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1 Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
2 Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
Correspondence
Zheng Xing
zxing{at}ucdavis.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Twigg, 2004). In influenza virus-infected hosts, mononuclear cells become infected after the primary target cells, respiratory ciliated epithelial cells (Fujisawa et al., 1987; Kaufmann et al., 2001). How macrophages function after influenza virus infection is critical in the host's immune response to the virus and, thus, to viral pathogenesis.
H9N2 viruses are among the most commonly occurring in domestic poultry populations, with several outbreaks reported in Asia and North America since 1990. H9N2 subtype viruses are classified as low-pathogenicity viruses both by molecular characterization and by pathotyping. This subtype is unique among low-pathogenicity avian influenza (LPAI) viruses in that they infect a wide variety of species including chickens, quail, turkeys, ducks, geese, pigs and humans (Alexander, 2000; Peiris et al., 2001; Tang et al., 1998). Because H9N2 viruses infect a number of species, they may be significant donors of genetic material to emerging human pathogens (Guo et al., 1999; Lee et al., 2000; Matrosovich et al., 2001; Peiris et al., 1999). There is no evidence to suggest that H9N2 subtype viruses can be transmitted from human to human (Uyeki et al., 2002). However, they could emerge as human pathogens through reassortment in intermediate hosts, such as pigs (Peiris et al., 2001) and in avian species, or through direct adaptation in the human host (Guan et al., 1999, 2000). The ability of H9N2 subtype viruses to spread widely in domestic poultry populations (Alexander, 2000; Chen et al., 1994; Naeem et al., 1999), to cross species barriers and potentially to impact on emerging viruses (Guo et al., 1999; Peiris et al., 1999) warrants further studies on the pathogenesis of H9N2 viruses in humans as well as in avian species.
Extensive studies on influenza virus-affected gene-expression profiling have been performed (Baskin et al., 2004; Degen et al., 2006; Diaz-Mitoma et al., 2004; Geiss et al., 2001, 2002; Huang et al., 2001; Kash et al., 2004, 2006; Kobasa et al., 2007; Marshall et al., 2005; Wei et al., 2006). It was indicated that the drastic increase in interleukin (IL)-6 in the lungs may be a key factor for pulmonary pathology. The data from A/PR/8/34 (H1N1) virus-infected lung epithelial cells also showed that non-structural gene 1 (NS1) is critical for suppression of selected genes involved in interferon (IFN) and IFN-inducible gene expression (Geiss et al., 2002). In contrast to the extensive work performed in mammalian model systems, little is known about the global gene-expression profiles associated with LPAI viruses, which mostly restrict their host ranges to avian species. How avian hosts survive LPAI virus infections remains unclear. In this study, we examined the susceptibility of the chicken HTC macrophage cell line to LPAI virus strains. Our interest was in examining the consequences of LPAI virus infections on the immune responses in macrophages and in chickens. Our results indicated that chicken macrophages were highly susceptible to infection with A/ph/CA/2373/98 (H9N2) virus resulting in apoptosis. In macrophages and lung tissues infected with A/ph/CA/2373/98 (H9N2), class II MHC antigens were downregulated. Whilst many pro-inflammatory cytokines, chemokines or their receptors were upregulated, some were selectively reduced, including IL-4 and the IL-4 receptor (IL-4R), both crucial in the activation of CD4+ helper T (Th) cells and humoral immunity. When chickens were challenged with A/ph/CA/2373/98 (H9N2), we found that the antibody responses in infected chickens were largely suppressed. Taken together, these findings suggest that some LPAI viruses may specifically modulate host adaptive immune responses negatively in avian species.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These cells were derived from peripheral blood monocytic/macrophage cells and have been spontaneously transformed. The HTC cells were grown under conditions described previously (Rath et al., 2003). Madin–Darby canine kidney (MDCK) cells were purchased from the ATCC and grown in Dulbecco's modified Eagle's medium containing 10 % fetal bovine serum. Chicken blood was from Colorado Serum Co. Fluorescein isothiocyanate (FITC)-conjugated anti-influenza A nucleoprotein (NP) monoclonal antibody was purchased from ViroStat. The In situ Cell Death Detection kit used for terminal dUTP nick-end labelling (TUNEL) assays was applied following the manufacturer's instructions (Roche Diagnostics).
