HERPES SIMPLEX VIRUS TYPE 2 INFECTION DURING PREGNANCY IS

INTERNATIONAL TRENDS IN IMMUNITY ISSN 2326-3121 (Print) ISSN 2326-313X (Online)

VOL.2 NO.1 http://www.researchpub.org/journal/iti/iti.html

JANUARY 2014

Herpes Simplex Virus Type 2 Infection during Pregnancy is Correlated with Elevated TLR9 and TNFα Expression in Cervical Cells Svitich Oxana A., MD1, Gankovskaya Ludmila V., MD2, Lavrov Viacheslav F., MD1, Grigor'eva Oxana Iu., PhD1, Karaulov Alexander V., MD 3, Zverev Vitalii V., MD 1

immunity is critically involved in early control of mucosal viral infections including HSV-2 [3, 5]. Innate immune system components, such as toll-like receptors (TLRs), expressed by epithelial cells of female reproductive tract, are able to recognize genomic signatures of viruses [6-11]. In particular, unmethylated CpG motifs of bacterial and viral nucleic acids bind to the endosomal TLR9 [12-14], leading to a strong innate immune response via activation of nuclear factor-κB (NF-κB)-dependent signaling pathway [15]. It is well established that NF-κB regulates the expression of various chemokines and pro-inflammatory cytokines, such as TNFα, IL-12 and IL-1 [11, 13, 15-19], which are presumably involved in preterm birth [20-22]. However, the relationship between the activation of TLR9-mediated signaling pathway in the cervix and preterm birth has not been addressed yet. Following this notion, we studied TLR9, TNFα and NF-κB gene expression in cervical epithelial cells in both healthy and genital HSV-2-infected pregnant women, and found an association between elevation in the expression of these genes in infected women and the occurrence of preterm birth.

Abstract—Activation of toll-like receptors (TLRs) by viruses induces secretion of pro-inflammatory cytokines that are suggested to be involved in preterm birth. In our study, we found that infection of cervical epithelial HeLa cells with herpes simplex virus type 2 (HSV-2) is accompanied by a dramatic elevation in TLR9 and TNFα gene expression in time- and dose-dependent manners. We also observed a significant elevation in TLR9, NF-κB and TNFα gene expression in cervical epithelial cells obtained from pregnant women with genital HSV-2 infection as compared to cells collected from healthy pregnant women. In addition, premature births were registered in 81% of pregnant women with genital HSV-2 infection, among which 38% exhibited a considerable increase in the cervical epithelial cell TLR9 gene expression. These data suggest involvement of TLR9, NF-κB and TNFα activation in HSV-2-mediated preterm labor. Keywords — Toll-like receptor, herpes, innate immunity, TNF, pregnancy.

II. MATERIALS AND METHODS I. INTRODUCTION

Cells and virus We used epithelial cell line HeLa generated from an adenocarcinoma of the human cervix [23]. Herpes Simplex Virus type 2 (HSV-2, strain MS) was obtained from the National Virus Collection (London, UK).

H

ERPES virus infection is one of the most widespread human viral infections. It is also known that genital herpes virus infection is a main cause for pregnancy complications [13]. Indeed, primary infection or reactivation of herpes virus infection (HSV-2) during pregnancy often results in preterm labor, prenatal death or intrauterine transmission of HSV-2 to fetus [4]. Mucosal surfaces serve as the entry sites for the majority of infectious agents, including HSV-2, and provide the first line of anti-infection defense. However, the mechanisms underlying anti-HSV-2 mucosal immunity remain poorly understood. Previous studies have shown that the innate

Clinical groups Cervical epithelial cells were obtained from pregnant women (n=16) with genital HSV-2 infection and normal pregnant women (n=10) in their third trimester of pregnancy (28-33 weeks of gestation), 24-34 years old. The pregnant women were the patients of the Department of Obstetrics and Gynecology in the Russian State Medical University. The university review board approved the protocol and statement of written informed consent, which was signed by each participant. Samples of vaginal epithelial cells from HSV-2 infected (seropositive) and non-infected participants were collected using swabs that were placed into vials containing 1 ml of polymerase chain reaction (PCR) transport medium and kept at

Received Sept 24th, 2013. Accepted with revision Dec. 10th, 2013 I.Mechnikov Research Institute of Vaccines and Sera RAMS Moscow, Russian Federation; 2 Pirogov Russian National Research Medical University, Moscow, Russian Federation; 3 I.M. Sechenov First Moscow State Medical University, Moscow, Russian Federation. *Correspondence to Svitich, Oxana A.(e-mail: [email protected]). 1

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– 200C until being processed for gene expression by PCR (see below).

