Comprehensive gene expression profiling of rat lung ...

Page 1 of 41 in PresS. Am J Physiol Lung Cell Mol Physiol (August 24, 2007). doi:10.1152/ajplung.00105.2007 Articles

Comprehensive gene expression profiling of rat lung reveals distinct acute and chronic responses to cigarette smoke inhalation Christopher S. Stevenson1, Cerys Docx1, Ruth Webster1, Cliff Battram1, Debra Hynx3, June Giddings1, Phillip R. Cooper4, Probir Chakravarty1, Irfan Rahman5, John A. Marwick1, Paul A. Kirkham1, Christine Charman1, Delwood L. Richardson2, N.R. Nirmala2, Paul Whittaker1, Keith Butler1 1

Respiratory Disease Area, Novartis Institutes for Biomedical Research, Horsham,

West Sussex, RH12 5AB, United Kingdom 2

Genome and Proteome Sciences, Novartis Institutes for Biomedical Research,

Cambridge, MA, 02139, USA 3

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058

Basel, Switzerland 4

Airway Disease, National Heart & Lung Institute, Department of Cardiothoracic

Surgery, Dovehouse Street, London, SW3 6LY, United Kingdom 5

Dept of Environmental Medicine, Division of Lung Biology and Disease, University

of Rochester Medical Center, MRBX 3.11106, 601 Elmwood Ave., Box 850, Rochester, NY 14642, USA. Running head: Acute and chronic responses to cigarette smoke in rat Address correspondence to: Christopher S. Stevenson Imperial College London – National Heart and Lung Institute Dovehouse Street London SW3 6LY [email protected]

Copyright © 2007 by the American Physiological Society.

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2 Abstract Chronic obstructive pulmonary disease is a smoking-related disease that lacks effective therapies due partly to the poor understanding of disease pathogenesis. The aim of this study was to identify molecular pathways which could be responsible for the damaging consequences of smoking. To do this, we employed Gene Set Enrichment Analysis to analyze differences in global gene expression, which we then related to the pathological changes induced by cigarette smoke (CS). SpragueDawley rats were exposed to whole-body CS for 1 day and for various periods up to 8 months. Gene Set Enrichment Analysis of microarray data identified that metabolic processes were most significantly increased early in the response to CS. Gene sets involved in stress response and inflammation were also up-regulated. CS exposure increased neutrophil chemokines, cytokines, and proteases (MMP-12) linked to the pathogenesis of COPD. After a transient acute response, the CS-exposed rats developed a distinct molecular signature after 2 weeks which was followed by the chronic phase of the response. During this phase, gene sets related to immunity and defence progressively increased and predominated at the later time-points in smokeexposed rats. Chronic CS inhalation recapitulated many of the phenotypic changes observed in COPD patients including oxidative damage to macrophages, a slowly resolving inflammation, epithelial damage, mucus hypersecretion, airway fibrosis, and emphysema. As such, it appears that metabolic pathways are central to dealing with the stress of CS exposure; however, over time, inflammation and stress response gene sets become the most significantly affected in the chronic response to CS.

Keywords: COPD, inflammation, metabolic pathways, oxidant stress

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3 Introduction Cigarette smoke (CS) is the main etiologic factor for developing chronic obstructive pulmonary disease (COPD), the 4th most common cause of chronic morbidity and mortality in the world (23). However, not all smokers develop COPD, suggesting there are additional genetic factors that influence disease susceptibility. The COPD phenotype is typified by an accelerated rate of age-related decline in lung function that is related with structural changes in the lung, including peribronchiolar fibrosis, emphysema, and mucus hypersecretion. The disease is defined by the WHO/NHBLI GOLD initiative as ‘a disease state characterized by airflow limitation not fully reversible…associated with an abnormal inflammatory response of the lungs to noxious particles and gases,’ (28). The inflammation is characterized by the infiltration of neutrophils, macrophages and lymphocytes into the lung tissue and airways that persists after smoking cessation. Unfortunately, much of what is known about the pathogenesis of COPD originates from clinical observations from a single time-point that, while they may imply potential mechanisms, cannot distinguish between causative and consequential factors of the disease process. Consequently, little is known about the sequence of events that leads to the resulting lung pathologies or the molecular pathways involved in effecting these changes. Because of these clinical limitations, animal models have played an important role in attempts to elucidate disease-related mechanisms. A variety of agents have been used to mimic COPD-like changes in the lungs of rodents, including elastase, sulphur dioxide, nitrogen dioxide, ozone, lipopolysaccharide, and coal dust (reviewed in 21). While some of these models have been important for understanding of how some of these pathological changes may occur, how these models correlate with the changes which occur in response to cigarette smoking remains unclear. There have

