Integration of reproductive endocrinology and innate immunity from the whole animal to the cell

Lead Research Organisation: Swansea University
Department Name: Institute of Life Science Medical School

Abstract

Infection of the uterus with bacteria is common in animals such as cattle, sheep, pigs and horses, and even in humans. These uterine infections occur during sexual intercourse, in pregnancy, or after parturition, and they cause infertility, abortion, pain and suffering. Uterine disease in animals is also difficult to prevent and costly to treat. The infertility is caused by damage to the lining of the uterus, by disrupting the development of the eggs within the ovary, and by altering the concentrations of the sex steroid hormones (progesterone and oestrogen) secreted by the ovary, which normally orchestrate the function of the uterus so that it is ready to nurture the fertilised egg. However, in an intriguing twist, the risk of infection depends on these sex steroids; progesterone suppresses immunity whilst oestrogen enhances it. Why this should be the case remains unclear, yet may be vital when developing treatments and developing prevention strategies. I am a vet with a scientific interest in uterine disease. I have focussed on cattle because infection is ubiquitous after parturition and is the most costly cause of infertility in the dairy industry. I, with my research group of 5 people, have successfully identified: 1. The bacteria which cause uterine diseases, 2. Systems to measure the severity of the disease, 3. The method of detection used by the immune system to recognise bacteria or bacterial toxins in the uterus and ovary. 4. That uterine and ovarian cell functions change when these detection systems are activated. While this work has already been both scientifically and clinically productive, the next stage is to test whether the concepts developed in cows apply across mammals, and we have preliminary data to support that idea. The key questions that have to be explored are: 1. Does activation of the immune detection system for bacteria in the genital tract disrupt uterine and ovarian function? In order to approach this issue, I will use specially bred mice in which the immune detection system is missing. I will also use novel techniques with cow tissues where the detection system can be suppressed. These approaches will allow me to compare the effect of bacteria on uterine and ovarian cell function when the immune detection system is intact or absent, and work out which cells are the key players for the immune response in the uterus and ovary. 2. How do ovarian steroids influence immunity? Here, I will test how steroids modify the immune response to bacteria in the uterus, and how they regulate the immune detection system. To work out which steroid hormone pathways are important within the cell, I will compare the immune response to uterine infection in mice that have been bred with parts of the steroid pathway missing . Finally, I will explore which parts of the immune pathway may interact with the steroid pathway inside the cells of the uterus. This work will be done at the Royal Veterinary College, which has all the necessary facilities. Training in the techniques required to complete the project will be provided by partnerships I have developed with researchers at the cutting-edge of science in the UK, USA and Germany. These studies should lead to the design of better treatments for uterine disease and infertility, and drugs that can enhance the immune response to disease, whilst avoiding side-effects associated with hormonal medicines. It may also enable strategies to be developed to avoid disease at times of adverse hormone status or to exploit the hormone status to resolve uterine infections. In the long term it may be possible to identify why cattle are particularly prone to uterine infection, and use this information to breed animals that are more resistant to uterine disease.

Technical Summary

Reproductive endocrinology and innate immunity are usually treated as separate entities. Yet there is a link as pathogenic organisms often infect the genital tract of domestic animals causing infertility, whilst the immune response is modulated by the stage of the ovarian cycle. I have used cattle to study this link as uterine infection is a costly cause of infertility in the dairy industry, disrupting uterine and ovarian function. Uterine cells express the immune receptor (TLR4) for the most common pathogen, E. coli. To test the paradigm that the endocrine and immune systems are integrated in mammals, I will use murine genetic models and employ novel laboratory techniques. To determine the role of the TLR4 pathway in the uterus and ovary, TLR4-/- mice will be used and siRNA to suppress TLR4 in bovine cells. The endocrine and inflammatory response to E. coli and LPS will be measured. Which cells are central to the uterine and ovarian inflammatory response will be explored using re-aggregation of cell types with different TLR4 expression. How and where uterine TLR4 expression is regulated during the ovarian cycle will be evaluated using molecular biology and histology. The effect of ovarian steroids on the immune and endocrine responses of uterine cells will be tested, using murine and bovine cells that lack functional TLR4. To test the role of steroid nuclear receptors, the response to LPS will also be examined using mice with functional disruptions of these receptors. Interaction of intracellular pathways following hormone and/or immune challenge will be examined by measuring components of the respective pathways, transcription factors and downstream endocrine and inflammation genes, using normal cells and cells that lack functional TLR4 or steroid nuclear receptors. These studies should lead to the design of better treatments for uterine disease and infertility, and drugs that can enhance immunity, whilst avoiding side-effects associated with hormonal medication.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/D02028X/1 01/01/2007 30/06/2008 £272,276
BB/D02028X/2 Transfer BB/D02028X/1 01/07/2008 31/12/2009 £144,657
 