Animals and viruses.
A/ph/CA/2373/98 (H9N2) was isolated from a domestically raised pheasant and A/ch/CA/1772/02 (H6N2) was isolated from a chicken during an LPAI outbreak in California in 2002–2003 (Woolcock et al., 2003). The eight genomic segments of both viral strains have been completely sequenced and analysed phylogenetically (J. Li, unpublished data). The viruses were grown in 10-day-old embryonated eggs from specific-pathogen-free hens (Charles River SPAFAS) and allantoic fluid was harvested 48 h after inoculation. Virus in the allantoic fluid was titrated using a standard haemagglutination test (Hirst, 1942) and infectious viral titres were determined in 10-day-old embryonated eggs (EID50) and in MDCK cells (TCID50).
Virus infection in macrophages and chickens.
HTC cells (5x106) were plated on 10 cm tissue culture plates 16 h before infection. In one-cycle infections, cells were infected with either A/ph/CA/2373/98 (H9N2) or A/ch/CA/1772/02 (H6N2) virus in allantoic fluid at an m.o.i. of 1 and incubated at 37 °C. Chickens from commercial egg production flocks were used in all challenge experiments. Both 20–30-month-old and 2–3-week-old hens were challenged with 107 EID50 virus delivered intranasally. In each challenge experiment, the birds were housed in groups in separate HEPA-filtered, negative-pressure isolators.
For microarray studies, 2–3-week-old birds were maintained in groups of ten; one group was an uninfected control group and the other group was inoculated with either H9N2 or H6N2 virus as described above. Two birds from each group were sacrificed on days 1, 3, 6, 9 and 12 and their lungs were taken for total RNA preparation. Both oropharyngeal and cloacal swabs were taken before challenge and on days 1, 3, 6, 9 and 12 post-challenge (p.c.). Swabs were placed immediately in virus transport medium on ice and subsequently used for virus isolation or viral RNA detection.
Additional challenge studies were carried out to determine the full range of antibody responses in H9N2- and H6N2-infected birds. Briefly, chickens were held in groups of 12. One group was maintained as an uninfected control group and two groups were challenged with either H9N2 or H6N2 virus. All chickens were bled and swabs were collected prior to challenge. After virus inoculation, the chickens were bled on days 3, 7, 10, 14, 17, 20 and 24, and both cloacal and oropharyngeal swabs were collected on days 2, 3, 4, 5, 6, 7, 11, 14 and 21. Chicken sera were prepared for haemagglutination inhibition (HI) antibody detection and swabs were handled as described above for examination of virus shedding. Two chickens from each group were euthanized, necropsied and examined for grossly evident lesions on days 3, 7 and 10. The challenge study was terminated on day 24 post-inoculation, with six chickens remaining in each group throughout the period of the challenge experiment.
Immunofluorescence staining and TUNEL assay.
HTC cells were grown on four-chamber slides, fixed and permeabilized with 2 % paraformaldehyde and 0.5 % Triton X-100, prior to incubation with FITC-labelled anti-NP antibody diluted 1 : 20 in PBS at 4 °C for 30 min. The cells were washed three times for 10 min each with PBS, after which they were air-dried and rewetted with glycerol and a cover slip applied. Cells were viewed under a Nikon Eclipse E400 fluorescence microscope. TUNEL staining was performed following the instructions of the In situ Cell Death Detection kit. To quantify apoptosis, infected and non-infected cells were trypsinized at certain time points p.i. (p.i.) using trypsin/EDTA. After two washes with PBS, the cells were stained with 7-amino-actinomycin D (7-AAD; Sigma-Aldrich) on ice for 30 min. The cells were washed three times with PBS containing 0.5 % BSA, followed by flow-cytometric analysis on a FACScan cell sorter (Benton Dickinson).
