Evaluation of porcine GM-CSF during PRRSV infection in vitro and in vivo indicating a protective role of GM-CSF related with M1 biased activation in alveolar macrophage during PRRSV infection

Abstract

Granulocyte-macrophage colony stimulating factor (GM-CSF), participates in diverse biological processes associated with innate and adaptive immunity, has unknown effects during PRRSV infection. Here, a double-antibody sandwich ELISA for pGM-CSF was developed in-house for evaluation of pGM-CSF level during PRRSV infection both in vitro and in vivo. In in vitro assay, it was notable that PRRSV-infected porcine alveolar macrophages (PAMs) yielded inconsistent pGM-CSF protein- and mRNA-level, suggesting a post-transcriptional inhibition of pGM-CSF mRNA was employed by PRRSV. Meanwhile, concurrent analysis of pGM-CSF levels in serum samples from PRRSV-infected piglets suggested that effect of PRRSV infection demonstrated minimum effect on pGM-CSF levels regardless of PRRSV virulence phenotypes. Moreover, in vitro treatment of PAMs with pGM-CSF prior PRRSV inoculation did not inhibit PRRSV replication in PAMs although genes downstream of pGM-CSF in PAMs could be upregulated by pGM-CSF treatment. Meanwhile, knockdown of pGM-CSF using siRNA did not enhance PRRSV replication as well. Intriguingly, therapeutic antibody treatment of HP-PRRSV-infected piglets led to significantly increased serum pGM-CSF levels, thus aligning with low pneumonia incidence and low intracellular PRRSV-RNA levels in PAMs of therapeutic antibody treated piglets. Furthermore, transcriptome analysis of PAMs from infected piglets revealed increased serum pGM-CSF levels correlated with activation of downstream signal of pGM-CSF in PAMs as evidenced by a M1-like phenotypes of gene expression pattern, implying a potential host-protective role played by pGM-CSF for PRRSV infection in vivo. In conclusion, our results demonstrated developments of a highly sensitive and specific ELISA for pGM-CSF and revealed a potential protective role conferred by pGM-CSF during PRRSV infection.

Keywords: ELISA; GM-CSF; PRRSV; immune response; macrophage activation.

Figure 1

Evaluation of specificity and repeatability for pGM-CSF double antibody sandwich ELISA. (A) The standard curve was generated by using recombinant pGM-CSF protein at the indicated concentrations, the formula and correlation coefficient were shown as well. (B) Evaluation of the assay specificity of pGM-CSF ELISA. Recombinant pGM-CSF along with IFN-α, IFN-β, IFN-γ, and IFN-λ3 at the concentration of 3ng/ml was used for pGM-CSF double antibody sandwich ELISA to evaluate the specificity the assay. (C) Evaluation of the pGM-CSF ELISA reactivity with mammalian expressed pGM-CSF. HEK-293T cells transfected with VenusC1-pGM-CSF or empty vector for 24 hours, then cells were lyzed using NP40 buffer and added as ELISA sample for pGM-CSF detection, and recombinant pGM-CSF protein (3ng) was included as positive control. Experiments were repeated at least three times. All data are expressed as mean ± SD and were subjected to Student’s t-test. Significant differences between indicated groups are marked by “***”, which means p< 0.001; ** means p< 0.01.

Figure 2

Evaluation of pGM-CSF mRNA and protein expression in PAMs infected by different PRRSV isolates. (A) PAMs were infected with infected with heterogeneous PRRSV isolates (PRRSV-JXA1, SD16, NADC30-like strain HNhx, vaccine strain TJM-F92 and classic strains VR2385 and VR2332) at 1 MOI for 24 hours. Then the cells were harvested for qPCR to evaluate the mRNA level of pGM-CSF. Non-infected PAMs served as controls (MOCK). All experiment was repeated at least for three times. (B) Cell culture supernatant collected from PAMs infected with heterogeneous PRRSV isolates (PRRSV-JXA1, SD16, NADC30-like strain HNhx, vaccine strain TJM-F92 and classic strains VR2385 and VR2332) at 1 MOI for 24 hours were harvested for ELISA analysis for pGM-CSF proteins. Supernatant from non-infected PAMs served as controls (MOCK). (C) Cell culture supernatant collected from PAMs infected with PRRSV-JXA1 at 1 MOI for different time hours were harvested for ELISA analysis for pGM-CSF proteins. Supernatant from non-infected PAMs served as controls (MOCK). (D) Cell culture supernatant collected from PAMs infected with PRRSV-JXA1 at different MOIs were harvested for ELISA analysis for pGM-CSF proteins. Supernatant from non-infected PAMs served as controls (MOCK). All experiment was repeated at least for three times and all data are expressed as mean ± SD.

