

Pro-inflammatory cytokines such as TNF-α and IL-1 are increased in conditions of low oxygen tension in placenta as in preeclampsia (PE).^[9],[10] High level of local TNF-α can affect trophoblastic apoptosis, syncytial shedding, and impair placental function. In addition, IL-6 has a pivotal role in injury and inflammatory processes by affecting genes involved in cellular differentiation, proliferation, and apoptosis; yet its role in the regulation of invasive behavior of EVT is still uncertain.^[5] Previous studies proposed that changes in decidual IL-6,-8 and TNF-α levels may impair trophoblastic invasion and remodeling of arteries and therefore contribute to defective placentation or even miscarriage.^[5]

IL-8 (CXCL8), also a chemokine with pro-inflammatory criteria, acts through the CXC-R1 and-R2 receptors.^[11],[12] It is a placental product of cells including trophoblasts, fibroblasts, and macrophages. It has been suggested that IL-8 recruits of neutrophils in placental bed.^[5] Changes in its expression have been reported in the placenta of miscarriage conditions.^[13]

Besides IL-10 and IL-13, IL-1Ra is a key cytokine with anti-inflammatory characteristics that can inhibit IL-1 β. It decreases inflammation induced by IL-1 β through completion at its receptor.^[14]

Our objective in the current study was to detect changes in these inflammatory cytokines in placental bed of creta cases compared to the normal placentas and whether there is a correlation between these changes and interstitial EVT number.

Subjects and Methods

Subjects

The current study is a case–control one that included 38 pregnant ladies. We obtained placentas from pregnant ladies having placenta creta (n = 19) and those with normal placenta (n = 19). They were recruited from the Obstetrics and Gynecology Department, Hospital of Mansoura University, in the period from the first of July 2018 to end of March 2019. All cases of this study signed an informed and written consent before conducting the study. In addition, the approval of carrying out this study (Proposal Code : R.18.10.318.R1) was issued on 2019/01/11 from the IRB Committee of Mansoura College of Medicine and according to the Declaration of Helsinki.

The identification of the gestational age was performed based on history the last cycle of menstruation, clinically as well as by ultrasonograph. All cases complaining of PE or any pregnancy-induced hypertension (HTN) and essential HTN were diagnosed then ruled out from the study. In addition, gestational diabetes, preexisting diabetes and those having evidence of infection, and fetuses with any chromosomal or structural abnormality were also from other exclusion criteria.

In our study, we collected postnatal data for each infant to exclude IUGR. In addition, we included ladies having infants who are proper for their gestational age (between 10^th and 90^th centile), while those with neonate large for their gestational age (more than 90^th centile) or small for gestational age (<10^th centile) infants were excluded. Delivery of all ladies were carried out vaginally or cesarean section by a consultant obstetrician (YM) [Supplementary Table 1].

Placental tissue manipulation for histological assessment

We collected placentas from each lady in both groups immediately after the delivery with weighing each infant and placentas. Placental bed biopsies were taken at site of the scar tissue then were cut into tiny pieces; we confirmed by light microscope that each specimen included in the study was from the placental bed of each group. Placental bed pieces were immediately preserved in liquid nitrogen for molecular evaluation; whereas other portions were prepared for paraffin sections by fixation in buffered formalin solution (10%).

To prepare slides for H and E staining and for immune-histochemical assessment, we cut paraffin sections at 3–5 μm thickness. In addition, we used ImageJ program (NIH, Bethesda, Maryland) for all morphometric assessments, and a light microscope (CX31) attached to a camera was used for all histopathological evaluations of the placental beds of both groups.

