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Physiol. Genomics 25: 336-345, 2006; doi:10.1152/physiolgenomics.00208.2005
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Received 15 August 2005; accepted in final form 13 February 2006.
Physiological Genomics 25:336-345 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Strain-dependent pulmonary gene expression profiles of a cystic fibrosis mouse model

Christina K. Haston1, Sean Cory2, Laurie Lafontaine1, Geneviève Dorion1 and Michael T. Hallett2

1 Meakins-Christie Laboratories and Department of Medicine, McGill University, Montreal, Quebec, Canada
2 McGill Centre for Bioinformatics, McGill University, Montreal, Quebec, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cystic fibrosis (CF) lung disease severity is influenced by unknown genetic factors apart from the disease causative gene, cystic fibrosis transmembrane conductance regulator (CFTR). Previous studies have shown the C57BL/6J congenic Cftr–/– (B6 CF) mouse to develop a fibrotic lung disease compared with both CF mice of the BALB/c background and wild-type animals. In this report, gene expression profiling with microarrays was used to identify genes differentially expressed in the lungs of B6 and BALB CF mice compared with non-CF littermates. Seven hundred two genes or expressed sequence tags (ESTs) were identified to be differentially expressed between the B6 CF and non-CF control lungs (P < 0.05), and, by Gene Ontology classification, the B6 CF response included the cell proliferation categories of DNA metabolism and mitosis. In the response of BALB mice to nonfunctional Cftr, 943 genes/ESTs were differentially expressed compared with controls. The biological processes of apoptosis and T and B cell proliferation were prominent in the gene list of the BALB CF strain. In support of this strain difference, increased T lymphocyte infiltration was evident in the lungs of BALB CF mice, through immunohistochemical staining, compared with the lungs from both B6 CF and non-CF control mice. Four hundred forty-four genes/ESTs were differentially expressed between B6 CF and BALB CF mice (P < 0.05, fold >2), including 56 that map to previously identified linkage intervals. These results suggest that the variable severity of CF lung disease in this mouse model is controlled by multiple genetic factors, including those of an immune response.

immunity; C57BL/6; BALB


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CYSTIC FIBROSIS (CF) is a disease in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to varied symptoms affecting principally the lung, intestine, liver, and pancreas (3). CF lung disease consists of damaging cycles of neutrophil-dominated inflammation and infection (usually by Pseudomonas aeruginosa) that ultimately destroy the airway and impede gas exchange, resulting in mortality (3). The mechanisms through which mutations in the chloride ion channel encoded by CFTR produce lung disease are not known. Furthermore, the extent of lung disease in CF is highly variable in the clinical population, and this variability does not correlate with CFTR genotype (17, 22, 35). Indeed, the subset of ~50% of CF patients who are homozygous for the {Delta}F508 mutation and who exhibit variable CF symptoms provides evidence that non-CFTR factors are involved in the development of this disease (22). The increased concordance of CF disease severity in monozygotic twins compared with dizygotic twins (20, 27) indicates that some of these non-CFTR factors are genetic (modifier genes). In support of this, clinical investigations (21) have identified associations between the genotypes of physiological candidate genes and the severity of CF lung disease.

The identification of CF modifier genes using the candidate gene approach can be limited by clinical sample size and variable environment, and by the need for a priori knowledge of the candidate. Investigations using genomic approaches and CF mouse strains of variable disease severity circumvent these limitations. In particular, it has been shown that the m1UNC/m1UNC mutation in CFTR (29) when carried in the C57BL/6J (B6) background results in mice that have a fibrotic lung disease (5, 8, 15) not evident when the mutation is carried in the BALB strain (8). The lung disease of 3-mo-old B6 CF mice consists of patchy inflammation, neutrophil influx, thickened alveolar walls, mucous in the bronchioles, postbronchiolar overinflation of alveoli, interstitial fibrosis, and an increased number of nonciliated cells compared with age-matched C57BL/6J Cftr+/+ controls and mixed genetic background UNC CF mice. The lungs of BALB CF mice, in contrast, develop a perpetual neutrophil influx evident from 4–12 wk of age with no further fibrotic pathology (8).