Statistical analysis Statistical differences of the gene expression levels were determined by using a Mann-Whitney U test. p < 0.05 was considered to be statistically significant. The results are shown as the mean ±SEM.

Infection of cells HeLa cells (6 x105 cells/ml) were seeded in 24-well tissue culture plates (Costar, USA) in DMEM (Sigma, USA) supplemented with 10% fetal calf serum (HyClone, USA), glutamine (PanEco, Russia) and antibiotics (PanEco, Russia), and incubated at 370C in a humidified atmosphere with 5% CO2 for 48 h and then infected with HSV-2 (2.5 and 3.5 TCD50/0.1ml). After different periods of time (1, 3, 6, 12 and 24 h post infection), the cells were harvested for RNA isolation.

III. RESULTS HSV-2 infection of HeLa cells induces TLR9 and TNFα gene expression Epithelial cells from the lower female reproductive tract express mRNA for human TLR1-9, suggesting a role in the local pro-inflammatory cytokine production during bacterial and viral infections that are associated with preterm birth [6, 24]. To clarify this hypothesis, we studied TLR9, TNFα and NF-κB gene expression in the cervical epithelial HeLa cell line and in primary cells collected from both healthy and genital HSV-2 infected pregnant women. First, we found that HSV-2 infection resulted in a dramatic elevation in TLR9 gene expression in HeLa cells already 1 h after infection; the most considerable elevation was observed 3 h after infection followed by its reduction maintaining a significant higher level as compared to uninfected cells (Fig. 1A). Of note, two applied HSV-2 doses revealed equal enhancing effect (Fig. 1A). Similarly to TLR9, TNFα gene expression was also higher in infected HeLa cells than in uninfected cells at all time points, reaching a peak 6 h after infection (Fig. 1B). However, the degree of elevation in TNFα gene expression, especially during the first 3 h post infection, was less pronounced as compared to TLR9 gene expression, thus demonstrating quantitative and kinetic differences in the expression of these two genes. In addition, unlike to TLR9, there was a clear dose-dependent effect of HSV-2 on TNFα gene expression (Fig. 1B). Noteworthy, TLR9 and TNFα gene expression levels were low and constant in uninfected cells (Fig. 1A,B). There was no difference in NF-κB expression between infected and uninfected cells (7.4 x 103 ±2.1 x 103 vs 4.6 x103 ± 0.31 x103 transcripts, respectively). In the cell cultures, infected HSV, the level of TNF protein is 467,0±63 pg/ml and in not infected - 20.0±10,0 pg/ml.

Isolation of RNA and RT- PCR Analysis Total RNA was isolated from HeLa cells and cervical epithelial cells by using a RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. For cDNA synthesis, 2 µl of total RNA was combined with random primers (Syntol, Russia) and reverse primers for the target genes (T9rev, NFrev, TNrev shown in Table 1). The mix was heated to 75oC for 3 min and then quick-chilled on ice; 3 µl of 10X Reverse Transcriptase Buffer (SibEnzime, Russia), 2 µl of 2.5mM dNTP (SibEnzime, Russia) and 10 µl of M-Multi Reverse Transcriptase (SibEnzime, Russia) were added to the mix (final volume of 30 µl). Afterwards, the mix was kept at 37oC for 60 min and at 95oC for 10 min. For quantitative analysis, real-time PCR was performed, using a SYBR Green Kit for qRT-PCR (Syntol, Russia), according to the manufacturer’s instructions. The primers and probes for the real-time PCR were synthesized by Syntol (Russia) and are listed in Table 1. The PCR reactions were carried out at 50°C for 2 min, 95°C for 2 min, followed by 35-40 cycles at the primer-specific annealing temperature (shown in Table 1) for 50s and 95°C for 20s. The mRNA expression levels of the target genes were normalized with the expression of the housekeeping gene β-actin.