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4 also been a number of descriptive studies investigating the acute and chronic effects of CS exposure in rodents (reviewed in 39 and 38, respectively). Most of these studies were largely focused on characterizing the inflammatory and/or the pathological changes that resulted, while only a few have focused on global molecular changes. In addition, few studies have characterized the effects of acute and chronic exposures within the same study (7). The use of knockout and transgenic animals in smoking studies has provided important insights into potential molecular mechanisms that are linked to disease pathogenesis (8, 19, 29). These studies are powerful, but limited to demonstrating the protective or destructive effects attributed to the abrogation or gross overexpression of a single gene. Because COPD susceptibility is likely to be heterogeneous and polygenic, understanding how macro changes at the level of the gene expression translate into pathological and physiological changes in response to CS will be essential for understanding how the disease develops. The aim of this study was to identify the molecular changes induced by CS inhalation that may drive the biological and pathological consequences, leading to disease. To do this, Sprague-Dawley rats were exposed to CS for 1 d and up to 34 w. In addition, we studied the effect of smoking cessation by exposing one group for 26 w followed by 8 w of no exposures. Using Gene Set Enrichment Analysis, global changes in gene expression were translated into alterations in sets of genes with common molecular functions. We then measured protein levels of some of the genes whose expression changed in response to CS. We also characterized inflammatory cell infiltration, oxidant-induced damage to cells in the lung, pathologies at every level of the airways, and lung function. Here, we demonstrate a clear distinction between the acute and chronic response to CS and provide a new hypothesis to explain why these changes occur, resulting in the pathologies linked to COPD.

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5 Materials and Methods Statement on Animal Welfare. Studies described herein were performed under a Project License issued by the United Kingdom Home Office and protocols were approved by the Local Ethical Review Process at the Novartis Institutes of Biomedical Research, Horsham. Animal maintenance conditions. Male, Sprague-Dawley rats (350-400g) (Charles River, Margate, UK) were housed in rooms maintained at constant temperature (21 ± 2°C) and humidity (55 ± 15%) with a 12 h light cycle and 15-20 air changes per h. Two animals were housed per cage containing 2 nest packs filled with grade 6 sawdust (Datesand, Manchester, UK), nesting material (Enviro-Dri, Lillico, UK), maxi fun tunnels and Aspen chew blocks (Lillico, UK ) to provide environmental enrichment. Animals were allowed food, RM1 Pellets, (SDS UK Ltd.) and water ad libitum. Cigarette Smoke Exposure. Animals were exposed to whole-body smoke as previously described (32) with some minor modification. Briefly, animals were exposed to 30 mLs of smoke every 60 s with fresh air being pumped in for the remaining time. The smoke was generated using 2R1 Research Cigarettes (University of Kentucky, Louisville, KY) and sham control animals were exposed to room-air only for the same duration of time (approximately 60 min per exposure period). Animals were sacrificed with an overdose of terminal anaesthetic (sodium pentobarbitone 200 mg i.p.) followed by exsanguinations 2 h after the last smoke exposure period. The carboxyhemaglobin levels were approximately ~5% at the time of termination. After 3 weeks of exposures, plasma cotinine levels varied from ~450 ng/mL 1 hour after exposure to ~150 ng/mL 22 hours after exposure. Animals were culled and changes were examined after 1, 3, and 5 d, 2, 3, 4, 6, 8, 12, 16, 26, and 34

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6 w of CS exposures. In addition, one group was exposed for 26 w followed by 8 w with no exposures to look at the effects of smoking cessation (referred to in the following text as the smoking cessation group). Microarray analysis. For microarray analysis, animals that did not undergo unconscious lung function manuevers or lavaged were used. The treatment and exposure regimen of the animals was otherwise identical. At the prescribed endpoint, animals were culled as described above and the lungs were removed and flash frozen in liquid nitrogen. Lung tissue was homogenized and RNA extracted using TRIZOL according to the manufacturer’s instructions. Protein contamination was checked at 260 and 280 nm and a ratio of < 1.6 was accepted. RNA was hybridized against Affymetrix RG_U34A GeneChips. The raw data was analysed using GeneSpring version 6. To derive differentially expressed genes a Welch t-test (parametric, not assuming equal variances) was used. The statistical cut-off used was 95% confidence. The fold regulation cut-off used was a > 2-fold up- or down-regulation comparing smoke- versus air-exposed controls. Gene lists for the 12 time-points were annotated computationally using DAVID, NetAffx, MAPPfinder, GenMAPP, and by systematic searches of PubMed. A non-redundant list of 340 genes that significantly changed between time-matched smoked and sham animals was generated by combining the gene lists from the 12 time-points. Data complied with MIAME standard and has been deposited in the GEO database (accession numbers GSM170276 - GSM170484). Gene set enrichment analysis. An in-house implementation of the Gene Set Enrichment Analysis method was used to analyze the data as previously described (24, 35). As input, GSEA requires microarray data from two conditions to be compared (eg., sham- versus smoke-exposed animals at each time-point). Genes with expression levels below 100 on more than 75% of the GeneChips were discarded.