We have used the difference in the propensity to develop lung disease between B6 CF and BALB CF mice to map factors of CF lung disease (8). In that study, the histological lung features of fibrosis and interstitial thickening were mapped to 11 significant or suggestive loci. Given this level of complexity, the identification of the actual modifier genes could require experimental approaches including the evaluation of congenic strains carrying these putative modifier loci and the integration of genomic gene expression analyses (32).

In the present report, we describe studies in which microarrays were used to measure the pulmonary gene expression profiles associated with the response to nonfunctional Cftr of B6 and BALB mice. This approach enables genes that may influence the inflammatory response in the CF disease-limiting tissue, in the absence of infection, to be identified without physiological assumptions. This analysis was coupled with an immunohistological assessment of the inflammatory phenotype, to elucidate expression changes potentially due to the altered cellular compositions of the CF lungs. In addition, to propose candidate CF lung modifier genes, a set of B6/BALB differentially expressed genes mapping to the previously defined pulmonary phenotype linkage intervals (8) was compiled.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice.
The mice of the C57BL/6J (B6) Cftr+/m1UNC (+/–) strain were provided by Dr. Danuta Radzioch of the Montreal General Hospital (QC, Canada), and mice of the BALBc/J (BALB) Cftr+/m1UNC strain were provided by Dr. Jim Hu of the Hospital for Sick Children (Toronto, ON, Canada). Homozygous B6 and BALB Cftr–/– (CF) mice were generated from matings of the heterozygote mice. For the litters produced, the tails of mice were clipped at 16 days of age, and genomic DNA was isolated for subsequent Cftr genotyping using a previously reported PCR assay (15). Because of the risk of death from intestinal obstruction, all CF mice were maintained on liquid diet (Peptamen; Nestlé, North York, ON, Canada) from the age of 18 days until death (16). All mice were housed in microisolator cages in a specific pathogen-free room of the animal facility at the McIntyre Building of McGill University. Mice were handled according to guidelines and regulations of the Canadian Council on Animal Care. Animal experiments were completed under a protocol approved by the McGill University Animal Care Committee.

To assess the pulmonary phenotype, the B6 and BALB CF mice were killed by pentobarbital sodium overdose at 12 wk of age, and lung tissue was removed for analysis. The left lung was immersion fixed in 10% neutral buffered formalin for subsequent histological processing, and the right lung was homogenized in 2 ml of Trizol reagent (Sigma-Aldrich, St. Louis, MO) and stored at –85°C until RNA isolation. For each strain, 3–5 CF and non-CF (Cftr+/+) male mice, and the same number of female mice, were killed. By routine serology surveillance performed on sentinel mice housed in the same room as the CF mice, no viral, mycoplasmal, fungal, or other respiratory pathogens were revealed.

Histology.
The left lung from each mouse was processed, sectioned at 3 µm, and stained with Masson's Trichrome for scoring of regions of increased collagen deposition. Each whole left lung section was given a score of 1–6 to indicate the severity of pathology as used in a prior study (8). All sections were scored by an observer blinded to mouse genotype.

To assess specific pulmonary cell types of CF mice, morphological analysis (neutrophils) and immunohistochemistry (macrophages and lymphocytes) were performed. One lung section per mouse, for each of three male CF and three male control mice, and the same number of female mice, was scored for each cell type, and the average of each of the cell counts was computed for each mouse. Macrophages were counted using a 400x magnification (10 fields = 2.3 mm2), whereas lymphocytes and neutrophils were observed using a 1,000x magnification (25 fields = 0.95 mm2). Differences in the histological and morphological scores between groups were assessed using t-tests.

