Performing Department
Population Health and Pathobiology
Non Technical Summary
Although several improvements in swine genetics, management and health have been achieved, porcine reproductive and respiratory syndrome (PRRS) remains one of the top priorities to U.S. pig industry. PRRS virus (PRRSV) affects mainly lung mononuclear phagocytes (MNP) and several comprehensive genomic profiles have been performed in lung alveolar macrophages during different PRRSV infections1. While a transcriptome profiling of primary lung parenchymal porcine macrophages (MΦ) and dendritic cell (DC) infected with different European PRRSV-1 are in progress (Crisci et al. 2019, in preparation), no high throughput transcriptional studies have been performed on primary MNP subsets upon U.S. PRRSV-2 infection.Therefore, the main goal of the proposed research is to dissect the lung pathogenesis involved during different PRRSV-2 infections. We will integrate immunological/functional phenotypes with the molecular genomic signature to identify specific cellular pathways altered during the infection and involved in the impairment of the immune responses. The innovative outcome of this project will be to produce a detailed transcriptome profile at the cell type level underling high and low virulent PRRSV-2 infections, coupled to functional validations of hypotheses generated after the joint analysis of the immunological and gene expression data. Since PRRSV-1 strains activated different MNP intracellular pathways depending on the isolate virulence, our hypothesis is that PRRSV-2 induces specific strain-dependent signature pathways involved in the virus-specific immune evasion strategy and different from PRRSV-1 viruses.Specific aim 1: Characterize the alterations of MNP subpopulations upon infection with different virulent PRRSV-2 strains, detailing both phenotypes and functions.Specific aim 2: Identify common and specific transcriptional signatures (coding and non coding RNAs) and the main regulatory pathways in PRRSV-2 infected MNP subpopulations.The knowledge generated from this project will help to refine future vaccine strategies considering specific PRRSV-2 isolates and the strain-dependent modulation of the immune responses.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The main goal of the proposed research is to dissect the lung pathogenesis involved during different PRRSV-2 infections. We will integrate immunological/functional phenotypes with the molecular genomic signature to identify specific cellular pathways altered during the infection and involved in the impairment of the immune responses. The innovative outcome of this project will be to produce a detailed transcriptome profile at the cell type level underling high and low virulent PRRSV-2 infections, coupled to functional validations of hypotheses generated after the joint analysis of the immunological and gene expression data. Since PRRSV-1 strains activated different MNP intracellular pathways depending on the isolate virulence, our hypothesis is that PRRSV-2 induces specific strain-dependent signature pathways involved in the virus-specific immune evasion strategy and different from PRRSV-1 viruses.Specific aim 1: Characterize the alterations of MNP subpopulations upon infection with different virulent PRRSV-2 strains, detailing both phenotypes and functions.Specific aim 2: Identify common and specific transcriptional signatures (coding and non coding RNAs) and the main regulatory pathways in PRRSV-2 infected MNP subpopulations.The knowledge generated from this project will help to refine future vaccine strategies considering specific PRRSV-2 isolates and the strain-dependent modulation of the immune responses.
Project Methods
We will isolate primary lung immune cells subpopulations belonging to the MNP system and investigate their interaction with diverse virulent PRRSV-2 strains using genomics and immunological approaches.Ex vivo lung respiratory system. Lung from conventionally bred pigs will be obtained from different sources. Lungs will be collected and broncho-alveolar lavage (BAL) and parenchyma (PAR) enzymatic digestion will be performed in both lungs.Virus production and titration. PRRSV-2 strains from different lineages will be used in the study. TCell sorting and flow cytometry analysis. Isolated PAR will be stained in blocking solution and antibodies will be added to the blocking solution for 30 min on ice and then washed in PBS/EDTA with 2% FCS. A multicolor flow cytometry staining will be performed as previously described 10. Primary antibodies (Abs): anti-CaM1 or anti-CD13 (unpublished data), anti-CD172a, anti-CD163, anti-CD1, anti-SLAII (MHCII) and anti-CD11c, followed by secondary Abs with a broad fluorochromes panel (FITC, PE, APC, PE-Cy7, Brillant Violet 421, Brillant Violet 480) and LIVE/DEAD Fixable Near-IR (635 nm) dead cells stain kit (Molecular Probes). MoFlo ASTRIOS sorter (Beckman-Coulter, Core facility) will be used to isolate the specific cell subpopulations and FlowJo software (version 10.1.0, Tree Star, OR) will be used to analyze the samples.Aim 1: Characterize the alterations of MNP subpopulations (AM, AM-like, cDC1, cDC2 and moDC) upon infection with different virulent PRRSV-2 strains, detailing both phenotypes and functions using real time RT-PCR, ELISAs and mixed lymphocyte reaction (MLR) assay.RNA extraction and RT-qPCR. Total RNA from sorted or MNP cells will be extracted using the Arcturus PicoPure RNA Isolation kit and reverse transcription will be made using high-capacity cDNA synthesis kit according to manufacturer's instructions. Relative mRNA expression was evaluated by qPCR using the Powerup SYBR Green. Relative gene expression will be assessed according to the ΔCt