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7 Differential expression for each probe-set was estimated by its expression ratio between pairs of conditions. Differentially modulated gene sets were identified using the Wilcoxon ranked sum test. Preparation of BAL fluid and lung tissue. Rats not used for microarray analysis were culled as above and lungs were lavaged with 3 x 4 mL aliquots of sterile PBS. All aliquots were combined for individual rats. BAL total cells counts and differentials were performed as previously described (32). The right lung lobes were tied off, snap frozen in liquid nitrogen and used for protein analysis. The trachea and left lung were inflated with 5 mLs of 10% neutral buffered formalin (NBF) for histological analysis. Immunohistochemistry for 4HNE. Immunohistochemistry for 4HNE was performed by microwave irradiation method. Briefly, lung sections were de-waxed in xylene, rehydrated and endogenous peroxidase inhibited with 0.5% hydrogen peroxide in methanol for 10 minutes. Sections were stained with anti-4HNE adduct (Calbiocherm) overnight at 4ºC. Immunodetection was performed using biotinylated rabbit anti-mouse Ig reagent (Dako Cytomation, Cambridgeshire, UK), sABC reagent (Dako Cytomation, Cambridgeshire, UK), and 3,3’-diaminobenzidine (DAB) (Sigma, Dorset, UK). The nuclei were counterstained with Cole’s haematoxylin solution. Tonsil was used as a positive control and for negative controls the primary antibody was omitted from one section of each of the samples. Cells were identified by both positive staining and standard morphological criteria. 4HNE staining intensity was assessed in alveolar macrophages semi-quantitatively in a blinded fashion and graded 0 = no staining, 1 = weak staining, 2 = moderate staining and 3 = strong staining. A Mann-Whitney non-parametric statistical test was performed to determine significance of data.

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8 BAL fluid and tissue cytokine analysis. Cell-free BAL and tissue homogenate supernatants were used to measure cytokine levels by ELISA (R&D Systems, Abingdon, UK, except for CINC-1/GRO; Amersham, UK). Tissue homogenate protein levels were measured using the Bio-Rad Protein assay (Bio-Rad Laboratories, Hertfordshire, UK) and cytokine values were normalized against protein levels for individual homogenate samples. Determination of BAL fluid mucin concentrations. Samples were assayed using a mucin enzyme-linked lectin assay (ELLA) as previously described (3, 15). Immunohistochemical staining of inflammatory cells. Macrophages and neutrophils were stained as previously described (22). Macrophages were stained for ED1 and neutrophils were identified with the substituted naphthol method for AS-D chloroacetate esterase. Cells were identified using an Axiohome Microscope by both positive staining and standard morphological criteria. Immunohistochemical staining of mucin and quantification of goblet cell density. Both tracheal and primary bronchus mucin were detected by a two stage immunoperoxidase method using Ulex europaeus Agglutinin-1 (UEA-1) (Sigma, Dorset, UK) validated by Jackson and colleagues (15) as previously described (32). Staining for collagen and smooth muscle. Sections (3µm) were incubated in 0.1% Sirius Red F3BA (VWR, Poole, UK) to identify collagen or with mouse anti-human smooth muscle actin, clone 1A4 (Dakocytomation, Ely, UK) (see supplementary methods for further detail). The areas of collagen and smooth muscle actin staining were assessed under a Zeiss Axioplan 2 microscope (x10 magnification) with an Imaging Associates KS400 image analyzer (Imaging Associates, Bicester, UK). Values were determined by measuring the inside perimeter of the airway and dividing it by the area of pigmented pixels, which was then converted into µm to give an