RNA preparation from cell cultures and chicken lungs.
An RNeasy RNA extraction kit (Qiagen) was used to prepare total RNA from HTC cell cultures and the lungs of chickens. Control and infected macrophages (5x106 total) were trypsinized and washed twice with PBS before being resuspended in 350 µl RLT buffer (Qiagen). Flash-frozen lung samples (
40 mg each) were placed into 600 µl RLT buffer before being homogenized with a MagNA Lyser instrument (Roche Diagnostics). The quantity and integrity of the resultant RNA were examined using an RNA Nano LabChip (Agilent Technologies) and the RNA preparations were used for probe labelling or RT-PCR.
cRNA probe amplification and labelling, and microarray analysis.
Reverse transcription, second-strand cDNA synthesis, and cRNA probe generation and biotin labelling were accomplished following standard Affymetrix protocols. cRNA probe hybridization to Chicken GeneChips (Affymetrix) and scanning were performed at the Genome Center, University of California, USA, following the Affymetrix Expression Analysis Technical Manual. Fluorescence intensity values (*.cel files generated from Microarray suite 5.0) from scanned GeneChips were subsequently subjected to analyses utilizing a DNA-Chip Analyser (dChip; http://www.dchip.org) (Schadt et al., 2001) and deposited in the GEO database (accession no. GSM158130). The intensity value of each sample was normalized. Briefly, by default, an array with median overall intensity was chosen as the baseline array against which other arrays were normalized at probe intensity level by dChip and the gene expression was calculated in a PM (perfect matching) model (Zhong et al., 2003). Changes in the level of mRNA of any gene were selected only when the following two criteria were met: (i) the alteration in expression was statistically significant (P value for paired Student's t-test of 0.05); and (ii) the change was at least 50 % (equivalent to a 1.5-fold change where the value for no change is 0) above or below the baseline expression level. The baseline was calculated as the expression level of the 0 h or day 0 for a particular probe set. Genes with significant transcriptional changes were analysed by the NetAffx Analysis Center (http://www.affymetrix.com/analysis/index.affx).
Real-time RT-PCR.
The presence of mRNAs for MHC classes I and II, IL-4, IL-1β, β-actin or glyceraldehyde-3-phospate dehydrogenase (GAPDH) in control and infected chicken macrophages was analysed using a two-step real-time RT-PCR. Total RNA (1 µg) was used for the reverse transcriptase reaction with a GeneAmp RNA PCR kit (Perkin Elmer) as described previously (Xing & Schat, 2000). Real-time PCR was carried out with 2.5 µl cDNA in a total volume of 25 µl using iQ SYBR Green Supermix (Bio-Rad) following the provided instructions. Relative expression values were normalized using either chicken β-actin or GAPDH. The fold change in relative gene-expression levels was calculated using the formula: 2(
Ct of gene–Ct of β-actin or GAPDH). Melting curves were analysed to determine the specificity. Reactions were conducted in duplicate for each sample and the mean value was calculated.
The sequences for the primers used for real-time RT-PCR were chosen using the web-based software Primer3 (http://frodo.wi.mit.edu/primer3/input.htm; v.0.4.0) as follows (forward and reverse primers, respectively): MHC class II B-LA, 5'-CTCGAGGTCATGATCAGCAA-3' and 5'-TGTAAACGTCTCCCCTTTGG-3'; MHC class II B-LB, 5'-GAGGTTTCTGGACAGGCAAA-3' and 5'-GTAGAAGCCCGTCACGTAGC-3'; MHC class I BF2, 5'-GGCTGAAGCAGTACCTGGAG-3' and 5'-AATCCAACACCAACCACGAT-3'; IL-4, 5'-GAGAGGTTTCCTGCGTCAAG-3' and 5'-TGGTGGAAGAAGGTACGTAGG-3'; IL-1β, 5'-GGCTCAACATTGCGCCTGTAC-3' and 5'-CCCACTTAGCTTGTAGGTGGC-3'; TLR-7, 5'-TGTGATGTGGAAGCCTTTGA-3' and 5'-ATTATCTTTGGGCCCCAGTC-3'; β-actin, 5'-CCATGAAACTACCTTCAACTCCA-3' and 5'-GATTCATCGTACTCCTGCTTGCT-3'; and GAPDH, 5'-CCTCTCTGGCAAAGTCCAAG-3' and 5'-CATCTGCCCATTTGATGTTG-3'.