Samples collection and preparations

Tissue samples were obtained from 38 women at Mansoura University Hospital. The criteria of involved patients of the study are displayed in [Supplementary Table 1]. We performed histopathological examination for samples collected from each group. Fixation of small tissue pieces was accomplished in formalin solution (10%) for 1–2 days, and paraffin blocks were prepared and sectioned (3–5 mm thick) to be stained by H and E and immune-histochemical assessment. We confirmed that the included samples in our work contained myometrial tissue, EVT at placental bed of the location of implantation by histopathological examination as well as immunohistochemistry as previously described.^[15]

Immuno-histochemistry

Dewaxing of sections was performed in xylene, then rehydration (through graded alcohol concentrations) was done and finally incubated in H₂O₂ (1%, for 10 min). In addition, we used the technique of avidin-biotin peroxidase to immunostain sections. EVT was specifically identified by cytokeratin-7 (CK7) antibodies (dilution 1:20, Mouse Monoclonal, Cat No NCL-L-CK7-OVTL). 0.01% H2O2 in di-amino-benzidine was employed (2–3 min) to obtain and visualize the brown reaction.

Histopathological examination of all slides was performed by 2 investigators (MS and AF) in a blind manner. The total number of EVT (at magnification of ×200) was evaluated in five isolated myometrial zones in each section. We evaluated fields (n = 190 in each group) from cases with placenta creta as well as those from normal placentation. In addition, EVT number in the interstitium per field (×200) was estimated in each group.

Real-time polymerase chain reaction (RT-PCR) for cytokine mRNA levels

We employed TRIZOL reagent to extract RNA totally from placental bed tissue (100 mg). This was followed by treatment by DNase guided by instructions of the manufacturer. We determined the purity and concentration of RNA by with NanoDrop^® ND-1000 to measure the optical density. Bioanalyzer (Agilent 2100) was employed to assess the RNA quality.^[16] We used a two-step real-time polymerase chain reaction (RTPCR) method to determine the expression levels of mRNA. The total amount of the RNA (1 μg) was reversely transcribed by empolying hexamers (50 ng), 2 mMdNTP mix, ribonuclease inhibitor (10 units), and reverse transcriptase enzyme (MMLV; 200 units) in 20 μl volume of reaction.

RTPCR has been accomplished by Predesigned TaqMan gene expression assays [Supplementary Table 2]. To detect the reference genes as regard optimal number and stability, geNORM version 3.4 (PrimerDesign Ltd) was used according to the described elsewhere.^[17] Glyceraldehyde 3-phosphate dehydrogenase and β-actin were the most stable housekeeping genes in our samples by the average gene-stability measure [Supplementary Material 1].

Assessment of placental bed cytokine protein levels

The role of local cytokines has been previously demonstrated in placental,^[18],[19] endometrial, tubal,^[20],[21] tissues, and extra-genital organs^[22],[23] both in experimental^[24],[25] and clinical studies. We evaluated IL-1β, IL-6 as well as TNF-α level in homogenates of placental bed (100 mL) by ELISA according to instructions of the manufacturer (Quantakine kits). Other cytokines (i.e., IL-1RA, IL-8, IL-10, and IL-13) were determined as described previously [Supplementary Material 2].^[26]

Statistical analysis

One-way analysis of variance was used to determine the statistical significance with detection of intergroup difference. Significance was detected in the findings when P < 0.05.

Results

Histopathological examination of the placenta revealed that extra-villous trophoblasts (EVT) was seen penetrating deeply the myometrium to reach serosa in the percreta cases. EVT had various histopathological arrangements from the mononuclear pattern to binuclear cells and less frequently multinucleated trophoblastic giant cells [Figure 1]. The invading trophoblasts were seen in perivascular, intramural, and intravascular areas of the deep myometrial blood vessels [Figure 2]. CK-7 expression specifically confirmed the presence of confluent groups and clumps of invading EVT in the myometrium of creta cases [Figure 3]. In addition, the number of interstitial EVT increased significantly (P < 0.05) in placentas of creta patients versus those of normal ones (21.4 ± 4.8 in creta group compared to 69.7 ± 5.2 in normal placentas).

