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Does tumour necrosis factor alpha-induced cyclooxygenase-2 expression lead to spontaneous abortion in Chlamydia trachomatis-infected women
Journal Pre-proof Does tumour necrosis factor alpha-induced cyclooxygenase-2 expression lead to spontaneous abortion in Chlamydia trachomatis-infected women Namita Singh, Priya Prasad, Banashree Das, Sangita Rastogi PII:
S0882-4010(19)30891-5
DOI:
https://doi.org/10.1016/j.micpath.2020.103994
Reference:
YMPAT 103994
To appear in:
Microbial Pathogenesis
Received Date: 20 May 2019 Revised Date:
31 December 2019
Accepted Date: 21 January 2020
Please cite this article as: Singh N, Prasad P, Das B, Rastogi S, Does tumour necrosis factor alphainduced cyclooxygenase-2 expression lead to spontaneous abortion in Chlamydia trachomatis-infected women, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/j.micpath.2020.103994. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Title Page
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Manuscript title: Does tumour necrosis factor alpha-induced cyclooxygenase-2 expression lead to spontaneous abortion in Chlamydia trachomatis-infected women
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Authors name and affiliations: Namita Singh,1 M.Sc., Ph.D.; Priya Prasad,1 M.Sc.; Ph.D.; Banashree Das,2 M.B.B.S., M.D.; Sangita Rastogi,1* M.Sc., M.Phil., Ph.D. 1
Microbiology laboratory, National Institute of Pathology (ICMR), Safdarjung
hospital campus, New Delhi-110 029, India. 2
Department of Obstetrics & Gynecology, Vardhaman Mahavir Medical College
(VMMC) & Safdarjung hospital, New Delhi-110 029, India.
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Corresponding author details: Dr. Sangita Rastogi, Scientist ‘G’ (Director Grade Scientist, Microbiology laboratory, National Institute of Pathology (ICMR), Sriramachari Bhawan, Safdarjung hospital campus, Post Box No. 4909, New Delhi 110 029, India. Tel. No.: + 91 – 011 – 26198402 - 406 (Ext. 314), Mobile no.: + 91 - 09810217260 Email: [email protected]
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ABSTRACT Cytokines might be involved in spontaneous abortion by triggering inflammatory mediators (Cyclooxygenases (Cox)) leading to spontaneous abortion in Chlamydia trachomatis (Ct)infected women. Study aimed to quantitate the expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-8) and Cox-2 in endometrial curettage tissue (ECT) of Spontaneous Aborters (SA). SA (n = 135) and 120 age-matched controls were enrolled from SJH, New Delhi, India. PCR was performed for detection of Ct MOMP gene (537 bp) in ECT. mRNA expression of pro-inflammatory cytokines and Cox-2 was assessed by real-time qPCR. Data was statistically evaluated. 14.8% SA were diagnosed as Ct-positive. Elevated expression of TNF-α, IFN-γ, IL-8 and Cox-2 was observed in Ct-infected SA. Ct-positive recurrent aborters showed significantly higher cytokine expression. Significant positive correlation was found between expression of Cox-2 and TNF-α in infected SA. Data suggested an increased expression of Th-1 cytokines, particularly TNF-α that induced Cox-2 expression in ECT, leading to spontaneous abortion in Ct-infected pregnant women.
Key words: Chlamydia trachomatis, spontaneous abortion, pro-inflammatory cytokines, cyclooxygenase-2, real time PCR
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1. Introduction During pregnancy, the risk of Chlamydia trachomatis (Ct) colonization increases, resulting in altered immune response and affects intra- and extra-uterine development in pregnant women causing abortion [1]. The adaptation of Ct to evade immune response allows organism to persist for extended periods within host epithelial cells, inducing a chronic inflammatory response [2]. Once Ct establishes infection, the innate immune response allows for production of pro-inflammatory cytokines such as interleukins (IL) (IL-1, IL-6, IL-8), and tumour necrosis factor alpha (TNF-α) [3]. Synergistic effect of IFN-γ and TNF-α also plays a role in the establishment of persistent chlamydial infection inside the host [4]. An abnormal production of pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6 and IL-8 was seen in placenta [5] and chorioamniotic membranes [6] in response to infection. Another study also revealed that administration of excess amount of Th1 cytokines induced abortion in mice [7] and low doses of anti-TNF-α antibodies reduced resorption rates in a murine abortion model [8]. In women, higher expression of proinflammatory cytokines was seen in recurrent miscarriage [9 - 11] and a significant expression of cytokines was also found in cultured Peripheral Blood Mononuclear Cells (PBMCs) of pregnant women with history of Recurrent Pregnancy Loss (RPL) [12]. An aberrant expression of pro-inflammatory cytokines during implantation window in women with idiopathic recurrent spontaneous miscarriage was also demonstrated [13]. A predominant Th1 type immunity was observed in recurrent spontaneous abortion [14, 15]. Furthermore, Cyclooxygenase-2 (Cox-2) is a key inflammatory response molecule, whose expression is induced by inflammatory stimuli such as IL-1β [16], TNF-α [17] and IFN-γ [18]. However, little is known about its role in infection-induced spontaneous abortions. Our recent studies demonstrated about the association of Ct infection with spontaneous abortion leading to the upregulation of Cox-2 in Ct-positive Recurrent
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Spontaneous Aborters (RSA) [19, 20]. Another study reported decreased expression of Cox-2 resulting in recurrent pregnancy loss [21]. In a murine model, it was found that abnormal expression of Cox-2, TNF-α and IL-6 resulted in recurrent spontaneous abortion [22]. It is apparent that till date, the immunological changes underlying the complex transition from uterine quiescence to spontaneous abortion are not clear in Ct-infected women. In our previous study, we found an elevated expression of circulatory cytokines; IFN-γ, TNF-α, IL-6, IL-17A in serum of Ct-positive aborters [23]. Hence in the present study, we hypothesized that altered level of pro-inflammatory Th1 type cytokines might be involved in Ct-infection induced spontaneous abortion further triggering the Cox-derived prostaglandins signalling pathway leading to abortion in women. Therefore, the study aimed to quantitate the expression of proinflammatory cytokines, viz.: TNF-α, IFN-γ and IL-8 in the endometrial curettage tissues (ECT) of in Ct-infected Spontaneous Aborters (SA) and attempted to find whether there was any correlation between cytokines and Cox-2 in Ctinfected SA.
2. Materials and methods 2.1. Study population and enrollment of patients The study had Hospital Ethical Committee permission and prior informed written consent was taken from each patient. 135 women experiencing incomplete spontaneous abortion in the first trimester of pregnancy and undergoing dilation and evacuation (Group I) at Department of Obstetrics and Gynaecology, Vardhman Mahavir Medical College (VMMC) & Safdarjung hospital, New Delhi/ India were enrolled in the study in consultation with the gynecologist. All patients in Group I were symptomatic and presented with bleeding per vagina, passage of clots, etc. Group I comprised of both sporadic spontaneous aborters (SSA) having a single abortion (n = 43) and recurrent spontaneous aborters (RSA), who had suffered 2 or more abortions (n = 92). 120 age-matched healthy asymptomatic first trimester 4
pregnant women undergoing induced abortion for medical termination of pregnancy (MTP) (Group II) were enrolled as controls. The reasons for MTP were varied, i.e. risk to the life of the pregnant woman or of grave injury to her physical or mental health, a substantial risk that if the child were born, it would suffer from such physical or mental abnormalities, or where any pregnancy occured as a result of failure of any device/ method used by any married woman/ her husband for the purpose of limiting the number of children. Subjects with local causes of bleeding/ history of recently treated sexually transmitted infection/ recent antibiotic therapy/ HIV/ Treponema pallidum/ TORCH (Toxoplasma gondii, Rubella, Cytomegalovirus, Herpes simplex virus)/ Neisseria gonorrhoeae/ Mycoplasma genitalium/ Trichomonas vaginalis/ Ureaplasma urealyticum-positivity/ genital tuberculosis were not included in the study. Information on obstetric/ medical/ gynaecological history including last menstrual period, age/ gravidity/ parity/ abortion history and past genital/ urinary tract infection was collected in standardized questionnaires from all enrolled patients.
2.2. Sample collection The endometrial curettage tissue (ECT) was collected from each patient in Groups I – II and transported in phosphate buffered saline (10 mM, pH- 7.4) on ice to the laboratory. One part of the tissue sample was used for DNA extraction and polymerase chain reaction (PCR) assay. The remaining tissue was stored in RNA later at -800C for RNA extraction and quantitative analysis of mRNA expression.
2.3. Isolation of genomic DNA in ECT DNA was isolated in the ECT utilizing a commercial kit (Wizard Genomic DNA Isolation kit, Promega, USA) as per the manufacturer’s recommendations. For this, the tissue was homogenized in liquid nitrogen, treated with lysis buffer and the lysate was, thereafter, treated with protein precipitation solution. Finally, the DNA was precipitated by isopropanol. 5
Qualitative and quantitative analysis of extracted DNA was performed before the PCR assay. The quality of DNA was checked for any shearing by running DNA samples on 0.8% agarose gel. DNA samples were further quantified on a nano-drop spectrophotometer (Fermentas-Thermo Fisher Scientific, Waltham, USA) at 260 nm. A 260:280 ratio of ~ 1.8 was considered as optimum.