The real-time RT-PCR with the TaqMan protocol for the identification of influenza virus was performed following a published protocol (Spackman et al., 2002). The primers used for detection of influenza A virus matrix (M) gene were 5'-AGATGAGTCTTCTAACCGAGGTCG-3' (forward), 5'-TGCAAAAACATCTTCAAGTCTCTG-3' (reverse) and the 5'-FAM-TCAGGCCCCCTCAAAGCCGA-TAMRA-3' (probe).
Flow-cytometric staining of macrophages.
Whole blood was taken from chickens inoculated with H6N2 or H9N2 virus on days 3 and 5 p.i. as described above. Peripheral blood mononuclear cells (PBMCs) were prepared with Ficoll (Mediatech) after low-speed centrifugation. A total of 106 cells was incubated with R-phycoerythrin-conjugated anti-chicken macrophage/monocyte, FITC-conjugated anti-chicken MHC class I or FITC-conjugated anti-chicken MHC class II antigen (Southern Biotech) on ice for 30 min. After three washes with PBS containing 0.5 % BSA, the cells were subject to flow-cytometric analysis using a FACScan cell sorter.
Virus shedding and HI assay.
Chickens were inoculated and samples were collected as described above. The virus load in the swab samples was determined by real-time RT-PCR. Antibody responses in uninfected and infected chickens were tested by HI assay based on Hirst's principle (Hirst, 1942). The serum was diluted 10-fold with saline before making an additional 2-fold dilution with PBS. Virus with haemagglutination titres of 1 : 8 to 1 : 16 was then added to each diluted serum sample and mixed for approximately 15 min. An equal volume (50 µl) of 0.5 % chicken red blood cells was added to the virus/serum mixture and incubated for 30–60 min before reading the results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Twelve hours after infection, the morphology of H9N2 virus-infected HTC macrophages altered. More dendrites were produced and the cells flattened and became more strongly adherent to the culture plates. After 24 h, these same cultures deteriorated (Fig. 1c). A large number of H9N2 virus-infected macrophages became apoptotic, as demonstrated by a TUNEL assay (Fig. 1i), which was positive as early as 12 h p.i. Comparatively fewer obvious morphological changes and less apoptosis were observed in cells infected with H6N2 virus (Fig. 1b and h). Quantitatively, at 18 h p.i., about 40 and 20 % of the cells infected with H9N2 and H6N2 viruses, respectively, underwent apoptosis. The number of apoptotic cells rose to 80 and 45 %, respectively, at 36 h p.i. (Fig. 1j).
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). MHC antigen genes, located in different spots with distinct probe-set IDs in the chips, were downregulated to various degrees in infected macrophages compared with uninfected cultures. Whilst expression of MHC class I antigens (B-FIV) were not changed (1.1–1.3-fold), expression of MHC class II antigens (B-LA and B-LB) detected at various loci was suppressed from 6 h p.i. MHC class II antigens were downregulated between 1.5- and 5.3-fold by 12 h p.i. (Fig. 2a and Table 1). It was notable that CD74, the MHC class II invariant peptide, was also downregulated by 1.2–2.0-fold.
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). Functionally, MHC class II antigens are responsible for presentation of antigenic peptides to activate CD4+ Th lymphocytes. The decrease in MHC class II antigens on the macrophages, together with the lowered CD74, could negatively impact on the activation of CD4+ Th cells, which may lead to a suppression of both Th1 and Th2 responses.