Figure 1: Representative photomicrographs of placental bed biopsy of creta cases showing chorionic villi (CH) with placental invasion by extensive extravillous trophoblasts (EVT) deeply into the myometrium (My). Less frequently multinucleated trophoblastic giant cells (MGTC, black arrow) are observed (H and E, ×400, Scale bar = 20 μm)

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Figure 2: Representative photomicrographs of placental bed biopsy of creta cases showing perivascular (black arrow), intramural (white arrow), and intravascular invasion (white arrowhead) of basal artery branches by EVT distributed in myometrium (My) (H and E, ×1000, Scale bar = 10 μm)

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Figure 3: Representative photomicrographs of placental bed biopsy of creta cases showing intense EVT infiltration of myometrium (My). They form confluent groups either mono-nucleated (black arrow) or bi-nucleated (white arrow) and scarcely multinucleated (black arrowhead) distributed all over the myometrium (My) (anti-CK-7, ×400, scale bar = 20 μm)

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Molecular assessment was performed to assess changes in expression of mRNA and proteins in bed of placenta creta compared to the normal ones. A significant increase (P < 0.05) was observed in the expression of TNF-α, IL-1 β, and IL6 without significant change in that of IL-1RA, IL-8, IL-10, and IL-13 in placental bed of creta cases vs. that of the normal placentas [Figure 4]. Similarly, a corresponding significantly higher protein level of TNF-α, IL-1 β, and IL-6 [Figure 5a] as well as an insignificant change in that of IL-1RA, IL-8, IL-10, and IL-13 [Figure 5]b was observed in placental bed in creta cases compared to the control ones.

Figure 4: Representative graphs of the mRNA levels in placental bed of creta cases compared to the control ones. A significant increase in the expression of TNF-α, IL-1β, and IL-6 with an insignificant change in that of IL-1RA, IL-8, IL-10, and IL-13 are observed in creta cases compared to the control ones. Data are represented as fold change ± SE in each group (n = 19). *Significance versus placentas of control group, Significance considered if P value < 0.05. TNF: Tumor necrosis factor, IL: Interleukin, SE: Standard error, NS: Not significant

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Figure 5: Graphs representing the protein levels in placental bed of creta cases compared to the control ones. A significant increase in the level of TNF-α, IL-1 β, and IL-6 (pg/mg, a) With an insignificant change in that of IL-1RA, IL-8, IL-10, and IL-13 (percent of control, b) Is observed in creta cases compared to the control ones. Data are expressed as means ± SD in every group in the study (n = 19), *Significance versus placentas of control group, P < 0.05 is significant. NS: Not significant, TNF: Tumor necrosis factor, IL: Interleukin, SD: Standard deviation

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Interestingly, a significant positive correlation (P < 0.05) was detected between interstitial EVT number and mRNA expression of almost all pro-inflammatory cytokines (r = 0.708, P = 0.022 for TNF-α; r = 0.634, P = 0.049 for IL-1β; and r = 0.875, P = 0.001 for IL6, respectively) in creta cases [Figure 6]. Nonetheless, a negative but insignificant correlation was observed between interstitial EVT number and mRNA expression of IL-8 (r = −0.559, P = 0.093), as well as the other anti-inflammatory cytokines [r = −0.535, P = 0.111 for IL-1RA; r = −0.286, P = 0.424 for IL-10; and r = −0.491, P = 0.150 for IL-13, respectively; [Figure 7]] in creta cases.

Figure 6: Representative graphs of the correlation between interstitial EVT number and various inflammatory cytokines in creta cases. A significant positive correlation is detected between EVT number and TNF-α, IL-1β as well as IL-6 mRNA expression (a-c, respectively). r: Spearman test, (n = 19) and significance considered if P < 0.05. TNF: Tumor necrosis factor, IL: Interleukin

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Figure 7: Graphs representing correlation between interstitial EVT number and IL-8 as well as other anti-inflammatory cytokines. An insignificant negative correlation is detected between EVT number and IL-1RA, IL-8, IL-10, as well as IL-13 (a-d, respectively) in the creta group. r: Spearman test, (n = 19) and P < 0.05 is significant. TNF: Tumor necrosis factor, IL: Interleukin

