Differential gene-expression and host-response profiles against avian ...

Journal of General Virology (2009), 90, 2134–2146

DOI 10.1099/vir.0.012401-0

Differential gene-expression and host-response profiles against avian influenza virus within the chicken lung due to anatomy and airflow Sylvia S. Reemers,1 Daphne A. van Haarlem,1 Marian J. Groot Koerkamp2 and Lonneke Vervelde1 Correspondence Lonneke Vervelde

1

Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands

[email protected]

2

Received 7 April 2009 Accepted 28 May 2009

Sampling the complete organ instead of defined parts might affect analysis at both the cellular and transcriptional levels. We defined host responses to H9N2 avian influenza virus (AIV) in trachea and different parts of the lung. Chickens were spray-inoculated with either saline or H9N2 AIV. Trachea and lung were sampled at 1 and 3 days post-inoculation (p.i.) for immunocytochemistry, real-time quantitative RT-PCR and gene-expression profiling. The trachea was divided into upper and lower parts and the lung into four segments, according to anatomy and airflow. Two segments contained the primary and secondary bronchi, cranial versus caudal (parts L1 and L3), and two segments contained the tertiary bronchi, cranial versus caudal (parts L2 and L4). Between the upper and lower trachea in both control and infected birds, minor differences in gene expression and host responses were found. In the lung of control birds, differences in anatomy were reflected in gene expression, and in the lung of infected birds, virus deposition enhanced the differences in gene expression. Differential gene expression in trachea and lung suggested common responses to a wide range of agents and site-specific responses. In trachea, site-specific responses were related to heat shock and lysozyme activity. In lung L1, which contained most virus, site-specific responses were related to genes involved in innate responses, interleukin activity and endocytosis. Our study indicates that the anatomy of the chicken lung must be taken into account when investigating in vivo responses to respiratory virus infections.

Genomics Laboratory, Department of Physiological Chemistry, Utrecht Medical Centre, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands

INTRODUCTION Avian influenza virus (AIV) infection is a continuing threat to both humans and birds worldwide. In order to control outbreaks, research into new intervention strategies and vaccines is ongoing. Therefore, studies into pathogenesis and host–virus interactions are being performed (Baskin et al., 2004; Kash et al., 2004; Degen et al., 2006). Host–virus interactions depend on the ability of the virus to enter host cells. The molecules used by avian and human influenza viruses to enter the respiratory tract are sialic acid linked to galactose by respectively an a-2,3 or an a-2,6 linkage, which are not distributed equally. Differences in virus entry generally correspond to variation in the type of sialic acid expressed in the respective host species (Shinya et al., 2006; Van Riel et al., 2007). The pattern of receptor distribution in chickens was shown to be less defined than in mammals Three supplementary tables showing expression of immune-related genes induced by H9N2 infection in upper trachea and lung L1 and L4 at 3 days p.i. are available with the online version of this paper.

2134

(Wan & Perez, 2005). In chickens, uneven spread of lowpathogenic AIV (LPAI) has been shown for H9N2, with a preference for infecting the upper part of the respiratory tract (Nili & Asasi, 2002). Difference in distribution of virus within the respiratory tract has an impact on the host responses, as shown for infected macaques (Baas et al., 2006). Host responses to respiratory virus infections have only been investigated in the whole lung, one part of the lung or a pool of samples, without taking the anatomy and airflow, bidirectional in mammals and unidirectional in birds, through the respiratory tract into account. Herein, we describe that gene expression within the lung of control chickens was affected by anatomy, and that airflow affected virus deposition and thereby the localized host responses. The trachea and lung of saline- and H9N2 AIVinoculated birds were sampled 1 and 3 days postinoculation (p.i.). The trachea was divided into upper and lower parts and the lung into four parts, based on airflow (Fig. 1). The airflow is unidirectional and ventilation is performed by the air sacs (Fedde, 1998). 012401 G 2009 SGM Printed in Great Britain

AIV distribution and host response differ within lung

Fig. 1. (a) Right chicken lung with a blunt probe indicating the localization of the primary bronchus. The lung was divided into four parts, L1–L4, according to airflow and lung anatomy, with the primary bronchus entering the lung in L1. The syrinx with the primary bronchus is shown on the left side. The white blocks indicate the part of the lung segments used for RNA isolation. The remainder of the segments were used for immunocytochemistry. (b, c) Illustration of the airflow pattern during (b) inspiration and (c) expiration in the avian lung. During inhalation, air flows through the primary bronchus into the caudal air sacs or through the mediodorsal and lateroventral secondary bronchi. This caudal-to-cranial flow pattern is also evident during expiration. 1, Primary bronchus; 2, lung; 3, clavicular air sac; 4, cranial thoracic air sac; 5, caudal thoracic air sac; 6, abdominal air sac. Adapted and reprinted from Kothlow & Kaspers (2008) with permission from Elsevier.

Between upper and lower trachea in both control and H9N2-infected birds, minor differences in gene expression were seen. In the different parts of the lung in control birds, gene expression differed significantly in that expression was similar in L1 and L3 and in L2 and L4. In infected birds, L1 and L3 contained larger and more virus-infected areas than L2 and L4, which enhanced the differences in gene expression already seen in control birds. Our data showed that anatomy and airflow in lung of both control and infected birds affect gene expression, virus deposition and subsequently host responses. The trachea and lung shared expression of genes involved in common host responses, but several genes were expressed in a site-specific manner. Our study suggests that anatomy and airflow, especially of the avian lung, must be taken into account when investigating in vivo host responses to respiratory virus infections.

trachea was divided into upper and lower parts and the lung was divided into four pieces (Fig. 1), L1–L4. Immunocytochemistry. Detection of virus nucleoprotein (NP) and

cellular influxes in cryosections was performed as described previously (Vervelde et al., 1996). NP was detected with a mouse mAb to NP of H9N2 (provided by Intervet Schering-Plough). KUL01+ cells (macrophages; Mast et al., 1998) and CD4+ cells were detected with monoclonal antibodies KUL-01 and CT-4 (Southern Biotech). For the detection of CD8a+ cells, a mixture of mAbs EP72 (Southern Biotech) and AV14 (a kind gift of Dr T. F. Davison, Institute for Animal Health, Compton, UK; Withers et al., 2005) was used to avoid differences in staining due to polymorphism in the chicken CD8a molecule (Breed et al., 1996; Luhtala et al., 1997). RNA isolation. Total RNA was isolated from upper and lower

METHODS

trachea (5 mm per part of the trachea) and all four segments of the lung (a 165 mm part per segment of the lung; Fig. 1a) by using an RNeasy Mini kit, and DNase-treated by using an RNase-free DNase set (both from Qiagen) according to the manufacturer’s instructions. All RNA samples were checked for quantity by using a spectrophotometer (Shimadzu) and for quality by using a 2100 Bioanalyzer (Agilent Technologies).

