Cytokine induction by immunostimulatory DNA in porcine PBMC is impaired by a hairpin forming sequence motif from the genome of Porcine Circovirus type 2 (PCV2)

Porcine Circovirus type 2 (PCV2) can cause postweaning multisystemic wasting syndrome (PMWS) in young pigs with severe immunosuppression as a major characteristic of the disease complex. Despite the dramatic involvement of the immune system, the interaction between PCV2 and the host is until date not well understood. The DNA genome of PCV2 contains sequences that in synthetic form (oligodeoxyribonucleotides; ODNs) can act immunomodulatory on porcine peripheral blood mononuclear cells (poPBMCs) in vitro. One such sequence (ODN PCV2/1) acts inhibitory on interferon (IFN)-α production induced by immunostimulatory DNA but not that induced by RNA, and the inhibitory activity is dependent on secondary structure formation. In the present study, the characteristic of ODN PCV2/1 was examined further by altering the nucleotide sequence to disrupt hairpin structure formation but still enable multimer structures through G-tetrads. This modification resulted in loss of IFN-α-inhibitory activity of the ODN and thus indicated the importance of hairpin structures. In addition, ODN PCV2/1 was compared to another inhibitory ODN (IRS 869) previously used in human and murine cells. In contrast to ODN PCV2/1, ODN IRS 869 did not inhibit IFN-α production induced by class A ODN 2216 but was a more efficient inhibitor of IFN-α production induced by plasmid DNA than ODN PCV2/1. In cultures induced by the RNA stimulator Poly I:C, however, a strong synergistic IFN-α stimulatory effect was seen in combination with ODN IRS 869. These results indicate that ODN PCV2/1 and ODN IRS 869 function through separate mechanisms to affect cytokine production by immune cells. The effect of ODN PCV2/1 was studied further by monitoring the expression of mRNA for IFN-α, IL-12p40, IL-10, IL-6, IFN-γ, IL-1β, TGF-β, and TNF-α in cultures of poPBMC stimulated with ODN 2216 or Poly I:C. Results from qPCR analyses showed that ODN PCV2/1 clearly inhibited the expression of IFN-α, IL-12p40, IL-10 and IL-6 when induced by ODN 2216, but did not seem to affect any of the cytokines examined when induced by Poly I:C. Initial studies using confocal microscopy and fluorochrome labelled ODNs indicate that ODN 2216 and ODN PCV2/1 co-localize in subpopulations of poPBMC.