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Discussion

Histological examination of placentas of the creta cases revealed CK-7⁺ confluent groups of extra-villous trophoblasts (EVT) penetrating deeply the myometrium by various histopathological arrangements and less frequent MTGC. The invading trophoblasts were seen in the deep myometrial blood vessels. Similar histological patterns of invasive trophoblastic proliferation in patients with creta placenta were described by former researches.^[27],[28] Hannon et al. (2012) proposed that aberrant characters of interstitial EVT are mainly observed locally in zones having deficient decidual tissue compared to areas of with normal deciduas.^[29]

Our finding of significantly numerous EVT interstitially in patients with placenta creta compared to those of normal ones was in accordance with previous studies.^[27] The increased interstitial EVT invading the myometrium with fewer MTGC is associated with defective decidualization.^[29] Interestingly, no change either in trophoblastic proliferation or apoptosis has been previously reported in creta placentas.^[27],[30]

While a significantly higher TNF-α, IL-1 β, and IL6 expression, an insignificant decrease in IL-1RA, IL-8, IL-10, and IL-13 both at mRNA and protein level was detected in creta cases compared to the control ones. Moreover, we demonstrated a significantly positive correlation between EVT number and mRNA expression of almost all pro-inflammatory cytokines in creta cases; nonetheless, a negative but insignificant correlation with the mRNA expression of anti-inflammatory cytokines. It was postulated that TNF-α may inhibit the trophoblastic invasion.^[31],[32] This effect may be attained through alteration in the level of matrix metalloproteinases which act as proteolytic enzymes^[5] or altering EVT apoptosis and proliferation.^[33] In our study, we suggest that the paradoxical increase in the pro-inflammatory cytokine level might be a compensatory mechanism induced locally in the scar to hinder the hyper-invasive phenotype of EVT.

Expression of TNF-α^[34] and that of IL-1^[9],[10] has been shown to be elevated by placental hypoxia and defective perfusion of PE. Moreover, it was reported that IL-6 does not much influence EVT invasive behavior in placental explants in the first trimester.^[35]. However, it was demonstrated that IL-6 can induce the behavior of invasiveness of the trophoblast-like cells by upregulating the trophoblastic integrin receptors which react with molecules of the extracellular matrix.^[36]

IL-1Ra competitively inhibits IL-1β, and their local level can determine which of the pro-inflammatory or anti-inflammatory effect will dominate.^[14] In addition, IL-1Ra has a protective effect against placental and developmental defects in maternal inflammatory conditions.^[37] However, in our study, the detected significantly higher expression the pro-inflammatory cytokines, TNF-α, IL-1, and-6, together with the insignificant decrease in the anti-inflammatory ones, IL-1Ra,-10 and-13, suggests a more relevant role of these pro-inflammatory mediators in creta pathogenesis.

In vitro study, IL-6 and IL-8 have been shown to disrupt uterine vascular remodeling;^[13] moreover, IL-8 can induce trophoblastic invasion probably through a crosstalk with NK cells.^[12],[38] However, we detected an insignificant change in IL-8 level, that of IL-1RA, IL-10, and IL-13 in our study with a negative but insignificant correlation their mRNA expression and interstitial EVT number. This may indicate suppression of anti-inflammatory cytokine in creta cases which again seems to have a minor role compared to pro-inflammatory ones.

Anti-inflammatory cytokines as IL-13 principally produced by Th2 cells^[39] and have many target cells in the placenta as endothelial cells and lymphocytes.^[40],[41] IL-10 is another anti-inflammatory that has antiapoptotic effect necessary for normal placentation;^[42],[43] accumulation apoptotic bodies may induce pro-inflammatory cytokine production by macrophages.^[44] This may explain the significant increase of most pro-inflammatory cytokines that accompanied the marginal decrease of anti-inflammatory ones in our study.