Immunohistochemistry.
For antibody staining, the paraffin tissue sections (5-µm thick) were initially deparaffinized and hydrated through graded ethanol. Antigen sites were unmasked by an antibody-specific antigen retrieval treatment. Macrophages were identified with a rat monoclonal anti-mouse F4/80 antigen (dilution 1:100), while lymphocytes were stained with rat anti-human CD3 (dilution 1:75; antibodies from Serotec, Raleigh, NC). Endogenous peroxidase activity was quenched using 3% (vol/vol) hydrogen peroxide in Tris-buffered saline (TBS) for 15 min. Nonspecific binding sites were blocked with 10% goat serum (Cedarlane Laboratories, Hornby, ON, Canada) in TBS for 20 min at 37°C. The sections were then incubated overnight at 4°C with each of the antibodies. After a washing, sections were incubated with 1:150 biotinylated goat anti-rat secondary antibody (DakoCytomation, Mississauga, ON, Canada) for 60 min at room temperature. Sections were then washed and incubated in an avidin-peroxidase solution (StreptABComplex/HRP, DakoCytomation) for 45 min. Sections were developed with 3,3-diaminobenzidine tetrahydrochloride (Liquid DAB Substrate Chromogen System, DakoCytomation) and then counterstained with Gill's hematoxylin. Finally, the sections were dehydrated and mounted with Cytoseal. For negative control preparations, the primary antibody was replaced by TBS.

Gene expression.
Total lung RNA was extracted according to the manufacturer's (Sigma-Aldrich) instructions. One sample of RNA for each mouse was processed through the RNeasy column (Qiagen, Mississauga, ON, Canada) and submitted for hybridization. RNA quality was assessed and confirmed using the Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). The responses of eight groups of mice, defined by sex, strain, and Cftr genotype (+/+ or –/–), were measured using three gene chips, one per mouse, per group, with two exceptions. The data for the B6 male CF group are from two chips, as the third was excluded after failing the quality control checks, and the data for the BALB CF males are from three chips representing the gene expression of two BALB CF males (as RNA was not obtained from the third); thus two of these chips are of RNA from each mouse and the third from the pooled RNA of the two mice. Lung tissue from both male and female mice was assessed because of the sex specificity of some of the previously mapped loci.

Microarray hybridization was performed by the Affymetrix Gene Chip Core facility at McGill University and Genome Quebec Innovation Centre. The probe synthesis, hybridization, and washing protocols used were as described in Ref. 25 and followed the standardized Affymetrix protocol. The starting material was 10 µl of total RNA. After hybridization to the murine MOE430 2.0 GeneChip (Affymetrix, Santa Clara, CA), the gene chips were automatically washed and stained with streptavidin-phycoerythrin by use of a fluidics system. The chips were scanned with a GeneArray Scanner (Agilent Technologies). The resultant gene expression profiles were then extracted and viewed using Microarray Suite 5.0 (MAS5, Affymetrix). The MOE430 2.0 GeneChip arrays contain 45,000 probe sets derived from sequence clusters contained in Build 107, June 2002, of UniGene that represent ~34,000 functionally annotated genes and a set of expressed sequence tags (ESTs).

Microarray data analysis.
Routines from Bioconductor version 1.5.12 (http://www.bioconductor.org) within the R version 2.1.0 statistical language (12) were used to assess quality control, normalization and differential expression. In particular, the quality of the raw microarray data was assessed by inspecting similarities between the intensity distribution and RNA digestion plot for each array. Normalization was performed using the robust probe level model (13). The data were filtered using MAS5 to exclude probe sets identified as "absent" in three or more chips within one group (defined by strain and Cftr genotype). Detection of differential expression was performed using the linear models for microarray data (LIMMA) package (18, 28) with a P value < 0.05. All analyses used standard false discovery rate approaches where applicable. Lists of significantly differentially expressed genes were generated for CF mice compared with control mice and for strain and sex differences in expression. The Gene Ontology categories significantly represented in each compiled gene list, relative to the list of genes on the chip, were determined using the GOhyperG function in Bioconductor (7). Raw and normalized expression data are available from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), accession number GSE3100 (http://www.ncbi.nlm.nih.gov/geo/). Gene function was ascertained using information retrieved from PubMed (http://www.ncbi.nlm.nih.gov/entrez/) and Mouse Genome Informatics (http://www.informatics.jax.org).

