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

Baumann A, Mateu E, Murtaugh MP, Summerfield A - Vet. Res. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Virology and Immunoprophylaxis (IVI), Sensemattstrasse 293, Mittelhäusern, 3147, Switzerland. artur.summerfield@ivi.admin.ch.

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.

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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), 25th and 75th 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.
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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), 25th and 75th 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.

Mentions: MARC-145 cells (ATCC, LGC Standards, Molsheim, France) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen, Switzerland) supplemented with 10% fetal bovine serum (FBS; Biowest, France). PAM were obtained from bronchoalveolar lung lavages[31]. Specific-free pathogen (SPF) pigs from 6 week- to 12 month-old were euthanized and lungs were aseptically removed. Briefly, lungs were filled up with approximately one to two liters of PBS containing a 2× concentrated penicillin/streptomycin (Pen/Strep) solution (Gibco, Invitrogen, Switzerland). The lavage was collected and cells were recovered by centrifugation (350 g, 10 min, 4°C), followed by three wash steps with 2 × Pen/Strep PBS and centrifugation at 350 g for 10 min. PAM were maintained in RPMI 1640 medium (Gibco) supplemented with Pen/Strep and 10% FBS or frozen in liquid nitrogen until use. MARC-145 cells and PAM were cultured at 37°C in a 5% CO2 atmosphere. MoDC were prepared using interleukin-4 (IL-4)/granulocyte–macrophage colony-stimulating factor (GM-CSF) as previously described[32]. For all experiment except in Figure 1D and1E, CD172a enrichment of pDC was performed as described earlier[33]. Briefly, peripheral blood mononuclear cells (PBMC) from 6 week- to 12 month-old pigs were isolated by Ficoll-Paque differential centrifugation[34] followed by CD172a (mAb 74-22-15a) enrichment using MACS sorting LD columns (Miltenyi Biotec GmbH, Germany) leading to > 80% of CD172a positive cells and 2-8% CD172lowCD4high pDC. The cells were cultured in DMEM with 10% FBS and 20 μM of β-mercaptoethanol (Invitrogen, Switzerland). For the experiment shown in Figure 1D and1E, PBMC were depleted of monocytes by anti-CD14 (mAb CAM36A, VMRD Inc., Washington, USA) followed by CD4 (mAb PT90A, VMRD Inc.) selection with MACS sorting LS column (Miltenyi Biotec GmbH, Germany). This sorting resulted in a pDC purity of 10%.


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

Baumann A, Mateu E, Murtaugh MP, Summerfield A - Vet. Res. (2013)

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), 25th and 75th 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.
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Related In: Results  -  Collection

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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), 25th and 75th 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.
Mentions: MARC-145 cells (ATCC, LGC Standards, Molsheim, France) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen, Switzerland) supplemented with 10% fetal bovine serum (FBS; Biowest, France). PAM were obtained from bronchoalveolar lung lavages[31]. Specific-free pathogen (SPF) pigs from 6 week- to 12 month-old were euthanized and lungs were aseptically removed. Briefly, lungs were filled up with approximately one to two liters of PBS containing a 2× concentrated penicillin/streptomycin (Pen/Strep) solution (Gibco, Invitrogen, Switzerland). The lavage was collected and cells were recovered by centrifugation (350 g, 10 min, 4°C), followed by three wash steps with 2 × Pen/Strep PBS and centrifugation at 350 g for 10 min. PAM were maintained in RPMI 1640 medium (Gibco) supplemented with Pen/Strep and 10% FBS or frozen in liquid nitrogen until use. MARC-145 cells and PAM were cultured at 37°C in a 5% CO2 atmosphere. MoDC were prepared using interleukin-4 (IL-4)/granulocyte–macrophage colony-stimulating factor (GM-CSF) as previously described[32]. For all experiment except in Figure 1D and1E, CD172a enrichment of pDC was performed as described earlier[33]. Briefly, peripheral blood mononuclear cells (PBMC) from 6 week- to 12 month-old pigs were isolated by Ficoll-Paque differential centrifugation[34] followed by CD172a (mAb 74-22-15a) enrichment using MACS sorting LD columns (Miltenyi Biotec GmbH, Germany) leading to > 80% of CD172a positive cells and 2-8% CD172lowCD4high pDC. The cells were cultured in DMEM with 10% FBS and 20 μM of β-mercaptoethanol (Invitrogen, Switzerland). For the experiment shown in Figure 1D and1E, PBMC were depleted of monocytes by anti-CD14 (mAb CAM36A, VMRD Inc., Washington, USA) followed by CD4 (mAb PT90A, VMRD Inc.) selection with MACS sorting LS column (Miltenyi Biotec GmbH, Germany). This sorting resulted in a pDC purity of 10%.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Virology and Immunoprophylaxis (IVI), Sensemattstrasse 293, Mittelhäusern, 3147, Switzerland. artur.summerfield@ivi.admin.ch.

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.

Show MeSH
Related in: MedlinePlus