method. Ribosomal protein S24 (RPS24) and/or GAPDH will be used as reference genes for normalization as previously described for pig lung8 for their stable expression among MNP subsets and upon infection. For PRRSV viral transcripts, primers were designed for ORF5 regions of NC 1-7-4, NC 1-3-4 and VR2332 (MLV). Primers used for cytokines and antiviral factors detection are described in 10. Limit of detection will be set at Ct<35.ELISAs. Cytokine levels in conditioned cell supernatants at different time points will be assassed by ELISA for porcine TNF-α, IL-6, IL-8 and IFN-α. For each ELISA, duplicate or triplicate wells of stimulated- or un-stimulated-cell supernatants will be used in the DuoSet® ELISA Development system (R&D Systems, UK) following manufacturer's instructions.Mixed Lymphocytes Reaction Assay. Mixed Lymphocytes Reaction (MLR) assays will be performed as previously described 10,13. Briefly, porcine peripheral blood mononuclear cells (PBMC) will be co-cultured with parenchymal gradient enriched MNP previously exposed to PRRSV or mock in complete RPMI. 1:6 or 1:8 ratio will be used for MNP:PBMC co-culture and after 3 days, total RNA will be extracted and gene expression analyzed by RT-qPCR. Four transcription factors (Tbet, GATA3, RORγT, and FOXP3) and cytokines (IFNγ, IL13, IL17, and TGFβ) expression will be used as indicators of T-helper polarization (respectively Th1, Th2, Th17, and Treg). The same PBMC will be CFSE stained to evaluate their proliferation rate. After 5 days of co-culture cells will be stained using anti-CD3 and CD4 antibodies and their corresponding isotype-specific secondary antibodies and analyzed by flow cytometry.Mitocondrial stress assay. The Seahorse XF Cell Mito Stress Test kit will be used for assessing mitochondrial function following manufacturer's instruction. Optimization of cell number and drugs will be performed and multiple parameters will be obtained: basal respiration, ATP production-coupled respiration, maximal and reserve capacities and non-mitochondrial respiration. We will seed a minimum of 3X105 cells/well in 80μl/well of alveolar and parenchyma MΦ. They will be infected at MOI 0.5 with the PRRSV strains in duplicates and mock. The cell will be Incubated at 37°C overnight and then the plate will be used in the Seahorse (Agilent, CA, USA). Spare capacity and OCR graphics will be used to compare the mytocrondrial function between treatments.Aim 2: Identify common and specific transcriptional signatures (coding and non coding RNAs) and the main regulatory pathways in PRRSV-2 infected MNP subpopulations.Immunogenomics (RNAseq). Cells will be isolated from the lungs of six controlled health status animals, housed in the CVM LAR for five days. The cells will be infected as previously described in the "in vitro infection" section. Infected (1-7-4, 1-3-4) and mock-inoculated BAL and PAR cells will be analyzed by deep sequencing (RNAseq). Total RNA from each cell subset (36 samples= 6 pigs X 3 conditions X 2 cell types) will be isolated as described above and RNA quantity and quality will be accessed using a pico sensitivity RNA chip on a Bioanalyzer 2100. For library construction and low input RNAseq we will rely on the expertise of Dr. Liu lab. For each library, amplified ds cDNA will be generated from 10ng of high quality (RIN> 9.0) total RNA using a SMART-Seq v4 Ultra low Input RNA kit (Clontech). The quality, quantity, and average size of the ds cDNA will be accessed using a high sensitivity DNA chip on a Bioanalyzer 2100). Next, 150pg of each ds cDNA sample will be used to generate sequencing libraries using a Nextera XT DNA library prep kit (Illumina). Library quality, quantity, and average size will be accessed using a high sensitivity DNA chip on a Bioanalyzer 2100. Libraries will be dual indexed to allow for multiplexing of all 36 libraries into a single pool. Libraries will all be normalized to 2nM and pooled. The library pool will be sequenced on a single S4 lane (150PE) on a NovaSeq6000 (Illumina) at Novogene. This will yield approximately 70M raw reads/sample. All pre-processing (i.e. adaptor removal and quality check), annotation (mapping to the latest updated reference pig genome (currently,Ensemble Sscrofa 11.1)), normalization, and differential expression analyses will be performed using the CLC Genomics Workbench (Qiagen). Functional analysis of expression data will be conducted with established tools (e.g. Ingenuity Pathway Analysis software-IPA) to identify common vs. specific pathways differentiating the cell subsets in response to PRRSV infection. Heatmap comparison analysis tool will be used to visualize clusters of biofunctions. Validation tests will be performed in new preparations of the target porcine cells. Depending on hypotheses, we will choose the best approach e.g. qPCR, pathway inhibitors experiments, gene silencing or functional studies (Crisci et al. 2019, in preparation) for validations.Statistical Analysis. Six animals will be used for genomics data. Since conventional statistics are not well designed for "omics methods" and machine learning approach can be additionally used for omics data, we will consider an N=6 appropriate for those type of analysis as previously described14. All the other data will be analyzed using GraphPad Prism 8 (GraphPad software, CA). Non-parametric approaches will be used when few number of samples will be available. The Mann-Whitney's test will be used to compare unpaired samples based on ranking. For paired samples, the Wilcoxon matched-pairs signed rank test will be used. Normally distributed data recorded throughout the study will be analyzed using a repeated-measures two-way ANOVA or Student T test. Differences will be defined significant (*) for P<0.05.