Quantitative real-time PCR.
To generate the cDNA for real-time PCR, 4–5 µg of total RNA from each right mouse lung were reverse transcribed with oligo(dT) primer using Superscript II RNase H– RT (Invitrogen, Carlsbad, CA) in a 20-µl total volume. Reverse transcription was performed at 65°C for 5 min, followed by the activation of SuperScript II RT at 42°C for 50 min. The reaction was inactivated by heating to 70°C for 15 min.

We performed Taqman relative quantification on the following five genes: Tnfaip3 (tumor necrosis factor-{alpha}-induced protein 3, NM_009397), Mmp9 (matrix metalloproteinase 9, NM_013599), Cftr (NM_021050), Cd84 (Cd84 antigen, Mm004488934_m1), and Sell (selectin, lymphocyte, NM_011346). Taqman probes were obtained from Applied Biosystems (Foster City, CA) as Assays-on-Demand. Relative quantification was performed with the Applied Biosystems 7500 Real-Time PCR System. Each 25-µl reaction on a 96-well plate contained 0.4–0.5 µg of cDNA template, 12.5 µl of TaqMan Universal PCR Master Mix (at a x2 concentration), and 1.25 µl of Assays-on-Demand Gene Expression Assay Mix, which contained forward and reverse primers and labeled probe. The manufacturer's default thermal cycling conditions for PCR were used. Relative gene expression data analysis was carried out with the standard curve method (33). In this analysis, the expression level of each of the target genes was determined relative to that of a reference gene chosen from the set of genes whose expression varied from 0.9 to 1.1 relative units across the 23 arrays. The expression of the reference gene Stx8, (syntaxin 8, NM_018768) was determined, by RT-PCR experiments, not to be regulated by the experimental conditions. Relative quantification values were obtained by using the Applied Biosystems software, and a calibrator sample was employed in each run to correct for run-to-run variation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of the lung disease phenotype in B6 and BALB CF mice.
To phenotypically characterize the lungs of B6 and BALB CF mice relative to non-CF mice, lung tissue was taken from mice at 12 wk of age, and histological evaluation and gene expression profiling were completed. As shown in Fig. 1, the B6 CF mice had marked alveolar interstitial thickening with collagen deposition at 12 wk of age compared with both BALB CF mice (P = 5.8 x 10–5) and non-CF littermates (P = 2.6 x 10–6), as has been previously reported (8). The lung histology of BALB mice did not differ by Cftr genotype for the fibrosis measure (P = 0.28). The CF strain differences in histology were evident in both sexes, as shown in Fig. 1, and no differences in semiquantitative pathology were determined by sex of the animal.


Figure 1
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  		Fig. 1. Lung phenotype of 12-wk-old cystic fibrosis (CF) and non-CF mice. A: semiquantitative histological scores for interstitial thickening with collagen deposition. Values are means ± SD; n = 3–5 mice/group. B6, C57BL/6J; BALB, BALBc/J. *Strain differences: P < 0.05. B: immunohistochemical staining of CD3-positive cells (dark brown staining) in lung tissue. Top: lung tissue from male CF mice. Bottom: lung tissue from non-CF littermates.

 
To further characterize the strain difference in CF lung disease, the inflammatory cell populations of 12-wk-old CF and non-CF mice were measured after immunohistochemical staining. There were significantly more lymphocytes in the lungs of BALB CF mice than in the lungs of BALB non-CF controls, for both male and female mice (see Table 1 and Fig. 1B), whereas macrophage and neutrophil counts were not different by Cftr genotype. No differences in inflammatory cell population by Cftr genotype were detected in B6 mice (as shown in Table 1); thus a strain difference in CF lung phenotype is evident, as a higher lymphocyte count was recorded for the lungs of BALB CF mice compared with B6 mice. The strain difference in lymphocyte infiltration was evident for both male and female mice (see Table 1), although the lungs from male mice were consistently more cellular than those from females.


