Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
(N/A)
Non Technical Summary
Inflammatory stimuli, such as infectious disease, incur significant costs to swine production. One important consequence of inflammation is the redirection of metabolic resources away from weight gain, resulting in significant production losses. CD163, an important receptor for porcine reproductive and respiratory syndrome virus (PRRSV) also participates in the regulation of inflammation. In preliminary work we showed that CD163 knockout (KO) pigs infected with a porcine circovirus type 2d (PCV2d), another important pig pathogen, showed resistance to infection. We propose that the absence of CD163 protects pigs by downregulating pathogen-mediated inflammatory responses. The proposed research project tests this hypothesis by measuring inflammatory responses in CD163 wild-type (WT) and KO pigs during infection with PCV2d. The experimental approach is the application of novel gene profiling and pathway analysis methods to map inflammatory responses in individual immune cell populations. Objective 1 is to compare gene profiles in macrophages from CD163 WT and KO pigs. Macrophages are chosen for study because they express CD163, and as a result are key mediators in the initiation and regulation of inflammation. The methods and results from Objective 1 will be applied to subsequent objectives. Objective 2 is to evaluate gene expression in CD163 WT and KO macrophages during infection with PCV2d and other macrophage-tropic viruses. Gene profiles will be constructed in macrophages during infection with PCV2d and a variety of important pig coronaviruses. The results will be used to understand the effect of PCV2d infection on the inflammatory response of macrophages and to determine if the response is a general property that can be applied to other pathogens. Objective 3 is to construct gene profiles of immune cell populations in WT and KO pigs during PCV2d infection. Gene profiles and inflammatory response pathways will be mapped in important cell populations sorted from the blood of pigs. Important immune cell populations investigated include monocytes, B cells, and T cells. Blocking CD163 function may protect pigs from inflammation caused by non-infectious stressors, such as weaning, environmental changes, and inflammatory cytokines produced in response to fast-growth diets. Altogether, the regulation of CD163 function would result in a pig that is better adapted to the modern production system.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
CD163 is an important receptor for porcine reproductive and respiratory syndrome virus (PRRSV). Pigs lacking CD163 fail to support PRRSV replication and are completely resistant to disease. Preliminary studies showed that pigs lacking CD163 are protected from the intense inflammatory response caused by another virus, porcine circovirus Type 2d (PCV2d), an important pathogen of pigs. PCV2d is a systemic pathogen that produces respiratory distress, a result of the release of pro-inflammatory cytokines. Since CD163 participates in the regulation of the inflammatory response, we propose that blocking CD163 function will downregulate inflammation resulting in protection from inflammatory stimuli produced by viruses and pathogenic bacteria. One important consequence of inflammation is the redirection of metabolic resources away from weight gain, resulting in significant production losses. Therefore, in a larger context, inhibition of CD163 function may enhance growth performance by protecting pigs from inflammation caused by other stressors, such as weaning, environmental changes, and the inflammatory cytokines produced in response to fast-growth diets. Altogether, the regulation of CD163 function would result in a pig that is better adapted to the environment present in the modern production system. The overall goal for this project is to understand the mechanism of how the removal of CD163 protects pigs from the inflammatory responses produced during infection with PCV2d. The experimental approach incorporates transcriptomic analysis of cytokine and other genes. The gene profiles in isolated cell populations are then used to construct host metabolic and immune response pathways. The hypothesis to be tested is that genomically modified pigs lacking CD163 will exhibit inflammatory response pathways that are downregulated during infection with PCV2d. Gene response pathways will also be linked to specific pathological changes associated with PCV2d infection. The first objective is to develop an in vitro system that can be used to investigate the response of cells.Objective 