Infection model. Avian influenza A virus, subtype H9N2, isolate A/

Real-time quantitative (q) RT-PCR. cDNA was generated from

Chicken/United Arab Emirates/99 was provided by Intervet ScheringPlough Animal Health. One-day-old White Leghorn chickens were housed under specificpathogen-free (SPF) conditions and all experiments were carried out according to protocols approved by the Intervet Animal Welfare Committee. Chickens were divided into two groups over two isolators, infected and non-infected, containing 20 birds per group. Fourteen-day-old chickens were inoculated via aerosol spray either with 20 ml 107.7 EID50 H9N2 AIV or with 20 ml saline per isolator. Of the 20 birds in each isolator, eight were used for the experiment described here. Chickens remained in the closed isolator for 10 min, after which the isolator was ventilated as before. At 1 and 3 days p.i., chickens were killed (n54 per time point per group) and trachea and left lung were isolated and stored in RNAlater (Ambion) at 280 uC for RNA isolation and in liquid nitrogen for immunocytochemistry. The http://vir.sgmjournals.org

500 ng RNA with reverse transcription using an iScript cDNA Synthesis kit (Bio-Rad). Real-time qRT-PCR was performed by using iQ SYBR Green Supermix (Bio-Rad) and TaqMan Universal PCR Master Mix (Applied Biosystems). Primers (Invitrogen Life Technologies) and probes (Applied Biosystems) were described by Degen et al. (2006) and Eldaghayes et al. (2006). Detection of glyceraldehyde-3-phosphate dehydrogenase and H9 haemagglutinin was described by Degen et al. (2006). Primers were used at 400 nM concentration. Detection of interleukins (IL), beta interferon (IFN-b) and 28S rRNA was as described by Ariaans et al. (2008). Primers were used at 600 nM and probes at 100 nM concentration. Corrections for variation in RNA preparation and sampling were performed according to Eldaghayes et al. (2006). A paired t-test was used to determine the statistical significance between upper and lower trachea samples from the same bird. To determine the statistical significance between parts of the lung, an 2135

S. S. Reemers and others ANOVA with a Tukey post-hoc test was used. A P-value ,0.05 was considered statistically significant.

independent vesicular transport, nuclear organization, energy metabolism and calcium binding.

Oligonucleotide microarray analysis. For microarray analysis, the

IL-1b, IL-6 and IFN-b mRNA expression was measured in the trachea of control birds at 3 days p.i. (Fig. 2b) and no significant differences in expression were found between upper and lower trachea. In the trachea of control birds, the lamina propria consisted of one to three cell layers with no lymphoid infiltrates. Few CD4+, CD8a+ and mainly KUL-01+ cells were found in the lamina propria of both upper and lower trachea (Fig. 2e).

Gallus gallus Roslin/ARK CoRe Array Ready Oligo Set v1.0 (Operon Biotechnologies) was used. The array was spotted onto Codelink activated slides (GE Healthcare) and contains 20 460 oligo probes representing chicken genes and 3828 control spots, used for quality control and normalization purposes (Van de Peppel et al., 2003). RNA amplification and labelling were performed according to Roepman et al. (2005). All hybridizations contained 2.5 mg cRNA per channel on a HS4800Pro hybstation (Tecan Benelux BVBA). All trachea and lung samples were co-hybridized with a trachea or lung reference sample, respectively. These reference samples consisted of RNA extracted from tracheas or lungs of four chickens that were not included in the infection experiment. Slides were scanned with a G2565AA scanner (Agilent Technologies) at 100 % laser power, 30 % photomultiplier tube sensitivity. Resulting image files were analysed by using Imagene 8.0 (BioDiscovery, Inc.). Within-slide normalization was performed with Printtip Loess (Yang et al., 2002) on mean data without background subtraction. Groups of replicates were analysed by using ANOVA (Wu et al., 2003). In a fixed-effect analysis, sample, array and dye effects were modelled. P-values were determined by a permutation F2 test, in which residuals were shuffled 5000 times globally. Genes with P,0.05 after family-wise error correction were considered to be statistically significantly differentially expressed and were selected to be included for further analysis. Visualization and cluster analysis were performed by using GeneSpring 7.2 (Agilent Technologies). Ensembl G. gallus (assembly: WASHUC2, May 2006; genebuild: Ensembl, Aug 2006; database version: 47.2e) was used for gene names, description and Gene Ontology (GO) annotations. Primary data are available in the public domain through Expression Array Manager (http://www.ebi.ac.uk/arrayexpress/ ?#ae=main[0]) under accession numbers E-TABM-637 for trachea and E-TABM-636 for lung.

RESULTS Responses in upper and lower trachea are similar Trachea of control birds at 3 days p.i. was used to analyse global and immune-related gene expression. The immunerelated category was based on the Gene Ontology (GO) terms host–pathogen interaction, external stimulus and immune response. Gene expressions were depicted in a scatter plot in which upper and lower trachea were plotted against each other (Fig. 2a). Only a few genes were statistically significantly expressed (P,0.05) with ¢2-fold difference (Table 1). These genes are involved in clathrin-