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and Table 1, both MHC Rfp-Y and CD1 antigens were downregulated between 1.2- and 4.8-fold.
Differential regulation of cytokines in H9N2 virus-infected avian macrophages
Pro-inflammatory cytokines IL-1β and IL-8, and chemokines K203, ah221 (CCL7), ah294, K60 (CXCL1), ligand 3 (CCL3), ligand 14 (CXCL14) and ligand 20 (CCL20) were all upregulated 1.3–26.7-fold. However, cytokines IL-4, IL-6, IL-16 and IL-17, as well as IL-4R 
-chain and IL-21R, were downregulated by between 1.2- and 10.8-fold (Fig. 2b and Table 1). The regulation of IL-1β and IL-4 expression was reproducible in the real-time RT-PCR analysis (Fig. 3b). Both IL-4 and IL-4R are critical to the Th2 response, and are required for activation and differentiation of Th2 CD4+ T cells. When considered together with the downregulation of MHC class II antigens, the activation of specific B lymphocytes to produce neutralizing antibodies in the Th2 response might be affected in A/ph/CA/2373/98 (H9N2) virus-infected chickens.
Regulation of IFN-related genes
Interferons were weakly induced in infected macrophages. As shown in Table 1, IFN-β expression was only increased up to 1.6-fold at 12 h p.i., whilst IFN- and -
expression did not change in H9N2-infected macrophages. Weak induction of IFNs probably contributed to differential regulation of IFN-inducible genes. 2'-5'-Oligoadenylate synthetase (OAS)-like gene was upregulated 6 h p.i. by 3.3-fold, but the Mx protein gene was unchanged or marginally downregulated (Table 1). Among the strongly downregulated genes was IFN-induced protein with RNA helicase domain 1 (IFIH1), also known as melanoma differentiation associated protein 5 (MDA-5), which is considered crucial in IFN activation and host defence (Kato et al., 2006; Kawai et al., 2005). Additionally, IFN regulatory factor (IRF)-8 was downregulated between 2.1- and 4.1-fold but IRF-1 was slightly upregulated (Table 1). Among the strongly upregulated genes was the IFN-inducible 58 kDa protein with tetratricopeptide repeats 5 (IFIT-5; upregulated 20.1-fold) at the early stage of infection (6 h p.i.). Interestingly, IFN-inducible protein IP-30, a lysosomal thiol reductase also involved in MHC class II antigen processing and functional in antigen-presenting cells (Arunachalam et al., 2000; Maric et al., 2001), was suppressed up to 2.4-fold.
Regulated expression of genes involved in immunity in the lungs of LPAI virus-infected chickens
Gene-expression profiling of the lungs of chickens infected with A/ph/CA/2373/98 (H9N2) was evaluated. As shown in Table 2, multiple MHC class I and II antigens were downregulated to variable degrees in the lungs of infected chickens. MHC class I antigens were reduced by between 1.3- and 7.4-fold, whilst MHC class II antigens (B-LBI and B-LBVI) were downregulated by between 1.3- and 30.3-fold. CD74 and cathepsin S were also downregulated by between 1.6- and 1.7-fold on day 6 p.i. Among the genes that were reduced were the Ig light chain precursor and the polymeric immunoglobulin receptor (Table 2). Functionally these two genes may recognize specific pathogen antigens on the surface of immune cells, such as B lymphocytes, and help in the endocytic process, ultimately resulting in antigenic presentation by MHC class II proteins and the activation of helper T cells.
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). The expression levels of most interleukins and chemokines remained unchanged in the lungs of virus-infected chickens (data not shown), suggesting that pro-inflammatory responses might all be inhibited.
