Marek's Disease Virus–Induced Immunosuppression: Array Analysis of Chicken Immune Response Gene Expression Profiling

Abstract

Marek's disease (MD) is a lymphoproliferative disease of chickens induced by a highly cell-associated oncogenic α-herpesvirus, Marek's disease virus (MDV). MDV replicates in chicken lymphocytes and establishes a latency infection within CD4⁺ T cells. Host-virus interaction, immune responses to infection, and transcriptional profiling of chicken gene expression in MD are poorly understood. In this study we conducted a global host gene expression analysis in the splenocytes of MDV-infected chickens using oligonucleotide-based Affymetrix GeneChip Chicken Genome Arrays. These arrays contain probes for more than 32,000 chicken transcripts and most of the known MDV genes and open reading frames. Two-week-old MD-susceptible chickens were inoculated with an oncogenic strain of MDV, and spleen samples were collected 5 and 15 days post-infection (dpi) for RNA isolation and microarray analysis. Array results displayed a significant differential pattern of immune response transcriptome between the two phases of MDV infection. The expression levels of more than 22 immune-response and related genes were downregulated, while the expression levels of at least 58 genes were increased at 5 dpi (cytolytic infection), compared to age-matched control birds. In comparison, out of 73 immune-response and related genes, 67 genes were downregulated, with only 6 genes having higher expression levels at 15 dpi (latency infection). Cytokines, chemokines, MHC molecules and related receptors, and adhesion molecules were among the many MDV-induced downregulated genes that are critical for an effective antiviral immune response. In addition, several apoptosis-associated genes were decreased in expression during latent infection, suggesting an MDV-induced blocking of initiation or progression of programmed cell death processes. These chicken arrays are valuable tools in understanding the molecular mechanisms behind viral pathogenesis and chicken gene expression patterns, and associated biological pathways in response to MDV infection.

Introduction

M arek's disease virus (MDV), a highly cell-associated oncogenic α-herpesvirus, is the etiological agent of Marek's disease (MD), a contagious T-cell lymphoproliferative disease of domestic chickens (13). Oncogenic strains of MDV induce an initial cytolytic phase of infection, mostly in B cells, between 3 and 6 days post infection (dpi), followed by a latent infection in the activated CD4⁺ T cells that lasts up to 2 wk prior to the reactivation and transformation phases of infection (11,52). The last phase of MDV infection is the reactivation of virus, characterized by a second cycle of lytic infection, immunosuppression, and development of lymphomas (58). The latently-infected CD4⁺CD8⁻ T cells carry the virus to the visceral organs, peripheral nerves, and feather follicle epithelium (FFE). FFE is the main anatomical site where infectious enveloped cell-free virus particles are produced and disseminated into the environment, and is therefore a common source of re-infection of birds sharing the same habitat (2,9,65). The shift from a lytic infection mainly in B cells, to a latent phase in the activated T cells, is likely regulated by the initial host immune responses to MDV infection (7). Establishment and maintenance of latency is a complex feature of the MDV life cycle, and it is speculated that host-virus interaction and subsequent immune responses play a critical role in its regulation, which leads to suppression of MDV genes with only limited viral antigen expression (64).

Innate and acquired immune responses, in addition to virus pathotype and genetic and physiological factors, are crucial in determining the outcome of MDV infection and the pathogenesis of MD (16). Studies have shown that impairment of immune responses during the early cytolytic infection delays establishment of latency with a prolonged lytic cycle and destruction of T and B cells, resulting in immunosuppressive effects (57). Macrophages, natural killer (NK) cells, cytokines, antibodies, and cytotoxic T lymphocytes, all play crucial roles in the outcome of MDV infection, and resistance or susceptibility of chickens to MD (1).

Recent advances in avian immunology and genetics has enabled researchers to investigate the roles of cytokines and chemokines in the innate and adaptive immune responses to MDV infection. Studies show that interferon (IFN)-γ expression is upregulated in the splenocytes of MDV-infected birds as early as 3 dpi (26). The transcriptional levels of interleukin (IL)-6 and IL-18 were also increased in the splenocytes of MD-susceptible chickens (26). The transcriptional activities of these proinflammatory cytokines, however, were not detected in MD-resistant birds. It was speculated that IL-6 and IL-18 are the driving factors behind the immune response to MDV infection, leading to T-cell transformation in the susceptible line, and the maintenance of latent infection in resistant birds. In addition to IFN-γ, the transcriptional level of IL-8 has also been shown to be upregulated in the splenocytes of MDV-infected chickens (58). The expression level of inducible nitric oxide synthase that catalyzes the production of nitric oxide (NO) is positively influenced by increased transcriptional activity of IL-8 and IFN-γ (36,62). It has been suggested that the production of NO has a direct inhibitory effect on MDV replication (58). Another study by Xing and Schat (69) revealed that the production level of NO was lowest in the splenocytes of MD-susceptible birds compared to a resistant line. Parvizi et al. (47) have also investigated the possible roles of cytokines in chicken susceptibility or resistance to MD, by analyzing the transcriptional activities of IL-6, IL-10, IL-18, and IFN-γ in CD4⁺ and CD8⁺ T cells. The gene expression analysis of the T-cell subsets of MDV-infected susceptible and resistant chickens at different time points post-inoculation did not provide conclusive evidence of an association between MD resistance or susceptibility and cytokine gene expression pattern.

Macrophages also play an essential role in defense against MDV infection. Macrophage depletion prior to MDV challenge, or continuous depletion during the disease, has resulted in an increased incidence of MD-related complications and tumor development (19). Activated macrophages from MDV-infected chickens produce NO that is involved in the inhibition of viral replication (58).