Upon infection, we found that the virus RNA level in upper trachea was significantly higher than in lower trachea at both 1 and 3 days p.i. (Fig. 2c). Samples from 3 days p.i. were used to analyse cytokine expression and global gene expression. The difference in virus RNA level did not result in significant differences in IL-1b, IL-6 and IFN-b mRNA expression between upper and lower trachea (Fig. 2b), but in infected birds, the mRNA levels of IL-1b and IL-6 mRNA increased by approximately 6 Ct values compared with control birds. IFN-b mRNA expression also increased, but to a lesser extent (approx. 2 Ct values). Gene expressions in upper and lower trachea of infected birds were plotted against each other in a scatter plot (Fig. 2d). Inoculation with H9N2 resulted in differential gene expression of approximately 1700 genes compared with control birds, but only a few genes were expressed with a statistically significant ¢2-fold difference (Table 1). Three genes were downregulated: cysteine- and glycine-rich protein 3, cardiac phospholamban and a chemokine gene. The upregulated genes were involved in metabolic processes, metallopeptidase activity, regulation of cytokinesis and endothelial-cell activation. At 1 and 3 days p.i., virus was located in the epithelial cells and the mucoid glands. The lamina propria was slightly swollen at 1 day p.i., with influxes of CD4+ and KUL-01+ cells, whereas at 3 days p.i., the lamina propria consisted of multiple cell layers due to cellular influxes. KUL-01+ cells were located in the epithelial cell layer and in the lamina propria; these cells had a round appearance, indicative of activation, whereas in the submucosa, these cells had a dendritic appearance with branched projections. Influxes of CD8a+ cells were found in the lamina propria from 3 days p.i. and were smaller than the CD4+ cell influxes. All cellular influxes were localized and not found

Fig. 2. (a) Scatter plots of global and immune-related genes in upper versus lower trachea of control birds at 3 days p.i. Genes in red were upregulated and those in green were downregulated. The middle diagonal line represents a 1-fold change and the two outer diagonal lines a 2-fold change difference between upper and lower trachea. (b) IL-1b, IL-6 and IFN-b mRNA expression in trachea at 3 days p.i. (c) Virus RNA levels in trachea of infected birds at 1 and 3 days p.i. Bars within a time point with different letters were significantly different (P,0.05). (d) Scatter plots of global and immune-related genes in upper versus lower trachea of infected birds at 3 days p.i. (e) Cryosections of trachea at 1 and 3 days p.i., stained for virus NP, KUL-01+, CD4+ and CD8a+ cells. Upon infection, KUL-01+ cells (macrophages) changed from dendritic (arrows) to round (arrowheads) morphology. Bar, 50 mm. 2136

Journal of General Virology 90


http://vir.sgmjournals.org

2137

S. S. Reemers and others

Table 1. Genes expressed significantly at ¢2-fold difference in upper versus lower trachea at 3 days p.i. in control and infected birds Gene description Control birds Syntaxin 8 Nesprin-1

Hypothetical protein LOC418424 AMP deaminase 1

Parvalbumin, thymic Infected birds Cysteine- and glycine-rich protein 3 Cardiac phospholamban Chemokine Potassium channel subfamily K member 2 Aminoacylase 1-like protein 2 Proline-rich protein 6

Coiled-coil domain containing 78 isoform 1 GTP-binding protein RAD Ankyrin repeat domain-containing protein 1

Ensembl ID

Fold change*

ENSGALT00000001879 ENSGALT00000021996 ENSGALG00000018428 genomic:Un_random+ 27728607-27728677 ENSGALESTG00000000019D ENSGALT00000024836 ENSGALT00000035596 ENSGALT00000003247 ENSGALT00000003550 ENSGALG00000023715 ENSGALT00000005489D

0.50 2.04 2.08 2.09 2.11 2.14 2.21 2.22 2.26 2.58 3.93

ENSGALT00000006436 ENSGALT00000024024 ENSGALT00000041379D ENSGALG00000009687 ENSGALT00000025446 ENSGALT00000025298 ENSGALT00000007174 ENSGALT00000007194 ENSGALT00000015324 ENSGALT00000003811 ENSGALT00000008251 ENSGALT00000010491

0.30 0.32 0.44 2.09 2.09 2.18 2.26 2.60 2.60 2.63 2.84 3.81

*Expression ratio in lower trachea dived by expression ratio in upper trachea. DImmune-related gene.

throughout the whole trachea, with no differences between upper and lower trachea (Fig. 2e).

clustering. L1 clustered with L3 and L2 clustered with L4 for both global and immune-related genes (Fig. 3a), coinciding with airflow and lung anatomy.

Differential responses in lung segments L1, L2, L3 and L4

No significant difference in IL-1b and IFN-b mRNA expression between L1, L2, L3 and L4 was found (Fig. 3b). However, IL-6 mRNA expression in L1 and L3 was significantly higher than in L2 and L4.

Lung was divided into four pieces according to unidirectional airflow and lung anatomy. In this study, the airflow through the air sacs is ignored for simplicity, because it does not affect the airflow through the lung. Global and immune-related gene expression in lung L1–L4 of control birds at 3 days p.i. was compared by hierarchical

In a non-infected lung, the KUL-01+, CD4+ and CD8a+ cells were distributed throughout the lung in the parabronchi, interparabronchial septa and lamina propria of larger airways. KUL-01+ and CD8a+ cells were found

Fig. 3. (a) Hierarchical clustering of expression of global and immune-related genes in lung L1–L4 in control birds at 3 days p.i. Genes in red were upregulated and those in green were downregulated. (b) IL-1b, IL-6 and IFN-b mRNA expression in lung L1– 4 at 3 days p.i. Bars with different letters were significantly different (P,0.05). (c) Virus RNA levels in infected birds at 1 and 3 days p.i. Bars within a time point with different letters were significantly different (P,0.05). (d) Hierarchical clustering of expression of global and immune-related genes in lung L1–L4 in infected birds at 3 days p.i. Genes in red were upregulated and those in green were downregulated. (e) Cryosections of lung L1–L4 stained for virus NP at 3 days p.i. The parabronchi of the different parts of the lung were affected to a different extent. L indicates the luminal side of a parabronchus. Bar, 75 mm. (f) Cryosections of lung L1 stained for virus NP and for KUL-01+, CD4+ and CD8a+ cells at 3 days p.i. Upon infection, KUL01+ cells (macrophages) change from dendritic (arrows) to round (arrowheads) morphology. Bar, 75 mm. 2138




