Inflammation and uterine disease in the postpartum dairy cow
Citation:BREWER, AMY ELIZABETH, Inflammation and uterine disease in the postpartum dairy cow, Trinity College Dublin.School of Biochemistry & Immunology, 2019
AB Thesis FINAL - with formatted margins (16-4-19).pdf (Thesis) 3.218Mb
Following calving, inflammation of the uterus is common during the first week postpartum and is essential for the reparative process of involution. However, whilst the majority of cows resolve this inflammation a significant proportion fail to do so, resulting in uterine disease and subfertility. Previous work by our group has identified transcriptomic differences in immune genes between cattle that resolve inflammation and those that develop uterine disease as early as 7 days postpartum (DPP), suggesting that the host immune response plays an important role in disease outcome. Here, we hypothesised that even in the context of early postpartum inflammation, cows that go on to develop uterine disease will be identified by a unique inflammatory signature in the circulation and in endometrial epithelial cells 7 DPP. In this study, we extensively characterised the local and systemic immune response of post-partum dairy cows phenotyped for both clinical and sub-clinical forms of disease. Sampling was carried out at 7 and 21 DPP and involved the collection of blood and endometrial epithelial cells from 112 cattle. Endometrial cells were collected using a cytobrush. Pre-calving bloods (40-60 days prepartum) were also acquired. Cattle were classified as healthy or with uterine disease (either cytological endometritis; CYTO, purulent vaginal discharge; PVD or both) on the basis of vaginal mucus score and >18% polymorphonuclear cell infiltrate into the endometrium at 21 DPP. Blood samples were used to analyse systemic metabolites, immune cells, acute phase proteins (APPs), vitamins and minerals. Endometrial epithelial cells were used for transcriptomic analysis at 7 DPP. Systemic analysis revealed that increased concentrations of APPs serum amyloid a (SAA) and haptoglobin (Hp) were present 7 DPP in cattle that subsequently developed PVD compared to cows that remained healthy. The same trend was observed for alpha-1-acid glycoprotein (AGP) and in cows with CYTO, though neither reached significance. Analysis of circulating immune cell populations identified a decline in eosinophil numbers in cattle that developed both CYTO and PVD. Interestingly, no difference in neutrophil numbers was detected between groups. Metabolite levels were also assessed, and whilst all cattle were in a state of negative energy balance 7 DPP, concentrations of glucose, urea, non-esterified fatty acids (NEFA) and betahydroxybuterate (BHB) did not differ between cows that remained healthy and cows that developed disease, which suggests that energy status was not contributing to the pathogenesis of uterine disease in this study. RNA-seq analysis of endometrial epithelial cells collected using cytobrushes identified differential expression of 294 genes (FDR <0.05) between cows that subsequently resolved inflammation (n=10) and those that developed disease (n=20). Pathway over-representation analysis of differentially expressed genes (DEG) identified significant changes in immune-related pathways, including the NOD-like receptor signalling pathway, cytokine-cytokine receptor interaction pathway and the Toll-like receptor signalling pathway which were up-regulated in cattle that subsequently developed disease. A major role for the cytokine IL-1 was also highlighted by over-representation of multiple IL-1 signalling pathways. The majority of the DEG were upregulated in cows that developed PVD, and included all genes upregulated in CYTO cows, suggesting a core inflammatory gene signature early post-partum contributes to the onset of uterine disease. This inflammatory signature was validated by qPCR in an independent group of cows (n=56) and included upregulation of pro-inflammatory genes (including TLR2, TLR4, NLRP3, IL1A, IL1B, IL8, and S100A8) at day 7 postpartum in cows that failed to resolve inflammation. Despite a large amount of inter-animal heterogeneity, these results suggest that excessive activation and inappropriate regulation of the inflammatory response early postpartum is a key feature of the subsequent development of uterine disease. In addition to the hyper-inflammatory response observed 7 DPP, cows that developed uterine disease also demonstrated down-regulation of genes associated with cell growth and adhesion when compared to healthy cows. Therefore we hypothesised that excessive inflammation prevents repair and remodelling of the endometrium, contributing to prolonged or unresolved inflammation (endometritis). One of the top differentially expressed genes upregulated in cows that developed both clinical and subclinical endometritis was pentraxin 3 (PTX3). PTX3 is an APP with important roles in the innate immune response and tissue repair. Recently, PTX3 has been found to play a dual role in the regulation of inflammation, either providing protection or amplifying tissue damage. Furthermore, recent evidence suggests PTX3 is capable of affecting epithelial barrier integrity. However, its effect on the endometrial epithelial barrier, particularly in the postpartum cow, has yet to be elucidated. Therefore we decided to investigate the effect of PTX3 on endometrial epithelial barrier repair in an in vitro model of the bovine endometrial epithelium designed to mirror the early post-partum uterus of the cow. In addition to confirming the presence of PTX3 in the uterus at protein level, we found that it had a detrimental effect on epithelial barrier repair, following disruption of the epithelium by calcium chelation. Deciphering the mechanisms underlying the effects of PTX3 on epithelial barrier repair could assist in new treatment or prevention strategies for tackling endometritis. In summary, this study has shown that despite a significant amount of inter-animal heterogeneity, both clinical and subclinical forms of uterine disease are preceded by excessive activation of the endometrial inflammatory response 7 DPP, which can be detected systemically by an increase in the APPs SAA and Hp and a decline in circulating eosinophils. Furthermore, integrity of the endometrial epithelial barrier is likely to play an important role in preventing disease onset. Not only have we enhanced our understanding of the mechanisms underlying the switch between physiological and pathological inflammation, but we have also paved the way for the development of novel biomarkers for the early detection of uterine disease.
Author: BREWER, AMY ELIZABETH
Publisher:Trinity College Dublin. School of Biochemistry & Immunology. Discipline of Biochemistry
Type of material:Thesis
Availability:Full text available