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  		Table 1. Lung tissue inflammatory cell counts for 12-wk-old B6 and BALB CF mice and non-CF littermates

 
Strain-specific gene expression response to nonfunctional Cftr.
To document the CF lung disease phenotype in B6 and BALB mice and to propose candidate genes that may influence its development, we completed gene expression analyses of 12-wk-old CF and non-CF mice of each strain.

To determine the set of genes of altered expression in the lungs of a B6 CF mouse, the data from five arrays representing the lung response of B6 CF male and female mice were compared with the data from six arrays of control lung tissue gene expression. Seven hundred two genes or ESTs were determined to be significantly differentially expressed in the lungs of CF mice compared with non-CF littermates, as shown in Supplemental Table S1, available at the Physiological Genomics web site1 (P < 0.05). The 702 differentially expressed genes or ESTs are derived from 952 probe sets. In this analysis, if one or more probe sets for a gene were determined to be differentially expressed, we then considered this gene to be differentially expressed. By Gene Ontology analysis, the biological processes most affected (P < 0.001) in the B6 CF mouse lung included cell proliferation (cytokinesis, DNA replication, cell cycle, mitosis) and T cell selection and activation (see Fig. 2 and Table 2). The genes most significantly differentially expressed in the lungs of B6 CF mice relative to non-CF littermates are listed in Table 3 and include nucleic acid metabolism (Rrm2, Tk1) and cytoskeletal genes (Myo10, Kif23). To assess whether pulmonary gene expression differed by the sex of the CF mice, we compared the gene expression profile of male B6 CF mice to that of B6 CF females, and 189 genes or ESTs were revealed to be differentially expressed by sex in the CF mice but not to differ in expression level by sex in B6 control mice. This set of genes is available in Supplemental Table S2.


Figure 2
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  		Fig. 2. Gene Ontology biological processes illustrate the difference between the C57BL/6J and BALBc/J pulmonary response to nonfunctional Cftr. Three categories (cellular physiological process, lymphocyte activation, and immune response) for which the contributing biological processes differed between the lung response of B6 CF mice, relative to B6 non-CF controls, and that of BALB CF mice, relative to the BALB control, are presented. Boxes, B6-specific CF processes; house symbols, BALB-specific responses; circles, responses common to both strains; diamonds, steps needed to complete the Gene Ontology pathways, but these processes were not significantly represented in the CF lung response of either strain.

 

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  		Table 2. Strain-specific GO biological processes significantly represented in the responses to Cftr deficiency

 

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  		Table 3. Genes most significantly differentially expressed in lungs of 12-wk-old Cftr–/– mice compared with Cftr+/+ littermates

 
A similar analysis of microarray data was completed to determine the set of genes involved in the pulmonary response of a BALB CF mouse. The data from six arrays of male and female CF mice were compared with the data from six arrays generated from lung tissue of control (BALB Cftr+/+) mice. Seven hundred genes were determined to be significantly differentially expressed in the lungs of BALB CF mice compared with non-CF littermates, as were 243 ESTs, as shown in Supplemental Table S3 (P < 0.05). The most significantly differentially expressed genes (ranked by P value) included Cftr, cell-signaling molecules Slamf6 and Evi2a, and the chemotaxis gene Ccr2, as shown in Table 3. By Gene Ontology analysis, the biological processes most affected (P < 0.001) in the BALB CF mouse lung, as shown in Fig. 2 and Table 2, included immune response (antigen presentation and immune cell migration, B cell proliferation and activation), apoptosis, and phagocytosis and endocytosis. When the response of the BALB CF male mice was compared with that of females, 39 genes or ESTs differed in expression level, including 9 genes of the X or Y chromosome, indicating a minimal sex difference in response in this strain.

One hundred twenty-six genes/ESTs (17.9% of the 702 genes/ESTs of the B6 CF response, 13.3% of the BALB CF response) were of altered expression in both CF mouse strains, indicating a significant B6/BALB strain difference in pulmonary response to nonfunctional Cftr. The genes common to the lung responses of the strains are given as Supplemental Table S4.

