ARS | Publication request: Effects of PRRSV infection on TLR-dependent induction of NOS

Submitted to: Meeting Abstract
Publication Type: Abstract
Publication Acceptance Date: September 25, 2008
Publication Date: N/A

Technical Abstract: Inducible nitric oxide synthase (iNOS) is an important effector enzyme in the macrophage arsenal against pathogens. This enzyme is produced in response to bacterial cell wall components such as lipopolysaccharide (LPS) and dsRNA--a by-product of viral replication, binding to Toll-like receptors (TLR). Because infection with PRRSV often results in morbidity/mortality due to secondary bacterial infections, the objective of our study was to determine if the expression of iNOS in porcine alveolar macrophages (PAM) was differentially affected if cells were stimulated with TLR ligands prior to or post-infection. PAM were isolated by lung lavage from 6- to 8-week-old pigs housed at the National Animal Disease Center. Cells were cultured overnight and non-adherent cells removed prior to treatment. Treatments included poly I:C as a TLR3 ligand and LPS from E. coli 055:B5 as a TLR4 ligand. Cells were treated prior to or post-infection with virus stocks of PRRSV VR-2332, and non-infected (mock) and unstimulated cells were included as controls. Supernatants were collected at 4 and 20 H post-infection and stored at -80°C until analyzed. A colorimetric assay utilizing the Griess reagent was used to quantify the concentration of nitrate in the supernatants as a measure of iNOS expression. There was a significant difference in NO production between mock- and PRRSV-infected PAM in both treatment designs. There was no significant difference in NO production between treatment of PAM with TLR ligands prior to or post-infection with PRRSV. Similar amounts of NO were produced when signaling occurred through either the TLR3 or the TLR4 pathways. Although not statistically different, treatment of PRRSV-infected PAM with TLR ligands increases NO production to levels greater than in PRRSV-infected PAM, but not as great as PAM treated with TLR ligands but not infected with PRRSV.