TABLE I

The primers and zonds, used in the work. Zonds 5’ -

Target gene

Primers 5’ -

TLR9

T9for

tgg-tgt-tga-agg-aca-gtt-ctc-tc

T9rev

cac-tcg-gag-gtt-tcc-cag-c

NFfor

gaa-ctc-ctc-cat-tgt-gga-acc-ca

NFrev

agc-att-gct-tgc-cca-cca-gac-t

TNfor

tgc-ttg-ttc-ctc-agc-ctc-tt

TNrev

tcg-aga-aga-tga-tct-gac-tg

Afor

gcg-gga-aat-cgt-gcg-tga-cat-t

Arev

gat-gga-gtt-gaa-ggt-agt-ttc-gt g

NF-kB

TNFa

β-actin

ROX-ggc-tga-atgcca-gtt-ggt-tcc-gt g-g-BHQ2

Anneali ng temperat ure 64°C

R6G-tac-ctg-gtg-a tc-gtg-gaa-cag-cct -aag-BHQ2

64°C

ROX-cgc-cac-cac -gct-ctt-ctg-cct-gc t-g-BHQ2

66°C

CY5-cct-gag-gcactc-ttc-cag-cct-tcc -tt BHQ2

62°C

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HSV-2 infection enhances TLR9, NF-κB and TNFα gene expression in cervical epithelial cells obtained from pregnant women. Significant enhancement in TLR9 and TNFα gene expression by HSV-2 infection of cultured HeLa cells (see Fig. 1) implies for a similar change in gene expression in the cervix of pregnant women during genital HSV-2 infection. Indeed, we observed a 2-fold elevation in TLR9 gene expression in the cervical epithelial cells of pregnant women with genital HSV-2 infection as compared to healthy pregnant women (1.4 x 10 6 ± 2.1 x 105 vs 0.69 x 106 ± 0.82 x 105 transcripts, respectively) (Fig. 2A). Upon examining NF-κB gene expression, we detected a 4.4-fold elevation in the group of genital 63



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HSV-2-infected pregnant women as compared with the control group (17 x 103 ±2.1 x 103 vs 3.9 x103 ±0.31 x103 transcripts, respectively) (Fig. 2B). Moreover, there was a 3-fold increase in TNFα gene expression in the cervical cells of genital HSV-2-infected pregnant women as compared with the control group (17 x 104 ±2.4 x 104 vs 5.4 x104 ±0.87 x104 transcripts, respectively) (Fig.2C). Importantly, clinical observations demonstrated premature births in 81% of pregnant women with genital HSV-2 infection, among which 38% revealed a considerable increase in TLR9 gene expression (4.7 x 106 ±5.2 x 105). We also show that the levels in the serum of patients with herpes viral infection rise 6,2 times. Importantly, clinical observations demonstrated premature births in 81% of pregnant women with genital HSV-2 infection, among which 38% revealed a considerable increase in TLR9 gene expression (4.7 x 106 ±5.2 x 105). We also show that the levels in the serum of patients with herpes viral infection rise 6,2 times.

IV. DISCUSSION Despite advances in the management of pregnancy complications, including premature birth, their incidence remains high and the underlying mechanisms are still poorly understood. Recent animal and clinical observations suggest involvement of the reproductive tract mucosal immunity and in particular TLRs activated by local bacterial and viral infections (Patni S, Flynn P, Wynen LP, et al. An introduction to Toll-like receptors and their possible role in initiation of labour. BJOG 2007; 114:1326-1334). For example, TLR3 agonist administration induces impairment of uterine vascular remodeling, leading to fetal losses in CBA × DBA/2 mice (Zhang J, Wei H, Wu D, Tian Z. Toll-like receptor 3 agonist induces impairment of uterine vascular remodeling and fetal losses in CBA × DBA/2 mice. J Reprod Immunol. 2007;74:61–67 ). Upon engagement of TLRs, the intracellular signaling adapter protein myeloid differentiation factor 88 (MyD88) is recruited, leading to a subsequent kinase cascade, which triggers the activation of NFκB pathway. A resultant production of pro-inflammatory cytokines may cause therefore

Figure 1. HSV-2-indused TLR9 (A) and TNFα (B) gene expression in the cervical epithelial cell line HeLa. Hela cells were cultured for 48 h and then were infected or not (control) with HSV-2 (2.5 and 3.5 TCD50/0,1ml). At indicated time points after infection, the cells were harvested and TLR9 and TNFα gene expression was analyzed by qRT-PCR. For normalization of the results, the house-keeping gene β-actin expression has been evaluated. The results are presented as the mean ± SEM (for TLR9 in log scale). * p < 0.05; ** p < 0.01.

