Impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells

Abstract

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infections are characterized by prolonged viremia and viral shedding consistent with incomplete immunity. Type I interferons (IFN) are essential for mounting efficient antiviral innate and adaptive immune responses, but in a recent study, North American PRRSV genotype 2 isolates did not induce, or even strongly inhibited, IFN-α in plasmacytoid dendritic cells (pDC), representing "professional IFN-α-producing cells". Since inhibition of IFN-α expression might initiate PRRSV pathogenesis, we further characterized PRRSV effects and host modifying factors on IFN-α responses of pDC. Surprisingly, a variety of type 1 and type 2 PRRSV directly stimulated IFN-α secretion by pDC. The effect did not require live virus and was mediated through the TLR7 pathway. Furthermore, both IFN-γ and IL-4 significantly enhanced the pDC production of IFN-α in response to PRRSV exposure. PRRSV inhibition of IFN-α responses from enriched pDC stimulated by CpG oligodeoxynucleotides was weak or absent. VR-2332, the prototype genotype 2 PRRSV, only suppressed the responses by 34%, and the highest level of suppression (51%) was induced by a Chinese highly pathogenic PRRSV isolate. Taken together, these findings demonstrate that pDC respond to PRRSV and suggest that suppressive activities on pDC, if any, are moderate and strain-dependent. Thus, pDC may be a source of systemic IFN-α responses reported in PRRSV-infected animals, further contributing to the puzzling immunopathogenesis of PRRS.

Figure 1

Effects of PRRSV genotype 1 and 2 isolates on IFN-α responses of enriched pDC. (A) PRRSV impact on CpG-induced IFN-α production by pDC. Enriched pDC were stimulated with CpG in presence of the indicated PRRSV isolates added at an MOI of 0.1 TCID₅₀/cell. (B) Induction of IFN-α by prototype 1 LV and LVP23 (MOI of 1 TCID₅₀/cell) in CD172a-enriched pDC (C) Comparative analysis of pDC IFN-α responses induced by various PRRSV strains (MOI of 1 TCID₅₀/cell). (D-E) PBMC (D) and CD14^-CD4⁺ monocyte-depleted enriched pDC (E) stimulated with the prototype of genotype 1 LV, LVP23 and CpG-ODN as control. IFN-α was determined by ELISA in supernatants harvested after 20 h. Boxplots in A, B and C indicate the median (middle line), 25^th and 75^th percentiles (boxes), maximum and minimum (whiskers) and the mean values (dotted line) calculated from at least three independent experiments with cells from different animals each performed in culture triplicates. Bars in (D) and (E) indicate culture triplicates ± 1 standard deviation. One of two representative experiments is shown. For (A) and (C), significance between isolates are indicated by different letters based on an ANOVA on Ranks and Dunn's Method pairwise multiple comparison (P < 0.05). In (A) not statistically significant suppression compared to mock-treated cells was determined by Mann–Whitney Rank Sum test (P < 0.02) and noted N.S. = not significant.

Figure 2

IFN-α is induced by CD172^lowCD4^high pDC. Intracelluar staining of IFN-α was performed in CD172a enriched cells exposed to mock, PRRSV (MOI of 1 TCID₅₀/cell) or CpG. Pseudo-color plot of pDC defined as CD172^lowCD4^high and CD172⁺CD4^- populations are gated (left panel) and density plot show that only gated pDC are positive for intracellular IFN-α (right panel) after PRRSV or CpG stimulation. Gate frequency is indicated as mean ± 1 standard deviation of one experiment performed in triplicate.

Figure 3

PRRSV sensing by pDC does not require live virus and is mediated via TLR7. (A) IFN-α induced by type 1 and 2 PRRSV (MOI of 1 TCID₅₀/cell) in enriched pDC does not require live virus and is potentiated synergistically by IFN-γ. (B) Production of IFN-α by enriched pDC exposed to PRRSV (MOI of 2.5 TCID₅₀/cell) is inhibited by IRS661, a TLR7 antagonist. IFN-α was determined by ELISA in supernatants harvested after 20 h. Bars indicate biological triplicates ± 1 standard deviation in one representative experiment out of two.

Figure 4

Cytokine-enhancement of pDC IFN-α production induced by type 1 and 2 PRRSV. LVP23 (A) or VR-2332 (B) at an MOI of 1 TCID₅₀/cell were incubated with pDC alone or in the presence of IFN-β (100 U/mL), IFN-γ (10 ng/mL), Flt3-L (100 U/mL), GM-CSF (100 U/mL) and IL-4 (100 U/mL). IFN-α was determined by ELISA in supernatants harvested after 20 h. Boxplots are marked by the median (middle line), 25^th and 75^th percentiles (boxes), maximum and minimum values (whiskers) and the mean (dotted line) representing three independent experiments each performed in triplicate cultures. Significant difference in the median value is indicated by an asterisk mark compared to untreated cells (“none”) using the Mann–Whitney Rank Sum test (P < 0.02).

Figure 5

pDC are resistant to PRRSV infection and can prevent infection of MoDC. (A) Nucleocapsid expression in CD172a^lowCD4⁺ pDC and CD172a^highCD4^- monocytes at 24, 48 and 72 h post infection with LV (black line histogram), 2982 (dotted line) or 3267 (dashed line) at an MOI of 2.5 TCID₅₀/cell. Mock-treated cell fluorescence is shown as grey filled histograms. PAM infected with LV (MOI of 1 TCID₅₀/cell) was used as positive control (upper panel). (B) IFN-α levels measured in the cultures shown in A. Bars represent means of triplicate cultures ± 1 standard deviation. A representative example from three independent experiments is shown. (C) Detection of PRRSV nucleocapsid in MoDC cultured alone or with enriched pDC at 24 and 48 h post infection with LV. One million MoDC co-cultured with one millions of enriched pDC or two millions MoDC alone were exposed to LV at an MOI of 1 TCID₅₀/cell. Histograms were obtained from forward/side scatter gated MoDC treated with mock control (tinted line) or LV (black line). (D) Time course of IFN-α secretion in co-cultures of MoDC and pDC shown in panel C. One representative experiment out of two is shown.