Kerstin Skovgaard, Karin T. Poulsen, Peter M. H. Heegaard
Technical University of Denmark, Denmark
MicroRNAs (miRNAs) are a highly evolutionarily conserved group of small non-coding RNA molecules, which regulate the activity of other genes at the post-transcriptional level. Recently it has become evident that miRNA plays an important role in modulating and fine tuning of the innate and adaptive immune responses. Still, little is known about the impact of miRNAs in the development and pathogenesis of lung infections. Expression of miRNA, known to be induced by bacterial (i.e., LPS) ligands and thus supposed to play a role in the regulation of antimicrobial defence, were studied in lung tissue from pigs experimentally infected with Actinobacillus pleuropneumoniae (AP) serotype 2 and 6. Circulating miRNAs were studied in blood from pigs infected with AP serotype 2 using real time-qPCR (RT-qPCR). Expression profiles of miRNA in blood of seven animals before and after infection, were also studied using miRCURY™ LNA arrays (Exiqon). Piglets were inoculated by dripping 1 ml bacterial suspension, into each nostril during inhalation. Most of the inoculated pigs revealed characteristic, well demarcated, lung lesions. No pathological changes were seen in lungs from control animals. All AP infected animals had a significantly higher level of mRNA coding for the acute-phase protein SAA-2 in the liver compared to the control group. Whole Blood samples were collected in PAXgene Blood RNA Tubes (PrenalytiX) before (control) and after infection of piglets (6 h., 12 h., 24 h. and 48 h.). Each time group was a different set of 4-6 pigs. Total RNA was extracted from blood samples using PAXgene™ Blood RNA kit (Qiagen/ PrenalytiX). Expression levels of selected miRNA were further studied in lung tissue collected at two time points (6 h. and 24 h.) after AP serotype 2 and 6 infection. 600 ng total RNA from blood samples before and after infection were labelled with Hy5™ and Hy3™ fluorescent label, respectively. Sample were hybridized to miRCURY™ LNA array version 11.0 (Exiqon, Denmark), which contains capture probes targeting all miRNAs for human, mouse and rat. A two-tailed T-test calculated between infected and control identified 10 of 1263 miRNA to be differentially expressed. MicroRNA expression in lung tissue over time in response to the two different serotypes was very similar. MiR-223 was found to be highly up regulated, followed by miR-146a and to a lesser degree miR-21 in lung tissue of the AP serotype 2 infected animals. MiR-223 was also found to be up regulated in blood based on both microarray and RT-qPCR, mir-223 is a negative regulator of neutrophil proliferation and activation and might act to limit the potentially harmful consequences of the accumulation of infiltrating neutrophils in AP infected lungs. More data of microRNA expression in blood of pigs infected with AP serotype 2 will be presented.
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