Figure 2. TLR9 (A), NF-κB (B) and TNFα (C) gene expression in the cervical epithelial cells of pregnant women with either healthy or with genital HSV-2 infection. Cervical epithelial cells were obtained from healthy pregnant women (n=10) and pregnant women with genital HSV-2 infection (n=16). TLR9, NF-κB and TNFα gene expression was analyzed by qRT-PCR. As a normalization control, the house-keeping β-actin gene expression was measured. The results are presented as the mean ±SEM. * p < 0.05; ** p < 0.01 .

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a fetal loss (Akira S. Toll-like receptor signaling. J Biol Chem. 2003;278:38105–38108; Thaxton JE, Nevers TA, Sharma S. TLR-mediated preterm birth in response to pathogeneic agens. Infect Dis Obstet Gynecol 2010; 2010:378472). Since genital epithelial cells are the first line of mucosal defense against bacterial and viral infections, we exploited the ability of these cells to express the innate immunity TLR9, NF-κB and TNFα genes and observed a significant elevation in TLR9 and TNFα gene expression in HeLa cells cultured in the presence of HSV-2. Interestingly, as compared to TNFα, TLR9 gene expression was found to be more pronounced and its elevation started earlier, confirming previously described sequence of triggered mucosal innate immunity events (Lee AJ and Ashkar AA. Herpes simplex virus-2 in the genital mucosa: insights into the mucosal host response and vaccine development. Curr Opin Infect Dis 2012; 25(1):92-9). Study of pregnant women revealed a significant elevation in TLR9, NF-κB and TNFα gene expression in women genitally infected with HSV-2 as compared to uninfected subjects, thus supporting also the involvement of the transcriptional factor NF-κB in the mucosal innate immunity activation following genital HSV-2 infection. This nuclear factor is present in unstimulated cells in its inactive form and after stimulation it translocates into the nucleus, after which it binds promoters of target genes [18], leading to transcription of pro-inflamatory cytokine genes, such as TNFα. Importantly, this cytokine along with its pro-inflammatory and cytotoxic activity against infected cells has been shown to play a role in pathology of different pregnancy complications [20-22], and its elevated levels are correlated with miscarriage and preterm labor [25]. In support, together with elevated levels of TLR9, NF-κB and TNFα gene expression in genital epithelial cells obtained from HSV-2 infected pregnant women, we also found that 81% of these women revealed premature births, among which 38% of the women demonstrated a considerable increase of TLR9 gene expression. In line with previous observations, we suggest a possible causal relationship between TLR9, NF-κB and TNFα activation following HSV-2 infection and preterm birth. Moreover, the results of the present study are consistent with our previous findings, demonstrating elevated TLR2 gene expression in cervical cells in pregnant women with genital bacterial infection as compared to healthy pregnant women. In addition, more than 70% of infected pregnant women revealed premature birth [26]. Collectively, our data suggest that the redundant expression of TLRs in genital epithelial cells in pregnant women with herpes virus infection or other types of genital infections results in increased production of pro-inflammatory cytokines, such as TNFα, thus leading to various pregnancy complications including preterm birth. Obviously, further studies on TLRs and their signaling pathways are necessary to elucidate the role of innate immunity in the development of pregnancy complications. Moreover, the capability of genital epithelial cells to be directly activated by both bacterial and viral TLR ligands to induce an innate anti-viral state [27] may help to develop new targets for the therapy of bacterial and viral infections during pregnancy.