Chickens were also infected with A/ch/CA/1772/02 (H6N2) virus. As shown in Table 2, both the Mx protein (1.6–2.7-fold) and 2'5'-OAS (1.8–1.9-fold) genes were downregulated. MHC class I antigens were downregulated by 2.2–6.3-fold between days 1 and 3 p.i., all of which were consistent with the results from A/ph/CA/2373/98 (H9N2)-infected chickens. However, in contrast to the findings with A/ph/CA/2373/98 (H9N2), the expression of MHC class II genes was either unchanged or upregulated (1.5–4.7-fold) in the lungs of H6N2-infected chickens.
Comparative analysis of H9N2 and H6N2 infection in chicken macrophages also indicated differences in the regulation of MHC class II antigens and IL-4 expression as revealed by the results of real-time RT-PCR (Fig. 4a). At 8 and 24 h p.i., the MHC class II antigens B-LA and B-LB were upregulated in H6N2-infected cells, whilst they were downregulated in H9N2-infected cells. A similar pattern was shown for IL-4 expression, but IL-1β expression was upregulated in both H6N2- and H9N2-infected macrophages (Fig. 4a).
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). Several chicken TLRs including TLR-1, TLR-4 and TLR-15, which are highly expressed in uninfected HTC macrophages, remained unchanged or changed only marginally after infection (data not shown). Interestingly, when the expression of TLR-7 was further analysed at the earlier stages of infection using real-time RT-PCR, we found that TLR-7 responded to infection and expression was actually upregulated up to 7.8-fold at 8 h p.i. (Fig. 4b). It appeared that expression of TLR-7 was only suppressed during the later stage of infection, which echoes the downregulation of expression of multiple cytokines during the later stages of infection.
Expression of MHC antigens on macrophages in infected chickens
PBMCs were prepared from whole blood taken from H6N2-infected, H9N2-infected or uninfected chickens on day 3 p.i. and stained with labelled anti-macrophage/monocyte and anti-MHC antigen antibodies (data not shown). MHC class I antigens were downregulated on the surface of monocytes from both H6N2- and H9N2-infected chickens. However, MHC class II antigens were upregulated in H6N2-infected birds, but downregulated in H9N2-infected birds.
Suppressed HI antibody responses in chickens challenged with H9N2 virus
We next examined how the host adaptive immunity responded in infected chickens. On day 7 p.c., HI antibodies were detectable in 100 % of H6N2-infected chickens but in only 20 % of H9N2 virus-infected birds became seropositive (Fig. 5a). Fifty per cent of chickens infected with H9N2 virus became seropositive on day 14 p.c. A comparison of the mean HI antibody titres between H6N2 and H9N2 virus-infected chickens is shown in Fig. 5(b). A vigorous HI antibody response was observed in H6N2-infected chickens starting at day 7, reaching its peak at day 10 and declining thereafter, whilst the antibody response in H9N2-infected chickens remained low throughout. The differences in the seroconversion and HI antibody titres between H9N2- and H6N2-infected chickens were significant (Student's t-test, P<0.01). The HI antibody titres are also shown in Table 3 for three pairs of chickens infected with either H6N2 or H9N2, which showed comparable virus shedding detected in oropharyngeal swabs using real-time RT-PCR but distinct antibody responses. All challenged birds survived infection, and necropsy of the birds showed that both H6N2- and H9N2-infected hens had no or mild grossly detectable lesions in the respiratory tract and lungs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kaufmann et al., 2001). Unlike epithelial cells, infected macrophages/monocytes are the main source of cytokine and chemokine production in response to influenza virus infection (Kaufmann et al., 2001). In this study, we found that the pro-inflammatory cytokines IL-1β, IL-8 and IL-18 and the chemokines CCL3, ah221 (CCL7), CCL20, ah294, K203, K60 (CXCL1) and CXCL14 were all upregulated, suggesting that chicken macrophages may play a key role in responding to LPAI virus infections. Among the genes downregulated in A/ph/CA/2373/98 (H9N2)-infected macrophages were IL-4, IL-4R, IL-17 and IL-21R. Both IL-4 and IL-4R are pivotal in the activation of Th2 responses and neutralizing-antibody production. IL-21R is related to IL-2Rβ in that it transduces the signals of IL-21, a member of the IL-2 cytokine family (Asao et al., 2001) and is important in the differentiation of T, B and NK cells (Strengell et al., 2002, 2003). Therefore, H9N2 virus-regulated changes in IL-4, IL-4R and IL-21R expression in macrophages may have an overall negative impact on the development of adaptive immunity in chickens.