RT-PCR verification of microarray data.
We investigated the reliability of the microarray data by assessing the expression levels of five genes (Mmp9, Cd84, Sell, Tnfaip3, and Cftr) using quantitative RT-PCR on RNA derived from lung tissue. The genes were chosen by relevance to CF (Cftr) and by putative linkage region map position (Sell, Cd84, Tnfaip3, and Mmp9). As shown in Fig. 3, the gene expression differences identified by microarray analysis were similar in direction and magnitude to those seen with RT-PCR. The strain difference in Cftr expression, evident by microarray, was also confirmed by RT-PCR analysis. By both methodologies, the expression level of Cftr was significantly higher in lung tissue from BALB Cftr+/+ control mice than B6 mice (relative expression level of BALB/B6 = 2.54, P = 1.6 x 10–4). Cftr gene expression in the BALB CF mice was similar to the low expression level of the B6 strain, which was determined to be independent of Cftr genotype (see Fig. 3).


Figure 3
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  		Fig. 3. Gene expression levels by RT-PCR and microarray experiments. Ratio of the pulmonary gene expression level in Cftr–/– mice relative to Cftr+/+ controls (±SD) is given by sex and strain for each assay. *Significant differences (P value < 0.05) between CF and control mouse pulmonary gene expression levels. RNA was from 12-wk-old mice; n = 3–5 mice/group.

 
Strain comparison of gene expression data.
Analyses were completed to identify the set of lung genes that differed in expression between B6 and BALB mice. The strain differences in gene expression in the CF mice were identified by comparing the data of five chips from B6 CF mice to the data set from six BALB CF chips. In a second analysis, B6 and BALB pulmonary gene expression levels in non-CF mice were evaluated (the data of 6 chips representing the expression of B6 control lung tissue were compared with those of 6 BALB control chips). Four hundred forty-four genes or ESTs were determined to be significantly differentially expressed in the lungs of B6 CF mice compared with BALB CF mice (P < 0.05, fold >2; Supplemental Table S5), and 391 genes/ESTs were altered in expression level between the strains in control mice (P < 0.05, fold >2; Supplemental Table S6). The majority of the genes (61.5%) of strain-dependent pulmonary expression in the CF mice were also differentially expressed between B6 and BALB control mice. The most significantly differentially expressed genes in B6 CF mice relative to BALB CF mice are listed in Table 4 and include genes of antigen presentation (H2-Ea, H2-D1) and transport (Pttg1) and a solute carrier (Slc15a2). The genes listed in Table 4 were also significantly differentially expressed by strain in the lungs of control mice. By Gene Ontology analysis, the biological processes most often represented in the set of B6/BALB CF differentially expressed genes included signal transduction, cell proliferation and migration, and cytoskeleton organization, which is consistent with the histological data. A complete list of all Gene Ontology categories significantly represented in the B6 and BALB strain responses to Cftr deficiency, and in the strain and sex differences in response, is given in Supplemental Table S7.


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  		Table 4. Genes most significantly differentially expressed in the lungs of 12-wk-old B6 Cftr–/– mice compared with BALB Cftr–/– mice

 
Comparisons among the lists of significantly differentially expressed genes were made to identify sex-specific strain differences in the gene expression response to nonfunctional Cftr. Five hundred ninety-six genes/ESTs were identified to be differentially expressed in lung tissue between B6 female CF mice and BALB female CF mice. Of these, 202 genes/ESTs (34% of 596) were not on the list of the strain-specific response independent of sex, presented above. Genes of differential expression between the congenic CF strains, for the female mice only, included the inflammatory signaling molecules chemokine ligands 12, -19, and -21a; five genes of the structural keratin complex (Krt1-13, Krt1-14, Krt1-15, Krt2-4, Krt2-5); and four serine proteinase inhibitors (Serpinb11, Serpinb12, Serpinb3c, Serpinb5). The biological processes of the genes significantly differentially expressed between B6 CF females and BALB CF females only, by Gene Ontology analysis, included natural killer cell-mediated toxicity, regulation of B cell proliferation and activation, and actin filament length regulation.

