Mitochondrial dysfunction in PRRSV-2-infected macrophages

Abstract

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically devastating viruses for the global swine industry. PRRSV has a known tropism for lung macrophages, where it causes impaired immune responses. This study evaluated the metabolic and immune profiles of primary porcine alveolar macrophages (PAMs) and pulmonary intravascular macrophages (PIMs) infected with different strains of PRRSV-2 isolated from North Carolina (NC) pig herds (NC134, NC18-9-7 referred to as NC174, and NC20-1 referred to as NC144), and VR2232, a PRRSV-2 prototype strain.

Materials and methods: Primary enriched mononuclear phagocytes were infected in vitro with NC134 and NC174, sorted, and processed. The total RNA was used for a transcriptomic approach; additionally, gene expression was further validated using RT-qPCR and NanoString technology. Complementary functional assays with additional NC strains were used to further investigate the mitochondrial and metabolic dysfunction, as well as the oxidative stress induced by PRRSV-2 infection.

Results: PAMs infected with both NC PRRSV-2 strains NC174 and NC134 showed similar transcriptomic profiles during the early stage of infection, with downregulation of genes involved in the oxidative phosphorylation and electron transport chain pathways. PIMs infected with both NC174 and NC134 strains showed limited alteration in the transcriptomic profiles compared to uninfected cells. Genetic reprogramming matched the PRRSV-2-induced mitochondrial impairment observed in functional assays performed using Seahorse technology. Mitochondrial respiration displayed slightly different profiles between PIMs and PAMs infected with the different PRRSV-2 strains, with PAMs showing a more substantial decrease in mitochondrial fitness compared to control cells. When reactive oxygen species (ROS) and nitric oxide (NO) production were evaluated, no differences were observed between PRRSV-2-infected PAMs and PIMs and control cells.

Conclusion: These results provide valuable insights into the pathogenetic mechanism of different NC PRRSV-2 strains by focusing on the alteration in mitochondrial function in lung macrophages during early infection and highlighting differences in lung macrophage responses to distinct PRRSV-2 strains.

Keywords: NanoString; PRRSV-2; macrophages; mitochondrial dysfunction; pig; seahorse technology; transcriptomics.

Figure 1

Experimental layout. Lungs were collected from influenza- and PRRSV-negative 8–12-week-old pigs. Mononuclear phagocytes (MNPs) from bronchoalveolar lavage (BAL) and parenchyma (PAR) were enriched using OptiPrep gradient. MNPs were infected with North Carolina (NC) PRRSV-2 strains NC134 and NC174 (MOI = 1) and control media for 12 h. Infected and control porcine alveolar macrophages (PAMs), pulmonary intravascular macrophages (PIMs), monocyte-derived dendritic cells (moDCs), and classical DCs (cDCs) were sorted, and RNA-seq was performed on each cell subset. Additional PAM and PIM sorted cells were used for qPCR and NanoString validations. For other assays, enriched MNPs were plated to allow macrophage adhesion for 2 h. PAMs and PIMs were subsequently infected with different NC PRRSV-2 strains (NC134, NC174, and NC144) or VR2332 prototype strain; uninfected cells were used as negative control. Infected macrophages were used in different assays: mitochondrial function assay (Seahorse Agilent technology) and ROS and NO production assays. Created in BioRender. Crisci, E (2025). https://BioRender.com/65x4hji. PRRSV, porcine reproductive and respiratory syndrome virus; MOI, multiplicity of infection; ROS, reactive oxygen species; NO, nitric oxide.

Figure 2

Cell type-specific responses of PAMs and PIMs to PRRSV-2 infection. RNA-seq analysis. (A) Principal component analysis of the PAM samples, PC1 vs. PC2. (B) Venn diagram showing the genes found upregulated in PAMs, with FDR < 0.05. (C) Venn diagram showing the genes found upregulated in PIMs, with FDR < 0.05. (D) Venn diagram showing the genes found downregulated in PAMs, with FDR < 0.05. (E) Venn diagram showing the genes found downregulated in PIMs, with FDR < 0.05. (F) Principal component analysis of the PIM samples, PC1 vs. PC2. (G) Functional annotation enrichment analysis. Heatmap showing selected functions found enriched in the MNPs infected with NC134 or NC174 as compared to control (Mock). Data represent four biological replicates for all the conditions. PAMs, porcine alveolar macrophages; PIMs, pulmonary intravascular macrophages; PRRSV, porcine reproductive and respiratory syndrome virus; NC, North Carolina; MNPs, mononuclear phagocytes.