2.4. Detection of Ct by MOMP gene-specific PCR in ECT PCR assay was performed for the diagnosis of Ct infection in both the groups. The Major Outer Membrane Protein (MOMP) gene of Ct [24] and the internal control gene, viz.: beta-globin [25] were detected in the ECT (Table 1). Amplification reaction was set up in 25 µl reaction volume using DNA Thermal Cycler (Applied Biosystems, USA) in 0.2 ml PCR tubes. The reaction mix consisted of 1 µg of genomic DNA, 0.2 mM of each dNTP (Fermentas, USA), 10 pM of each oligonucleotide (Fermentas, USA), 2.5 µl of 10X PCR buffer (Fermentas, USA) and 1 unit of Taq DNA polymerase (Fermentas, USA). The genomic DNA which was isolated from ECT of enrolled subjects, was initially denatured at 95oC for 5 minutes, followed by 30 cycles at 95oC for 30 seconds while primer annealing was done at 52.4oC for beta-globin and at 45oC for 1 minute for MOMP primer and extension at 72oC for 45 seconds. Final extension was carried out at 72oC for 10 minutes to obtain a product of 268 bp and 537 bp for beta-globin and Ct MOMP gene, respectively. A Ctpositive control (Vircell Microbiologist, Granada, Spain) and negative control (nuclease-free water) were also amplified along with the DNA samples. The amplified product was confirmed by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized under UV Transilluminator.
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2.5. Isolation of RNA in ECT The total RNA was isolated from the same ECT (from which the DNA was isolated) by using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, disruption of the tissue sample was done by taking 20 - 30 mg of the ECT and thereafter, homogenization was done with 1 ml Trizol reagent with 200µl chloroform. The lysate was centrifuged at 40oC and 75% ethanol was added to the supernatant. The tube was recentrifuged and the pellet was air-dried. 30µl of RNase-free water was added and the resulting RNA was stored at -80oC. The purity was checked by running the samples on 1% agarose gel and by taking the nanodrop O.D. A 260/ 280 ratio, i.e. 1.9 - 2.0 was considered as optimum.
2.6. Synthesis of cDNA from RNA For cDNA synthesis, total RNA (1.5 µg) was used to synthesize first strand cDNA using High Capacity cDNA Reverse Transcription Kit with the treatment of RNase Inhibitor (Applied Biosystems, USA) by One-Step Reverse Transcription-PCR as per the manufacturer’s instructions (briefly, 10x RT buffer- 10 µl, 25x dNTP mix (100 mM)- 2 µl, 10x RT random primers- 2 µl, RNase inhibitor- 1 µl, template RNA- 1.5 µg and nucleasefree water) and cDNA synthesis was performed on PCR Thermal Cycler (Applied Biosystems, Foster City, CA). The PCR conditions were as follows: initial denaturation for 10 minutes at 94o C, annealing at 50o-68o C for 120 minutes, extension for 5 minutes at 85o C and then 4o C for α time. 2.7. Detection of Ct by quantitative real-time PCR Quantitative real-time PCR (q-PCR) assay was performed on Step-one plus real-time PCR system (Applied Biosystems, USA) to detect the chlamydial load in the PCR-positive 7
ECT samples. To quantitate the copy number of Ct in ECT, first, serial dilutions (10000, 1000, 100, 10, 0.1 µl) of AMPLIRUN Ct DNA control (Vircell Microbiologists, Granada, Spain) were prepared as per the manufacturer’s instructions. Briefly, 10µl of SYBR green master mix, 1 µl of plasmid gene (200 bp) (Table 1) forward primer and 1 µl of plasmid gene (200 bp) reverse primer [26], 5 µl of Vircell Ct diluted DNA (Vircell Microbiologists, Granada, Spain) of each concentration and 3 µl of nuclease-free water were mixed to make up a final volume of 20 µl. The reactions were set in duplicates for each concentration, and standard curve was drawn. By using this standard curve, the chlamydial load was calculated in each ECT sample. The threshold cycle (CT) value was calculated as an average CT target genes for each sample.
2.8. Quantitative analysis of cytokine genes in ECT Taqman quantitative Real Time PCR (q-PCR) assay was performed on 7000 Realtime PCR system (Applied Biosystems, USA) for studying the quantitative expression of selected cytokine genes, viz.: TNF-α, IFN-γ and IL-8 in the ECT from each enrolled patient (viz.: Ct-positive controls, Ct-negative controls and SA). Commercialized human genes probes (‘Assay on Demand’) (Applied Biosystems, ThermoFisher Scientific, Waltham, USA) were used for the real-time assays. These genes were utilized for standard gene expression experiments because of their small size and detected maximum number of transcripts for the gene of interest. The assay IDs for GAPDH, β-actin, TNF-α, IFN-γ and IL-8 genes are listed in Table 2. 1.5 µg/ µl concentration of c-DNA was used for q-PCR. 10µl of Taqman PCR master mix (Applied Biosystems, USA), 1µl of probe, 4µl of c-DNA and 5µl of nuclease-free water (Ambion, Thermo Fisher Scientific, USA) were mixed to make up a final volume of 20µl. Finally, the reaction mix was loaded into real time optical PCR tubes (Applied Biosystems, USA). The assay was standardized for the internal control gene glyceraldehyde 3-
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phosphate dehydrogenase (GAPDH) and beta actin (β-actin) in the ECT samples and those showing consistency with both GAPDH and β-actin were selected for real time PCR studies. The Threshold Cycle (CT) value was calculated as average ∆CT target genes for each sample by using SDS software and the relative quantification was used to measure gene expression by relating the PCR signal.
2.9. Quantitative analysis of Cox-2 gene expression in ECT Quantitative Real Time PCR (q-PCR) assay was performed on 7000 Real-time PCR system (Applied Biosystems, USA) for studying the quantitative expression of Cox-2 gene (Table 2) in ECT as described previously [19].
2.10. Statistical analysis Statistical analysis was performed using the GraphPad prism software (version 5.0, San Diego, California, USA). Fisher’s Exact test and Mann-Whitney test/ Welch’s test were used to calculate the significance between the groups. Spearman’s rank correlation coefficient was used to calculate the correlation between the expression levels of cytokines and Cox-2.
3. Results 3.1. Detection of Ct infection in ECT SA (n = 135) constituted the study group (Group I) while 120 aborters comprised the control group (Group II). The average gestational age of first trimester aborters in Groups I II was 8 weeks. Table 3 shows the clinical characteristics of enrolled aborters (N = 255). Overall, 14.8% (n = 20) women experiencing spontaneous abortion were diagnosed as Ctpositive for the MOMP gene by PCR assay in Group I; in the latter, 13.9% (6/ 43) SSA and 15.2% (14/ 92) RSA were positive for Ct infection. In Group II controls, 1.4% (n = 2) women were found to be positive for Ct infection (Fisher’s Exact test; ‘p’ < 0.0001). For 9
experimental analysis, comparisons were made between Ct-positive SA (n = 20) versus Ctnegative controls (n = 118), Ct-positive SA (n = 20) versus Chlamydia-positive controls (n = 2), Ct-positive SA (n = 20) versus Ct-negative SA (n = 115). Also, CT values were analysed between Ct-positive SA (n = 20) versus Ct-negative controls (n = 118) and between Ctpositive SSA (n = 6) versus Ct-positive RSA (n = 14). Furthermore, q-PCR was performed by using the SYBR green-based chemistry to quantitate the Ct copies in ECT of Groups I – II. Standard curve was obtained by preparing the serial dilutions of Ct-positive control. Cycle threshold (CT) values of all the samples were calculated by q-PCR and the copy load of Ct in each sample was evaluated by using the standard curve, plotted by using the known concentrations of Ct-positive control. A total of 1 - 4 and 4 – 12 copies were detected in the ECT of Ct-positive SSA and RSA, respectively.