For the male CF mice, 545 genes/ESTs were identified to be differentially expressed by strain, and 178 of these elements (33% of the response) were specific to this sex. Immunoglobulin genes (Igk-V1 Igk-V8 Igl-V1, Fcer1g) and genes of solute carrier family members (Slc11a1, Slc28a2 Slc2a3) were among those of altered expression between B6 CF male mice and BALB CF male mice. Consistent with this list of genes, biological processes of innate immune response, inflammatory response, and regulation of apoptosis were significantly represented in the strain difference of pulmonary response in CF male mice.

Differentially expressed genes specific to loci.
To propose candidate CF modifiers, the set of genes that were differentially expressed in the lungs of B6 CF mice relative to BALB CF mice, and that map to the previously defined linkage intervals (8), was documented. This analysis was completed with sex-specific gene expression comparisons for the loci mapped in only male or female mice. Of the 2,090 genes or ESTs in the combined linkage intervals, 56 (2.7%) were significantly differentially expressed (P < 0.05, B6/BALB relative expression >2) and are therefore positional and expression-derived candidates as CF lung disease modifier genes. These data are given in Supplemental Table S8, and a subset of this data, with the most significantly differentially expressed genes by P value, is given in Table 5. These candidate genes are from immunity (Ifi202b), ion-carrying (Atp1a2), and signal transduction (Dusp16, Tulp4, Pim1) pathways.


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  		Table 5. Linkage region genes most significantly differentially expressed in the lungs of 12-wk-old B6 Cftr–/– mice compared with BALB Cftr–/– mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the studies reported here, we have identified a mouse strain-dependent pulmonary gene expression response to Cftr deficiency in which the profile of B6 mice features genes of cell proliferation and T cell activation, while the BALB strain response includes apoptosis, cell migration, and T and B cell proliferation genes. Because these distinct gene expression profiles were measured in uninfected CF mouse strains that differ in their histological lung phenotypes, they may be useful in understanding the clinical inflammatory "priming" or excessive inflammatory response to pathogens (24, 26) and immune dysfunction features (23) of CF lung disease.

The gene expression phenotype suggests that the absence of Cftr triggers separate responses in the lungs of B6 and BALB CF mice, and this difference may provide a relevant model for dissecting the causes of CF-related adaptive immunity deficiencies [whose existence is inferred from the chronic infection status of CF patients (23)]. Specifically, the B6 and BALB CF strains were shown to differ in the expression of lymphocyte activation genes, among other genes of their expression profiles, in response to nonfunctional Cftr, and a strain-dependent difference in T lymphocyte influx was evident. T cell and B cell aggregates have been reported by Hubeau et al. (11) as part of the clinical CF lung disease course, and this airway leukocyte trafficking was proposed by Moss (23) to influence the earliest stages of inflammatory CF lung disease.

In this study, mice were assessed at the age of 12 wk; therefore, the phenotypes presented represent the strain adaptation to nonfunctional Cftr, in the lungs, and are thus a composite of CF-induced changes and the tissue response to this genetic deficiency. Because the pulmonary phenotype of B6 wild-type mice after exposure to agents such as P. aeruginosa (30), cyclophosphamide (10), and bleomycin (9) is distinct from that of BALB, these data may indicate an inherent difference in wound healing between these strains. The gene expression profiles and cell type staining measured here indicate, at least in part, a contributing difference in immune response, cell proliferation, and lymphocyte activation to the lung phenotype. The genetic factors influencing the strain-specific adaptation or repair response may be important CF lung modifiers, perhaps mechanistically linked to the CF lung modifier gene transforming growth factor (TGF)ß (4) and, furthermore, may have functional consequences in the established exaggerated inflammatory response of CF mice to Staphylococcus aureus (2) and P. aeruginosa exposure (31), which, in the latter case, has been shown to be independent of Cftr mutation genotype. The pulmonary phenotypes presented may also have been influenced by an undetected pathogen, but such a finding would not alter the observation of strain-specific CF lung responses.