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V. CONCLUSIONS premature births were registered in 81% of pregnant women with genital HSV-2 infection, among which 38% exhibited a considerable increase in the cervical epithelial cell TLR9 gene expression. These data suggest involvement of TLR9, NF-κB and TNFα activation in HSV-2-mediated preterm labor. REFERENCES [1] Duerst RJ, Morrison LA. Innate immunity to herpes simplex virus type 2. Viral Immunol 2003;16(4):475-490. [2] Beauman JG. Genital herpes: a review. Am Fam Physician 2005;72(8):1527-1534. [3] Lavrov VF, Koval'chuk LV, Gankovskaia LV, Barkevich OA, Kuzin SN. Natural immunity and herpes infection. Vopr Virusol 2006;1(3):4-9. [4] Avgil M, Ornoy A. Herpes simplex virus and Epstein-Barr virus infections in pregnancy: consequences of neonatal or intrauterine infection Reprod Toxicol 2006;21(4):436-45. [5] Gankovskaia OA, Zverev VV. Interaction of viruses and Toll-like receptors. Zh Mikrobiol Epidemiol Immunobiol 2010;Mar-Apr(2):101-5. [6] Fazeli A, Bruce C, Anumba DO. Characterization of Toll-like receptors in the female reproductive tract in humans. Mol Hum Reprod 2005;20(5):1372-78. [7] Sandor F, Buc M. Toll-like receptors. I. Structure, function and their ligands. Folia Biol (Praha) 2005; 51(5):148-57. [8] Takeda K, Akira S. Toll-like receptors in innate immunity. Int Immunol 2005;17(1):1-14. [9] Carpenter S, O'Neill LA. How important are Toll-like receptors for antimicrobial responses? Cell Microbiol 2007;9(8):1891-901. [10] Janssens S, Beyaert R. Role of Toll-like receptors in pathogen recognition. Clin Microbiol Rev 2003;16(4):637-46. [11] Kawai T, Akira S. TLR signaling. Cell Death Differ 2006;13(5):816-25. [12] Bowie AG. Translational mini-review series on Toll-like receptors: recent advances in understanding the role of Toll-like receptors in anti-viral immunity. Clin Exp Immunol 2007;147(2):217-26. [13] Krishnan J, Selvarajoo K, Tsuchiya M, Lee G, Choi S. Toll-like receptor signal transduction. Exp Mol Med 2007;39(4):421-38. [14] Wagner H, Bauer S. All is not Toll: new pathways in DNA recognition. J Exp Med 2006;203(2):265-8. [15] Sandor F, Buc M. Toll-like receptors. II. Distribution and pathways involved in TLR signaling. Folia Biol (Praha) 2005;51(6):188-97. [16] Parker LC, Prince LR, Sabroe I. Translational mini-review series on Toll-like receptors: networks regulated by Toll-like receptors mediate innate and adaptive immunity. Clin Exp Immunol 2007;147(2):199-207. [17] Gill N, Deacon PM, Lichty B, Mossman KL, Ashkar AA. Induction of innate immunity against herpes simplex virus type 2 infection via local delivery of Toll-like receptor 65



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[24] Andersen JM, Al-Khairy D, Ingalls RR. Innate immunity at the mucosal surface: role of toll-like receptor 3 and toll-like receptor 9 in cervical epithelial cell responses to microbial pathogens. Biol Reprod 2006;74(5):824-31. [25] Vogel I, Goepfert AR, Thorsen P, Skogstrand K, Hougaard DM, Curry AH, Cliver S, Andrews WW. Early second-trimester inflammatory markers and short cervical length and the risk of recurrent preterm birth. J Reprod Immunol 2007;75(2):133-40. [26] Gankovskaia OA, Koval'chuk LV, Gankovskaia LV, Lavrov VF, Romanovskaia VV, Kartashov DD, Fenzeleva VA. Role of Toll-like receptors and defensins in antimicrobial protection of urogenital tract in females. Zh Mikrobiol Epidemiol Immunobiol 2008;Jan-Feb(1):46-50. [27] NazliA, Yao XD, Smieja M, Rosenthal KL, Ashkar AA, Kaushic C. Differential induction of anti-viral responses by TLR ligands against herpes simplex virus, type 2, infection in primary genital epithelium in women. Antiviral Res 2009;81(2):103-1.

HERPES SIMPLEX VIRUS TYPE 2 INFECTION DURING PREGNANCY IS

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