3.2. Quantitative analysis of mRNA expression for pro-inflammatory cytokines (TNF-α, IFN-γ, IL-8) by q-PCR assay in ECT In order to investigate whether altered mRNA expression of cytokines was associated with spontaneous abortion in women found infected with Ct, expression of mRNA-encoding cytokines (TNF-α, IFN-γ, IL-8) was studied in the ECT by the q–PCR. These assays were designed to allow quantitative comparisons of mRNA abundance between different cytokines by comparing mean CT values for control ECT. At transcript level, the analysis of mRNA expression for TNF-α, IFN-γ and IL-8 genes within the ECT was compared among patient group as a ratio to the expression of constitutively expressed GAPDH and β-actin gene by performing q-PCR. The abundance of mRNA expression of Th1 cytokines, viz.: TNF-α, IFNγ and IL-8, demonstrated a significant increase in Ct-positive SA in comparison to Ctnegative SA/ Ct-negative controls (non-parametric Mann-Whitney test; Fig. 1 A- C). In the 2 control subjects who were Ct-positive, the cytokine profile (viz.: TNF-α, IFN-γ and IL-8) was
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found to be significantly decreased in comparison to the Ct-positive SA (non-parametric Welch’s test, Fig. 1 A - C). Upon analysis of CT values, the TNF-α, IFN-γ and IL-8 genes were found to be significantly upregulated (3.0-fold, 4.5-fold and 2.0-fold, respectively) in Ct-infected SA versus Ct-negative controls (Table 4). The mRNA expression of the pro-inflammatory Th1 genes was also compared between the Ct-positive SSA versus Ct-infected RSA and it was found that TNF-α, IFN-γ and IL-8 genes’ expression was significantly higher in the latter in comparison to the former (Mann-Whitney test; Fig. 2 A - C). CT values were further analysed and it was found that in the Ct-positive RSA, all 3 cytokine genes, viz.: TNF-α, IFN-γ and IL-8 were upregulated (3.5-fold, 5-fold and 1.2-fold, respectively), while, the fold-change in Ct-positive SSA was 1.5-fold, 2.8-fold and 1-fold, respectively.
3.3. Quantitative analysis of mRNA expression for Cox-2 gene in ECT Analysis of mRNA expression of Cox-2 gene within the ECT by q–PCR was compared among study group as a ratio to the expression of the constitutively expressed GAPDH. Cox-2 mRNA expression demonstrated a significant increase in Ct-positive SA in comparison to Ct-negative SA/ Ct-negative controls (non-parametric Mann-Whitney test; Fig. 3 A). In the 2 control subjects who were Ct-positive, the expression of Cox-2 gene was found to be significantly decreased in comparison to the Ct-positive aborters (non-parametric Welch’s test, ***‘p’ < 0.0001; Fig. 3 A). Analysis of CT values showed that the Cox-2 gene was 2.3-fold upregulated in Ct-positive SA versus Ct-negative controls. Also, Cox-2 was significantly increased in Ct-infected RSA in comparison to the Ct-positive SSA (MannWhitney test; ‘p’ < 0.0001; Fig. 3 B).
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3.4. Correlation between Cox-2 and cytokines in SA infected with Ct In infected SA, a statistically significant positive correlation was found between the expression of Cox-2 and TNF-α in the ECT (Spearman rank correlation; r = 0.77, ***‘p’ < 0.0001; Fig. 4 A), however, the correlation between Cox-2 and IFN-γ was not statistically significant (Spearman rank correlation; r = 0.04; ‘p’ = 0.85) (Fig. 4 B). The IL-8 cytokine expression in the ECT was also found to be insignificant in correlation with Cox-2 (Spearman rank correlation; r = 0.39; ‘p’ = 0.09) (Fig. 4 C).
4. Discussion The causes of pregnancy loss are not well understood, but infection and immune mechanisms are thought to be involved. Besides Ct infection, there are also other known reasons for abortion in women such as chromosomal, genetic, endocrine, infectious, autoimmune, etc. In our study, patients with any anatomical abnormality and diabetes mellitus were excluded. Furthermore, women with local causes of bleeding/ history of recently treated sexually transmitted infection/ recent antibiotic therapy/ HIV/ Treponema pallidum/ TORCH (Toxoplasma gondii, Rubella, Cytomegalovirus, Herpes simplex virus)/ Neisseria gonorrhoeae/ Mycoplasma genitalium/ Trichomonas vaginalis/ Ureaplasma urealyticum-positivity/ genital tuberculosis were not included in the study. One limitation of our study was that chromosomal abnormalities could not be excluded. Also, environmental factors and other unexplained etiologies could be one of the reasons for spontaneous abortions in Ct-negative aborters. Furthermore, the complex interplay between maternal and foetal immune mechanisms changes temporally as pregnancy progresses. If this delicate balance is adversely affected, immunoregulatory mechanisms may be insufficient to restore homeostasis and this may lead to pregnancy failure. Successful pregnancy may depend, at least in part, on the bias of the maternal immune response shifting away from Th1 type responses towards a Th2 phenotype, 12
both in murine models and humans. Recent attention has focused on elucidating the immunobiological role of cytokines in normal human pregnancy following complex cytokine activity within uteroplacental tissues [27, 28] Numerous in vivo and in vitro studies have reported a strong, maternal Th1 type reactivity deleterious to pregnancy in mice [8, 29] and likewise, in vitro studies in the human system also [14, 30]. The elaboration of proinflammatory cytokines such as TNF-α and IL-1β, typical of a Th1 cell-mediated immune response, is hypothesized to be detrimental to the survival of the developing conceptus. TNFα is an important immunoregulatory cytokine, which may be produced in Th1 or Th2 type responses and is known to have different effects depending on gestational age [31]. TNF-α production by PBMCs was suppressed at the mRNA level during early pregnancy while its production in late pregnancy is implicated in the induction of labour in humans [32]. Also, TNF-α production is enhanced at the onset of labour and spontaneous abortion [33]. Higher concentrations of Th-1 cytokines (TNF-α) were demonstrated systemically in the serum of patients with idiopathic/ unexplained recurrent miscarriages than normal controls [34]. It has also been reported that cytokines such as IFN-γ and TNF-α are harmful for pregnancy [14, 35]. Our study also showed a statistically significant increased expression of proinflammatory cytokines, viz.: TNF-α, IFN-γ and IL-8 in Ct-positive SA in comparison to the Ct-infected SA and uninfected/ infected control groups. Significant levels of the Th-1 cytokines, IFN-γ and IL-2 have been detected at the maternal–foetal junction in cases of spontaneously aborting conceptus by other groups also [36]. In response to infection, an abnormal production of pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, and IL-8 was found in the placenta [5] and chorioamniotic membranes [6], which might have eventually led to spontaneous abortion. Our results further revealed a high expression of TNF-α, IFN-γ and IL-8 cytokines in the Chlamydia-infected RSA in comparison to infected SSA. High level of Th1 cytokines,
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viz.: TNF-α and IFN-γ were earlier detected in RSA [11, 37]. In another study, TNF-α was found abundantly in decidua of RPL women who had miscarried [38]. Elevated values of TNF-α and IFN-γ were reported in recurrent miscarriage group [39]. In another study, the expression of IL-6 and IL-8 in cervical mucus was found to be significantly higher in idiopathic recurrent miscarriage cases who subsequently miscarried than in those who had a live birth [40]. Increased level of IL-8 in amniotic fluid was found in cases of preterm labour with histologically confirmed chorioamnionitis [41]. The increased level of IL-6 and IL-8 were also observed in women with second trimester abortions [42, 43]. It is apparent in our study that pro-inflammatory cytokines constitute an important component of the host immune response to infection with Chlamydia, particularly IFN-γ, which restricts chlamydial growth [44]. The generation of an inflammatory response to the control infection alters the delicate cytokine balance in the placenta, resulting in abortion [45]. Indeed, inflammatory cytokines such as IFN-γ, TNF-α and IL-2 have an abortigenic effect [45] and abundant expression of these cytokines at the materno-fetal interface was found associated with abortion [46]. It was also studied that the production of TNF-α by fetal macrophages may be of considerable significance in the pathogenesis of abortion in pregnant sheep infected with C. psittaci [47]. Furthermore, a higher expression of Cox-2 during implantation window in the endometrial tissue of women with idiopathic recurrent spontaneous miscarriage may be attributed to the exaggerated expression of proinflammatory cytokines such as TNF-α and IFN-γ [13]. IL-6 and TNF-α can induce the expression of Cox-2 leading to an increased production of prostaglandins (PG). An ascending infection into the uterus during pregnancy is followed by local increase of Cox-2 activity, and consequently elevated PG production. Lipopolysaccharide (LPS)-induced decidual prostanoid production via increased Cox-2 expression and Cox-2 mediated eicosanoid production is probably a key pathophysiologic event in the LPS-mediated fetal death [48]. Other in vitro
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studies provide evidence that in cells infected with chlamydiae, TNF-α induces the production of PGE2 much more effectively than in uninfected cells and PGs mediate symptoms of gram-negative septic shock, stimulate myometrium and are capable abortifacients and they are likely to be important mediators of LPS-induced pregnancy loss. In our previous studies, it was found that chlamydial infection leads to upregulation of Cox-2 in Ct-positive RSA which probably mediated an increased PG synthesis [19]. In the present study, mRNA expression of Cox-2 was also found to be significantly increased in the Ct-positive SA in comparison to Ct-uninfected SA and uninfected/ infected controls. We found a positive correlation only between Cox-2 and TNF-α among the Ctpositive SA. The pro-inflammatory cytokines can induce the expression of Cox-2 and cause increased PG production. Other studies have reported a positive correlation between TNF-α and IL-6 while no correlation was found between Cox-2, TNF-α and IL-6 in a recent study [22]. It has also been suggested that when normal levels of IL-6 and TNF-α expression are present, Cox-2 can form the dynamic equilibrium, which can ensure the safe implantation of embryo sac and avoid abortion [49]. In our results, we found a statistically non-significant correlation between Cox-2/ IFN-γ and Cox-2/ IL-8. The non-significant finding between Cox-2 and IFN-γ might be the result of the high expression of IFN-γ level (4.5-fold) in comparison to Cox-2 (2.3-fold). IFN-γ levels were earlier found to be significantly higher in cervical washes of women with recurrent infection. IFN-γ is known for its requirement in resolution of chlamydial infection, and has been shown to inhibit the growth of Chlamydia in cell culture. IFN-γ showed the greatest fold-change among all the differentially induced mRNA in Ct-infected vaginal tissues in mice suggesting that IFN-γ production is being stimulated by other immunoregulatory factors at this later stage of infection [50]. The expression of IL-8 (2.0fold) was also non-significantly correlated with Cox-2. It was studied previously that in the
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presence of Ct infection, the increased IL-8 production by the trophoblasts might lead to increased neutrophils at the maternal-fetal interface and this may have a negative impact on the pregnancy outcome. The continued high expression of the IL-8 and IFN-γ can lead to an increase/ aggregation of the inflammatory cytokine concentration, and the interaction between cells can also cause cytotoxic effect, and can lead to the imbalance of immune tolerance and then induced SA. IL-8 and IFN-γ can induce the expression of Cox-2 and then cause increased PGs which can eventually result in miscarriage. Apparently, spontaneous abortion is a pleiotropic condition with several as yet unidentified causes; however, an aberration in cytokine production also constitutes an important contributing factor. Our data support the hypothesis of Th1 cytokine involvement, particularly TNF-α in the pathogenesis of SA.