1. Analyze gene expression profiles in porcine alveolar macrophages (PAMs) and monocyte-derived macrophages (MDMs) from CD163 WT and KO pigs. As a first experiment we will characterize differences in the transcriptome profiles of freshly isolated PAMs from CD163 WT and CD163 KO pigs. PAMs from WT pigs are about 90% positive for CD163, providing a rich source of CD163-positive cells for use in this study. A second source of CD163-positive cells, monocyte-derived macrophages (MDMs), are derived from peripheral blood mononuclear cells (PBMCs) following stimulation with granulocyte-monocyte colony stimulating factor (GM-CSF). Both sources of CD163-positive macrophages are easily infected with PCV2d and other viruses. A second goal under this objective is to compare the gene expression profiles of MDMs versus PAMs. If the profiles are similar, MDMs will provide a convenient substitute for PAMs. The experiments under this and the other objectives incorporate RNA-seq and workflow analysis to identify unique gene expression patterns or biomarkers that can distinguish WT and CD163 KO pigs and yield baseline data for objectives 2 and 3.Objective 2. Study the role of CD163 in macrophages after infection with PCV2d and other macrophage-tropic viruses. Based on the results from Objective 1 we will incorporate CD163-positive cells obtained from the lung or MDMs. Since the collection of PAMs requires the sacrifice of the pig, MDMs provide a convenient source of CD163-positive cells. The purpose of this objective is to investigate the role of CD163 in the response of macrophages to infection. The experimental approach is to analyze gene expression profiles in macrophages infected with PCV2d, transmissible gastroenteritis virus (TGEV), or porcine deltacoronavirus (PDCoV). The results will provide an in vitro model system that can be used to study the function of CD163 without infecting the pig. There is a large amount of data showing that PCV2d infection stimulates the production of inflammatory cytokines and genes in alveolar macrophages. The hypothesis to be tested is that macrophages lacking CD163 will show an altered gene profile compared to infected WT macrophages.Objective 3. Investigate gene expressions profiles in immune cell populations following infection of pigs with PCV2d. Preliminary work indicates that the absence of CD163 provides disease protection during infection with PCV2d. Under this objective we will explore a mechanistic basis for reductions in disease and viremia during PCV2d infection. The hypothesis is that pigs lacking CD163 will show suppressed pro-inflammatory responses following PCV2d infection. Four groups of pigs will be evaluated: 1) CD163 WT non-infected, 2) CD163 WT infected, 3) CD163 KO non-infected, and 4) CD163 KO infected. The experimental approach is to measure immunity by evaluating gene expression profiles in individual immune cell populations, including CD4-positive helper T cells, CD8-positive effector cells, CD4-CD8 double-positive memory T cells, monocytes, and B cells. Immune effector function will be evaluated by measuring virus neutralization activity in serum along with measurements of viremia and total antibody.
Project Methods
CD163 KO and WT pigs. The CD163-modified and WT pigs are from a pathogen-free source, free of PRRSV, PCV2 and other pathogens. Pigs are provided by Randy Prather at the University of Missouri, who is the director of the National Swine Resource and Research Center. CD163 WT and KO pigs used in the experiments are from the same litters. The breeding process incorporates crossing CD163+/- females with CD163-/- males. The result is an expected offspring ratio of 50% KO (CD163-/-) and 50% heterozygous WT (CD163+/-). The heterozygous pigs retain a WT phenotype.Isolation of macrophages.Porcine alveolar macrophages (PAMs) are from lungs obtained from euthanized pigs. Lung lavage by pouring 100 ml of cold PBS into the trachea. The lungs are gently massaged and contents poured into 50 ml centrifuge tubes and stored on ice.PAMs are sedimented by centrifugation. Monocyte-derived macrophages (MDMs) are from from peripheral blood mononuclear cells (PBMCs), which are plated and allowed to differentiate into MDMs GM-CSF.Virus infection of macrophages. Approximately 105 macrophages per well are added to a 12 well plate and incubated overnight then washed to remove non-adherent cells. Serial 1:10 dilutions of each virus in media are added. The PCV2d used in this project is a field isolate from an infected pig. Coronaviruses include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PCov). Virus-infected cells are detected by fluorescent antibody