Abstract
Dysregulation of cytokine responses plays a major role in the pathogenesis of severe and life-threatening infectious diseases like septicemia or viral hemorrhagic fevers. In pigs, diseases like African and classical swine fever are known to show exaggerated cytokine releases. To study these responses and their impact on disease severity and outcome in detail, reliable, highly specific and sensitive methods are needed. For cytokine research on the molecular level, real-time RT-PCRs have been proven to be suitable. Yet, the currently available and most commonly used SYBR Green I assays or heterogeneous gel-based RT-PCRs for swine show a significant lack of specificity and sensitivity. The latter is however absolutely essential for an accurate quantification of rare cytokine transcripts as well as for detection of small changes in gene expressions. For this reason, a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines was designed and validated within the presented study. Cytokines were chosen to represent different immunological pathways and targets known to be involved in the pathogenesis of the above mentioned porcine diseases, namely interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-α. Beta-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as reference genes for normalization. For absolute quantification a synthetic standard plasmid was constructed comprising all target cytokines and reference genes within a single molecule allowing the generation of positive control RNA. The standard as well as positive RNAs from samples, and additionally more than 400 clinical samples, which were collected from animal trials, were included in the validation process to assess analytical sensitivity and applicability under routine conditions. The resulting assay allows the reliable assessment of gene expression profiles and provides a broad applicability to any kind of immunological research in swine.
Highlights
Cytokines are important mediators that orchestrate cellular functions including inflammatory responses and innate immune reactions
Excessive activation of the innate immune system in response to pathogens can lead to pathological inflammatory consequences [1], and dysregulation of cytokine responses plays a major role in the pathogenesis of severe and lifethreatening infectious diseases including viral haemorrhagic fevers [2]
Cytokine profiles, especially when targeting a set of cytokines expressed within a certain microenvironment [4,5], can provide important insights into the development of infectious diseases, which are characterised by an immune pathogenesis such as classical swine fever (CSF), a severe porcine infection that can be accompanied by haemorrhagic lesions
Summary
Cytokines are important mediators that orchestrate cellular functions including inflammatory responses and innate immune reactions. Excessive activation of the innate immune system in response to pathogens can lead to pathological inflammatory consequences [1], and dysregulation of cytokine responses plays a major role in the pathogenesis of severe and lifethreatening infectious diseases including viral haemorrhagic fevers [2]. Another example is the ‘‘cytokine storm’’ that is held responsible for the exceptionally high morbidity and mortality in human highly pathogenic influenza virus infections [3]. Similar responses are known for African swine fever (ASF) [7], a disease that recently gained importance through its introduction into several Eastern European countries [8,9]
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