5. Conclusions The present study concludes that modulation of Th1 dominance may bring about an immunological milieu that is more conducive to successful pregnancy. An overall understanding of cytokine production in SA during Ct infection will contribute to the immunopathogenesis of disease and in the development of better management and therapeutic intervention for a successful pregnancy. Further large studies on relationship among inflammation, cytokine production and treatment strategy in abortion are clearly needed.
Conflict of interest The authors declare no conflicts of interest.
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Acknowledgments NS is thankful to University Grants Commission, New Delhi, India for the award of Senior Research Fellowship (SRF) and also acknowledges National institute of Pathology (ICMR), New Delhi, India for providing SRF. PP gratefully acknowledges the award of Junior Research Fellowship from Council of Scientific & Industrial Research, New Delhi, India and also acknowledges with thanks National institute of Pathology (ICMR), New Delhi, India for providing SRF.
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Legends to figures: Fig. 1. A-C. Significantly high expression of pro-inflammatory cytokine genes (viz.: TNF-α/ IFN-γ/ IL-8) in endometrial curettage tissue of Chlamydia trachomatis (Ct)-positive spontaneous aborters (SA) versus Ct-negative SA and uninfected/ infected controls by quantitative real time PCR (non-parametric Mann-Whitney test). ‘p’ values: * < 0.05; ** < 0.001; *** < 0.0001
Fig. 2. mRNA expression of pro-inflammatory cytokines in endometrial curettage tissue of spontaneous aborters (SA), viz.: sporadic SA (SSA, Sub-group Ia) versus recurrent SA (RSA, Sub-group Ib) by quantitative real time PCR (non-parametric Mann-Whitney test). A. TNF-α expression was significantly high in Chlamydia trachomatis (Ct)-positive RSA versus Ctpositive SSA (***‘p’< 0.0001). B-C. Expression of IFN-γ and IL-8 was significantly high in Ct-positive RSA versus Ct-positive SSA (** < 0.001).
Fig. 3. Expression of Cyclooxygenase-2 (Cox-2) in endometrial curettage tissue of Chlamydia trachomatis (Ct)-positive spontaneous aborters (SA) by real time PCR (nonparametric Mann-Whitney test): A. Expression of Cox-2 gene was significantly high in Ctpositive SA versus Chlamydia–negative SA/ Ct-negative controls/ Ct-positive controls. B. Cox-2 mRNA expression was significantly high in Ct-positive recurrent SA versus Chlamydia-positive sporadic SA. ‘p’ values: ** p < 0.001; ***p < 0.0001)
Fig. 4. A- C. Correlation between Cox-2 and TNF-α/ IFN-γ/ IL-8 in Chlamydia trachomatispositive spontaneous aborters (Spearman rank correlation test; ‘r’ = 0.77, 0.04 and 0.39, respectively).
Table 1: Primers used for molecular diagnosis of Chlamydia trachomatis in the endometrial curettage tissue of aborters
Name of gene
Primer Sequence: 5'-3'
MOMP gene of Ct
F: TAT ACA AAA ATG GCT CTC TGC TTT
Size of Reference amplicon (bp) 537 bp
24
268 bp
25
200 bp
26
AT β-globin
R: CCC ATT TGG AAT TCT TTA TTC ACA TC F: CAA CTT CAT CCA CGT TCA CC R: GAA GAG CCA AGG ACA GGT AC
Plasmid gene of Ct
F: CTA GGC GTT TGT ACT CCG TCA R: TCC TCA GGA GTT TAT GCA CT
Abbreviations: MOMP, major outer membrane protein; Ct, Chlamydia trachomatis, β-globin, beta globin.
Table 2: Probes used for quantitative expression of cytokines and cyclooxygenase by real time PCR assays
Gene name
Genes probes (‘Assay on Demand’) Amplicon (Gene ID) size (bp)
GAPDH
Hs02758991_g1
93
β-actin
Hs01060665_g1
63
TNF-α
Hs00174128_m1
80
IFN-γ
Hs00989291_m1
73
IL-8
Hs00174103_m1
101
Cox-2
Hs00153133_m1
75
Abbreviations: GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; β-actin, beta actin; TNF-α, tumour necrosis factor alpha; IFN-γ, interferon gamma; IL-8, interleukin-8; Cox-2, cyclooxygenase-2
Table 3. Demographic characteristics of the enrolled participants
Clinical Characteristics
Group I (Spontaneous aborters) n= 135 (%)
Group II (Controls) n= 120 (%)
‘p’-value
Age (years) <20 20-25 26-30 31-35 35-40
12 50 28 27 18
(8.9) (37.0) (20.7) (20.0) (13.3)
10 42 24 28 16
(8.3) (35.0) (20.0) (23.3) (13.3)
1.00 0.90 1.00 0.65 1.00
35 38 47
(26.6) (34.6) (38.6)
0.89 1.00 0.81
Mean age: 25 ± 4.0 Gravidity 01 02 >2
42 44 49
(32.7) (30.7) (36.6)
Average gravidity: 2 Average gestational age: 8 weeks
Parity 0-1 2->2
88 47
(64.7) (35.3)
29 (30.6) 91 (69.3)
0.001 0.005
No. of Abortions 0-1 2- >2
43 (32.7) 92 (70.6)
0 0
< 0.0001 < 0.0001
Smoking status Non-smoker Smoker
131 (97.03) 04 (2.9)
120 (100) 0
0.125
Education Literate Illiterate
51 (37.8) 84 (62.2)
79 (65.8) 41 (34.17)
0.001
39 (28.9) 96 (71.1)
82 (68.3) 38 (31.7)
0.001
Residence Urban Rural
Previously used contraceptive method Pills Condom Other Never used contraceptive
54 12 28 41
(40) (88.8) (20.7) (30.4)
26 16 10 68
(21.7) (13.3) (7.4) (56.7)
0.0019 0.316 0.007 0.001
Marital status Married Unmarried
135 (100) 0
120 (100) 0
1.000
Table 4. Fold-change values of TNF-α, IFN-γ, IL-8 in the endometrial curettage tissue
of
Chlamydia
trachomatis-infected
spontaneous
aborters
versus
C.
trachomatis-negative controls.
Cytokines
Ct-negative controls Avg. ∆CT
Foldchange
‘p’value
TNF-α
Ct-positive spontaneous aborters Avg. ∆CT 6.36
7.93
3.0
< 0.0001
IFN-γ
8.32
10.6
4.5
< 0.0001
IL-8
6.83
7.82
2.0
< 0.0001
Abbreviations: Ct, Chlamydia trachomatis; ∆CT, Cycle threshold
Fig. 1. A
B
C
Fig. 2. A
B
C
t+ v
B t-v
t+ v
C
e
e
SA
SA
co nt ro ls
co nt ro ls
Relative expression of Cox-2 4
C
e
tve
C
C
Fig. 3. A
*** *** **
3
2
1
0
Fig. 4. A
B
C
•
Study revealed effect of proinflammatory cytokines on C. trachomatis-positive SA
•
Increased expression of TNF-α, IFN-γ, IL-8 in ECT of C. trachomatis-positive SA
•
TNF-α, IFN-γ and IL-8 expression was significantly higher in RSA versus SSA
•
TNF-α and COX-2 were positively correlated with each other in infected aborters
•
Aberration in TNF-α and IFN-γ can induce COX-2 leading to immunopathogenesis of SA
Author Statement
Kindly note that our research work has neither been published previously nor is under consideration for publication elsewhere and its submission and publication is agreed and approved by all authors and authorities where the work was carried out; and if accepted, it will not be published elsewhere in the same form in English or in any other language, without written consent of the publisher.