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9 average thickness surrounding the airway. To avoid bias on slant cuts, where the airway wall is thicker, a constant pixel count away from the airway was always measured when calculating the area. This prevented scoring of pseudo-area of pigmented tissue owed to more staining on obliquely cut airways. Collagen and SMA were quantified on areas of the upper, upper-middle, lower-middle and lower segments of the lung. The mean thickness for each sample was determined and then the group (smoke- or air-exposed) was averaged to give a mean and standard error of the mean (SEM) for each time-point. A Mann-Whitney non-parametric statistical test was performed to determine significance of data. Quantifying mean linear intercept. Mean linear intercept was measured manually using a method previously described (37) using a reticule with a Thurlbeck grid. See supplementary text file for details. Conscious lung function measurements by whole-body plethysmography. Rats were placed in a plethysmograph chamber (Buxco, Wilmington, NC, USA) and left to acclimatize for 5 min. Bias flow (air removed from chamber) was set at 1 L/min. The pressure wave forms generated by respiration of the rats were analyzed by Buxco (USA) software. PenH, area under the curve (PenH AUC), peak expiratory flow (PEF-ml/s) and breathing frequency (f) were calculated on a breath by breath basis as previously described (2, 5, 17) and averaged every 30s. All measurements were calculated over a 5 minute recording period. Unconscious lung function measurements using forced manoeuvres. Respiratory function was measured using methods similar to those described previously (1, 4). See supplementary text for details. Statistical analysis. All data are presented as mean + standard error of the mean (SEM). Unless otherwise stated, statistical significance of data was determined using

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10 a two-tailed T-test with each group being treated as an independent group and significance measured only against the time-matched air-exposed controls. A result was considered significant if the p value was < 0.05.

Results Cigarette smoke-induced changes in gene expression. Principal component analysis (PCA) was used to identify the major set of expression profiles within the 340 genes identified as being significantly different between smoke- and air-exposed rats from pair wise comparisons. The tool characterises the most abundant themes or building blocks that occur in many genes within the experiment. PCA identified 9 distinct expression profiles over the course of the study (see supplementary figure 1). Of the 9, 2 clusters were clearly either preferentially down-regulated or up-regulated in response to CS exposure, while there were 4 others that demonstrated gradual increases in expression. For clustering analyses, gene chips from sham- and CS-exposed samples at each time-point were clustered using the Pearson correlation as the similarity measure. This process attempts to group the most similar chips together based solely on the expression patterns and values. Figure 1 shows the results of the experimental clustering analysis. Earlier time-points (1 d to 5 d) show no separation of the two treatment groups based on global gene expression. Starting from 2 w, the two treatment groups begin to form distinct clusters based on treatment. Once the sham and CS-exposed groups have separated at 2 w, they remain so during the rest of the experiment. This clear pattern of differential gene expression gained in intensity over the course of the model. In the smoking cessation group the distinction between

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11 sham- and smoke-exposed rats largely remained, apart from one CS-exposed rat that grouped with the sham animals (figure 1). Pathway changes in response to smoke. During the first week, metabolic functions, in particular genes involved in oxidative phosphorylation and glycolysis (figure 2 and supplementary figure 2), were most significantly elevated by smoke exposure. Signal transduction gene sets were significantly reduced. Several metabolic pathways remained up-regulated over the course of the study (eg., PPARsignalling and carbohydrate metabolism); however, by 3 w pathways related to immunity and defence and proteolysis were most significantly increased (figure 3 and supplementary figure 3). Both inflammation and stress response/detoxification genes were also up-regulated acutely. Genes including the rat GRO homologues, IL-1 , and nuclear factor-erythroid 2-related factor 2 (NF-E2 factor 2 or Nrf2) were increased from 3 d and stayed elevated over the study. Many of these changes became more pronounced as the duration of exposures increased (figure 3). In particular those genes associated with macrophage function (eg., MMP-12) and phagocytosis (eg., CD36), progressively increased over the course of the model. This was also true for genes associated with the lymphocyte function such as Ly6-C antigen, IgG, and immunoglobulin receptor expression. By 34 w, gene sets related to immunity and defence and B cell-mediated immunity were the most significantly increased (figure 3 and supplementary figure 4). MHC class II- and macrophage-mediated immunity pathways were also very significantly elevated. Although slightly lower than at earlier time-points, inflammation and stress response/detoxification gene sets remained increased in the smoking cessation group (figure 3). In contrast, most the gene sets involved in metabolic processes returned to control levels (figure 2).