Recent studies provide evidence for the critical role of NK cells in controlling herpesvirus replication and infection (15,31). Sharma and Coulson (59) have reported that NK cells have cytotoxic activities against MDCC-MSB1 cells. It has also been shown that MD-resistant chickens have higher NK activity than susceptible birds. This reduced biological activity was more pronounced in susceptible birds that had developed MD-induced tumors (23,60). In a recent study, Sarson et al. (53) reported an upregulation of NK lysine, granzyme A, and other cytolytic-associated transcripts during the early stages of MDV infection.

Although immune responses against viral infections are predominantly mediated by cytotoxic T lymphocytes, humoral immune responses also play an important role in the pathogenesis of herpesviruses. Antibody-mediated virus neutralization, and antibody-dependent cell cytotoxicity (ADCC) are two essential components of humoral immune responses against viral infections (37). Even though MDV-infected chickens develop precipitating and neutralizing antibodies, most are not relevant for a protective antiviral immune response. Considering the cell-associated nature of MDV, antibodies are of critical importance only when cell-free viruses infect chickens, or MDV antigens are expressed on cell surfaces (10,23). Antibody-dependent complement activation and ADCC will lead to the destruction of infected cells, and consequently control of viral replication or infection (1).

Cell-mediated immunity has been recognized as the most important protective immune response mechanism developed against MDV infection (45,59). Due to the lymphotropic and highly cell-associated nature of MDV, it has been rather difficult to clearly determine the specific roles of cytotoxic and helper T cells in MD pathogenesis. Depletion of either CD4⁺ or CD8⁺ T lymphocytes by neonatal thymectomy, and injection of antibodies against CD4 and CD8 molecules in herpesvirus of turkeys (HVT)-vaccinated and MDV-challenged chickens, was shown to prevent MDV-induced tumor development (40). Schat et al. (56) and Pratt et al. (48) developed a new assay system using reticuloendotheliosis virus-transformed cell lines expressing specific MDV genes to study cytotoxic responses against MDV. The results obtained from these and other studies indicate that the vaccines induce specific cytotoxic CD8⁺ T-cell activity against several MDV antigens (45,46).

The goal of this study was to further investigate immunological responses to MD by examining the transcriptional profiling of immune response genes and related proteins in the spleen tissues of chickens infected with a highly pathogenic strain of MDV during lytic and latent infection. Comparative analysis of host gene expression profiling between the infected and control birds indicates that a severe immune suppression involving different aspects of the innate and adaptive immune responses is induced during latent infection by a very virulent plus (vv+) strain of MDV. To our knowledge this is the first study to provide direct evidence for MDV-induced immunosuppression in chickens during latent infection by gene expression profiling. The transcriptional profiling of MDV genes resulting from this study has been previously published (21).

Materials and Methods

Experimental chickens

The chickens were F₁ progeny (15I₅ × 7₁) of Avian Disease and Oncology Laboratory (ADOL) line 15I₅ males and 7₁ females. The 15I₅ × 7₁ birds were from unvaccinated breeder hens and carried no maternal antibodies to MDV or HVT. The chicks were hatched at an ADOL poultry facility and housed in modified Horsfall-Bauer isolation units for the duration of the experiment.

Virus

A vv+ strain of MDV, 648A-p8 (passage 8), which is propagated and maintained in our laboratory, was used in this experiment (66).

RNA extraction and array processing

Total RNA was isolated from the spleen tissues of three MDV-inoculated and three control birds at 5 and 15 dpi (three biological replications) by TRIzol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Residual DNA was digested by DNase treatment of RNA samples using Ambion's RNase-free DNase kit (Austin, TX). The cDNA synthesis, labeling, hybridization, and scanning processes were all carried out at the Michigan State University Research Support Technology Facility (RSTF) according to the protocol provided by Affymetrix. Raw data were provided as CEL or Excel files for statistical analysis.

Real-time quantitative PCR for validation of microarray data

Real-time quantitative PCR analysis of relative quantification of selected chicken genes was carried out at the RSTF of Michigan State University in East Lansing, Michigan. Briefly, 4 μg of total RNA was used for cDNA synthesis using the SuperScript III First-Strand Synthesis System according to the manufacturer's protocol (Invitrogen). Each reaction consisted of 2.5 μL of diluted oligo-dT-based RT product (equivalent to 120 ng of starting material). In addition, 300 nM of each for specific sense and antisense primers were used in the presence of 5 μL of SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). The amplification program was as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 sec, followed by 60°C for 1 min. All the reactions were run in duplicate in a 7900HT Sequence Detection System (Applied Biosystems). The primers for chicken genes were designed using MacVector software (Accelrys, San Diego, CA). All the primers were synthesized by Operon Biotechnologies, Inc. (Huntsville, AL). The primer sequences and their amplicons are listed in Table 1. Relative quantification of select chicken genes for verification of array data was determined using the 2^−ΔΔCT method (34). The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Table 1.

Primers Used for Validation of Microarray Data Analysis

Name	Direction	Sequence	Product (bp)
MMP-13	Forward	5′-CCCAAAACATCCCAAAACGC-3′	108
	Reverse	5′-CGCCAGAAAAACCTGTCCTTG-3′
Bu1	Forward	5′-TTCGTCTGGAGAAAACTGTGCC-3′	140
	Reverse	5′-TGCCAAGATGTAGATGAACCACG-3′
IL-18	Forward	5′-GAGGTGAAATCTGGCAGTGG-3′	184
	Reverse	5′-GAATGTCTTTGGGAACTTCTCC-3′
IL-8	Forward	5′-CTCTGTCGCAAGGTAAGTGAATCC-3′	077
	Reverse	5′-CACACATCTCAGCAAGTGCCAAG-3′
IFN-α	Forward	5′-TACGGCATCCTGCTGCTCAC-3′	093
	Reverse	5′-AGAGAAGGTGGCATCCTGGG-3′
TLR-15	Forward	5′-TGGTGCTAACGGGCTTATGC-3′	141
	Reverse	5′-GCTCGCTGTATGAAATGAAGGC-3′
K203	Forward	5′-ATCCAACGGGAAGATGAAGC-3′	203
	Reverse	5′-TGATGAAGATGATGGCAGGC-3′
IL-6	Forward	5′-GACGAGGAGAAATGCCTGACG-3′	190
	Reverse	5′-CCGAGTCTGGGATGACCACTTC-3′
IFN-γ	Forward	5′-AAGTCAAAGCCGCACATCAAAC-3′	132
	Reverse	5′-CTGGATTCTCAAGTCGTTCATCG-3′
Mx1	Forward	5′-TGTAGGAGTTTTTGGGGTGCC-3′	130
	Reverse	5′-CTCTTCTCTGTCATTCAGCCTCTTG-3′
GAPDH	Forward	5′-TGCCATCACAGCCACACAGAAG-3′	123
	Reverse	5′-ACTTTCCCCACAGCCTTAGCAG-3′