S0882-4010(19)30891-5
DOI:
https://doi.org/10.1016/j.micpath.2020.103994
Reference:
YMPAT 103994
To appear in:
Microbial Pathogenesis
Received Date: 20 May 2019 Revised Date:
31 December 2019
Accepted Date: 21 January 2020
Please cite this article as: Singh N, Prasad P, Das B, Rastogi S, Does tumour necrosis factor alphainduced cyclooxygenase-2 expression lead to spontaneous abortion in Chlamydia trachomatis-infected women, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/j.micpath.2020.103994. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Title Page
(a)
Manuscript title: Does tumour necrosis factor alpha-induced cyclooxygenase-2 expression lead to spontaneous abortion in Chlamydia trachomatis-infected women
(b)
Authors name and affiliations: Namita Singh,1 M.Sc., Ph.D.; Priya Prasad,1 M.Sc.; Ph.D.; Banashree Das,2 M.B.B.S., M.D.; Sangita Rastogi,1* M.Sc., M.Phil., Ph.D. 1
Microbiology laboratory, National Institute of Pathology (ICMR), Safdarjung
hospital campus, New Delhi-110 029, India. 2
Department of Obstetrics & Gynecology, Vardhaman Mahavir Medical College
(VMMC) & Safdarjung hospital, New Delhi-110 029, India.
(c)
Corresponding author details: Dr. Sangita Rastogi, Scientist ‘G’ (Director Grade Scientist, Microbiology laboratory, National Institute of Pathology (ICMR), Sriramachari Bhawan, Safdarjung hospital campus, Post Box No. 4909, New Delhi 110 029, India. Tel. No.: + 91 – 011 – 26198402 - 406 (Ext. 314), Mobile no.: + 91 - 09810217260 Email: [email protected]
1
ABSTRACT Cytokines might be involved in spontaneous abortion by triggering inflammatory mediators (Cyclooxygenases (Cox)) leading to spontaneous abortion in Chlamydia trachomatis (Ct)infected women. Study aimed to quantitate the expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-8) and Cox-2 in endometrial curettage tissue (ECT) of Spontaneous Aborters (SA). SA (n = 135) and 120 age-matched controls were enrolled from SJH, New Delhi, India. PCR was performed for detection of Ct MOMP gene (537 bp) in ECT. mRNA expression of pro-inflammatory cytokines and Cox-2 was assessed by real-time qPCR. Data was statistically evaluated. 14.8% SA were diagnosed as Ct-positive. Elevated expression of TNF-α, IFN-γ, IL-8 and Cox-2 was observed in Ct-infected SA. Ct-positive recurrent aborters showed significantly higher cytokine expression. Significant positive correlation was found between expression of Cox-2 and TNF-α in infected SA. Data suggested an increased expression of Th-1 cytokines, particularly TNF-α that induced Cox-2 expression in ECT, leading to spontaneous abortion in Ct-infected pregnant women.
Key words: Chlamydia trachomatis, spontaneous abortion, pro-inflammatory cytokines, cyclooxygenase-2, real time PCR
2
1. Introduction During pregnancy, the risk of Chlamydia trachomatis (Ct) colonization increases, resulting in altered immune response and affects intra- and extra-uterine development in pregnant women causing abortion [1]. The adaptation of Ct to evade immune response allows organism to persist for extended periods within host epithelial cells, inducing a chronic inflammatory response [2]. Once Ct establishes infection, the innate immune response allows for production of pro-inflammatory cytokines such as interleukins (IL) (IL-1, IL-6, IL-8), and tumour necrosis factor alpha (TNF-α) [3]. Synergistic effect of IFN-γ and TNF-α also plays a role in the establishment of persistent chlamydial infection inside the host [4]. An abnormal production of pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6 and IL-8 was seen in placenta [5] and chorioamniotic membranes [6] in response to infection. Another study also revealed that administration of excess amount of Th1 cytokines induced abortion in mice [7] and low doses of anti-TNF-α antibodies reduced resorption rates in a murine abortion model [8]. In women, higher expression of proinflammatory cytokines was seen in recurrent miscarriage [9 - 11] and a significant expression of cytokines was also found in cultured Peripheral Blood Mononuclear Cells (PBMCs) of pregnant women with history of Recurrent Pregnancy Loss (RPL) [12]. An aberrant expression of pro-inflammatory cytokines during implantation window in women with idiopathic recurrent spontaneous miscarriage was also demonstrated [13]. A predominant Th1 type immunity was observed in recurrent spontaneous abortion [14, 15]. Furthermore, Cyclooxygenase-2 (Cox-2) is a key inflammatory response molecule, whose expression is induced by inflammatory stimuli such as IL-1β [16], TNF-α [17] and IFN-γ [18]. However, little is known about its role in infection-induced spontaneous abortions. Our recent studies demonstrated about the association of Ct infection with spontaneous abortion leading to the upregulation of Cox-2 in Ct-positive Recurrent
3
Spontaneous Aborters (RSA) [19, 20]. Another study reported decreased expression of Cox-2 resulting in recurrent pregnancy loss [21]. In a murine model, it was found that abnormal expression of Cox-2, TNF-α and IL-6 resulted in recurrent spontaneous abortion [22]. It is apparent that till date, the immunological changes underlying the complex transition from uterine quiescence to spontaneous abortion are not clear in Ct-infected women. In our previous study, we found an elevated expression of circulatory cytokines; IFN-γ, TNF-α, IL-6, IL-17A in serum of Ct-positive aborters [23]. Hence in the present study, we hypothesized that altered level of pro-inflammatory Th1 type cytokines might be involved in Ct-infection induced spontaneous abortion further triggering the Cox-derived prostaglandins signalling pathway leading to abortion in women. Therefore, the study aimed to quantitate the expression of proinflammatory cytokines, viz.: TNF-α, IFN-γ and IL-8 in the endometrial curettage tissues (ECT) of in Ct-infected Spontaneous Aborters (SA) and attempted to find whether there was any correlation between cytokines and Cox-2 in Ctinfected SA.
2. Materials and methods 2.1. Study population and enrollment of patients The study had Hospital Ethical Committee permission and prior informed written consent was taken from each patient. 135 women experiencing incomplete spontaneous abortion in the first trimester of pregnancy and undergoing dilation and evacuation (Group I) at Department of Obstetrics and Gynaecology, Vardhman Mahavir Medical College (VMMC) & Safdarjung hospital, New Delhi/ India were enrolled in the study in consultation with the gynecologist. All patients in Group I were symptomatic and presented with bleeding per vagina, passage of clots, etc. Group I comprised of both sporadic spontaneous aborters (SSA) having a single abortion (n = 43) and recurrent spontaneous aborters (RSA), who had suffered 2 or more abortions (n = 92). 120 age-matched healthy asymptomatic first trimester 4
pregnant women undergoing induced abortion for medical termination of pregnancy (MTP) (Group II) were enrolled as controls. The reasons for MTP were varied, i.e. risk to the life of the pregnant woman or of grave injury to her physical or mental health, a substantial risk that if the child were born, it would suffer from such physical or mental abnormalities, or where any pregnancy occured as a result of failure of any device/ method used by any married woman/ her husband for the purpose of limiting the number of children. Subjects with local causes of bleeding/ history of recently treated sexually transmitted infection/ recent antibiotic therapy/ HIV/ Treponema pallidum/ TORCH (Toxoplasma gondii, Rubella, Cytomegalovirus, Herpes simplex virus)/ Neisseria gonorrhoeae/ Mycoplasma genitalium/ Trichomonas vaginalis/ Ureaplasma urealyticum-positivity/ genital tuberculosis were not included in the study. Information on obstetric/ medical/ gynaecological history including last menstrual period, age/ gravidity/ parity/ abortion history and past genital/ urinary tract infection was collected in standardized questionnaires from all enrolled patients.
2.2. Sample collection The endometrial curettage tissue (ECT) was collected from each patient in Groups I – II and transported in phosphate buffered saline (10 mM, pH- 7.4) on ice to the laboratory. One part of the tissue sample was used for DNA extraction and polymerase chain reaction (PCR) assay. The remaining tissue was stored in RNA later at -800C for RNA extraction and quantitative analysis of mRNA expression.
2.3. Isolation of genomic DNA in ECT DNA was isolated in the ECT utilizing a commercial kit (Wizard Genomic DNA Isolation kit, Promega, USA) as per the manufacturer’s recommendations. For this, the tissue was homogenized in liquid nitrogen, treated with lysis buffer and the lysate was, thereafter, treated with protein precipitation solution. Finally, the DNA was precipitated by isopropanol. 5
Qualitative and quantitative analysis of extracted DNA was performed before the PCR assay. The quality of DNA was checked for any shearing by running DNA samples on 0.8% agarose gel. DNA samples were further quantified on a nano-drop spectrophotometer (Fermentas-Thermo Fisher Scientific, Waltham, USA) at 260 nm. A 260:280 ratio of ~ 1.8 was considered as optimum.
