The dysregulation of leukemia inhibitory factor and its implications for endometriosis pathophysiology

Abstract

Endometriosis is an estrogen dominant, chronic inflammatory disease characterized by the growth of endometrial-like tissue outside of the uterus. The most common symptoms experienced by patients include manifestations of chronic pelvic pain- such as pain with urination, menstruation, or defecation, and infertility. Alterations to Leukemia Inhibitory Factor (LIF), a cytokine produced by the luminal and glandular epithelium of the endometrium that is imperative for successful pregnancy, have been postulated to contribute to infertility. Conditions such as recurrent implantation failure, unexplained infertility, and infertility associated diseases such as adenomyosis and endometriosis, have demonstrated reduced LIF production in the endometrium of infertile patients compared to fertile counterparts. While this highlights the potential involvement of LIF in infertility, LIF is a multifaceted cytokine which plays additional roles in the maintenance of cell stemness and immunomodulation. Thus, we sought to explore the implications of LIF production within ectopic lesions on endometriosis pathophysiology. Through immunohistochemistry of an endometrioma tissue microarray and ELISA of tissue protein extract and peritoneal fluid samples, we identify LIF protein expression in the ectopic lesion microenvironment. Targeted RT qPCR for LIF and associated signaling transcripts, identify LIF to be significantly downregulated in the ectopic tissue compared to eutopic and control while its receptor, LIFR, is upregulated, highlighting a discordance in ectopic protein and mRNA LIF expression. In vitro treatment of endometriosis representative cell lines (12Z and hESC) with LIF increased production of immune-recruiting cytokines (MCP-1, MCP-3) and the angiogenic factor, VEGF, as well as stimulated tube formation in human umbilical vein endothelial cells (HUVECs). Finally, LIF treatment in a syngeneic mouse model of endometriosis induced both local and peripheral alterations to immune cell phenotypes, ultimately reducing immunoregulatory CD206+ small peritoneal macrophages and T regulatory cells. These findings suggest that LIF is present in the ectopic lesions of endometriosis patients and could be contributing to lesion vascularization and immunomodulation.

Uterine secretome initiates growth of gynecologic tissues in ectopic locations

“…To demonstrate the role of a known uterine secretome factor, we supplemented tissue injections with recombinant LIF, which statistically increased oviduct and endometrium implantation in the presence of hormonally synchronized endometrium. Gene expression studies in embryo implantation [28,29,40], endometriosis [10,11] and ovarian cancer metastasis [41] have all found altered expression of LIF, and other genes such as CD44 and miR 99a-5p [42,43], supporting our hypothesis that the uterine secretome is important in benign and malignant processes. Similar miRNA has been reported in these settings [44,45].…”

“…Other factors at play in the development of EM may depend on factors present in the peritoneal fluid, likely migrating from the uterus. A multitude of studies have identified increased levels of a variety of cytokines and growth factors, including, interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNF-α), epidermal growth factor (EGF), Fibroblast Growth Factors (FGFs), vascular endothelial growth factor (VEGF), and leukemia inhibitory factor (LIF) in peritoneal fluid of women with EM [8–11]. Interestingly, many of these factors are essential to the process of implantation, including LIF [12].…”

The dysregulated IL-23/TH17 axis in endometriosis pathophysiology

“…Total RNA was extracted from ectopic (n=9) and eutopic (n=8) endometrium of patients, and normal healthy endometrium (n=9) using a total RNA purification kit (17200, Norgen Biotek Corp., Canada) as per manufacturer’s protocol. RNA quality was assessed and cDNA synthesized as previously detailed 34 . RT qPCR was conducted with custom array plates (330171, Qiagen, Canada) using the LightCycler 480 Real-Time PCR system (Roche Molecular Systems Inc., Switzerland) with RT 2 SYBR Green qPCR Mastermix (330501, Qiagen, Canada) and 200ng cDNA, as per manufacturer’s protocol.…”

“…An endometrioma tissue microarray (TMA) was constructed using patient tissue samples from Kingston General Hospital (Kingston, ON, Canada), as previously described 35 . Matched patient ectopic and eutopic tissues (n=17) as well as normal endometrium controls (n=10) were subjected to immunohistochemistry (IHC) staining using a polyclonal IL-23 antibody (1:200; ab45420, Abcam, UK) and analysis using HALO image analysis software (Indica Labs, USA) was conducted as previously outlined 34 .…”

“…EECCs were maintained in DMEM (D6429, Sigma-Aldrich, Canada) supplemented with 10% FBS (FBS; 97068-085, VWR, USA) and 1% penicillin and streptomycin (15140122, ThermoFisher, Canada). Cell line cultivation as well as 12Z, HUVEC, and hESC maintenance was conducted as previously described 34 .…”