2139


closer to the lumen than CD4+ cells, and the number of KUL-01+ cells exceeded those of CD4+ and CD8a+ cells (Fig. 3f). After inoculation (Fig. 3c) at both 1 and 3 days p.i., no significant differences in virus RNA levels were found between L1 and L3 or between L2 and L4. However, at both time points, virus RNA level was higher in L1 and L3 than in L2 and L4, being significantly different at 1 day p.i. for L2 and at both time points for L4. The effect of uneven virus distribution on IL-1b, IL-6 and IFN-b mRNA expression is shown at 3 days p.i. (Fig. 3b). IL-1b and IL-6 mRNA levels were similar in L1 and L3, but significantly higher than in L2 and L4. In all four parts of the lung, IL-1b and IL-6 mRNA expression was significantly higher in infected birds than in control birds. The IFN-b mRNA levels in infected birds were low and not significantly different between L1, L2, L3 and L4, nor were they different in control birds. A hierarchical clustering of both global and immunerelated gene expression was performed on infected birds at 3 days p.i. (Fig. 3d). In infected birds, the same clustering was observed as in control birds, but the clustering of L1/ L3 and of L2/L4 in infected birds was even stronger for both global and immune-related genes, as the branches leading to the L1/L3 and L2/L4 clusters were longer and the branches leading to the individual lung parts were shorter compared with the cluster tree in control birds. By using immunocytochemistry, virus deposition and cellular influxes in the lung parts were studied (Fig. 3e). In both L1 and L3, virus-infected areas were found in the primary and secondary bronchi and adjacent parabronchi. At 3 days p.i., more virus was located in the parabronchi than at 1 day p.i. In L2 and L4, infected areas were located in the parabronchi and an increase over time was seen. The largest infected areas were found in L1, followed by L3; L4 and L2 were similar and contained fewer and smaller infected areas (Fig. 3e). At 1 day p.i., influxes of KUL-01+, CD4+ and CD8a+ cells were seen in both L1 and L4 in the bronchi and parabronchi (Fig. 3f). Cellular influxes colocalized with virus-infected areas, and the number of cells correlated positively with the size of the virus-infected areas. At 1 day p.i., KUL-01+ cells in infected areas were mainly round, whereas in non-infected areas they were dendritic in morphology. The number of KUL-01+, CD4+ and CD8a+ cell influxes increased over time. Differences between lung L1 and L4 In both control and infected birds, gene expression differed most between L1 and L4. These significantly differentially expressed genes are depicted in a graph showing three major GO categories and an immune-related category (Fig. 4a). In control birds, more genes were expressed at a higher rate in L1 than in L4 for all four categories. Immunerelated genes that differed significantly between L1 and L4 are depicted in a heat map (Fig. 4b). Genes expressed at a 2140

higher rate in L1 were mainly involved in cell adhesion and motility, e.g. VTN and TSPAN1, T cell- and B cell-related, e.g. ITFG1 and PIGR, or involved in chemotactic activities, e.g. CCL20, CX3CR1 and CHIA. In infected birds, more genes were expressed at a higher rate in L4 than in L1 for the molecular function, cellular component and biological processes categories, but not for immune-related genes. Immune-related genes that differed significantly between L1 and L4 are depicted in a heat map (Fig. 4c). These genes were mainly involved in lymphocyte activation, apoptosis, chemokine and IL activity. Genes expressed at a higher rate in L1 were mainly involved in lymphocyte activation, mainly T cell-related, such as CD3E, CD28 and ICOS, and chemokine and IL activity, e.g. CCL19, SOCS1, CXCLi1, IL-1b, IL-7Ra and IL-16. In L4, mainly genes involved in apoptosis, stress responses and major histocompatibility complex (MHC) pathways were expressed at a higher level than in L1, e.g. BCLX, BNIP3L, HSP70, HSPB2 and MHC class II. Induction of host responses against H9N2 in upper trachea and lung L1 and L4 To quantify chicken responses against H9N2 in the respiratory tract at the transcriptional level, immunerelated genes that were significantly differentially expressed between control and infected birds were analysed. Most genes were significantly differentially expressed in trachea and lung L1 (respectively 244 and 272 immune-related genes), whereas in lung L4, there were 86 significantly differentially expressed immunerelated genes. Genes were divided into functional groups based on GO interpretations, of which the groups containing most genes are depicted in Fig. 5. The complete gene sets are listed in Supplementary Tables S1–S3, available in JGV Online. A shortened gene list, indicating the location in the respiratory tract at which genes were significantly induced due to H9N2 infection, is depicted in Table 2. This list is based on the functional groups shown in Fig. 5 and depicts a global overview of the location of expression and expression rate of genes (up or down) within a functional group. The most striking facts are that many fewer genes were expressed differentially in lung L4, and most genes were upregulated independently of the location and functional group. In trachea and both lung L1 and L4, genes involved in chemokine and cytokine signalling were most commonly expressed, with the different regions sampled sharing many similar genes. In contrast to the upregulation of chemokine-related genes, several genes related to growth factors were downregulated. Of the genes involved in innate responses and IFN signalling, only the genes involved negatively in IFN signalling (FLN29, USP18) and related to IFN-c signalling (IRF10) were expressed in trachea, lung L1 and L4. Other Journal of General Virology 90


DEDD MHC B-G PCNA IAP SRF DAP TSPAN3 LY6E LGALS3BP CX3CR1 ATRN TRAF3IP3 RASSF2 CHIA DOCK2 PBEF1 IL-28RA TRAF3IP1 VTN PIGR TSPAN1 CCL20 MSLN CD34 Q9PU42_CHICK Ig lambda chain C region IRF6 LIMS1 CIDEA STYXL1 ITFG1

TRAF5 Cytokine CBLB STK17A SH2D1A IGJ MSLN Ig lambda chain C region TSPAN1 SFTPA1 CCL20 MMP7 STK17B IL-1B Bu-1 PDL2 CCL19 LITAF CXCLi2 IL-7Ra IL-6 CXCLi1 TRAT1 PDL1 PRDX1 CIITA TNFRSF18 IL-16 CD38 CD3D CD3E CD8B SPIC ZAP70 CTLA4 BCL11A

ICOS CD28 Chemokine CD8A IL-21R RASSF2 DcR3 CCR8 VTN CXCR4 RFTN1 RGS1 IL-18 IRF10 TNFRSF4 IKZF1 VCAM1 SOCS1 STAT4 GIMAP4 C-type lectin-like receptor MAP3K7IP2 CD55 HSPB2 MHC B-G CISH CD200R-B1 CMAP27 CSF3R HSP70 BCLX MHC class II MHC class IV IFRD1 PTDSR SH2B3 BNIP3L RBM5 CASP9