Figure 3

The pGM-CSF cannot inhibit PRRSV replication in PAMs in vitro. (A) PAMs were either treated with 40ng recombinant pGM-CSF for 24 hours or left untreated (MOCK). Next, PAMs were harvested by TRIzol agent and reverse transcribed. The mRNA level of indicated genes was evaluated using qPCR. Experiments were repeated for at least three times. All data are expressed as mean ± SD and were subjected to Student’s t-test. Significant differences between indicated groups are marked by “*” (p < 0.05). (B) PAMs were treated with different dose of recombinant pGM-CSF for 24 hours or left untreated (0ng). Next, PAMs were harvested by TRIzol agent and reverse transcribed. The mRNA level of iNOS, IFN-γ and IL-13 was evaluated using qPCR. Experiments were repeated for at least three times. All data are expressed as mean ± SD and subjected to Student’s t-test. Significant differences between indicated groups are marked by “*” (p < 0.05). (C) PAMs were either treated with 40ng recombinant pGM-CSF protein for 24 hours or left un-treated (MOCK) before inoculated with PRRSV-JXA1 strains (0.1 MOI) for additional 24 hours. PAMs were harvested for SDS-PAGE followed by western blotting assay use anti-PRRSV-N specific mAb-6D10. Tubulin was probed from the same membrane as a loading control. (D) PAMs were either treated with 40ng recombinant pGM-CSF protein for 24 hours or left un-treated (MOCK) before inoculated with PRRSV-JXA1 strains (0.1 MOI) for additional 24 hours. Next, PAMs were harvested by TRIzol agent and reverse transcribed for evaluating PRRSV-RNA level using qPCR. Experiments were repeated for at least three times and presented as mean ± SD. (E) PAMs were either treated with 40ng recombinant pGM-CSF protein for 24 hours or left un-treated (MOCK) before inoculated with PRRSV-JXA1 strains (0.1 MOI) for additional 24 hours. Next, cell culture supernatant of PAMs were harvested for PRRSV titration and determined as TCID₅₀. Experiments were repeated for at least three times and presented as mean ± SD.

Figure 4

Knock-down of pGM-CSF in BM-DCs did not promote PRRSV replication in vitro. (A) HEK-293T cells were transfected with pVenusC1-pGM-CSF plasmids for 5 hours, then followed by transfection of 1μg pGM-CSF siRNAs or control siRNA for additional 24 hours. Next, the cells were observed directly under microscope of harvested for western blot using pGM-CSF specific mAb-2A4H11. Tubulin was probed from the same samples and included as protein loading control. (B) BM-DCs were either transfected with 1μg pGM-CSF siRNA-1 or control siRNA for 24 hours or left as un-transfected cells before inoculated with PRRSV-JXA1 strains (0.1 MOI) for additional 24 hours. Next, cells were harvested for SDS-PAGE followed by western blotting assay using anti-PRRSV-N specific mAb-6D10. Tubulin was probed from the same membrane as a loading control.

Figure 5

Infection of piglets using PRRSV strains with different virulence cannot induce pGM-CSF induction. (A) All piglets were inoculated with 1.0×10⁵ TCID50 of HP-PRRSV-XJA1, or two vaccines strains (MLV and TJM-F92) via both intramuscular and intranasal routes. Clinical signs and survival rates were monitored daily for a total of 21 days. (B) Representative ventral and dorsal lung images from surviving piglets of each group were captured immediately after piglets were autopsied at 21 dpi.

Figure 6

Serum pGM-CSF level in PRRSV infected piglets correlated with disease. (A) Lung gross pathological examination via hematoxylin and eosin (H&E) staining was conducted for piglets infected with PRRSV-JXA1(JXA1), PRRSV-infected but therapeutic mAb treated piglets (JXA1+therapeutic mAb), or PRRSV-infected but control mAb treated piglets (JXA1+control mAb). Lung tissue sample from non-infected piglets (Mock) was includes as control. Arrow indicated thickening of interlobular septal or infiltration of inflammatory cells around bronchiole or within or around alveolus and bronchus. Triangle indicates inflammatory cells, necrotic debris and exfoliated epithelial cells infiltrate in the bronchiole. Hollow arrow indicated hemorrhage or infiltration of inflammatory cells within alveolar septa, and alveolar spaces. (B) Evaluation of serum pGM-CSF, IFN-γ and IL-13 concentration from the piglets inoculated with PRRSV-JXA1 but treated with therapeutic mAb at different time points (7 dpi, 14dpi and 21dpi) using home-made pGM-CSF ELISA method and commercial ELISA kits for IFN-γ and IL-13. Animal number (64) demonstrated no detectable pGM-CSF was marked by “*”. (C) Serum samples from piglets were harvested by TRIzol agent for RNA extraction and reverse transcription. The serum PRRSV-RNA copied numbers was evaluated using Taqman probe in qPCR. (D) PAMs from piglets were harvested by TRIzol agent for RNA extraction and reverse transcription. The intracellular PRRSV-RNA copied numbers was evaluated using Taqman probe in qPCR. (E) PAMs from piglets were harvested by TRIzol for RNA extraction and reverse transcription. The mRNA level of pGM-CSF was evaluated using qPCR. Experiments for above qPCR were repeated at least three times (representing three different animals). All data are presented as mean ± SD and were subjected to Student’s t-test. Significant differences between indicated groups are marked by “*” (p < 0.05), whereas “ns” means no sense.

Figure 7

Transcriptome profiling of pGM-CSF activated genes in PAMs isolated from PRRSV-JXA1-infected piglets with or without therapeutic mAb treatment. Heatmap analysis of genes downstream of pGM-CSF from PAMs collected from PRRSV-JXA1 infected piglets (JXA1) and PAMs from PRRSV-JXA1 infected but treated with therapeutic mAb (JXA1_mAb). The health piglets inoculated with PBS was included as control (Mock).