| dc.description.abstract | Beta-defensins are a class of host defence peptides with antibacterial, antiviral and immunomodulatory roles. The bovine genome contains up to 57 putative beta-defensin genes in four syntenic clusters on different chromosomes. The regulation of expression of these pleiotropic molecules is still poorly explored. As beta-defensins are involved in bovine fertility, the overall aim of this project was to investigate the regulation of expression of beta-defensins in this context.
We have bioinformatically analysed the proximal promoter regions of chromosome 13 beta-defensin genes from the bovine and other mammals to identify potential regulators of gene transcription for further investigation. Transcription factor binding site over-representation analysis of a beta-defensin cluster which is highly expressed in the male reproductive tract indicates enrichment of DNA binding sites for transcription factors important in regulating gene expression at this site. These include androgen receptor, glucocorticoid receptor and retinoic acid receptor. Alignment of over 300kb of this beta-defensin gene region from five distantly-related mammals highlighted three putative intergenic enhancers within the cluster. A candidate enhancer proximal to DEFB126, which is associated with male fertility, contains binding sites for androgen receptor and AP-2alpha, a chromatin-opening transcription factor known to co-operate with androgen receptor in the murine epididymis. We hypothesise that these potential enhancers may act as distal regulatory elements to control expression of one or more male reproductive tract-specific beta-defensins.
Beta-defensin 3, encoded by the gene DEFB103, has been attributed roles in direct antimicrobial defence, immunomodulation and wound healing. As this gene is associated with an inflammatory gene signature in the post-partum bovine uterus, in which endometritis is both prevalent and economically important, we were interested in identifying stimuli driving DEFB103 expression. Application of our transcription factor binding site over-representation approach to the promoters of DEFB103 orthologs indicates AP-1 regulation of this gene at the transcriptional level. AP-1 mediates the response to a number of inflammatory stimuli and regulates DEFB103 in some human cell types. Therefore we investigated inflammation-driven beta-defensin expression in a bovine reproductive tract-derived primary cell culture model. Cultured primary endometrial stromal cells isolated from reproductive tracts of recently slaughtered cows constitutively express DEFB103. A primer set targeting the ruminant expansion of DEFB4A (eDEFB4A), consisting of eight chromosome 27 beta-defensins with high sequence similarity, revealed expression of at least one of these genes in stromal cells. As IL-1beta and IL-17A are implicated as drivers of human beta-defensin expression, they were used to stimulate bovine endometrial stromal cells. IL-1beta upregulated expression of DEFB103 and eDEFB4A. IL-17A also upregulates DEFB103. Upregulation of beta-defensins by pro-inflammatory cytokines may be an important link between post-partum inflammation and beta-defensin expression.
Viral infection of the cow reproductive tract contributes to prolonged post-partum inflammation, with associated economic and welfare implications. Here, we profile nucleic acid-mediated beta-defensin expression in endometrial stromal cells. The dsRNA molecule poly(I:C) upregulates DEFB103 (n=6; p=0.0041), while eDEFB4A is downregulated (p=0.0139). Similarly, poly(dA:dT) is a synthetic dsDNA analog which upregulates transcription of DEFB103 (n=6; p=0.0001) but not eDEFB4A. Thus, viral infection may prolong inflammation in the post-partum uterus.
As a dietary hormone with the ability to modulate HDP expression, 1,25-dihydroxyvitamin D (1,25D) is a candidate therapeutic immunomodulator. In the endometrial stromal cell model, 1,25D induces expression of eDEFB4A (n=6; 25.51?11.48 fold, p=0.0005). To investigate whether 1,25D could modulate the viral-mediated differential regulation of beta-defensins, stromal cells were stimulated with viral ligands in the presence of 1,25D. 1,25D blunts poly(dA:dT)-induced DEFB103 expression. Conversely, 1,25D reverses poly(I:C)-mediated downregulation of eDEFB4A.
In conclusion, we have used computational and primary cell culture techniques to identify multiple potential regulatory mechanisms driving fertility-related beta-defensin expression. As stromal cells of the endometrium experience both systemic inflammatory signals and local infection, induction of these pleiotropic beta-defensins in response to such stimuli may be a key driver of pathological inflammation in the post-partum uterus. In addition, hormonally-active vitamin D can modulate beta-defensin expression, which is of potential therapeutic relevance in the context of endometritis. | en |