Fig. 4. Differences in responses between lung L1 and L4. (a) Genes that differed significantly (P,0.05) in expression between L1 and L4 in control and infected birds at 3 days p.i. are categorized into three major GO terms and an immune-related category. (b, c) Heat maps of immune-related genes significantly (P,0.05) differentially expressed in L1 compared with L4 in (b) control birds and (c) infected birds. Genes expressed at a higher rate in L1 are shown in red and those expressed at a higher rate in L4 in green.

genes involved in IFN-c signalling (IFI35, MIME, IFI30) and type I IFN signalling (IRFs) were expressed at certain locations, rather than in trachea and both lung segments. Genes involved in NF-kB signalling were expressed in trachea and L1, but in L4, no genes related to NF-kB signalling were found. Genes involved in antigen presentation were all upregulated. MHC class IV or B-G, which is only expressed in lung L1, and ASB2, which was only expressed in trachea, were downregulated.

trachea. Several genes were expressed exclusively in L1 and were involved in co-stimulation (CD86, CTLA4, PDL2), T-cell activation (ZAP70, TXK), T-cell proliferation (IGSF2, TNFRSF4) and CD3 signal transduction (TRAT1). Many genes involved in B-cell activity were expressed especially in lung L1 and trachea. All B cell-related genes were upregulated except for BRAG, which was expressed only in trachea, and PIGR, which were both downregulated. In lung L4, genes related to T- and Bcell responses were rarely expressed.

Upregulation of genes that supported an increased T-cell response was mostly observed in lung L1 and

Several genes involved in cell migration and adhesion were expressed exclusively in trachea (CD9, THBS4) or in lung


2141


disease virus, an increase in virus-specific IgA antibodyforming cells (AFCs) in the HG is found (Russell & Koch, 1993; Van Ginkel et al., 2008), whereas most IgG-AFCs reside in the spleen (Russell & Koch, 1993). In mice, intranasal immunization with influenza virus results in local, long-term specific antibody production. These AFCs reside in larger numbers in the diffuse region, rather than in the organized region, of the nasal-associated lymphoid tissue (Liang et al., 2001; Etchart et al., 2006), showing that, also within the nasal compartment of mice, sampling of specific parts of the tissue will result in a different outcome, as shown for antibody production, but which will probably also be found at the host transcriptional level.

Fig. 5. Host responses against H9N2 infection in upper trachea and lung L1 and L4. Immune-related gene sets, based upon GO interpretation, significantly (P,0.05) differentially expressed in infected compared with control birds are shown.

L1 (MAEA, VCAM1). Genes involved in monocyte activity were equally expressed in all three parts of the respiratory tract, sharing similar genes. Infection with H9N2 mostly caused upregulation of genes involved in the regulation of apoptosis, mainly via caspase activity. Most genes involved in apoptosis were expressed in trachea and lung L1, whereas in lung L4, few apoptosisrelated genes were expressed. Several genes involving oxidative stress were upregulated, but mainly in trachea and lung L1. Lung L4 only expressed p40-phox. Gene expression related to complement activity mainly seemed to occur via the classical route. In L4, only CD93 and ITGB2 were expressed.

DISCUSSION To be able to study host responses to respiratory viruses, we first wanted to know whether sampling certain locations would affect the outcome of the study. The aim of this study was firstly to determine whether gene expression differed between upper and lower trachea and between different parts of the lung in control birds and whether this was affected by H9N2 AIV infection, and secondly to study the early host responses against LPAI H9N2. Besides the trachea and lung, other structures, such as the paranasal organs, paraocular Harderian gland (HG) and the conjunctiva-associated lymphoid tissue, come into contact with aerosolized virus or particles (Corbanie et al., 2006). Although cells capable of rapid responses against pathogens are present in these tissues (reviewed by Kothlow & Kaspers, 2008), only the humoral responses to pathogens have been described for the chicken. Upon infection with infectious bronchitis virus or Newcastle 2142

In our study, the trachea was divided into upper and lower parts, as H9N2 LPAI has a preference for infecting the upper part of the respiratory tract (Nili & Asasi, 2002), which coincided with our data at both 1 and 3 days p.i. However, only minor differences in gene expression were seen between upper and lower trachea in both control and infected birds, suggesting that gene expression within the trachea was not affected by a difference in virus deposition. Lung was divided into four pieces according to unidirectional airflow and lung anatomy. Lung L1 was most similar to L3 and L2 resembled L4. In this paper, we report that the differences in anatomy and airflow were reflected upon gene expression in control birds and, upon infection with H9N2, host responses in the different parts of the lung were even more diverse. Furthermore, airflow and anatomy affected virus deposition within the lung. During inhalation, air flows through the primary bronchus, bypassing the cranially located openings of the medioventral secondary bronchi, and flows into the caudal air sacs or through the mediodorsal and lateroventral secondary bronchi (Fig. 1). This caudal-to-cranial flow pattern is also evident during expiration. In the caudal part of L1 and L3, which contains the bifurcations to the mediodorsal and lateroventral secondary bronchi, virus RNA levels were significantly higher and virus-infected areas were larger than in L2 and L4. This correlates with results of studies on deposition of airborne microspheres in the avian respiratory tract, which indicated that airborne particles are not distributed uniformly in the lung and deposition is influenced by particle size and size of the airway, and that particles are intercepted at bifurcations within the respiratory tract (Hayter & Besch, 1974; Mensah & Brain, 1982; Corbanie et al., 2006). Differences in virus deposition in infected birds enhanced the differences in gene expression already seen in control birds. This coincided with the finding in the lung of macaques that differences in H1N1 influenza virus mRNA result in differences in gene expression (Baas et al., 2006). A functional consequence of the flow pattern in the avian lung, resulting in an uneven spread of virus, may explain the absence of bronchusassociated lymphoid tissue (BALT) in the cranial part of the lung. Although organized BALT nodules are not observed before the third week after hatching, a similar time-course of BALT development in SPF and convenJournal of General Virology 90


Table 2. Immune-related genes induced by H9N2 infection in upper trachea (UT) and lung L1 and L4 at 3 days p.i. ID, Ensembl ID; E in the ID column indicates ENSGALG000000. + or 2 represents up- or downregulated gene expression in infected birds compared with control birds. Gene

