The influence of ovarian hormones, bacteria and uterine health on the innate immune response in the equine uterus

© 2016 Dr. Christina Deborah Anna MarthPersistent mating-induced endometritis (PMIE) is a major problem in the equine breeding industry. While transient inflammation is a physiological reaction to uterine contamination during breeding, its persistence is one of the main causes for decreased pregnancy rates in affected horses. The research described in this thesis focussed on identifying the molecular mechanisms that underpin inflammatory processes in the uterus. To obtain results with a minimal amount of biological variation, a model of bacterial inoculation at different stages of the equine oestrous cycle was established. Results were then compared between a larger group of mares resistant and susceptible to PMIE. The initial objectives of the studies described here were to characterise uterine transcriptome profiles in clinically healthy horses during different stages of the oestrous cycle and to investigate how the uterine transcriptome is influenced by a bacterial challenge in different steroid hormone environments. Global gene expression analyses identified a set of genes associated with the innate immune response that were further investigated in resistant and susceptible mares. Five Standardbred mares were shown to be resistant to persistent mating-induced endometritis in a preliminary experiment. They were inoculated with Escherichia coli (E. coli) once during oestrus, the cycle stage in which oestrogen is the predominant hormone and which prepares the uterus for the elimination of foreign material introduced during breeding. In the next cycle, they were inoculated during dioestrus, when high progesterone levels dampen both the immune response and uterine contractility. Biopsy samples were obtained before and at several time points after inoculation. Samples taken before and three hours after inoculation were analysed using the next-generation sequencing technique RNA-Seq. A subset of genes showing increased expression after the introduction of bacteria was investigated at time points up to three days after inoculation with E. coli using quantitative real-time PCR. These included pathogen recognition receptors, chemokines and antimicrobial peptides. The same group of genes was also analysed in samples from 21 PMIE-resistant and 19 PMIE-susceptible mares and evaluated as diagnostic markers for the disease. These investigations demonstrated that a large number of genes were expressed at differing levels in oestrus and dioestrus. However, the differences were small compared to the up-regulation of the majority of genes associated with the immune system after the introduction of E. coli into the uterine environment. Repeated assays of the expression of a subset of genes involved in the innate immune response demonstrated that peak expression levels for these genes were reached 3 to 12 hours after inoculation with E coli. Expression often returned to baseline levels within 24 to 48 hours. Comparisons of uterine gene expression levels in mares resistant and susceptible to PMIE revealed that susceptibility to this disease was associated with higher expression levels of genes for three chemokines and three antimicrobial peptides. Assays of expression of the genes for the three antimicrobial peptides had high levels of sensitivity and specificity as diagnostic markers for PMIE. These studies have shown that both changes in hormone levels over the oestrous cycle and the introduction of E. coli into the uterus alter the levels of expression of endometrial genes in healthy mares. After bacteria are added, differences associated with the oestrous cycle stage are negligible. A subset of genes encoding pathogen recognition receptors, chemokines and most analysed antimicrobial peptides seem to be good markers for infectious endometritis. Additionally, genes for three antimicrobial peptides have considerable potential as diagnostic markers for PMIE