Description Uterine infection after parturition is a common endemic disease of dairy cattle, leading to infertility, increased culling, pain and suffering. Dairy cattle are notable amongst mammals because bacterial contamination of the uterus is ubiquitous after calving and up to 40% of animals develop uterine disease when infected by specific bacteria - such as Escherichia coli and Arcanobacterium pyogenes. The cost of uterine disease to the UK dairy industry is about £193 million per annum. The present project aimed to identify the molecular and cellular mechanisms underlying the infertility associated with uterine infection in dairy cattle.

The main findings during the project were:
1. The endometrium collected in vivo from postpartum cows that subsequently develop uterine disease had a greater expression of genes associated with inflammation such as IL1B, IL6 and IL8, than infected cows that rapidly recovered from infection. In addition, there was greater expression of the gene encoding the TLR4 innate immune receptor that detects the cell wall component of E. coli, known as lipopolysaccharide (LPS, endotoxin). Thus, innate immunity is likely central to combating infection in the female genital tract.
2. Innate immunity was driven by the epithelial and stromal cells of the endometrium, and the granulosa cells in the ovary that surround the egg. These cells expressed TLR4 protein, and produced an inflammatory response to LPS, via key intracellular pathways of the innate immune system (ERK1/2, p38, NFkB, PKC). On the other hand fertilization of eggs and embryo development were resistant to LPS or inflammatory mediators. The implications of these data are that the cells lining the uterus and nurturing the egg have innate immune capabilities.
3. Knocking down the TLR4 pathway using siRNA prevented the inflammatory response. So, in collaboration with industry, inhibitors of the innate immune system were identified that could limit the severity of inflammation in endometrial and ovarian cells treated with LPS.

Impact/Benefit/Knowledge Transfer
a) Industry: The industrial partner now has a proprietary programme to take our work forward to develop products that limit the incidence and/or severity of uterine disease. Reducing the incidence of uterine disease will also increase the profitability of the 16,400 SMEs that keep dairy cattle in the UK.
b) KT: We have generated 9 peer-review publications as well as multiple media open-access approaches (popular journals, website, presentations) for knowledge transfer to veterinarians, who are the key stakeholders for translating our findings for the treatment of uterine disease in cattle.
c) Environment: Reducing the incidence of uterine disease will benefit animal welfare in the UK. In addition, fewer animals need to be kept to replace infertile cows, so reducing the greenhouse gases and water degradation associated with dairy farming.
d) Funding: We developed a research consortium including Glasgow, Hannover, INRA and Pfizer Global Alliances and we have been awarded an EURO 3.2 million ERA-Net to further explore the mechanisms of uterine disease and test potential strategies to limit the severity and incidence of disease.
e) Bioscientist research training: Two postdoctoral researchers were trained during the project, and the work has spawned two BBSRC PhD studentships.

Project Duration
01/06/2007 to 15/11/2010 (3 years with gap for transfer between RVC and Swansea University)
Exploitation Route The project and the relationship with Pfizer Animal Health, lead to an ERANET award with Zoetis, including development of IP.
Sectors Agriculture, Food and Drink