ID

UT

L1

+ + +

+ + + 2 + + + +

+ +

+ +

Gene

ID

UT

L1

PTX3 E09694 + USP18 E13057 + + Cell migration, macrophage responses CAD11 E05278 2 2 CD34 E01177 2 2 CD9 E17274 2 CSF3R E02112 + + THBS4 E14804 2 VCAM1 E05257 + ZYX E14688 + + CSTA E14412 + + IGSF6 E07059 + + MAEA E13310 2 RGS18 E21143 + + IL1B E00534 + + IL13RA2 E20316 + + IL18 E07874 + LY75 E11153 + + LY86 E12801 + LY96 E15648 + + Antigen presentation (MHC pathways) ASB2 E10881 2 CD74 E04594 + + CTSB E16666 + + CTSC E17239 + + DMA E00158 + + MHC B-G E24357 2 MHC I E00178 + + E00141 + MHC II b TSPAN8 E10152 + +

L4 + 2

+

+ + + +

+ +

+ + + +

Gene

ID

TLR and NF-kB signalling TLR1 E17485 TLR3 E13468 TLR4 E07001 TLR7 E16590 TLR15 E08166 CARD9 E01889 NFKBIA E10063 NFKBIE E10171 TNIP2 E15666 TNIP3 E11961 TRAF1 E01583 TRAF3 E11389 TRAF5 E09864 TRAIP E02908 B- and T-cell responses BCAP29 E08009 BLNK E06973 PIGR E00919 RGS1 E02549 BRAG E09704 CD28 E08669 CD3D E07418 CD86 E14362 CTLA4 E08666 GIMAP7 E05136 IGSF2 E15467 IL2RG E05638 IL18R1 E16786 PDL2 E15032

UT

L1

L4

+ +

+

+

+ + + +

+ + +

+ + + + + +

+ + + 2 + 2 + +

+ + +

+ + + + + +

+ + 2 + + + + + + + + + +

+

+

Gene

ID

SLA E16234 TNFRSF4 E01875 TRAT1 E15362 TXK E14127 ZAP70 E01486 Apoptosis, oxidative stress, BCL2A1 E06511 BCL2L14 E11544 CASP8 E08355 CIDEA E00018 DAP E13002 GZMA E13548 PANX1 E17213 RASSF2 E00206 RIPK1 E12827 TFIP8 E02196 TNFAIP8L1 E04266 ERO1L E12404 MAFF E12277 NCF1 E01189 NOXO1 E05572 p40-phox E12520 SOD3 E18557 C1QB E21569 C1QC E23605 C1S E14603 C3AR1 E13218 CD59 E24468 CD93 E08359 ITGB2 E07511

UT

L1

+

+ + + + + complement + + + + + 2 2 2 2 + + + + + + + + + + + + + + + + + + + + + + + + + + + 2 +

+

L4 +

+

+ + +

+

2 +

2143


Cytokines and chemokines BAFF E16852 + + CCL4 E00951 + + CCLi3 E00956 + CCLi7 E14585 + + CCR5 E11732 + + CXCLi2 E11668 + + GHR E14855 2 2 NMI E12480 + + SOCS3 E07189 + + STAT4 E07651 + + TGFB3 E10346 2 2 Innate immune and IFN response AVID E23622 + AVR2 E02441 + CLL1 E16113 2 FLN29 E04802 + + GAL4 E19843 + IFI30 E03389 + IFI35 E02832 + + IRF1 E06785 + + IRF10 E06448 + + IRF3 E14297 + IRF8 E05757 + + MARCO E12119 + + MDA5 E11089 + + MIME E04732 2 2 MMP7 E17184 + + MX E16142 + + OASL E13723 + +

L4


tional chickens was found (Fagerland & Arp, 1993) and infections with pathogenic micro-organisms increased the number of BALT nodules significantly (Van Alstine & Arp, 1988). This probably resulted in more differential expression with a higher fold change of genes involved in cell adhesion and motility, chemotactic and IL activities and T and B cell-related genes in L1 compared with L4, in both control and infected birds. By analysing host responses in lung L1 and L4 separately, gene-expression patterns were not diluted and could be used to define overall and site-specific responses to H9N2 infection. After comparing gene expression in trachea, lung L1 and L4, we found that most changes occurred in trachea and L1. Most genes expressed in L4 were also expressed in trachea and L1, indicating these were part of a common host response, independent of the amount of virus RNA. Most of these shared responses have been described previously as being part of a common response to pathogens (Jenner & Young, 2005; Pennings et al., 2008) and involve upregulation of genes related to chemokine activity and inflammatory responses, as described in primate and rodent influenza models (Pennings et al., 2008). These correspond with increased IL-1b and IL-6 mRNA expression and the massive influx of KUL-01+ cells in trachea and lung of infected birds. Trachea and L1 had an overlap in gene expression that was not shared by L4, indicating that these responses were affected by virus load. Several site-specific responses were measured upon infection. Toll-like receptors (TLRs) contribute to innate responses, of which TLRs 3 and 7–9 recognize nucleosides that are important in virus recognition. In chicken, TLR9 has not been identified, but it is speculated that the two TLR7 splice variants, TLR15 or TLR21, may elicit responses to mammalian TLR9 ligands (Philbin et al., 2005; Jenkins et al., 2009). After infection with H9N2, upregulation of TLR3 was found in trachea, as was also found at the mRNA level in lung and brain of chickens upon infection with high-pathogenic AIV (Karpala et al., 2008). TLR1 and TLR7 were upregulated in trachea, lung L1 and L4. Both TLR1 and TLR7 are upregulated in human primary macrophages infected with influenza or Sendai viruses (Miettinen et al., 2001), indicating that upregulation of these genes is probably part of a common host response to viruses. TLR15 expression has been reported to remain unchanged after in vitro H9N2 infection (Xing et al., 2008), in contrast to our data, which show that, upon H9N2 infection in vivo, TLR15 was upregulated in L1. Several genes were especially expressed in L1 and were involved in innate responses, IL activity and vesicle trafficking. Molecules involved in innate immunity to pathogens, such as CLL1, PTX3 and GAL4, were only upregulated in L1. C-type lectins and PTX3 are known to have antiviral activity against influenza virus (Hogenkamp et al., 2008; Reading et al., 2008). In mice, b-defensins such 2144