We have previously reported the B6 strain histological response to nonfunctional Cftr to differ from that of BALB/c mice and used this strain difference to map loci of a CF fibrotic lung phenotype (8). By combining the linkage data with the present gene expression analyses, we have identified a set of position- and expression-derived candidate CF lung modifier genes. The gene expression experiment provides one criterion by which the (linkage derived) positional candidate genes can be ranked to facilitate further analyses, given that a (2-fold) change in expression is not a necessary condition for implication of phenotypic causality in a candidate gene. The gene expression data enable the set of positional candidate genes that are expressed in lung tissue (very likely a key condition of a CF lung disease modifier gene) to be known, and the utility of such data in the investigations to define causal genetic factors of complex traits has been substantiated (6). Further evidence to rank the candidate genes as CF modifier genes, apart from clinical investigation, could come from studies of specifically bred congenic mice and the identification of B6/BALB functional DNA sequence variations.

On the basis of CF strain-dependent differential expression and map position, interferon-{gamma} response genes and transcription factor genes were among the candidates identified as CF lung disease modifiers in this mouse model. Specifically, interferon-activated genes 202b and 203 were identified to be differentially expressed between B6 and BALB CF mice, and the strain-dependent expression of these genes, which are regulators of cell proliferation and differentiation thought to be involved in autoimmunity (19), in the presence of the CF defect could alter the lung phenotype. Similarly, the CF-induced altered expression of transcription factors, such as Atf3 or Nfe2l2, could initiate a response modifying the inflammatory state of the CF lung. Cho et al. (1) have identified Nfe2l2, whose function is to activate antioxidant enzymes to influence murine susceptibility to pulmonary fibrosis, whereas Atf3, a proapoptotic transcription factor, has been shown to be upregulated in the inflammatory condition of pancreatitis (14).

The evaluation of congenic strains and the use of a more probe-dense microarray in the present study permitted the identification of CF response genes in greater numbers than in the profile reported by Xu et al. (34), although a few specific genes common to the three largely distinct strain-dependent expression profiles were revealed. In their report, Xu et al. studied pulmonary tissue from UNC CF mice in a mixed B6/FVB background and Cftr+/– controls, of three ages and both sexes, and identified a set of 54 CF differentially expressed genes. Forty-three of the fifty-four genes were evaluated with the chip used in the present study, and a change in the expression levels of four genes was common to the B6, BALB, and B6/FVB CF lung response. These genes are potassium inwardly rectifying channel, subfamily J, member 15 (Kcnj15); adenylate cyclase 4 (Adcy4); tumor necrosis factor, {alpha}-induced protein 3 (Tnfaip3); and colony-stimulating factor 3 receptor (Csf3r). Two of the genes of this CF lung response independent of mouse strain, sex, and age may indicate an adaptation to the loss of the cAMP-regulated chloride channel function of Cftr, as the expression of the potassium channel gene Kcnj15 and of the cAMP biosynthesis gene Adcy4 was altered in CF mice. The second theme of the common response genes is inflammation, as the neutrophil chemotaxis factor gene Csf3r and the apoptosis-related transcription factor Tnfaip3 were upregulated in CF mice. If confirmed to be of altered expression or activity in CF lungs, these genes could be involved in the inflammatory priming component of CF lung disease.

In conclusion, we have used gene expression analysis and immunohistochemistry to document the CF lung responses of the B6 and BALB mouse strains. Genes of transcription regulation, intracellular signaling, and immunity were identified to be among those differentially expressed in the lungs of B6 CF mice relative to BALB CF mice and to map to previously reported linkage intervals. Further study of these genes may yield specific candidates for subsequent clinical investigation for factors influencing pulmonary response to the absence of Cftr and the enhanced inflammatory response of CF lungs.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by funding from the Canadian Cystic Fibrosis Foundation and from Fonds de la Recherche en Santé Québec.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: C. Haston, Dept. of Medicine, McGill Univ., Meakins-Christie Laboratories, 3626 rue St. Urbain, Montreal, Quebec H2X 2P2, Canada (e-mail: christina.haston{at}mcgill.ca).

1 The Supplemental Material for this article (Supplemental Tables S1–S8) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00208.2005/DC1. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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