Evaluation of Toll-like receptor 3 (TLR3) signaling pathway genes and its genetic polymorphisms in ectopic and eutopic endometrium of women with endometriosis

Abstract

Objective

Toll-like receptors (TLRs, as members of the innate immune system) are expressed in the human endometrium and their aberrant regulation and expression are involved in the pathogenesis of endometrial diseases. This study is aimed at evaluation of TLR3 signaling pathway genes and its genetic changes in endometriosis patients.

Materials and methods

Blood samples were collected from 83 endometriosis patients and 93 healthy fertile women and PCR was performed in blood-derived DNA for detection of SNP of TLR3. Also, ectopic (EC) and eutopic (EU) endometrial biopsies were obtained from endometriosis patients (n = 20), as well as endometrium from healthy women (n = 16, CE). Q-PCR was performed for determination of mRNA expression level of TLR3 signaling pathway genes (TLR3, TICAM, NF-kB1A, CXCL10, IRF3, IFN-B1, IL-6 and IL-8). Also, serum protein levels of TLR3, IFN-β, IL-6 and IL-8 were determined using ELISA.

Results

The mRNA expression levels of TLR3, NF-kB1A, IFN-B1, IRF3, TICAM1, IL-6 and IL-8 were significantly higher in EU compared to ectopic ones and also compared to CE. SNPs frequency (rs3775291 and rs3775290) was not significantly different between patients and controls. Serum protein levels of TLR3, IFN-β, IL-6 and IL-8 were significantly increased in endometriosis patients.

Conclusion

Significant changes were observed in the expression of IL-6 and IL-8 cytokines and other genes in TLR3 cascade in diseased EU, demonstrating that EU similarly to EC is in an intensive inflammatory state. These fundamental alterations in the concept of immune response in EU may lead to its activation, escapes from apoptosis, and displaced implantation of the endometrium.

Introduction

Endometriosis is regarded as a condition in which a benign tumor develops in women of reproductive age. In this disease, endometrial tissue grows outside of its original location (i.e. the uterus); endometriosis is associated with symptoms such as chronic pelvic pain, dyspareunia (painful intercourse) and dysmenorrhea (menstrual pain) and infertility [1, 2]. The main cause of endometriosis is not fully understood, but among the proposed etiologies, the Sampson's theory is acceptable. According to this theory, menstrual blood can return to the pelvic cavity through the fallopian tubes thus, endometrial cells can grow on the ovaries in the peritoneum. Although in 70–90% of women, blood is returned, but the disease occurs only in 10% of cases [3]. Noteworthy, an important and significant role has been suggested for the immune system in the pathogenesis of endometriosis, so that its cellular, molecular and genetic defects can lead to the inability of immune cells including lymphocytes, macrophages and natural killer (NK) cells, to destroy endometriotic tissue which consequently results in its implantation and survival outside the uterus [4, 5].

Changes including reducing the number of NK cells and their cytotoxic ability, diminishing the strength of the chemotactic responses of neutrophils and also rising the number of activated macrophages and thereby enhancement of the inflammatory and pro-inflammatory factors, growth and angiogenesis in the peritoneal fluid, can result in the planting and development of the endometriotic tissue. For example, interleukin-8 (IL-8) is an inflammatory cytokine that enhances the matrix metalloproteinases and as a result, facilitates the adhesion of endometrial cells to the peritoneal surface and development of the endometriosis [4, 6, 7].

It is believed that endometriosis is a sterile inflammatory condition in which initial bacterial infection in the peritoneal fluid and endometrium, and subsequently intracellular contents released from damaged cells into the extracellular space (as endogenous ligands) would be lead to oxidative stress and activation of Toll-like receptors (TLRs) [8]. These responses associated with increased levels of inflammatory regulators such as IL-1, 6 and 8 and the recruitment of peripheral blood mononuclear cells (PBMCs) and neutrophils to remove cellular debris in the pelvic cavity [9, 10]. TLRs are a main family of pattern recognition receptors that have pivotal roles in the innate immune system for identification of molecules associated with pathogens. So far, 13 TLR have been identified in humans (i.e. TLR1 to TLR13) [11]. TLR3 (CD283) is one of the key members of the family that plays a fundamental role in the innate immunity and by identifying endogenous and exogenous ligands, it is involved in different processes such as cell proliferation, apoptosis, angiogenesis, tissue remodeling and repair [12]. In addition, TIR-domain-containing adapter-inducing interferon-β (TRIF) is an essential protein for TLR3 signaling. Considering TRIF's role, TLR3 signaling pathway is directed toward two paths as follows. One path leads to activation of nuclear factor kappa beta (NF-κB) and AP-1 transcription factor and the other pathway, activates interferon regulatory factor3 (IRF3) and IRF-7 [13, 14]. Using TRAF6, TRIF activates TAK1 through a series of adapter proteins. TAK1 can activate kinases and MAPK. IKKs activate NF-κB via phosphorylation and degradation of its inhibitor (i.e. IκB). Then, IκB and NF-κB are transferred to the nucleus and induce transcription of inflammatory cytokine genes such as IL-6 and IL-8 [15, 16].

Moreover, using TRAF3, TRIF can activate the interferons and subsequently, it enhances type 1 and type 3 interferons (IFN) production and also induces the transcription of a series of IFN-induced genes. TLR3 is expressed in endometrial tissue, dendritic cell, macrophage, fibroblast, and Creatine Blast, as well as endothelial and epithelial cells.

This study attempt to evaluate the relation between TLR3 and endometriosis by investigating TLR3 signaling pathway (TICAM1 (TRIF), NF-kB1A, IRF3, CXCL10, IFN-B1, IL-6 and IL-8 genes) as well as the genetic changes in the TLR3 active site as it plays a vital role in the activity and signaling of this receptor.