Experimental design

One-day-old chicks were randomly distributed into two groups of 34 birds each in separate isolators (A and B). The birds in group A were inoculated subcutaneously with 10,000 plaque-forming units of 648A-p8 MDV at 2 wk of age. The birds in group B served as negative non-inoculated controls. Three birds from the infected and control groups were euthanized by CO₂ inhalation and necropsied at 5 and 15 dpi (for cytolytic and latency infection, respectively). The spleen tissues were collected and immediately stored in RNAlater at −20°C to prevent RNA degradation (Ambion).

Statistical analysis

The analysis of differential gene expression requires normalization within and between arrays to remove technological sources of variations. The normalization estimate of microarray data was based on a set of host-encoded housekeeping genes. These were triplicate spots of β-actin, GAPDH, and eukaryotic elongation factor-1α1 (EEF1α1). The data were normalized by the publically available software R v.2.4.1 using gcrma (68), followed by linear model analysis, to identify differentially-expressed genes between infected and control birds at 5 and 15 dpi. The list of genes differentially expressed was ranked based on the moderated t-statistics introduced by Smyth (61). In this approach, a gene-wise linear model is fitted based on the experimental design. For each gene, a moderated t-statistic is calculated using posterior standard deviation to extract those with maximal differential expression. The p values generated by this analysis were adjusted for multiple comparisons to control the false discovery rate, which is the expected proportion of false discoveries among the rejected hypotheses.

Results

A comparative global gene expression profile analysis was conducted between the infected and control chickens during the cytolytic and latent infection of MDV. Selection of the birds for microarray analysis was random for the lytic phase, and based on clinical signs (e.g., transient paralysis, depression, and crippling) for latency. The spleen tissues from three MDV-infected and three control chickens at 5 and 15 dpi were used for RNA isolation and array analysis (three biological replications). The RNA samples of the control birds were used as reference or baseline for detection of signal intensity in the array analysis. Although several thousand genes were differentially expressed in the infected chickens during both phases of infection compared to the corresponding age-matched uninfected control birds, we decided to concentrate only on those genes that play a direct or distant role in immune responses to infection. The chicken genome sequence is incomplete, and majority of the sequenced genes are not annotated and their functions are unknown. Listing of all these non-annotated genes with obscure functions here is unnecessary, and would have little-to-no effect on this discussion. The differentially-expressed immune-related genes identified at both phases of MDV infection were grouped into seven different categories based on their speculative function and homology (Tables 2 and 3).

Table 2.

Chicken Immune Response Genes Expression Profiling During Cytolytic Infection: Comparative Analysis Between Infected and Control Spleen Tissues

