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Home > Resources > Polyplus-transfection Database > Genotype 2 strains of porcine reproductive and respiratory syndrome virus dysregulate alveolar macrophage cytokine production via the unfolded protein response
Genotype 2 strains of porcine reproductive and respiratory syndrome virus dysregulate alveolar macrophage cytokine production via the unfolded protein response
Citation
Authors: Chen, W. Y., Schniztlein, W. M., Calzada-Nova, G., Zuckermann, F. A.
Year: 2017
Journal: J Virol
Applications:in vitro / DNA / jetPEI-Macrophage
Cell type: ZMAC-1 Description: Pig sus scrofa lung cell line.
Known as: UIMAC FBAL-A; FBAL-A; ZMAC.
Method
To deliver poly I:C into the cytoplasm, in some experiments ZMAC-1 cells were transfected with poly I: C using jetPEI-Macrophage. Briefly, 400 μg of poly I:C in 50 μl volume was mixed with 50 μl of a 150 mM NaCl solution containing 1 μl of jetPEI -Macrophage. Following a 30 -minute incubation at room temperature (RT) , this mixture was added to a well of a 24 well -plate containing 2.5x10^5 ZMAC-1 cells in a 0.5 mL volume.
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) infects alveolar macrophages (AMPhi) causing dysregulated interferon (IFN)-alpha and tumor necrosis factor (TNF)-alpha production through a mechanism(s) yet to be resolved. Here, we show that AMPhi infected with PRRSV secreted a reduced quantity of IFN-alpha following the cell exposure to synthetic dsRNA. This reduction did not correlate with reduced IFNA1 gene transcription. Rather, it coincided with two events that occurred late during infection and were indicative of translational attenuation, specifically, the activation of eukaryotic translation initiation factor 2alpha (eIF2alpha), and the appearance of stress granules. Notably, the typical rapid production of TNF-alpha by AMPhi exposed to lipopolysaccharide (LPS) was suppressed or enhanced by PRRSV depending on when the LPS exposure occurred after virus infection. If exposure was delayed until 6 h post-infection (hpi) so that the development of the cytokine response coincided with the time in which phosphorylation of eIF2alpha by the stress sensor PERK (protein kinase RNA (PKR)-like ER kinase) occurred, inhibition of TNF-alpha production was observed. However, if LPS exposure occurred at 2 hpi, prior to a detectable onset of eIF2alpha phosphorylation, a synergistic response was observed due to the earlier NF-kappaB activation via the stress sensor IRE1alpha (inositol-requiring kinase 1alpha). These results suggest that the asynchronous actions of two branches of the unfolded protein response (UPR), namely IRE1alpha, and PERK, activated by ER stress resulting from the virus infection, are associated with enhancement or suppression of TNF-alpha production, respectively.IMPORTANCE The activation of AMPhi is controlled by its microenvironment to deter excessive pro-inflammatory cytokine responses to microbes that could impair lung function. However, viral pneumonias frequently become complicated by secondary bacterial infections triggering severe inflammation, lung dysfunction, and death. Although dysregulated cytokine production is considered an integral component of the exacerbated inflammatory response in viral-bacterial co-infections, the mechanism responsible for this event is unknown. Here, we show that PRRSV replication in porcine AMPhi triggers activation of the IRE1alpha branch of the UPR, which causes a synergistic TNF-alpha response to LPS exposure. Thus, the severe pneumonias typically observed in pigs afflicted with PRRSV-bacterial co-infections could result from dysregulated, overly robust TNF-alpha production to opportunistic pathogens that is not commensurate with the typical restrained reaction by uninfected AMPhi. This notion could help design therapies to mitigate the severity of viral and bacterial co-infections.