Figure 3

Similarity in the responses of PIMs and PAMs to PRRSV-2 infection. (A) Analysis of the pathways and gene sets of interest enriched in PAMs upon NC134 and NC174 infections by high-throughput GSEA using BubbleGUM. (B) Analysis of the pathways and gene sets of interest enriched in PIMs upon NC134 and NC174 infections using BubbleGUM. The Hallmark gene sets from MSigDB (H.), together with lists of DEGs in PAMs or PIMs in response to NC134 or NC174 infection, were used to assess their enrichments in the comparisons between macrophage subsets in control (Mock) vs. NC134 or NC174 conditions using the BubbleMap module of BubbleGUM. Gene numbers per gene set are written in parentheses. (C) Genes located in the mitochondrial membrane that are enriched in the mitochondrial dysfunction pathway or in the downregulation of the oxidative phosphorylation in NC174-infected PAMs. Data represent four biological replicates for all the conditions. PAMs, porcine alveolar macrophages; PIMs, pulmonary intravascular macrophages; PRRSV, porcine reproductive and respiratory syndrome virus; GSEA, Gene Set Enrichment Analysis; DEGs, differentially expressed genes.

Figure 4

Mitochondrial dysfunction in PRRSV-2-infected porcine alveolar macrophages (PAMs). PAMs were infected for 12–16 h with different PRRSV-2 strains and washed, and mitochondrial dysfunction was measured using the Cell Mito Stress Test kit (Agilent, 103010-100). Top figures correspond to representative figures of the key components of mitochondrial respiration in the Seahorse Cell Mito Stress assay, including oxygen consumption rate (OCR) (Left) profiles and extracellular acidification rate (ECAR) (Right) in infected and control PAMs. Oligomycin (Oligo) reduces the oxygen consumption ratio (OCR) through inhibition of ATP synthase, a critical facilitator of oxidative phosphorylation of ADP into ATP. Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) potently collapses the proton gradient at the inner mitochondrial membrane, simulating a maximal energy demand in the cells. Rotenone and antimycin A are complex I and complex III inhibitors, respectively, of the electron transport chain (ETC). The profile of mitochondrial respiration in the test includes basal respiration (A), maximal respiratory capacity (B), spare capacity (C), non-mitochondrial oxygen consumption (D), ATP-linked respiration (E), and coupling efficiency (F). (G) Cell respiratory control. (H) ATP-linked respiration to the maximal respiration as described in Divakaruni et al. (53). N = 7–9. Data were analyzed in Wave software (Agilent) and visualized using GraphPad Prism v10. Statistics: ordinary one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. PRRSV, porcine reproductive and respiratory syndrome virus.

Figure 5

Mitochondrial dysfunction in PRRSV-2-infected pulmonary intravascular macrophages (PIMs). After enriched parenchyma cell adhesion for 2 h and removal of non-adherent cells, PIMs were infected for 12–16 h with different PRRSV-2 strains and washed, and mitochondrial dysfunction was measured using the Cell Mito Stress Test kit (Agilent, 103010-100). Top figures correspond to representative figures of the key components of mitochondrial respiration in the Seahorse Cell Mito Stress assay, including oxygen consumption rate (OCR) (Left) profiles and extracellular acidification rate (ECAR) (Right) in infected and control PAMs. Oligomycin (Oligo) reduces the oxygen consumption ratio (OCR) through inhibition of ATP synthase, a critical facilitator of oxidative phosphorylation of ADP into ATP. Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) potently collapses the proton gradient at the inner mitochondrial membrane, simulating a maximal energy demand in the cells. Rotenone and antimycin A are complex I and complex III inhibitors, respectively, of the electron transport chain (ETC). The profile of mitochondrial respiration in the test includes basal respiration (A), maximal respiratory capacity (B), spare capacity (C), non-mitochondrial oxygen consumption (D), ATP-linked respiration (E), and coupling efficiency (F). (G) Cell respiratory control. (H) ATP-linked respiration to the maximal respiration as described in Divakaruni et al. (53). N = 7. Data were analyzed in Wave software (Agilent) and visualized using GraphPad Prism v10. Statistics: ordinary one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001. PRRSV, porcine reproductive and respiratory syndrome virus.

Figure 6

Reactive oxygen species (ROS) and nitric oxide (NO) production in PRRSV-2-infected macrophages. Macrophages were plated and infected with different strains of PRRSV-2. ROS production was measured using DHR-123 from 0 to 72 h. Figures show ROS production at 48 h post-infection with different MOIs (1 and 3). Data are expressed in relative fluorescence units (RFU) measured at 540 nm. N = 5–8. Results were analyzed using one-way ordinary ANOVA with Tukey’s multiple-comparisons test and visualized using GraphPad Prism v10. The measurement of nitrite concentration as an index of nitric oxide (NO) production was analyzed using Griess reaction system following manufacturer’s instructions (Promega). Absorbance was measured at 540 nm and proportionally correlated to the standard, ranging from 0 to 100 µM. N = 3–9. Statistics: ordinary one-way ANOVA. *p < 0.05. PRRSV, porcine reproductive and respiratory syndrome virus; MOIs, multiplicities of infection.