	Probe ID	Grouping of genes based on speculative function	Fold change
Leukocyte transendothelial migration
1	Gga.10714.1.S1_at	Adhesion molecule with Ig-like domain 2	−5.97
2	Gga.398.1.S1_at	Matrix metalloproteinase-13	−4.83
3	Gga.10186.1.S1_at	Cell adhesion molecule 2	−3.21
4	Gga.16689.1.S1_s_at	Parvin, beta	2.05
5	Gga.11828.2.S1_a_at	Ninjurin 1	2.08
Cytokines, chemokines, receptors, and related proteins
6	GgaAffx.7251.1.S1_at	Chemokine (C-C motif ) receptor 6	−2.87
7	GgaAffx.7467.1.S1_at	C-C chemokine receptor 8-like	−2.27
8	Gga.2535.1.S1_at	Latent transforming growth factor beta binding protein 1	−2.18
9	GgaAffx.7468.1.S1_at	Chemokine (C motif ) receptor 1	−2.13
10	Gga.986.1.S1_s_at	Tumor necrosis factor receptor superfamily, member 18	2.00
11	GgaAffx.7940.1.S1_at	Cytokine receptor common beta chain precursor-human	2.01
12	Gga.9882.1.S1_at	Similar to GM-CSF receptor	2.21
13	Gga.158.1.S1_a_at	Interferon regulatory factor 10	2.37
14	Gga.512.1.S1_at	K60 protein	2.45
15	Gga.801.1.S1_at	Interferon (alpha)	2.50
16	GgaAffx.11933.1.S1_s_at	Pre-B-cell colony enhancing factor 1	2.77
17	Gga.296.1.S1_at	Chemokine (C-C motif ) ligand 1 (CCL1)	2.82
18	Gga.11252.1.S1_at	Similar to small inducible cytokine A19 precursor (MIP-3-beta)	2.97
19	Gga.915.1.S1_at	Suppressor of cytokine signaling 3	3.11
20	GgaAffx.26468.1.S1_at	Chemokine binding protein 2	3.18
21	Gga.8774.1.S1_s_at	Tumor necrosis factor (ligand) superfamily, member 10	3.48
22	GgaAffx.8807.1.S1_s_at	Interleukin 22 receptor, alpha 2	3.49
23	GgaAffx.9569.1.S1_at	Cytokine receptor binding protein	3.64
24	Gga.9133.1.S1_at	Similar to chemokine ah221	3.92
25	Gga.255.1.S1_at	Chemokine K203	5.55
26	Gga.2769.1.S1_at	Interleukin 6 (interferon, beta 2)	5.62
27	Gga.6492.1.S1_at	Similar to chemokine CXCL13/BCA-1	5.68
28	Gga.4409.1.S1_a_at	Chemokine (C-C motif ) ligand 4	5.76
29	Gga.916.1.S1_at	Interferon gamma	6.23
Cell surface molecules/cluster of differentiation
30	GgaAffx.7930.2.S1_at	CD1d molecule	−4.24
31	Gga.6031.2.S1_s_at	Hepatitis A virus cellular receptor 1	−3.70
32	Gga.3652.1.S1_at	C-type lectin domain family 3, member B	−3.05
33	GgaAffx.12136.1.S1_s_at	B6.3 protein (Bu-1)	−2.94
34	GgaAffx.151.1.S1_s_at	CD79b molecule, immunoglobulin-associated beta	−2.71
35	GgaAffx.2421.1.S1_s_at	T-cell immunoglobulin and mucin domain containing 4	−2.70
36	Gga.475.1.S2_at	Follicle-stimulating hormone receptor	−2.21
37	GgaAffx.9118.1.S1_at	CD86 molecule	2.13
38	Gga.972.1.S1_at	Cytotoxic and regulatory T-cell molecule	2.29
39	Gga.4973.3.S1_x_at	Major histocompatibility complex class I, B-F minor heavy chain	2.33
40	Gga.17678.1.S1_x_at	Similar to immunoglobulin-like receptor CHIR-A	2.57
41	Gga.4973.1.S1_x_at	Major histocompatibility complex class I glycoprotein	2.58
42	GgaAffx.7930.1.S1_at	CD1a molecule	2.68
43	Gga.5069.1.S1_at	MHC class I antigen B-F minor heavy chain	3.22
44	GgaAffx.5458.1.S1_at	CTLA-4: cytotoxic T-lymphocyte-associated protein 4	3.48
45	GgaAffx.12647.1.S1_at	Similar to immunoglobulin-like receptor CHIR-B2 precursor	3.94
46	GgaAffx.10012.1.S1_at	Coxsackievirus and adenovirus receptor	4.02
Components of the innate and adaptive immune responses
47	Gga.7886.1.S1_at	C1q and tumor necrosis factor related protein 2	−3.95
48	Gga.713.2.A1_at	Lysozyme	−2.45
49	GgaAffx.24534.1.S1_at	C7 complement component 7	−2.43
50	GgaAffx.7505.1.S1_at	C1q and tumor necrosis factor related protein 1	−2.25
51	GgaAffx.21842.1.S1_s_at	Gal 7 (beta-defensin7)	2.05
52	GgaAffx.9256.1.S1_s_at	Complement component 1, s subcomponent	2.24
53	Gga.17647.1.S1_at	Neutrophil cytosolic factor 2	2.42
54	GgaAffx.8628.1.S1_at	Granzyme K (granzyme 3; tryptase II)	2.60
55	Gga.495.1.S1_at	Gallinacin 2	2.63
56	Gga.11658.1.S1_at	Bactericidal/permeability-increasing protein	2.66
57	GgaAffx.8585.3.S1_s_at	Toll-like receptor 3	3.09
58	GgaAffx.5126.1.S1_at	Toll-like receptor 15	3.09
59	GgaAffx.24484.2.S1_s_at	Complement component 1, r subcomponent	4.25
60	Gga.6713.1.S1_at	Granzyme A (T-lymphocyte serine esterase 3)	5.57
61	Gga.9103.1.S1_at	Lysozyme G-like 2	5.79
Apoptosis
62	Gga.9216.1.S1_at	Similar to proapoptotic caspase adaptor	−2.24
63	GgaAffx.23578.1.S1_at	Apoptotic peptidase activating factor 1	2.10
64	Gga.5164.1.S2_at	BCL2-related protein A1	2.50
65	GgaAffx.7315.1.S1_at	BCL2-like 14 (apoptosis facilitator)	4.27
66	Gga.16457.1.S1_s_at	Interferon induced with helicase C domain 1	4.66
Transcription factor, cell communication, signal transduction, and related proteins
67	Gga.6172.1.S1_at	Inositol polyphosphate-5-phosphatase F	−2.19
68	Gga.276.2.S1_a_at	Early B-cell factor 1	−2.12
69	Gga.18894.1.S1_s_at	Programmed cell death 1	2.23
70	GgaAffx.20135.1.S1_at	Signal transducer and activator of transcription 3	2.46
71	Gga.11597.2.S1_s_at	Signal transducer and activator of transcription 1	4.56
Tumorigenesis, antivirus, others
72	GgaAffx.21278.1.S1_at	B-cell CLL/lymphoma 11A (zinc-finger protein)	−3.87
73	Gga.9136.2.S1_a_at	Tumor-associated calcium signal transducer 1	2.11
74	Gga.12326.1.S1_s_at	MAX interactor 1	2.15
75	GgaAffx.1654.1.S1_at	Mucosa-associated lymphoid tissue lymphoma translocation gene 1	2.46
76	Gga.1094.1.S1_at	Cathepsin D	2.72
77	GgaAffx.11358.1.S1_s_at	Zinc-finger CCCH-type, antiviral 1	2.73
78	Gga.10787.1.S1_at	Similar to Jun dimerization protein p21SNFT	2.94
79	GgaAffx.21915.1.S1_at	Interferon-induced protein with tetratricopeptide repeats 5	4.34
80	Gga.131.1.S1_at	Mx1: myxovirus (influenza virus) resistance 1	7.09

The differentially expressed immune-related genes identified at both phases of MDV infection are grouped into seven different categories based on their speculative function and homology.