2.4. Detection of Ct by MOMP gene-specific PCR in ECT PCR assay was performed for the diagnosis of Ct infection in both the groups. The Major Outer Membrane Protein (MOMP) gene of Ct [24] and the internal control gene, viz.: beta-globin [25] were detected in the ECT (Table 1). Amplification reaction was set up in 25 µl reaction volume using DNA Thermal Cycler (Applied Biosystems, USA) in 0.2 ml PCR tubes. The reaction mix consisted of 1 µg of genomic DNA, 0.2 mM of each dNTP (Fermentas, USA), 10 pM of each oligonucleotide (Fermentas, USA), 2.5 µl of 10X PCR buffer (Fermentas, USA) and 1 unit of Taq DNA polymerase (Fermentas, USA). The genomic DNA which was isolated from ECT of enrolled subjects, was initially denatured at 95oC for 5 minutes, followed by 30 cycles at 95oC for 30 seconds while primer annealing was done at 52.4oC for beta-globin and at 45oC for 1 minute for MOMP primer and extension at 72oC for 45 seconds. Final extension was carried out at 72oC for 10 minutes to obtain a product of 268 bp and 537 bp for beta-globin and Ct MOMP gene, respectively. A Ctpositive control (Vircell Microbiologist, Granada, Spain) and negative control (nuclease-free water) were also amplified along with the DNA samples. The amplified product was confirmed by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized under UV Transilluminator.
6
2.5. Isolation of RNA in ECT The total RNA was isolated from the same ECT (from which the DNA was isolated) by using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, disruption of the tissue sample was done by taking 20 - 30 mg of the ECT and thereafter, homogenization was done with 1 ml Trizol reagent with 200µl chloroform. The lysate was centrifuged at 40oC and 75% ethanol was added to the supernatant. The tube was recentrifuged and the pellet was air-dried. 30µl of RNase-free water was added and the resulting RNA was stored at -80oC. The purity was checked by running the samples on 1% agarose gel and by taking the nanodrop O.D. A 260/ 280 ratio, i.e. 1.9 - 2.0 was considered as optimum.
2.6. Synthesis of cDNA from RNA For cDNA synthesis, total RNA (1.5 µg) was used to synthesize first strand cDNA using High Capacity cDNA Reverse Transcription Kit with the treatment of RNase Inhibitor (Applied Biosystems, USA) by One-Step Reverse Transcription-PCR as per the manufacturer’s instructions (briefly, 10x RT buffer- 10 µl, 25x dNTP mix (100 mM)- 2 µl, 10x RT random primers- 2 µl, RNase inhibitor- 1 µl, template RNA- 1.5 µg and nucleasefree water) and cDNA synthesis was performed on PCR Thermal Cycler (Applied Biosystems, Foster City, CA). The PCR conditions were as follows: initial denaturation for 10 minutes at 94o C, annealing at 50o-68o C for 120 minutes, extension for 5 minutes at 85o C and then 4o C for α time. 2.7. Detection of Ct by quantitative real-time PCR Quantitative real-time PCR (q-PCR) assay was performed on Step-one plus real-time PCR system (Applied Biosystems, USA) to detect the chlamydial load in the PCR-positive 7
ECT samples. To quantitate the copy number of Ct in ECT, first, serial dilutions (10000, 1000, 100, 10, 0.1 µl) of AMPLIRUN Ct DNA control (Vircell Microbiologists, Granada, Spain) were prepared as per the manufacturer’s instructions. Briefly, 10µl of SYBR green master mix, 1 µl of plasmid gene (200 bp) (Table 1) forward primer and 1 µl of plasmid gene (200 bp) reverse primer [26], 5 µl of Vircell Ct diluted DNA (Vircell Microbiologists, Granada, Spain) of each concentration and 3 µl of nuclease-free water were mixed to make up a final volume of 20 µl. The reactions were set in duplicates for each concentration, and standard curve was drawn. By using this standard curve, the chlamydial load was calculated in each ECT sample. The threshold cycle (CT) value was calculated as an average CT target genes for each sample.
2.8. Quantitative analysis of cytokine genes in ECT Taqman quantitative Real Time PCR (q-PCR) assay was performed on 7000 Realtime PCR system (Applied Biosystems, USA) for studying the quantitative expression of selected cytokine genes, viz.: TNF-α, IFN-γ and IL-8 in the ECT from each enrolled patient (viz.: Ct-positive controls, Ct-negative controls and SA). Commercialized human genes probes (‘Assay on Demand’) (Applied Biosystems, ThermoFisher Scientific, Waltham, USA) were used for the real-time assays. These genes were utilized for standard gene expression experiments because of their small size and detected maximum number of transcripts for the gene of interest. The assay IDs for GAPDH, β-actin, TNF-α, IFN-γ and IL-8 genes are listed in Table 2. 1.5 µg/ µl concentration of c-DNA was used for q-PCR. 10µl of Taqman PCR master mix (Applied Biosystems, USA), 1µl of probe, 4µl of c-DNA and 5µl of nuclease-free water (Ambion, Thermo Fisher Scientific, USA) were mixed to make up a final volume of 20µl. Finally, the reaction mix was loaded into real time optical PCR tubes (Applied Biosystems, USA). The assay was standardized for the internal control gene glyceraldehyde 3-
8
phosphate dehydrogenase (GAPDH) and beta actin (β-actin) in the ECT samples and those showing consistency with both GAPDH and β-actin were selected for real time PCR studies. The Threshold Cycle (CT) value was calculated as average ∆CT target genes for each sample by using SDS software and the relative quantification was used to measure gene expression by relating the PCR signal.
2.9. Quantitative analysis of Cox-2 gene expression in ECT Quantitative Real Time PCR (q-PCR) assay was performed on 7000 Real-time PCR system (Applied Biosystems, USA) for studying the quantitative expression of Cox-2 gene (Table 2) in ECT as described previously [19].
2.10. Statistical analysis Statistical analysis was performed using the GraphPad prism software (version 5.0, San Diego, California, USA). Fisher’s Exact test and Mann-Whitney test/ Welch’s test were used to calculate the significance between the groups. Spearman’s rank correlation coefficient was used to calculate the correlation between the expression levels of cytokines and Cox-2.
3. Results 3.1. Detection of Ct infection in ECT SA (n = 135) constituted the study group (Group I) while 120 aborters comprised the control group (Group II). The average gestational age of first trimester aborters in Groups I II was 8 weeks. Table 3 shows the clinical characteristics of enrolled aborters (N = 255). Overall, 14.8% (n = 20) women experiencing spontaneous abortion were diagnosed as Ctpositive for the MOMP gene by PCR assay in Group I; in the latter, 13.9% (6/ 43) SSA and 15.2% (14/ 92) RSA were positive for Ct infection. In Group II controls, 1.4% (n = 2) women were found to be positive for Ct infection (Fisher’s Exact test; ‘p’ < 0.0001). For 9
experimental analysis, comparisons were made between Ct-positive SA (n = 20) versus Ctnegative controls (n = 118), Ct-positive SA (n = 20) versus Chlamydia-positive controls (n = 2), Ct-positive SA (n = 20) versus Ct-negative SA (n = 115). Also, CT values were analysed between Ct-positive SA (n = 20) versus Ct-negative controls (n = 118) and between Ctpositive SSA (n = 6) versus Ct-positive RSA (n = 14). Furthermore, q-PCR was performed by using the SYBR green-based chemistry to quantitate the Ct copies in ECT of Groups I – II. Standard curve was obtained by preparing the serial dilutions of Ct-positive control. Cycle threshold (CT) values of all the samples were calculated by q-PCR and the copy load of Ct in each sample was evaluated by using the standard curve, plotted by using the known concentrations of Ct-positive control. A total of 1 - 4 and 4 – 12 copies were detected in the ECT of Ct-positive SSA and RSA, respectively.