staining.Cell phenotyping and sorting. Approximately 1X106 cells are used for staining. A minimum of 10,000 cells are analyzed for each sample. Cell sorting is performed on a Sony SH800 Cell sorter. Sorting is performed by personnel in the immunology core facility, using protocols developed during the first year of the project.PCV2d infection of pigs. Four-week-old WT and KO pigs receiving the same treatment are maintained in the same room throughout the study period. Personnel are blind as to the genotype of each pig. The PCV2d isolate used in these studies was derived from a pig with severe PCVAD. For infection, one half of the PCV2d inoculum containing approximately 105 virions is administered intramuscularly and the other half of the inoculum administered intranasally. Control pigs receive culture media used to propagate the virus. Under Objective 3, four groups of pigs, five pigs per group, are incorporated into each study: 1) uninfected CD163 WT, 2) uninfected CD163 KO, 3) infected WT, and 4) infected KO. WT and KO pigs with the same infection status are maintained in the same room, for a total of 10 pigs per room. This design ensures that all pigs with the same infection status pigs, regardless of genotype, are maintained under the same environmental conditions. Pigs are followed for 42 days after infection and then necropsied. PBMC are obtained from blood collected on a weekly basis. PCV2d viremia in serum is measured using PCR. PCV2d-specific antibodies are measured using a virus neutralization assay. Pigs are followed for clinical signs throughout the study. At the end of the study, lungs and tissues are removed and assessed for gross and micro-histological changes and scored for pathology. Blood samples are collected weekly until the end of the study, at 42 days. The lungs are scored for gross pathology. Tissue samples collected from lung, tracheobronchial lymph node, inguinal lymph node, and other affected organs are removed and immediately placed in 10% neutral buffered formalin. The fixed tissues are processed and evaluated for pathology. PCV2d viremia is measured in serum.Measurement of PCV2d nucleic acid in serum. For the measurement of PCV2d viremia, viral DNA is extracted from serum or tissue using Ambion's MagMAX 96 Viral Isolation Kit (Applied Biosystems) in accordance with the manufacturer's instructions. PCV2d DNA is quantified using a SYBR Green PCR method developed in our lab. Plasmid DNA containing a positive control template is used to prepare standards. The PCR assay results are reported as log10 PCV2d DNA starting quantity per 20 μl reaction volume.Isolation of mRNA from cells and tissues and sequencing. Cell pellets are homogenized and RNA extracted using the RNeasy kit (Qiagen). RNA quality is assessed using a bioanalyzer prior to library preparation. Poly-A RNA selection for enrichment of mRNA prior to construction of strand specific RNA library using a TruSeq RNA Library Kit (Illumina). Library quality is assessed and then sequenced on an Illumina NovaSeq 6000. A depth of 20 million reads is targeted for each library.Gene expression profiling. The approach for comparing inflammatory immune responses in cell populations from CD163 KO and WT cells is RNA-Seq followed by a novel analytical workflow designed by Dr. Gaulke, CoPD. The workflow maximizes speed, accuracy, and statistical rigor by using highly benchmarked RNA-Seq analysis tools. In summary, quality filter reads are prepared and aligned to the pig genome (Sscrofa11.1) using the STAR RNA-Seq aligner. Count tables are input into DESeq2, which is used to quantify differential gene expression. To identify sets of genes or biomarkers that will stratify groups principal components are calculated and the genes that most strongly correlate with the top axes (the axes that explains the most variation in gene expression) are collected (custom R scripts). These genes are then used to identify the functional pathways that differ between groups. Gene set enrichment analysis (GSEA) is performed using gprofiler2. QPCR is performed to confirm the RNA-Seq results. The PrimePCR SYBR Green Assay incorporates forward and reverse gene-specific primers. Expression is normalized to the beta-2-microglobulin gene.Statistical analysis. Descriptive statistics include mean and standard deviation. Statistical analysis is performed using the GraphPad Prism software package. Data incorporating repeated measures are analyzed by one way analysis of variance followed by the Tukey post-test. Differences at specific time points are analyzed by the Kruskal-Wallis test. Comparison of two groups is performed using the Mann-Whitney U test.