Table 3.

Chicken Immune Response Gene Expression Profiling During Latent Infection: Comparative Analysis Between Infected and Control Spleen Tissues

	Probe ID	Grouping of genes based on speculative function	Fold change
Leukocyte transendothelial migration
1	Gga.9915.2.S1_at	Platelet/endothelial cell-adhesion molecule (CD31 antigen)	−5.88
2	Gga.398.1.S1_at	Matrix metallopeptidase 13 (collagenase 3)	−4.68
3	GgaAffx.1733.5.S1_s_at	Selectin P (granule membrane protein)	−4.48
4	GgaAffx.4681.1.S1_at	Integrin alpha FG-GAP repeat, containing 3	−3.57
5	Gga.1784.1.S1_at	Integrin, alpha 8	−3.17
6	Gga.2967.1.S1_at	Integrin, alpha 6	−3.10
7	GgaAffx.20275.1.S1_s_at	Activated leukocyte cell-adhesion molecule	−2.99
8	Gga.4999.1.S1_a_at	Integrin, alpha 4 (antigen CD49D)	−2.93
9	GgaAffx.24760.1.S1_at	Junctional adhesion molecule 2	−2.74
10	GgaAffx.9093.1.S1_at	Integrin alpha FG-GAP repeat, containing 2	−2.64
11	Gga.16689.1.S1_s_at	Parvin, beta	−2.55
12	Gga.11915.2.S1_a_at	CD99 molecule-like 2	−2.30
13	Gga.815.1.S1_at	Integrin, alpha V (antigen CD51)	−2.20
Cytokines, chemokines, receptors, and related proteins
14	GgaAffx.11258.4.S1_s_at	Attractin	−5.30
15	Gga.804.1.S1_at	Transforming growth factor, beta receptor III	−5.28
16	GgaAffx.7262.1.S1_at	Tumor necrosis factor, alpha-induced protein 2	−4.53
17	GgaAffx.11679.1.S1_s_at	Similar to interleukin-4 receptor alpha-chain	−4.51
18	GgaAffx.10863.1.S1_at	Tumor necrosis factor (ligand) superfamily, member 11	−4.14
19	GgaAffx.2425.1.S1_at	Chemokine (C-C motif ) receptor 7	−4.12
20	Gga.13989.1.S1_s_at	Interferon (alpha, beta and omega) receptor 2	−3.98
21	Gga.10579.1.S1_s_at	Interleukin 17 receptor A	−3.90
22	Gga.9513.1.S1_a_at	Chemokine (C-X-C motif ) ligand 12	−3.83
23	GgaAffx.21754.1.S1_s_at	Interleukin 18	−3.27
24	Gga.2305.1.S1_at	Chemokine (C-X-C motif ) receptor 4	−3.24
25	GgaAffx.7940.2.S1_s_at	Similar to cytokine receptor common beta chain	−3.15
26	GgaAffx.10742.1.S1_at	Interleukin 1 receptor, type II	−3.07
27	Gga.910.1.S1_at	IL-2 alpha receptor	−3.05
28	GgaAffx.24185.1.S1_at	Interleukin 7 receptor, alpha chain	−2.97
29	GgaAffx.6294.1.S1_at	Suppressor of cytokine signaling 5	−2.96
30	Gga.826.1.S1_s_at	Interleukin 8	−2.81
31	Gga.12516.1.S1_s_at	Interleukin 20 receptor, alpha	−2.63
32	GgaAffx.7251.1.S1_at	Chemokine (C-C motif ) receptor 6	−2.61
33	GgaAffx.7467.1.S1_at	C-C chemokine receptor 8 like	−2.52
34	GgaAffx.22628.1.S1_at	Interleukin 17 receptor E-like	−2.51
35	Gga.4359.1.S1_s_at	Tumor necrosis factor (ligand) superfamily, member 13b	−2.49
36	GgaAffx.11933.1.S1_s_at	Pre-B-cell colony enhancing factor 1	−2.46
37	GgaAffx.21450.2.S1_s_at	Transforming growth factor, beta 3	−2.45
38	GgaAffx.21772.1.S1_s_at	Interleukin 10 receptor, beta	−2.42
39	GgaAffx.13085.1.S1_at	Chemokine (C-C motif ) receptor 8	2.04
40	GgaAffx.8729.1.S1_s_at	Suppressor of cytokine signaling 7	2.15
Cell surface molecule/cluster of differentiation
41	Gga.576.1.S1_at	Inducible T-cell co-stimulator ligand	−6.69
42	GgaAffx.10882.1.S1_at	Cysteinyl leukotriene receptor 2	−5.35
43	GgaAffx.8892.1.S1_at	Similar to immunoglobulin-like receptor CHIR-A2	−4.92
44	GgaAffx.7930.2.S1_at	CD1d molecule	−4.14
45	Gga.5153.2.S1_at	Similar to MHC Rfp-Y class I alpha chain	−3.92
46	GgaAffx.12136.1.S1_s_at	B6.3 protein (Bu-1)	−3.81
47	GgaAffx.512.1.S1_at	Platelet-activating-factor receptor	−3.53
48	Gga.4900.6.S1_x_at	CD69 molecule	−3.17
49	GgaAffx.26062.1.S1_s_at	Similar to immunoglobulin-like receptor CHIR-B3	−2.84
50	Gga.518.1.S1_x_at	MHC class II antigen B-F minor heavy chain	−2.66
51	GgaAffx.9855.1.S1_at	CD80 molecule	−2.52
52	Gga.4569.1.S1_at	Polymeric immunoglobulin receptor	2.08
Components of the innate and adaptive immune responses
53	GgaAffx.7943.1.S1_s_at	Neutrophil cytosolic factor 4	−3.23
54	Gga.11057.1.S1_s_at	Similar to MHC class II transactivator	−2.91
55	GgaAffx.23341.5.S1_s_at	Complement component (3d/Epstein-Barr virus) receptor 2	−2.41
56	GgaAffx.11201.2.S1_s_at	Toll-like receptor 1	−2.39
57	Gga.6713.1.S1_at	Granzyme A (T-lymphocyte-associated serine esterase 3)	3.82
Apoptosis
58	Gga.19441.1.S1_s_at	Bcl2-associated transcription factor 1	−3.66
59	Gga.504.1.S1_at	Caspase 2	−2.60
60	Gga.4916.1.S1_s_at	Programmed cell death 4	−2.33
Transcription factor, cell communication, signal transduction, and related proteins
61	GgaAffx.11846.1.S1_s_at	Janus kinase 1	−5.40
62	Gga.13269.1.S1_at	Endothelial differentiation, sphingosine-1-phosphate receptor 1	−3.33
63	Gga.12733.1.S1_a_at	TNF receptor-associated factor 3 interacting protein 1	−3.20
64	Gga.14057.1.S1_a_at	Similar to immune costimulatory protein B7-H4	−2.58
65	Gga.276.2.S1_a_at	Early B-cell factor 1	−2.45
66	Gga.12961.1.S1_at	Programmed cell death 1	2.76
Tumorigenesis, antivirus, others
67	GgaAffx.4921.7.S1_s_at	B-cell CLL/lymphoma 11A (zinc finger protein)	−4.27
68	GgaAffx.10979.1.S1_s_at	LATS, large tumor suppressor	−3.18
69	GgaAffx.12967.1.S1_s_at	Heat shock protein 90-kDa alpha	−2.49
70	Gga.13140.2.S1_at	Influenza virus NS1A binding protein	−2.48
71	GgaAffx.21053.1.S1_at	BCL6 co-repressor	−2.32
72	GgaAffx.21710.1.S1_s_at	Interferon-related developmental regulator 1	−2.20
73	Gga.131.1.S1_at	Myxovirus (influenza virus) resistance 1	4.55