as GAL4 are upregulated in the airway epithelial cells due to influenza virus infection (Chong et al., 2008). ANXA6, DDEF2, PICALM and ZFYVE20 are involved in vesicle trafficking, such as endocytosis and phagocytosis, and were only expressed in L1; in contrast to most genes, they were downregulated. Changes in expression of these genes have not previously been described in a virusinfection model, although the endocytosis pathway is known to play an important role in the entry of influenza virus into host cells (Lakadamyali et al., 2004). Whether downregulation of these genes was induced by host cells to inhibit virus entry or by antigen-presenting cells to control dissemination of virus remains to be investigated. Although not described in this paper, the avian respiratory tract also contains multiple air sacs that work as bellows to enable ventilation of the lungs. Upon inoculation with infectious bronchitis virus or influenza virus, reduced lucency of the air sacs and cellular influxes containing CD4+, CD8+, cd-TCR+ and KUL-01+ cells and heterophil infiltrates are found (Perkins & Swayne, 2002; Matthijs et al., 2009). It is highly likely that the airflow also affects responses in the air sacs, because only certain air sacs will be exposed to the inhaled air containing the virus (Fig. 1). To our knowledge, we are the first to give an overview of host responses to AIV at transcriptional level in the trachea and in the lung, taking into account the effect of anatomy and airflow through the respiratory tract. Gene expression within the lungs of control birds already differed significantly. Moreover, airflow affected virus deposition and subsequently gene expression, indicating that sampling at specific sites within the lung affects the outcome of studies of respiratory infections in chickens. Changes in gene expression and cellular influxes were significantly more pronounced in the parts of the respiratory tract where virus deposition was highest. These findings suggest not only that responses against AIV are affected locally, but also that most pathogens that are not distributed evenly throughout an organ will induce localized responses; careful sampling of the organ will be essential.

ACKNOWLEDGEMENTS We thank Peter van de Haar, Dik van Leenen, Cheuk Ko and Linda Bakker for their technical assistance and Dr Winfried Degen for collaboration on the in vivo experiment. This work was supported by a BSIK VIRGO consortium grant (no. 03012), the Netherlands.

REFERENCES Ariaans, M. P., Matthijs, M. G., van Haarlem, D., van de Haar, P., van Eck, J. H., Hensen, E. J. & Vervelde, L. (2008). The role of phagocytic

cells in enhanced susceptibility of broilers to colibacillosis after infectious bronchitis virus infection. Vet Immunol Immunopathol 123, 240–250. Baas, T., Baskin, C. R., Diamond, D. L., Garcia-Sastre, A., BielefeldtOhmann, H., Tumpey, T. M., Thomas, M. J., Carter, V. S., Teal, T. H. & other authors (2006). Integrated molecular signature of disease:


AIV distribution and host response differ within lung analysis of influenza virus-infected macaques through functional genomics and proteomics. J Virol 80, 10813–10828.

Lakadamyali, M., Rust, M. J. & Zhuang, X. (2004). Endocytosis of

Baskin, C. R., Garcıá-Sastre, A., Tumpey, T. M., Bielefeldt-Ohmann, H., Carter, V. S., Nistal-Villań, E. & Katze, M. G. (2004). Integration of

Liang, B., Hyland, L. & Hou, S. (2001). Nasal-associated lymphoid tissue is a site of long-term virus-specific antibody production following respiratory virus infection of mice. J Virol 75, 5416–5420.

clinical data, pathology, and cDNA microarrays in influenza virusinfected pigtailed macaques (Macaca nemestrina). J Virol 78, 10420– 10432. Breed, D. G., Carr, P. & Vermeulen, A. N. (1996). Differential binding of two monoclonal antibodies directed against the chicken CD8a

influenza viruses. Microbes Infect 6, 929–936.

Luhtala, M., Tregaskes, C. A., Young, J. R. & Vainio, O. (1997). Polymorphism of chicken CD8-a, but not CD8-b. Immunogenetics 46,

396–401.

molecule. Vet Immunol Immunopathol 52, 117–125.

Mast, J., Goddeeris, B. M., Peeters, K., Vandesande, F. & Berghman, L. R. (1998). Characterisation of chicken monocytes, macrophages

Chong, K. T., Thangavel, R. R. & Tang, X. (2008). Enhanced expression of murine b-defensins (MBD-1, -2,- 3, and -4) in upper

and interdigitating cells by the monoclonal antibody KUL01. Vet Immunol Immunopathol 61, 343–357.

and lower airway mucosa of influenza virus infected mice. Virology 380, 136–143. Corbanie, E. A., Matthijs, M. G., van Eck, J. H., Remon, J. P., Landman, W. J. & Vervaet, C. (2006). Deposition of differently sized airborne

microspheres in the respiratory tract of chickens. Avian Pathol 35, 475–485. Degen, W. G., Smith, J., Simmelink, B., Glass, E. J., Burt, D. W. & Schijns, V. E. (2006). Molecular immunophenotyping of lungs and

spleens in control and vaccinated chickens early after pulmonary avian influenza A (H9N2) virus infection. Vaccine 24, 6096–6109. Eldaghayes, I., Rothwell, L., Williams, A., Withers, D., Balu, S., Davison, F. & Kaiser, P. (2006). Infectious bursal disease virus:

strains that differ in virulence differentially modulate the innate immune response to infection in the chicken bursa. Viral Immunol 19, 83–91. Etchart, N., Baaten, B., Andersen, S. R., Hyland, L., Wong, S. Y. & Hou, S. (2006). Intranasal immunisation with inactivated RSV and

bacterial adjuvants induces mucosal protection and abrogates eosinophilia upon challenge. Eur J Immunol 36, 1136–1144. Fagerland, J. A. & Arp, L. H. (1993). Structure and development of

bronchus-associated lymphoid tissue in conventionally reared broiler chickens. Avian Dis 37, 10–18. Fedde, M. R. (1998). Relationship of structure and function of the

avian respiratory system to disease susceptibility. Poult Sci 77, 1130– 1138. Hayter, R. B. & Besch, E. L. (1974). Airborne-particle deposition in

the respiratory tract of chickens. Poult Sci 53, 1507–1511. Hogenkamp, A., Isohadouten, N., Reemers, S. S., Romijn, R. A., Hemrika, W., White, M. R., Tefsen, B., Vervelde, L., van Eijk, M. & other authors (2008). Chicken lung lectin is a functional C-type

lectin and inhibits haemagglutination by influenza A virus. Vet Microbiol 130, 37–46. Jenkins, K. A., Lowenthal, J. W., Kimpton, W. & Bean, A. G. (2009).

