Transcriptional profiling of Bovine Herpesvirus 4 infected bovine endometrial stromal cells

ABSTRACT Bovine postpartum uterine diseases affect half of all dairy cattle after parturition, causing infertility by disrupting uterine and ovarian function. Female genital tract infections are a primary economic problem for efficient performance of dairy herds, as they cause reduced pregnancies as well as reduced milk production. Although the etiology of bovine uterine diseases is mainly ascribed to bacterial pathogens, such as Escherichia coli and Arcanobacterium pyogenes, more extensive diagnostics have in many cases also detected the presence of Bovine Herpesvirus 4 (BoHV-4) in affected cattle, implying that co-infection may be involved in a possible vicious circle. Following recognition of bacterial lipopolysaccharide (LPS) by the Toll-like receptor 4 (TLR4) expressed at the bovine endometrial epithelium level, expression of immune factors such as prostaglandin E (PGE) and Tumor necrosis factor alpha (TNF-α) are activated. This cytokine cascade triggers BoHV-4 replication by binding specific sites within the immediate early IE2 gene promoter. Conversely, Interferon gamma (IFN-γ) produced by the cells of the immune system, inhibits viral replication, always acting at the level of the IE2 gene promoter. Although BoHV-4 is often isolated from animals affected by these uterine pathologies, and for this reason, consistently associated with postpartum metritis and chronic infertility in cattle, it is difficult to directly correlate the presence of the virus to the pathology in progress, partly because, the cellular pathways and the molecular mechanisms involved are not well defined, as well as which extracellular stimuli related to the intrauterine environment could influence the BoHV-4 replicative cycle. In order to shed light on the true pathogenic role and to better investigate the molecular mechanisms of the physio-pathological response underlying the pathogen-host interaction, an experimental BoHV-4 infection was carried out with the aim to optimize infection conditions and a transcriptomic analysis of a pure bovine endometrial stromal cells population (BESCs) infected with BoHV-4 was performed. The RNA-seq technology used for this purpose allowed to discover a set of differentially expressed genes (DE genes), among which 417 were up-regulated while 118 were down-regulated during BoHV-4 infection. DE genes have been functionally classified through enrichment functional analysis on the basis of their involvement in various cellular pathways. Many pathways, related to cell proliferation and cell surface integrity, were found to be affected by BoHV-4 infection. Some DE genes that were found to be up-regulated also play a key role in pathogenesis as in the case of IL-8 and MMP-1. MMP-1 up-regulation was not unexpected, as it belongs to the zinc-dependent endo-peptidases family involved in numerous and important cellular networks. Thanks to the ECM remodeling and immuno-modulation processes, MMPs assume a relevant connotation in the particular context of metritis, where under suitable conditions favor pathogen eradication, inflammatory state resolution as well as tissue anatomical and functional restoration. Alternatively, pathogenic dysregulation and MMPs hyper-activation leads to host morbidity and mortality thereby favoring pathogen persistence and dissemination, preventing tissue healing and finally defining an immuno-pathological status. By virtue of the fact that these enzymes play a pivotal role in maintaining the delicate balance between normal and hyper-activated immune response and for the putative involvement of MMP-1 in the bovine uterine diseases development, it was selected as a candidate gene for further studies as a key host factor involved in BoHV-4 pathogenesis. The in silico observations were further corroborated by reverse transcription PCR, real time PCR, Western immunoblotting and finally a luciferase assay where a bovine MMP-1 specific promoter reporter construct was exploited. BESC cells transfected with the reporter construct and subsequently infected with BoHV-4 showed an increasing luciferase expression with increasing viral load. BoHV-4 replication is promoted by IE2 gene expression, while the IE2 protein product RTA/50 is able to transactivate both viral and host cell genes, such as those coding for the chemokine IL-8. IL-8 expression in infected BESCs was shown to increase in a time- and dose-dependent manner. This suggested a potential relationship between MMP-1 up-regulation, IE2 gene expression and viral replication. Following BESCs transfection with an IE2 over-expressing construct, a direct correlation between RTA/50 over-expression and MMP-1 up-regulation was observed. This justified the abnormally high metalloproteinase levels in tissues, and suggested a possible connection to the defective endometrium healing and unresolved inflammation state. These data, combined with those obtained by transcriptomic profile analysis, represent significant steps toward understanding the vicious cycle mentioned above, more precisely the existing interactions between virus, endometrial layer and host immune response, which sustain viral replication thereby eliciting tissue damage. Elucidation of the molecular mechanisms and host cell pathways involved in this pathogenesis is central to the discovery of new targets for novel therapeutic treatments based on MMP-1 down-regulation.