The differentially expressed immune-related genes identified at both phases of MDV infections are grouped in 7 different categories based on their speculative function and homology.

Chicken immune response gene expression profiling

Table 2 depicts the normalized expression profiles of the chicken immune response and related genes during cytolytic infection (5 dpi). Out of 80 genes differentially expressed during this phase of infection, at least 22 genes were downregulated (2−5.97-fold difference) in comparison to age-matched negative controls. In addition, the expression levels of 58 genes were increased (2−7.09-fold difference) in the infected birds compared to uninfected control chickens. Table 3 depicts the normalized expression profiles of the chicken immune response and related genes during latent infection (15 dpi). In comparison to the lytic phase of infection, there seems to be generalized suppression of the expression levels of most immune-response genes and related proteins. Out of 73 differentially-expressed genes in this stage of infection, only six genes had higher expression levels (2−4.55-fold difference) in the infected birds compared to the uninfected control chickens. At least 67 genes showed lower levels of expression (2−6.69-fold difference) in the infected chickens than in the un-inoculated control birds. For comparative analysis, the data from the cytolytic and latent infection were compared to those of the age-matched control chickens as the baseline.

Real-time PCR validation of array data

Table 4 depicts the expression analysis of selective chicken genes that were differentially expressed based on the array data during the lytic and latent infection. Total RNA from the spleen tissues of three MDV-infected and three age-matched control birds were used for SYBR Green-based real-time PCR analysis of 10 differentially expressed genes at 5 and 15 dpi. All the samples were run in duplicate, and the mean C_T values obtained were used to determine the fold changes in gene expression using a chicken GAPDH gene for normalization. For the genes that were downregulated during either phase of infection (MMP-3, Bu-1, IL-18, and IL-8), the fold differences were calculated using the infected samples as the reference or baseline. For the upregulated genes, the control samples were used as the baseline. The real-time PCR analysis of the expression pattern of this set of randomly-selected genes provides strong evidence for the validity of the array data.

Table 4.

Validation of Array Data Using the Comparative C_T Method: Relative Fold Differences in Select Chicken Gene Expression at 5 dpi, 15 dpi, or Both Time Points, Based on Their Expression Pattern in the Array Data

Genes	Days post-infection	C_T mean ± SD infected	C_T mean ± SD control	ΔΔC_T ± SD	Fold difference (real-time PCR)	Fold difference (array analysis)
MMP-13	5	25.07 ± 0.51	21.32 ± 0.51	−4.15 ± 0.72	17.8^a	−4.83
	15	25.58 ± 0.47	21.42 ± 0.09	−3.54 ± 0.48	11.6^a	−4.68
Bu1	5	19.99 ± 0.58	17.42 ± 0.57	−2.97 ± 0.82	07.8^a	−2.94
	15	21.34 ± 0.51	17.56 ± 0.15	−3.15 ± 0.53	08.9^a	−3.81
IL-18	15	23.31 ± 0.74	20.13 ± 0.20	−2.55 ± 0.77	05.9^a	−3.27
IL-8	15	28.42 ± 0.38	24.74 ± 0.31	−3.05 ± 0.49	08.3^a	−2.81
IFN-α	5	19.65 ± 1.46	23.42 ± 0.69	−3.37 ± 1.61	10.34^b	+2.50
TLR-15	5	19.21 ± 0.72	21.16 ± 0.57	−1.55 ± 0.84	02.9^b	+3.09
K203	5	17.72 ± 0.68	23.88 ± 0.90	−5.75 ± 1.13	54.2^b	+5.55
IL-6	5	−21.45 ± 0.84	26.46 ± 0.74	−4.61 ± 1.12	24.4^b	+5.62
IFN-γ	5	21.06 ± 0.55	26.02 ± 0.62	−4.56 ± 0.82	23.6^b	+6.23
Mx1	5	19.02 ± 0.88	24.23 ± 0.56	−4.72 ± 1.04	26.4^b	+7.09
	15	22.79 ± 0.64	25.37 ± 0.32	−3.21 ± 0.71	09.3^b	+4.55
GAPDH	5	14.13 ± 0.43	14.53 ± 0.62
	15	15.75 ± 0.61	15.12 ± 0.07

For the downregulated genes (MMP-13, Bu1, IL-18, and IL-8; with higher Ct values in the infected tissues), the fold differences were calculated using the infected samples as the reference or baseline. In these genes the fold difference values obtained represent higher expression levels in the control tissues in comparison to the infected ones.

