Uterine Epithelial Cells Specifically Induce Interferon-Stimulated Genes in Response to Polyinosinic-Polycytidylic Acid Independently of Estradiol

Wira CR (2012) Uterine Epithelial Cells Specifically Induce Interferon-Stimulated Genes in Response to Polyinosinic- Polycytidylic Acid Independently of Estradiol. PLoS ONE 7(4): e35654. doi:10.1371/journal.pone.0035654 Uterine Epithelial Cells Specifically Induce Interferon- Stimulated Genes in Response to Polyinosinic- Polycytidylic Acid Independently of Estradiol Mickey V. Patel 0 Mimi Ghosh 0 John V. Fahey 0 Charles R. Wira 0 Santanu Bose, The University of Texas Health Science Center at San Antonio, United States of America 0 Department of Physiology and Neurobiology, Dartmouth Medical School , Lebanon, New Hampshire , United States of America Interferon b (IFNb) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNb and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I:C) induced the mRNA expression of IFNb, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNb or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNb upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I:C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I:C), had no effect on IFNb or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNb partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I:C) had no effect on IFNb and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens. - Funding: This work was supported by AI51877 and AI071761 (awarded to Dr. Wira) from National Institute of Health (www.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The female reproductive tract (FRT) is a unique mucosal site that must reconcile two competing functions: host defense versus reproduction. It is the primary site of infection by sexually transmitted diseases (STDs) including Herpes Simplex Virus (HSV), Human Immunodeficiency Virus (HIV), Neisseria gonorrhoeae and Chlamydia trachomatis, which cause morbidity and mortality in large numbers of women. The innate immune response in the FRT is a key component of host defense against these incoming pathogens about which much still remains to be elucidated. Epithelial cells are essential for mucosal defense providing a physical and structural barrier against pathogen entry into the body. They are the first cells exposed to invading pathogens and thus, their responses could determine whether an infection occurs. Human uterine epithelial cells (UEC) express a full complement of pattern recognition receptors (PRR) including Toll-like receptors (TLR) 19, RIG-like receptors (RLR) and NOD-like receptors (NLR) [1,2] that recognize unique and conserved pathogenassociated molecular patterns (PAMPs) allowing them to detect a plethora of infectious agents. TLRs 1, 2, 4, 5 and 6 recognize bacterial and fungal PAMPs [3]. TLR3 recognizes viral doublestranded RNA (dsRNA) while TLR7 and 8 recognize imiquidizalones such as imiquimod (IQ) and resiquimod as well as singlestranded RNA (ssRNA) [3]. TLR9 binds to unmethylated CpG DNA which is relatively rare in the vertebrate genome [3]. PAMP binding to a TLR activates multiple signaling pathways that allow a cell to generate a protective response. In previous studies we have demonstrated that UEC respond to bacterial exposure by secreting a panel of proinflammatory cytokines [1,4]. Type I interferons (IFNs) are a family of cytokines that can rapidly induce a protective antiviral response and create a restrictive environment for pathogen survival [5]. Several subtypes of Type I IFN exist in humans including 13 isoforms of IFNa and single forms of IFNb, IFNk, IFNv and IFNe [6]. Type I IFNs are secreted into the external environment and signal in an autocrine and paracrine loop through a heterodimeric receptor complex consisting of IFN receptor 1 and IFN receptor 2 (IFNAR1 & 2) that activate a conserved JAK/STAT signaling pathway leading to the upregulation of hundreds of interferon-stimulated genes (ISG) [7]. ISG involved in inhibiting the viral lifecycle include Myxovirus A (MxA), 29-59 Oligoadenylate Synthetase (OAS) and Protein Kinase R (PKR). MxA, a high molecular weight cytoplasmic GTPase, is believed to exert its antiviral function by binding viral nucleocapsid proteins, forming aggregates and thus preventing mature virion formation and release [8]. OAS exists as 3 isoforms (OAS1-3) and catalyses the conversion of adenosine triphosphate (ATP) into long chains of 29-59 linked oligoadenylates [9,10]. These molecules activate the endogenous ribonuclease, RNaseL, which digests viral RNA. Inactive PKR is present in the cytoplasm in monomeric form. Upon activation by viral RNA, it dimerizes and undergoes autophosphorlyation [9,10]. In turn, activated PKR phosphorylates eukaryotic initiation factor 2 alpha (eIF2a) thus leading to a general suppression in protein synthesis [11]. Together, these three ISG represent a crucial mechanism behind Type I IFN-mediated defense the ability to protect the cell by simultaneously inhibiting multiple stages of the viral lifecycle and are capable of inhibiting a range of different viruses including Hepatitis C virus, HIV and HSV [12,13,14]. Every cell in the premenopausal FRT exists in an environment that is constantly exposed to varying levels of the sex hormones estradiol and progesterone. Previously, we have shown that estradiol regulates several aspects of the innate immune system in the FRT. In human UEC estradiol stimulates the production of antimicrobial compounds such as elafin and secretory leukocyte protease inhibitor (SLPI) that are essential in restricting viral and bacterial infections [15,16]. However, estradiol can also be inhibitory. For example, it represses the TLR3-induced secretion of cytokines such as IL-6 and IL-8 in UEC [17]. Whether estradiol directly regulates the Type I IFN pathway, and thus alters intracellular innate immune protection ha (...truncated)