Deep sequencing of the uterine immune response to bacteria during the equine oestrous cycle

Abstract

BackgroundThe steroid hormone environment in healthy horses seems to have a significant impact on the efficiency of their uterine immune response. The objective of this study was to characterize the changes in gene expression in the equine endometrium in response to the introduction of bacterial pathogens and the influence of steroid hormone concentrations on this expression.MethodsEndometrial biopsies were collected from five horses before and 3 h after the inoculation of Escherichia coli once in oestrus (follicle >35 mm in diameter) and once in dioestrus (5 days after ovulation) and analysed using high-throughput RNA sequencing techniques (RNA-Seq).ResultsComparison between time points revealed that 2422 genes were expressed at significantly higher levels and 2191 genes at significantly lower levels 3 h post inoculation in oestrus in comparison to pre-inoculation levels. In dioestrus, the expression of 1476 genes was up-regulated and 383 genes were down-regulated post inoculation. Many immune related genes were found to be up-regulated after the introduction of E. coli. These include pathogen recognition receptors, particularly toll-like receptors TLR2 and 4 and NOD-like receptor NLRC5. In addition, several interleukins including IL1B, IL6, IL8 and IL1ra were significantly up-regulated. Genes for chemokines, including CCL 2, CXCL 6, 9, 10, 11 and 16 and those for antimicrobial peptides, including secretory phospholipase sPLA2, lipocalin 2, lysozyme and equine β-defensin 1, as well as the gene for tissue inhibitor for metalloproteinases TIMP-1 were also up-regulated post inoculation.ConclusionThe results of this study emphasize the complexity of an effective uterine immune response during acute endometritis and the tight balance between pro- and anti-inflammatory factors required for efficient elimination of bacteria. It is one of the first high-throughput analyses of the uterine inflammatory response in any species and several new potential targets for treatment of inflammatory diseases of the equine uterus have been identified.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2139-3) contains supplementary material, which is available to authorized users.

Oestrous cycle-dependent equine uterine immune response to induced infectious endometritis

“…Our previous next-generation sequencing analysis revealed that a large number of immune response genes were up-regulated within the first 3 h after the introduction of E. coli into the uterus [18]. To investigate the progression of the inflammatory response after this time point, expression of a panel of genes was followed by qPCR until 72 h after inoculation.…”

“…Genes were chosen for further analysis by PCR based on the analysis of next-generation sequencing data published previously [18]. These included the genes for the Toll-like receptors TLR2 and 4, the NOD-like receptor NLRC5, the chemokines CCL2, CXCL9, CXCL10 and CXCL11, as well as the antimicrobial peptides equine β-defensin 1 (EBD 1), lysozyme, secretory leukoprotease inhibitor (SLPI), secreted phospholipase A 2 (sPLA 2 ), lipocalin 2 (LCN 2), lactoferrin, uteroferrin and the uterocalin precursor (P19).…”

“…Inoculation of E. coli into the uterus of healthy mares during oestrus [7] results in a much less pronounced inflammation than in dioestrus [17], in which purulent or serohaemorrhagic vaginal discharge, heavy bacterial growth and acute systemic inflammation were detected. A high-throughput sequencing study identified 1500 genes with significant differences when directly comparing samples taken 3 h after inoculation of E. coli in oestrus with those taken at the same time point in the same horses in dioestrus [18]. Overall, these studies suggest that hormone levels affect the immune response in the equine uterus.…”

“…To gain an in-depth understanding of the innate immune cascade in the equine uterus, a next generation sequencing analysis was performed on samples taken from the equine uterus before and 3 h after inoculation of healthy mares with E. coli . It identified a number of immune response genes that were up-regulated after the introduction of E. coli , particularly in the pathogen recognition receptor (PRR) family, the chemokine and cytokine families and the antimicrobial peptide group [18]. …”

“…These included PRRs, as well as several antimicrobial peptides, chemokines, cytokines and MMPs [18]. The results of this RNA-Seq study were used to select a range of genes for analysis at later time points.…”