Conclusion

In summary, the expression of most of the pro-inflammatory cytokines is upregulated in creta placenta, unlike the anti-inflammatory ones which were marginally downregulated. The upregulated pro-inflammatory cytokines were positively correlated with the increased interstitial EVT. This provides a supporting evidence of a potentially more relevant role of pro-inflammatory cytokines in the pathogenesis of placenta creta rather than the anti-inflammatory ones.

Compliance with ethical standards

Approval of this work (Proposal Code: R.18.10.318.R1) was obtained from IRB Committee of Mansoura College of Medicine.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Supplementary Materials

Supplementary Material 1: Determination of cytokine mRNA levels

Real-time polymerase chain reaction (PCR) was accomplished by predesigned TaqMan gene expression assays (Applied Biosystems) for all evaluated and housekeeping genes on a MX3005p sequence detection [Table 1]. To determine the stability and optimal number of housekeeping genes, we used geNORM version 3.4 (PrimerDesign Ltd.) according to the manufacturer's instructions (Vandesompele et al. 2002) and tested the used reference genes from different functional classes in 10 samples from each test group. (Vandesompele et al. 2002) The average gene-stability measure ranked β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the most stable genes in our samples.

The PCR efficiency was tested over 5-log dilution series and confirmed that all target genes and housekeeping genes had similar amplification efficiencies. For each primer/probe set, the PCR was performed using of cDNA (10 μL, dilution 1:10). To eliminate nonspecific amplification, each qPCR plate included “no template” and “no reverse transcriptase” controls. To confirm specificity, one sample from each target gene was run on a gel and all samples were run in triplicate manner. The geometric mean of β-actin and GAPDH to normalize data of target gene qPCR to and then expression were displayed relative to the control samples in the 2^–2(ΔΔCt) manner, with considering ΔΔCt = (Ct target – Ct internal control) subject – (Ct target – Ct internal control) control. The Ct internal control equals the geometric mean of β-actin and GAPDH Ct values. Exclusion of the outliers was performed if the normalized (ΔCt) values were more than double standard deviations from the group mean. The levels of relative expression were determined as the fold change, and values of ΔCt were used for subsequent statistical analysis (Applied Biosystems) (Pandey et al. 2018).

Supplementary Material 2: Assessment of placental bed cytokine protein levels

Concisely, equal volumes of membrane fractions isolated by this procedure (30 μg of protein in 20 μL) were loaded onto 4%–12% (weight per volume) Bis-Tris gel and then electrophoresed. The blots were initially developed using the polyclonal primary antibodies (1:1000 dilution) and subsequently using horseradish-peroxidase-linked secondary antirabbit antibody (1:3000 dilution). The membranes were stripped using stripping solution (Chemicon International) and probed by β-actin monoclonal primary (1:5000 dilution; Sigma Chemical Co.) and anti-mouse secondary antibody (1:5000 dilution). The levels of inflammatory cytokine proteins were calculated as a ratio of the optical density of the primary antibody to the optical density of β-actin antibody (Dwivedi and Pandey, 2000). The polyclonal antibodies interleukin-8 (IL-8) (molecular weight 8 kDa), IL-10 (molecular weight 20 kDa), IL-13 (molecular weight 13 kDa), and IL-1RA (molecular weight 25 kDa) were purchased from Santa Cruz Biotechnology, Inc.

References

Dwivedi Y, Pandey GN. Adrenal glucocorticoids modulate [3H] cyclic AMP binding to protein kinase A (PKA), cyclic AMP-dependent PKA activity, and protein levels of selective regulatory and catalytic subunit isoforms of PKA in rat brain. J Pharmacol Exp Ther 2000;294:103-16.

Pandey GN, Rizavi HS, Zhang H, Bhaumik R, Ren X. Abnormal protein and mRNA expression of inflammatory cytokines in the prefrontal cortex of depressed individuals who died by suicide J Psychiatry Neurosci 2018;43:376-85.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3:RESEARCH0034.

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