The in vitro and in ovo responses of chickens to TLR9 subfamily ligands. Dev Comp Immunol 33, 660–667. Jenner, R. G. & Young, R. A. (2005). Insights into host responses

against pathogens from transcriptional profiling. Nat Rev Microbiol 3, 281–294. Karpala, A. J., Lowenthal, J. W. & Bean, A. G. (2008). Activation of the TLR3 pathway regulates IFNb production in chickens. Dev Comp

Immunol 32, 435–444. Kash, J. C., Basler, C. F., Garcıá-Sastre, A., Carter, V., Billharz, R., Swayne, D. E., Przygodzki, R. M., Taubenberger, J. K., Katze, M. G. & Tumpey, T. M. (2004). Global host immune response: pathogenesis

and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol 78, 9499–9511. Kothlow, S. & Kaspers, B. (2008). The avian respiratory immune

system. In Avian Immunology, 1st edn, pp. 273–288. Edited by F. Davison, B. Kaspers & K. Schat. London: Academic Press. http://vir.sgmjournals.org

Matthijs, M. G., Ariaans, M. P., Dwars, R. M., van Eck, J. H., Bouma, A., Stegeman, A. & Vervelde, L. (2009). Course of infection and

immune responses in the respiratory tract of IBV infected broilers after superinfection with E. coli. Vet Immunol Immunopathol 127, 77– 84. Mensah, G. A. & Brain, J. D. (1982). Deposition and clearance of inhaled aerosol in the respiratory tract of chickens. J Appl Physiol 53, 1423–1428. Miettinen, M., Sareneva, T., Julkunen, I. & Matikainen, S. (2001).

IFNs activate Toll-like receptor gene expression in viral infections. Genes Immun 2, 349–355. Nili, H. & Asasi, K. (2002). Natural cases and an experimental study of

H9N2 avian influenza in commercial broiler chickens of Iran. Avian Pathol 31, 247–252. Pennings, J. L., Kimman, T. G. & Janssen, R. (2008). Identification of

a common gene expression response in different lung inflammatory diseases in rodents and macaques. PLoS One 3, e2596. Perkins, L. E. & Swayne, D. E. (2002). Susceptibility of laughing gulls (Larus atricilla) to H5N1 and H5N3 highly pathogenic avian influenza viruses. Avian Dis 46, 877–885. Philbin, V. J., Iqbal, M., Boyd, Y., Goodchild, M. J., Beal, R. K., Bumstead, N., Young, J. & Smith, A. L. (2005). Identification and

characterization of a functional, alternatively spliced Toll-like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens. Immunology 114, 507–521. Reading, P. C., Bozza, S., Gilbertson, B., Tate, M., Moretti, S., Job, E. R., Crouch, E. C., Brooks, A. G., Brown, L. E. & other authors (2008). Antiviral activity of the long chain pentraxin PTX3 against

influenza viruses. J Immunol 180, 3391–3398. Roepman, P., Wessels, L. F., Kettelarij, N., Kemmeren, P., Miles, A. J., Lijnzaad, P., Tilanus, M. G., Koole, R., Hordijk, G. J. & other authors (2005). An expression profile for diagnosis of lymph node metastases

from primary head and neck squamous cell carcinomas. Nat Genet 37, 182–186. Russell, P. H. & Koch, G. (1993). Local antibody forming cell

responses to the Hitchner B1 and Ulster strains of Newcastle disease virus. Vet Immunol Immunopathol 37, 165–180. Shinya, K., Ebina, M., Yamada, S., Ono, M., Kasai, N. & Kawaoka, Y. (2006). Avian flu: influenza virus receptors in the human airway.

Nature 440, 435–436. Van Alstine, W. G. & Arp, L. H. (1988). Histologic evaluation of lung

and bronchus-associated lymphoid tissue in young turkeys infected with Bordetella avium. Am J Vet Res 49, 835–839. Van de Peppel, J., Kemmeren, P., van Bakel, H., Radonjic, M., van Leenen, D. & Holstege, F. C. (2003). Monitoring global messenger

RNA changes in externally controlled microarray experiments. EMBO Rep 4, 387–393. Van Ginkel, F. W., van Santen, V. L., Gulley, S. L. & Toro, H. (2008).

Infectious bronchitis virus in the chicken Harderian gland and 2145

S. S. Reemers and others lachrymal fluid: viral load, infectivity, immune cell responses, and effects of viral immunodeficiency. Avian Dis 52, 608–617.

with the presence of undifferentiated follicles in the recovering bursa. Viral Immunol 18, 127–137.

Van Riel, D., Munster, V. J., de Wit, E., Rimmelzwaan, G. F., Fouchier, R. A., Osterhaus, A. D. & Kuiken, T. (2007). Human and

Wu, H., Kerr, M., Cui, X. & Churchill, G. (2003).

avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals. Am J Pathol 171, 1215– 1223. Vervelde, L., Vermeulen, A. N. & Jeurissen, S. H. (1996). In situ

characterization of leucocyte subpopulations after infection with Eimeria tenella in chickens. Parasite Immunol 18, 247–256.

MAANOVA: a software package for the analysis of spotted cDNA microarray experiments. In The Analysis of Gene Expression Data: Methods and Software, pp. 313– 341. Edited by G. Parmigiani, E. S. Garrett, R. A. Irizarry & S. L. Zeger. New York: Springer.

Xing, Z., Cardona, C. J., Li, J., Dao, N., Tran, T. & Andrada, J. (2008). Modulation of the immune responses in chickens by

Wan, H. & Perez, D. R. (2005). Quail carry sialic acid receptors

low-pathogenicity avian influenza virus H9N2. J Gen Virol 89, 1288–1299.

compatible with binding of avian and human influenza viruses. Virology 346, 278–286.

Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J. & Speed, T. P. (2002). Normalization for cDNA microarray data: a robust

Withers, D. R., Young, J. R. & Davison, T. F. (2005). Infectious bursal

composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30, e15.

disease virus-induced immunosuppression in the chick is associated

2146