3.2. Quantitative analysis of mRNA expression for pro-inflammatory cytokines (TNF-α, IFN-γ, IL-8) by q-PCR assay in ECT In order to investigate whether altered mRNA expression of cytokines was associated with spontaneous abortion in women found infected with Ct, expression of mRNA-encoding cytokines (TNF-α, IFN-γ, IL-8) was studied in the ECT by the q–PCR. These assays were designed to allow quantitative comparisons of mRNA abundance between different cytokines by comparing mean CT values for control ECT. At transcript level, the analysis of mRNA expression for TNF-α, IFN-γ and IL-8 genes within the ECT was compared among patient group as a ratio to the expression of constitutively expressed GAPDH and β-actin gene by performing q-PCR. The abundance of mRNA expression of Th1 cytokines, viz.: TNF-α, IFNγ and IL-8, demonstrated a significant increase in Ct-positive SA in comparison to Ctnegative SA/ Ct-negative controls (non-parametric Mann-Whitney test; Fig. 1 A- C). In the 2 control subjects who were Ct-positive, the cytokine profile (viz.: TNF-α, IFN-γ and IL-8) was
10
found to be significantly decreased in comparison to the Ct-positive SA (non-parametric Welch’s test, Fig. 1 A - C). Upon analysis of CT values, the TNF-α, IFN-γ and IL-8 genes were found to be significantly upregulated (3.0-fold, 4.5-fold and 2.0-fold, respectively) in Ct-infected SA versus Ct-negative controls (Table 4). The mRNA expression of the pro-inflammatory Th1 genes was also compared between the Ct-positive SSA versus Ct-infected RSA and it was found that TNF-α, IFN-γ and IL-8 genes’ expression was significantly higher in the latter in comparison to the former (Mann-Whitney test; Fig. 2 A - C). CT values were further analysed and it was found that in the Ct-positive RSA, all 3 cytokine genes, viz.: TNF-α, IFN-γ and IL-8 were upregulated (3.5-fold, 5-fold and 1.2-fold, respectively), while, the fold-change in Ct-positive SSA was 1.5-fold, 2.8-fold and 1-fold, respectively.
3.3. Quantitative analysis of mRNA expression for Cox-2 gene in ECT Analysis of mRNA expression of Cox-2 gene within the ECT by q–PCR was compared among study group as a ratio to the expression of the constitutively expressed GAPDH. Cox-2 mRNA expression demonstrated a significant increase in Ct-positive SA in comparison to Ct-negative SA/ Ct-negative controls (non-parametric Mann-Whitney test; Fig. 3 A). In the 2 control subjects who were Ct-positive, the expression of Cox-2 gene was found to be significantly decreased in comparison to the Ct-positive aborters (non-parametric Welch’s test, ***‘p’ < 0.0001; Fig. 3 A). Analysis of CT values showed that the Cox-2 gene was 2.3-fold upregulated in Ct-positive SA versus Ct-negative controls. Also, Cox-2 was significantly increased in Ct-infected RSA in comparison to the Ct-positive SSA (MannWhitney test; ‘p’ < 0.0001; Fig. 3 B).
11
3.4. Correlation between Cox-2 and cytokines in SA infected with Ct In infected SA, a statistically significant positive correlation was found between the expression of Cox-2 and TNF-α in the ECT (Spearman rank correlation; r = 0.77, ***‘p’ < 0.0001; Fig. 4 A), however, the correlation between Cox-2 and IFN-γ was not statistically significant (Spearman rank correlation; r = 0.04; ‘p’ = 0.85) (Fig. 4 B). The IL-8 cytokine expression in the ECT was also found to be insignificant in correlation with Cox-2 (Spearman rank correlation; r = 0.39; ‘p’ = 0.09) (Fig. 4 C).
4. Discussion The causes of pregnancy loss are not well understood, but infection and immune mechanisms are thought to be involved. Besides Ct infection, there are also other known reasons for abortion in women such as chromosomal, genetic, endocrine, infectious, autoimmune, etc. In our study, patients with any anatomical abnormality and diabetes mellitus were excluded. Furthermore, women with local causes of bleeding/ history of recently treated sexually transmitted infection/ recent antibiotic therapy/ HIV/ Treponema pallidum/ TORCH (Toxoplasma gondii, Rubella, Cytomegalovirus, Herpes simplex virus)/ Neisseria gonorrhoeae/ Mycoplasma genitalium/ Trichomonas vaginalis/ Ureaplasma urealyticum-positivity/ genital tuberculosis were not included in the study. One limitation of our study was that chromosomal abnormalities could not be excluded. Also, environmental factors and other unexplained etiologies could be one of the reasons for spontaneous abortions in Ct-negative aborters. Furthermore, the complex interplay between maternal and foetal immune mechanisms changes temporally as pregnancy progresses. If this delicate balance is adversely affected, immunoregulatory mechanisms may be insufficient to restore homeostasis and this may lead to pregnancy failure. Successful pregnancy may depend, at least in part, on the bias of the maternal immune response shifting away from Th1 type responses towards a Th2 phenotype, 12
both in murine models and humans. Recent attention has focused on elucidating the immunobiological role of cytokines in normal human pregnancy following complex cytokine activity within uteroplacental tissues [27, 28] Numerous in vivo and in vitro studies have reported a strong, maternal Th1 type reactivity deleterious to pregnancy in mice [8, 29] and likewise, in vitro studies in the human system also [14, 30]. The elaboration of proinflammatory cytokines such as TNF-α and IL-1β, typical of a Th1 cell-mediated immune response, is hypothesized to be detrimental to the survival of the developing conceptus. TNFα is an important immunoregulatory cytokine, which may be produced in Th1 or Th2 type responses and is known to have different effects depending on gestational age [31]. TNF-α production by PBMCs was suppressed at the mRNA level during early pregnancy while its production in late pregnancy is implicated in the induction of labour in humans [32]. Also, TNF-α production is enhanced at the onset of labour and spontaneous abortion [33]. Higher concentrations of Th-1 cytokines (TNF-α) were demonstrated systemically in the serum of patients with idiopathic/ unexplained recurrent miscarriages than normal controls [34]. It has also been reported that cytokines such as IFN-γ and TNF-α are harmful for pregnancy [14, 35]. Our study also showed a statistically significant increased expression of proinflammatory cytokines, viz.: TNF-α, IFN-γ and IL-8 in Ct-positive SA in comparison to the Ct-infected SA and uninfected/ infected control groups. Significant levels of the Th-1 cytokines, IFN-γ and IL-2 have been detected at the maternal–foetal junction in cases of spontaneously aborting conceptus by other groups also [36]. In response to infection, an abnormal production of pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, and IL-8 was found in the placenta [5] and chorioamniotic membranes [6], which might have eventually led to spontaneous abortion. Our results further revealed a high expression of TNF-α, IFN-γ and IL-8 cytokines in the Chlamydia-infected RSA in comparison to infected SSA. High level of Th1 cytokines,
13
viz.: TNF-α and IFN-γ were earlier detected in RSA [11, 37]. In another study, TNF-α was found abundantly in decidua of RPL women who had miscarried [38]. Elevated values of TNF-α and IFN-γ were reported in recurrent miscarriage group [39]. In another study, the expression of IL-6 and IL-8 in cervical mucus was found to be significantly higher in idiopathic recurrent miscarriage cases who subsequently miscarried than in those who had a live birth [40]. Increased level of IL-8 in amniotic fluid was found in cases of preterm labour with histologically confirmed chorioamnionitis [41]. The increased level of IL-6 and IL-8 were also observed in women with second trimester abortions [42, 43]. It is apparent in our study that pro-inflammatory cytokines constitute an important component of the host immune response to infection with Chlamydia, particularly IFN-γ, which restricts chlamydial growth [44]. The generation of an inflammatory response to the control infection alters the delicate cytokine balance in the placenta, resulting in abortion [45]. Indeed, inflammatory cytokines such as IFN-γ, TNF-α and IL-2 have an abortigenic effect [45] and abundant expression of these cytokines at the materno-fetal interface was found associated with abortion [46]. It was also studied that the production of TNF-α by fetal macrophages may be of considerable significance in the pathogenesis of abortion in pregnant sheep infected with C. psittaci [47]. Furthermore, a higher expression of Cox-2 during implantation window in the endometrial tissue of women with idiopathic recurrent spontaneous miscarriage may be attributed to the exaggerated expression of proinflammatory cytokines such as TNF-α and IFN-γ [13]. IL-6 and TNF-α can induce the expression of Cox-2 leading to an increased production of prostaglandins (PG). An ascending infection into the uterus during pregnancy is followed by local increase of Cox-2 activity, and consequently elevated PG production. Lipopolysaccharide (LPS)-induced decidual prostanoid production via increased Cox-2 expression and Cox-2 mediated eicosanoid production is probably a key pathophysiologic event in the LPS-mediated fetal death [48]. Other in vitro
14
studies provide evidence that in cells infected with chlamydiae, TNF-α induces the production of PGE2 much more effectively than in uninfected cells and PGs mediate symptoms of gram-negative septic shock, stimulate myometrium and are capable abortifacients and they are likely to be important mediators of LPS-induced pregnancy loss. In our previous studies, it was found that chlamydial infection leads to upregulation of Cox-2 in Ct-positive RSA which probably mediated an increased PG synthesis [19]. In the present study, mRNA expression of Cox-2 was also found to be significantly increased in the Ct-positive SA in comparison to Ct-uninfected SA and uninfected/ infected controls. We found a positive correlation only between Cox-2 and TNF-α among the Ctpositive SA. The pro-inflammatory cytokines can induce the expression of Cox-2 and cause increased PG production. Other studies have reported a positive correlation between TNF-α and IL-6 while no correlation was found between Cox-2, TNF-α and IL-6 in a recent study [22]. It has also been suggested that when normal levels of IL-6 and TNF-α expression are present, Cox-2 can form the dynamic equilibrium, which can ensure the safe implantation of embryo sac and avoid abortion [49]. In our results, we found a statistically non-significant correlation between Cox-2/ IFN-γ and Cox-2/ IL-8. The non-significant finding between Cox-2 and IFN-γ might be the result of the high expression of IFN-γ level (4.5-fold) in comparison to Cox-2 (2.3-fold). IFN-γ levels were earlier found to be significantly higher in cervical washes of women with recurrent infection. IFN-γ is known for its requirement in resolution of chlamydial infection, and has been shown to inhibit the growth of Chlamydia in cell culture. IFN-γ showed the greatest fold-change among all the differentially induced mRNA in Ct-infected vaginal tissues in mice suggesting that IFN-γ production is being stimulated by other immunoregulatory factors at this later stage of infection [50]. The expression of IL-8 (2.0fold) was also non-significantly correlated with Cox-2. It was studied previously that in the
15
presence of Ct infection, the increased IL-8 production by the trophoblasts might lead to increased neutrophils at the maternal-fetal interface and this may have a negative impact on the pregnancy outcome. The continued high expression of the IL-8 and IFN-γ can lead to an increase/ aggregation of the inflammatory cytokine concentration, and the interaction between cells can also cause cytotoxic effect, and can lead to the imbalance of immune tolerance and then induced SA. IL-8 and IFN-γ can induce the expression of Cox-2 and then cause increased PGs which can eventually result in miscarriage. Apparently, spontaneous abortion is a pleiotropic condition with several as yet unidentified causes; however, an aberration in cytokine production also constitutes an important contributing factor. Our data support the hypothesis of Th1 cytokine involvement, particularly TNF-α in the pathogenesis of SA.