Among the key findings of the current analysis was that the MHC antigens, and the MHC class II antigens in particular, were extensively downregulated by A/ph/CA/2373/98 (H9N2). In fact, MHC class II -chain (B-LA), β-chain (B-LB), -chain 1B locus, β1 domain (B-LBI) and β1 domain (B-LBVI) were all downregulated at the various time points examined following infection, making it one of the most consistent effects of this LPAI virus in infected chicken macrophages. The MHC class I antigens B-FIV, ID B-F2 minor, YFV (MHC class I heavy chain) and Rfp-Y -chain were also downregulated, some, at least transiently, at certain time points p.i. In addition, CD74 and cathepsin S also had reduced expression. CD74 functions mainly as an MHC class II chaperone, promoting MHC class II antigen exit from the endoplasmic reticulum and entry into endocytic compartments. It also contributes to peptide editing in the MHC class II compartment (Stumptner-Cuvelette & Benaroch, 2002). The impact of MHC class II antigen downregulation, considered in light of the decrease in IL-4 and IL-4R, is likely to suppress adaptive immune responses, especially humoral immunity, in infected birds.
Although it was a clear effect of the H9N2 virus used in these studies, the suppression of MHC antigens is not a consistent finding for all LPAI viruses. Our data with A/ch/CA/1772/02 (H6N2) infection indicated that, in infected macrophages and lungs, MHC class II antigen expression was upregulated to varying degrees. Although we were unable to confirm that lymphocyte activation is inhibited directly at this stage, we did observe that MHC class II antigens were regulated in a similar manner on macrophages/monocytes from infected chickens (Fig. 4). The generalized observations that humoral responses may be suppressed by A/ph/CA/2373/98 (H9N2) were confirmed by differences in the virus-specific antibody responses in chickens (Fig. 5). Only half of the commercial chickens challenged with A/ph/CA/2373/98 (H9N2) developed detectable antibodies, which were at low levels in the same time frame, whilst chickens inoculated with A/ch/CA/1772/02 (H6N2) developed a robust humoral response. These findings bring into question the role of humoral immunity in controlling some LPAI virus infections in birds.
At this stage, we do not know how A/ph/CA/2373/98 (H9N2) downregulates MHC antigens. Studies have identified some viral proteins that are responsible for the regulation of specific host proteins and defence mechanisms (Alexander, 2000; Guan, 2000; Marshall et al., 2005; Taylor et al., 2005; Wei et al., 2006). Critically, NS1 plays a special role in the suppression of IFN-induced genes and the 1918 pandemic viral NS1 exhibited more efficient inhibition (Geiss et al., 2002). Using the genetically engineered A/Texas/36/91 (H1N1) virus in pigtailed macaques, the induction of a number of adaptive immunity-related genes including HLA-A, HLA-C (both MHC class I) and MHC class II DOβ were also observed in the lungs and tracheobronchial lymph nodes (Baskin et al., 2004). It has been noted that HLA-C (MHC class I) was indeed upregulated in an NS1-deficient mutant A/PR/8/34 (H1N1), but was almost unchanged in wt A/PR/8/34-infected A549 cells (Geiss et al., 2002), indicating that the wt NS1 essentially suppressed the induction of HLA-C. A recent study in macaques infected with the 1918 pandemic influenza virus strain indicated that MHC class II antigens (HLA-DRB1, HLA-DRB3 and HLA-DRB5) were downregulated among almost all upregulated genes involved in both the innate and adaptive host defence including MHC class I antigens (Kobasa et al., 2007). It is rather unusual to observe the similar decrease in the MHC class II genes in birds shown by our data. We are currently generating NS1-mutant recombinant viruses using a reverse-genetics approach to elucidate the underlying mechanism.