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12 Cigarette smoke-induced oxidative damage. Immunohistochemical analysis of the rat lungs for 4HNE protein modification showed that there was no difference in the general staining pattern and intensities between the CS-exposed and sham-exposed rat lungs. However, CS-exposed lungs exhibited stronger staining in the alveolar macrophages compared to the sham-exposed lungs samples (figure 4). In addition, in the 26 w CS-exposed rat lung, the terminal bronchial epithelium associated with macrophage lesions displayed increased 4HNE staining. The alveolar macrophages in the 8 w and 26 w CS-exposed rat lung also appeared to be clumped together in foci around the alveolar airspaces (figure 4D). Effect of CS exposure on inflammatory mediator levels. The neutrophil chemokines, CINC-1 and CINC-2 (rat GRO homologues) were increased in response to CS exposure in a bi-phasic fashion in both bronchoalveolar lavage (BAL) fluid (figure 5A-B) and lung homogenate supernatant (figure 5C-D). The initial peak appeared at 3 d, followed by a small decrease in the levels of these chemokines until 4 w. Subsequently, the second phase of CINC production occurred, peaking between 12 and 16 w. The CINC-1 response returned to baseline levels after smoking cessation, whilst, in contrast, CINC-2 levels remained significantly elevated in the tissue (p < 0.05) although much lower than earlier time points. Interleukin-1 was also increased in response to CS, however only in the lung homogenate supernatants (see supplementary figure 5). The levels were elevated and greatest after 3 d of exposure, increasing from 173 + 25 pg/mg tissue in sham animals to 375 + 57 pg/mg tissue in the CS-exposed rats (p < 0.01). The levels remained significantly increased over the course of the study, but returned to control levels after smoking cessation. In addition, IL-6, IFN- , MCP-1, TNF- , and IL-4 were assayed

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13 for, but were below the levels of detection. Levels of IL-13 were measured and, while variable, appeared to be increased at some but not all time points (data not shown). Effect of CS exposure on inflammatory cell infiltration. CS increased the number of neutrophils in the BAL fluid in a bi-phasic pattern similar to that of the neutrophil chemokines (figure 6A). After an initial peak at 5 d, the number of neutrophils increased again starting from 6 w and reached their greatest levels at 34 w, increasing from 0.5 + 0.1 x 105 in the sham animals to 14.7 + 2.3 x 105 neutrophils recovered in the BAL fluid of CS-exposed rats. A similar bi-phasic pattern of neutrophil infiltration was observed in the lung tissue (figure 6B). In both the BAL and tissue, the numbers of neutrophils decreased from their peak levels in the cessation group; however, neutrophil numbers remained elevated after smoking cessation in BAL fluid (p < 0.001). Macrophage numbers in the BAL fluid were significantly decreased in response to CS exposure starting from 3 d, continuing over the duration of the study, and stayed significantly lower in the smoking cessation group (p < 0.05) (figure 6C). Conversely, macrophage numbers in the tissue increased starting from 5 d and slowly, but progressively increased over the course of the study (figure 6D). They reached a maximum 3.7-fold increase at 34 w (p < 0.001). Again, the number of macrophages in the tissue remained significantly elevated after 8 w of smoking cessation (p < 0.01) although they were lower than earlier time points. No consistent increases in T lymphocytes were detected in the BAL fluid or tissue (data not shown); however, these cells appeared to be up-regulated in the CS-exposed animals, primarily localizing to areas containing macrophage aggregations and vessels that were excluded from the counting method.

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14 The most prominent inflammatory changes occurred in the alveoli, where pigmented, some bi-nucleated, macrophages began to accumulate by 3 d. These aggregations of macrophages began to fill alveolar spaces by 3 w, initially in those spaces adjacent to the terminal bronchioles (figure 7B). At 6-8 w, the aggregates progressively spread into surrounding spaces and began to incorporate neutrophils (figure 6C). Many of these cells showed signs of degeneration and cell death as determined by nuclear pyknosis and debris. These inflammatory lesions progressively worsened over the duration of the study (figure 7D). The interalveolar walls were multifocally distended with aggregates of lymphocytes and macrophages. In addition, by 8 w, a moderate perivascular lymphocytic inflammation was present in all CS-exposed animals (data not shown). There were also some mild inflammatory changes around the smaller airways in the CS-exposed rats. Acutely, around the bronchioles, there was a mild intra- and subepithelial neutrophilic inflammation that decreased after 2 w and was not present at 34 w. There were also mild peribronchiolar lymphocytic infiltrations in some CSexposed animals after 3 w (data not shown). Effect of CS exposure on the epithelium of the central airways. CS induced epithelial hypertrophy and hyperplasia (increased number of mitotic bodies) that peaked at 5 d, but was subsequently attenuated thereafter (figure 8). These changes were also associated with a significant increase in the number of mucin containing cells in both the trachea (figure 8A-D) and the primary bronchus. The area of epithelium stained positive for mucin in both the trachea and the bronchus was increased from 3 d and progressively increased over the course of the study, reaching a plateau by 16 w (figure 8E). These changes in mucus levels in the lung tissue correlated closely to the amount of mucin detected in the BAL fluid (figure 8F).