The fold differences for the remaining genes (upregulated) were calculated using the control tissues as the baseline.

The Ct values represent the average of three biological replications (three chickens) each run in duplicates.

Discussion

MDV, the etiological agent of MD, is an α-herpesvirus that causes various clinical syndromes, including T-cell lymphomas in chickens. To provide insights into the molecular mechanism of virus pathogenesis and chicken immune responses to MDV infection, we conducted a microarray experiment using Affymetrix chicken genome arrays. These DNA chips contain probes for both chicken and MDV transcripts. The MDV gene expression profiling of this study has previously been published (21). Despite the differential expression of more than 3,250 chicken genes during both phases of infection, here we will only discuss the transcriptional activities of immune-response genes and related proteins (see the materials and methods section for further details).

Table 4 shows the results of real-time quantitative PCR analysis of the set of chicken gene expression used for validation of array data. Ten differentially-expressed genes from both phases of infection were randomly selected and analyzed for expression pattern at 5 and 15 dpi using the same RNA samples that were used for microarray study. The expression levels obtained for these selected chicken genes follow the same pattern of expression observed in the array study (Table 4, two far right columns), providing evidence for validation of the array data. It should be mentioned that in theory the fold change differences for the differentially-expressed genes should be the same in both real-time PCR and array analysis. The fold changes in our array study, however, represent log-based data normalized to three different housekeeping genes (β-actin, GAPDH, and EEF1α1), while the fold changes obtained in real-time PCR analysis are simply the raw data normalized to a single housekeeping gene (GAPDH).

MDV-induced immune suppression can be divided into a transient phase associated with the early cytolytic infection, and a more permanent phase following the reactivation and tumorigensis stage of MDV infection. The cytolytic infection of MDV involves a burst of productive/restrictive infection mainly in B cells, followed by latent infection in CD4⁺ T cells that eventually results in a significant reduction of lymphocyte populations and an irreversible bursal/thymic atrophy (11,52,66,67). Destruction of B and T lymphocytes has direct negative impact on the chicken adaptive immune responses, that consequently leads to severe immunosuppression (38). Apoptosis is the likely mechanism of MDV-induced lymphocyte destruction (39). It has been reported that monocytes/macrophages can also become infected by vv+ strains of MDV. It is not clear, however, whether this event leads to a productive infection in the phagocytic cells (4). In addition to the destruction of CD4⁺ T cells that selectively undergo apoptosis, downregulation of CD8 antigen on CD4⁺CD8⁺ and CD4⁻CD8⁺ lymphocytes has also been reported during MDV cytolytic infection (39). CD8 antigen interacts with invariant components of the MHC I molecule and plays a critical role in the process of antigen recognition. MDV-induced downregulation of CD8 will lead to reduced biological activity of cytotoxic T cells, and consequently immune suppression in the cell-mediated immune response (39,58).

Modulation of MHC molecules is another immunosuppressive and immune surveillance evasion strategy of MDV. The downregulation of MHC I in a OU2 fibroblast cell line and chicken embryonic fibroblasts have been reported (24,33). It is speculated that MHC I downregulation is due to MDV-induced impediment of peptide transport or retention of MHC molecules in the endoplasmic reticulum (24). Unlike other herpesviruses, MDV induces an upregulation of MHC II in infected cells (42). The authors speculate that the upregulation of MHC II may contribute to virus spread by increasing the chance of contact between the infected cells and the activated susceptible T cells.

The final stage of MDV infection is reactivation of virus replication, further destruction of lymphocytes, tumor development, and permanent immune suppression (10). The immunosuppressive nature of tumor cells was investigated by some early mitogen stimulation assays. It was shown that the presence of MDV-transformed lymphoblastoid cells prevented the lymphoproliferative responses of normal spleen cells to mitogen stimulation (50). One suggested mechanism for the immunosuppressive nature of tumor cells is the decreased expression of MHC I antigen after activation that leads to viral antigen expression (24). Downregulation of CD28 (a co-stimulatory molecule essential for the activation of T cells) on lymphoma cells has been suggested as another potential mechanism of the immune suppressive/evasive activities of tumor cells (6). And finally, it has been speculated that high expression levels of CD30 on tumor cells might play a role in the immunosuppressive nature of tumor cells by induction of a Th-2 type immune response in MDV-infected chickens (55).

In the present study, the results obtained from array analysis of chicken gene expression profiling at 5 dpi revealed that at least 22 host genes were reduced in expression in comparison to uninfected control birds. In addition, close to 58 immune-response genes and related proteins were increased during this early phase of MDV infection. Of the downregulated genes seen during both phases of infection, adhesion molecules and matrix metalloproteinases (MMPs) are among the genes that play critical roles in the transmigration of leukocytes and the host defense against infection. The recruitment of leukocytes from the circulation into infected tissues is mediated by adhesion molecules and their corresponding receptors between leukocytes and endothelial cells (8). A virally-induced suppression of adhesion molecules or complete lack of expression due to a genetic mutation will have detrimental consequences for the afflicted animals. Leukocyte adhesion deficiency syndrome is an autosomal recessive congenital disease characterized by recurrent bacterial infection associated with marked persistent neutrophilia, impaired inflammatory responses, delayed would healing, and stunted growth. The molecular basis for this congenital disease is a point mutation in the CD18 gene (a subunit of β2-integrin) that leads to a severe reduction of the biological activity of neutrophils, including adherence, chemotaxis, and phagocytosis (5,28,41). MMPs, on the other hand, have been implicated in many physiological and pathological processes, including the migratory capacity of NK cells and the extracellular matrix proteolysis and remodeling that mediates NK cell extravasation to tumor sites (18,29).

