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Regulation of innate immunity in the female reproductive tract

Final Report Summary - RIIFERT (Regulation of innate immunity in the female reproductive tract)

Summary description of the project objectives

The 'Regulation of innate immunity in the female reproductive tract' (RIIFERT) project aims to obtain data that will help to generate an in silico model for prediction of alterations of the innate immune response (more specifically toll-like receptor 5 (TRL5)) in response to sex hormones and the presence of the embryo in endometrial cells. These data can help in prediction, diagnoses and treatment of embryo implantation failure.

Description of the work performed since the beginning of the project

Implantation failure after natural mating or in vitro fertilisation (IVF) may be due to either embryo or endometrial factors. Recent evidence suggests that sub-clinical infections, inflammation or malfunctions of the immune system in the female reproductive tract (FRT) all contribute to implantation failure. One of the main regulators of the immune response is the TRL family (TLR). TLRs are the main family of pattern recognition receptors (PRRs) of the innate immune system. This family of receptors have been seen to be expressed in human endometrial tissue and trophoblasts and are known to have a key role in the modulation of immune and inflammatory responses in mammals. We have recently shown that TLR5 activation results in a decrease of trophoblast cells binding to endometrial cells in an in vitro model of human implantation. TLR5 recognises the flagellin protein component of bacterial flagella. After the engagement of TLR5 with flagellin, MyD88 recruits the IL-1 receptor associated kinases IRAK1 and IRAK4. IRAK4 is activated and is essential in the activation of mitogen-activated protein kinases (MAPK) and the nuclear factor ?B transcription factor (NF-?B). NF-?B critically regulates innate immunity and inflammation and has a key role in cell division and cell death. In the absence of stimulus NF-?B proteins are bound to I?B-alpha and I?B-epsilon in the cytoplasm, preventing its translocation to the nucleus.

There are two main factors that can affect TLR5 functionality during the early stages of pregnancy. The first one is the presence of the embryo and the second one is the changes hormones concentrations. To evaluate how these two factors affect TLR5 functionality, we developed a set of reporter plasmids and deoxyribonucleic acid (DNA) constructs that allowed us to study TLR5 expression and activation of downstream transcription factors, such as NF-?B and AP-1. Then, we tried to answer whether the presence of the embryo exerts a local effect on the endometrial immune response in the early stages of pregnancy. We showed that the addition of trophoblasts to an endometrial monolayer produce an increase in NF-?B activation, modulating the innate immune response of the endometrial epithelium. Thus, it seems that embryo arrival to the uterus may influence innate immune mediation in the FRT, and any alteration of this cross-talk between the embryo and the endometrium may lead to pregnancy failure.

In a second period, we tried to evaluate the effect of different sex hormones on TLR5 expression and functionality. We used an in vitro model to evaluate endometrial function in the presence of different concentrations of hormones. To evaluate TLR5 function, we have to take into account that TLR5 signalling can activate different transcription factors, including NF-?B and AP-1. Moreover, the effect of estradiol signalling in the endometrium is highly complex, since involves the action of different estrogen receptors (ER-alpha, ER-beta and / or GPR30), which can have a diverse effect on cell function. We were able to show how estradiol modifies NF-?B and AP-1 transcripiton activity through different estrogen receptors. Moreover, using single cell confocal live imaging and luminescence microscopy we have shown that estradiol affects P65 (a subunit of NF-?B) translocation from the cytoplasm to the nucleus.

Description of the main results achieved so far

The results obtained by Dr Caballero show that trophoblasts produce an increase in NF-?B activation, modulating the innate immune response of the endometrial epithelium; these results suggest that embryo arrival to the uterus may influence innate immune mediation in the FRT. Moreover, we have observed that implantation failure caused by intrauterine infections could be associated with abnormal levels of NF-?B activation. We have also shown that sex hormones can modulate TLR5 function, which could affect embryo implantation and pregnancy. To understand the plasticity of cells, it is vital to measure quantitatively and dynamically the molecular processes that underlie cell-fate decisions in single cells. The data obtained from single cell live imaging is of great value since populations of cells are almost always heterogeneous in function and fate. We have recorded data from single cells that will help to construct and validate a mathematical model to predict alterations in TLR5 function.

Expected final results and their potential impact and use

The work carried out during this project demonstrates that both the embryo and sex hormones have an effect on the endometrial innate immune system. Our data provides an important addition to the present literature regarding the modulation of TRL function. We clearly show that the hormonal environment influences the functionality of TLR5, changing the transcriptional activity of both NF-?B and AP-1 transcription factors. Since TLR5 activation has been shown to lead to implantation failure, changes in the hormonal environment, especially in women treated for IVF, could affect TLR5 response against pathogens in those women, leading to implantation failure.

The information gained in this project not only increases our knowledge on mediation of immune system in the FRT but also are important to immunologists and cancer research, since TLRs, and more specifically TLR5, have been related to autoimmune diseases and cancer proliferation. The extra knowledge acquired will help to develop diagnostic tools and therapies that can directly help and improve the health and well-being of patients in European Member States.