Previous studies have found that LPAI viruses induced apoptosis to variable degrees in human and mouse macrophages (Brydon, 2005; Mok et al., 2007). Chicken macrophages are susceptible to infection with the LPAI H9N2 and H6N2 subtype viruses used in these studies. In our study, infection in the absence of trypsin led to aggressive apoptosis in A/ph/CA/2373/98 (H9N2) virus-infected macrophages. This is in contrast to reports in which productive infection in mammalian macrophages with H1N1 and H3N2 subtype viruses did not result in apoptosis (Seo et al., 2004; Tyner et al., 2005). It has been postulated that macrophages have evolved mechanisms to allow them to remain viable during all types of infection. In keeping with this strategy, it has been reported that the upregulation of chemokine ligand CCL5 and the induced downstream pathways for survival signals by respiratory syncytial virus and human influenza virus (H1N1) infection are critical in preventing apoptosis (Tyner et al., 2005). In the chicken microarray chips used in this study, CCL5 was absent. In order to determine whether there was a CCL5 homologue in chickens, we compared the sequences of a number of chicken chemokines and found that chemokine ah294 may be the chicken counterpart of CCL5. A sequence comparison showed that ah294 had the highest sequence identity with duck (77 %), pig (68 %), and mouse and human (64 %) CCL5 (data not shown). In this study, ah294 was upregulated up to 26.7-fold in A/ph/CA/2373/98 (H9N2)-infected macrophages (Table 1), although this clearly did not prevent the cells from becoming apoptotic. More evidence is needed to determine whether ah294 is indeed the counterpart of CCL5 in mammals and whether it plays a role in apoptosis in chicken macrophages.
It has been suggested that T lymphocytes or CD8+ T cells from H9N2-infected chickens may be sufficient to prevent chickens from disease caused by H5N1 infection through cross-reactive cell-mediated immunity in the absence of cross-reactive neutralizing or HI antibodies (Seo & Webster, 2001). Specific neutralizing or HI antibodies are still considered protective and can clear the virus in birds and humans. Frankly, we know little about how birds recover from LPAI infections. In this study, we observed mixed responses among IFN and IFN-inducible genes and β-defensins in response to infection in chickens. Whilst 2'5'-OAS was upregulated, the Mx protein gene was marginally downregulated in A/ph/CA/2373/98 (H9N2)-infected macrophages. However, they were both downregulated in infected chicken lungs. IFIT-5 was highly induced in macrophages, but this induction appeared only on day 1 and started to decline thereafter in chicken lungs. Differential regulation of IFN-inducible genes is probably due to weak IFN induction in H9N2-infected macrophages and the lung tissues (Table 1). Interestingly, IFIH1 (MDA-5) was highly downregulated up to 6.9-fold and is considered to be critical, like RIG-1, as an RNA virus sensor in IFN-β induction (Kato et al., 2006; Kawai et al., 2005). Differential regulation of IFN-inducible genes was also observed in other microarray studies, including one using macaques (Baas et al., 2006). In the lungs of A/Texas/36/91 (H1N1)-infected macaques on day 2 p.i. in this study, OAS1, OAS3, MX1 and IFIT2 were upregulated, whilst MX2, GBP-1 and GBP-2 were downregulated. Our data clearly showed that TLR-7 is upregulated as the sensor responsive to avian influenza virus in chicken macrophages at the earlier stages of infection, but is suppressed at later times. We consider that downregulation of TLR-7 may be one of the mechanisms causing the downregulation of IFNs and IFN-inducible genes including MHC antigens. Suppression of IFNs and IFN-inducible genes in influenza virus-infected cells and in the host is unique among virus infections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 8 August 2007;
accepted 10 January 2008.
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