5. Conclusions The present study concludes that modulation of Th1 dominance may bring about an immunological milieu that is more conducive to successful pregnancy. An overall understanding of cytokine production in SA during Ct infection will contribute to the immunopathogenesis of disease and in the development of better management and therapeutic intervention for a successful pregnancy. Further large studies on relationship among inflammation, cytokine production and treatment strategy in abortion are clearly needed.
Conflict of interest The authors declare no conflicts of interest.
16
Acknowledgments NS is thankful to University Grants Commission, New Delhi, India for the award of Senior Research Fellowship (SRF) and also acknowledges National institute of Pathology (ICMR), New Delhi, India for providing SRF. PP gratefully acknowledges the award of Junior Research Fellowship from Council of Scientific & Industrial Research, New Delhi, India and also acknowledges with thanks National institute of Pathology (ICMR), New Delhi, India for providing SRF.
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23
Legends to figures: Fig. 1. A-C. Significantly high expression of pro-inflammatory cytokine genes (viz.: TNF-α/ IFN-γ/ IL-8) in endometrial curettage tissue of Chlamydia trachomatis (Ct)-positive spontaneous aborters (SA) versus Ct-negative SA and uninfected/ infected controls by quantitative real time PCR (non-parametric Mann-Whitney test). ‘p’ values: * < 0.05; ** < 0.001; *** < 0.0001
Fig. 2. mRNA expression of pro-inflammatory cytokines in endometrial curettage tissue of spontaneous aborters (SA), viz.: sporadic SA (SSA, Sub-group Ia) versus recurrent SA (RSA, Sub-group Ib) by quantitative real time PCR (non-parametric Mann-Whitney test). A. TNF-α expression was significantly high in Chlamydia trachomatis (Ct)-positive RSA versus Ctpositive SSA (***‘p’< 0.0001). B-C. Expression of IFN-γ and IL-8 was significantly high in Ct-positive RSA versus Ct-positive SSA (** < 0.001).
Fig. 3. Expression of Cyclooxygenase-2 (Cox-2) in endometrial curettage tissue of Chlamydia trachomatis (Ct)-positive spontaneous aborters (SA) by real time PCR (nonparametric Mann-Whitney test): A. Expression of Cox-2 gene was significantly high in Ctpositive SA versus Chlamydia–negative SA/ Ct-negative controls/ Ct-positive controls. B. Cox-2 mRNA expression was significantly high in Ct-positive recurrent SA versus Chlamydia-positive sporadic SA. ‘p’ values: ** p < 0.001; ***p < 0.0001)
Fig. 4. A- C. Correlation between Cox-2 and TNF-α/ IFN-γ/ IL-8 in Chlamydia trachomatispositive spontaneous aborters (Spearman rank correlation test; ‘r’ = 0.77, 0.04 and 0.39, respectively).
Table 1: Primers used for molecular diagnosis of Chlamydia trachomatis in the endometrial curettage tissue of aborters
Name of gene
Primer Sequence: 5'-3'
MOMP gene of Ct
F: TAT ACA AAA ATG GCT CTC TGC TTT
Size of Reference amplicon (bp) 537 bp
24
268 bp
25
200 bp
26
AT β-globin
R: CCC ATT TGG AAT TCT TTA TTC ACA TC F: CAA CTT CAT CCA CGT TCA CC R: GAA GAG CCA AGG ACA GGT AC
Plasmid gene of Ct
F: CTA GGC GTT TGT ACT CCG TCA R: TCC TCA GGA GTT TAT GCA CT
Abbreviations: MOMP, major outer membrane protein; Ct, Chlamydia trachomatis, β-globin, beta globin.
Table 2: Probes used for quantitative expression of cytokines and cyclooxygenase by real time PCR assays
Gene name
Genes probes (‘Assay on Demand’) Amplicon (Gene ID) size (bp)
GAPDH
Hs02758991_g1
93
β-actin
Hs01060665_g1
63
TNF-α
Hs00174128_m1
80
IFN-γ
Hs00989291_m1
73
IL-8
Hs00174103_m1
101
Cox-2
Hs00153133_m1
75
Abbreviations: GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; β-actin, beta actin; TNF-α, tumour necrosis factor alpha; IFN-γ, interferon gamma; IL-8, interleukin-8; Cox-2, cyclooxygenase-2
Table 3. Demographic characteristics of the enrolled participants
Clinical Characteristics
Group I (Spontaneous aborters) n= 135 (%)
Group II (Controls) n= 120 (%)
‘p’-value
Age (years) <20 20-25 26-30 31-35 35-40
12 50 28 27 18
(8.9) (37.0) (20.7) (20.0) (13.3)
10 42 24 28 16
(8.3) (35.0) (20.0) (23.3) (13.3)
1.00 0.90 1.00 0.65 1.00
35 38 47
(26.6) (34.6) (38.6)
0.89 1.00 0.81
Mean age: 25 ± 4.0 Gravidity 01 02 >2
42 44 49
(32.7) (30.7) (36.6)
Average gravidity: 2 Average gestational age: 8 weeks
Parity 0-1 2->2
88 47
(64.7) (35.3)
29 (30.6) 91 (69.3)
0.001 0.005
No. of Abortions 0-1 2- >2
43 (32.7) 92 (70.6)
0 0
< 0.0001 < 0.0001
Smoking status Non-smoker Smoker
131 (97.03) 04 (2.9)
120 (100) 0
0.125
Education Literate Illiterate
51 (37.8) 84 (62.2)
79 (65.8) 41 (34.17)
0.001
39 (28.9) 96 (71.1)
82 (68.3) 38 (31.7)
0.001
Residence Urban Rural
Previously used contraceptive method Pills Condom Other Never used contraceptive
54 12 28 41
(40) (88.8) (20.7) (30.4)
26 16 10 68
(21.7) (13.3) (7.4) (56.7)
0.0019 0.316 0.007 0.001
Marital status Married Unmarried
135 (100) 0
120 (100) 0
1.000
Table 4. Fold-change values of TNF-α, IFN-γ, IL-8 in the endometrial curettage tissue
of
Chlamydia
trachomatis-infected
spontaneous
aborters
versus
C.
trachomatis-negative controls.
Cytokines
Ct-negative controls Avg. ∆CT
Foldchange
‘p’value
TNF-α
Ct-positive spontaneous aborters Avg. ∆CT 6.36
7.93
3.0
< 0.0001
IFN-γ
8.32
10.6
4.5
< 0.0001
IL-8
6.83
7.82
2.0
< 0.0001
Abbreviations: Ct, Chlamydia trachomatis; ∆CT, Cycle threshold
Fig. 1. A
B
C
Fig. 2. A
B
C
t+ v
B t-v
t+ v
C
e
e
SA
SA
co nt ro ls
co nt ro ls
Relative expression of Cox-2 4
C
e
tve
C
C
Fig. 3. A
*** *** **
3
2
1
0
Fig. 4. A
B
C
•
Study revealed effect of proinflammatory cytokines on C. trachomatis-positive SA
•
Increased expression of TNF-α, IFN-γ, IL-8 in ECT of C. trachomatis-positive SA
•
TNF-α, IFN-γ and IL-8 expression was significantly higher in RSA versus SSA
•
TNF-α and COX-2 were positively correlated with each other in infected aborters
•
Aberration in TNF-α and IFN-γ can induce COX-2 leading to immunopathogenesis of SA
Author Statement
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