In addition to adhesion molecules and MMP-13, chemokine receptors, components of the complement system, Bu-1, and lysozyme have also been found to be reduced in expression during the lytic phase of infection. Bu-1 is a B-cell surface molecule (63) that is reduced in expression, likely due to early infection and destruction of B cells. In a microarray analysis of immune-specific gene expression profiling, Sarson et al. (54) also reported a significant downregulation of Bu-1 B-cell surface marker in MDV-susceptible chickens.

The genes that have been found to be upregulated during the cytolytic phase of MDV infection include several chemokines (CXCL13, K60, and K203), cytokines (IL-6, IFN-α, and IFN-γ), toll-like receptors (TLR-3 and TLR-15), MHC I, MHC II, granzymes, and antimicrobial peptides (Gal2 and Gal7). A comparative gene expression analysis of a panel of chicken cytokines/chemokines and related proteins between cytolytic and latent infections of MDV has been previously documented and is a validation of the array data (22). Another gene that has the highest level of expression in both phases of the MDV life cycle (Tables 2 and 3) is Mx1 (myxovirus resistant 1) gene. Mx proteins are members of a large family of interferon-inducible GTPases found in many organisms that function as important components of innate immunity against a wide range of viruses, including influenza viruses (20). Whether Mx1 protein plays an active role against MDV infection is not clear and remains to be investigated.

Table 3 depicts the list of genes that have been differentially expressed during the latent infection of MDV. The suppression of various components of the immune system is evident from the significant reduction in the expression of many host proteins associated with the innate and adaptive immune responses. The significance of MDV-induced downregulation of adhesion molecules and MMPs has already been discussed. In addition, many cytokines, chemokines, cytokine/chemokine receptors, and associated proteins have been suppressed in their transcriptional activities. Of the cytokine genes downregulated at this stage of infection, IL-18 is of critical importance. IL-18 is a proinflammatory cytokine and a potent inducer of IFN-γ production in NK and T cells (43). The proinflammatory function of this cytokine is mediated by production of TNF-α and IL-1β (49). IL-18 also induces production of IL-8, a strong chemoattractant and activator of neutrophils (32). The suppression of IL-18 expression could be one explanation for the downregulation of IL-8 (no. 23 and 30 in Table 3).

The data presented here clearly indicate that there is a generalized subversion of immune responses induced by a highly pathogenic strain of MDV during latency. It is intriguing to realize that this severe suppression of chicken immune responses is induced during a rather inactive phase of the MDV life cycle, in which only a few MDV genes are transcriptionally active (21). The most direct cause of viral-induced immunosuppression is the destruction of B and T lymphocytes or their precursors. MDV cytolytic infection is characterized by depletion of B and T cells that is likely induced by apoptotic-associated cell death (39). It is conceivable that MDV strains with very high virulence (vv+) do not establish a latent infection or initiate the reactivation phase of infection faster than less virulent strains of MDV (12). This continuous phase of active infection and replication undoubtedly leads to a massive destruction of lymphocytes, and consequently prolonged immunosuppression.

The more logical explanation for this extended immune response suppression is that one or more of the limited number of MDV genes that are active during latency functions as an immune-suppressive agent. This assumption becomes a more likely scenario when the antiapoptotic nature of MDV is considered. Herpesviruses not only can trigger apoptosis during productive infection, but they also have the ability to block it during the latent and reactivation phases of infection (17). Our data also indicate that several genes that are directly or indirectly associated with apoptotic processes are reduced in expression. These include Bcl2-associated transcriptional factor 1, caspase 2, and programmed cell death 4, and TNF-associated proteins (Table 3). Caspase 2 is one of the well-conserved mammalian caspases that has been implicated in p53-mediated apoptosis in different experimental systems. Biochemical analysis provides evidence that caspase 2 acts as an initiator of apoptosis, with an increased expression level and biological activity during the process of programmed cell death (3,30). Programmed cell death 4 is a recently discovered tumor suppressor gene with increased expression following the onset of apoptosis (14,25). It has also been shown that overexpression of Bcl2-associated transcriptional factor 1 results in apoptosis and transcriptional repression (27). Among the many known physiological inducers of programmed cell death, TNF is the most potent and well studied. In addition to TNF, many other members of its superfamily are also known to be involved in the process of apoptosis under different physiological conditions (51). Despite its misleading name, programmed cell death 1, which is upregulated at 15 dpi (no. 66 in Table 3), appears to play no role in apoptosis, and instead functions as an inhibitor of lymphocyte activation, which on its own is an indication of the immunosuppressive nature of MDV (44). In addition, meq oncogene, one of the few MDV genes active during all three phases of MDV infection, is known to act as an antiapoptotic oncoprotein (35). Furthermore, our preliminary studies indicate that a meq-deleted mutant MDV is less immune suppressive than a parental wild-type virus.

In summary, the data presented here provide evidence that highly pathogenic strains of MDV induce severe and prolonged immune suppression by repression of the transcriptional activities of many genes that are critical components of both the innate and adaptive immune responses. Although only a limited number of genes associated with apoptosis were annotated and identified in this study, there are indications that a vv+ MDV also blocks the initiation or progression of apoptosis during latent infection, likely by an unknown meq oncoprotein-mediated mechanism that leads to the suppression of many genes that play essential roles in programmed cell death.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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