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15 Effect of CS exposure on smaller airways. The acute response to CS in the epithelium was associated with an increased number of mitotic bodies found in the epithelial cells in the small airways (data not shown). The epithelium in sham animals were a single cell layer of flattened to cuboidal cells; however, in the CS-exposed rats, the epithelium was multilayered containing cuboidal to prismatic cells. The hyperplastic response began to attenuate after 3 d, but remained elevated throughout the study. After 16 w of CS exposure, collagen deposition around the airways (other than primary bronchus) increased from 26.9 + 1.7 µm to 56.0 + 2.5 µm (p < 0.0001) (figure 9A). As time progressed, the amount of collagen around the airways in sham animals increased. Although there was still more collagen around the airways of CSexposed animals, the increase was no longer significant. The amount of smooth muscle around the airways was not significantly changed compared to air-exposed controls after 16 or 34 w (figure 9B). Effect of CS exposure on alveolar structure. In the alveoli, the alveolar walls of CSexposed rats were lined with cuboidal type II pneumocytes by 6 w. The mean linear intercept of the alveoli of CS-exposed animals increased after 34 w of exposures from 50.4 + 2.0 µm in control animals to 61.7 + 3.9 µm in CS-exposed (p < 0.05) (figure 10A-C). This translates into an increase of approximately 20%. No difference was detected between the groups which received 26 w of either air or CS exposures (data not shown). Effect of CS exposure on lung function. CS exposure caused very acute changes in lung function, as measured by conscious whole-body plethysmography (see supplementary figure 6). Small increases in PenH and decreases in peak expiratory flow (PEF) were detected; however, the changes were transient and lung function returned to normal within 6 h. The increased breathing frequency in CS-exposed

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16 animals during 6 h measurements was due to differences in the exploratory behaviour of the animals at this time. There were also no consistent, significant changes in FEV100, FVC, or TLC as measured by unconscious forced maneuvers (see supplementary figure 7).

Discussion The response to CS exposure in rat was comprised of two distinct phases – an acute phase that peaked at the end of the first week and a second, more chronic phase that started after a transcriptional shift in the gene expression pattern of the CSexposed rats, which occurred after 2 w of exposures. Many of the molecular changes induced by cigarette smoke were progressive and present during both phases. However, most of the lung pathology associated with smoking was apparent in the chronic phase. The major features of the acute phase included an immediate increase in the expression of metabolic gene sets and stress response genes, inflammation, and changes to the epithelium. Increased expression of metabolic genes sets was a new finding that has not been previously reported in the response to smoke. We propose that these changes are due to increased energy demands for repairing smoke-mediated damage. In addition, these pathways are critical for producing important reducing equivalents that may be needed to protect cells from the oxidizing effects of smoke. There is evidence to support the concept that energy transfer pathways and oxygen detoxification pathways co-evolved and are increased in concert in response to environmental stresses such as CS (33). Stress response genes were also increased during the acute phase. Enzymes such as heme oxygenase 1 (HO-1), thioredoxin, -GCS, MnSOD, and the

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17 transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf-2), were immediately up-regulated, consistent with previous reports (9). These genes appear to be critical in the response to smoke and have been implicated in the pathogenesis of COPD. HO-1 expression is up-regulated in alveolar macrophages of smokers (20) and promoter polymorphisms for this gene have been identified in certain COPD patients (40). Further, over-expression of HO-1 protects against lung injury in elastase and cigarette smoke models (31, 36). HO-1 and other antioxidant enzymes are regulated by Nrf-2 (6) which is essential for protection against CS-induced inflammation and emphysema (29). The inflammation in this acute phase was primarily neutrophilic in nature. The observed decrease in BAL macrophage numbers is likely to be due to the activation of these cells. In addition, oxidant modification to matrix proteins by cigarette smoke extract has also been proposed to enhance the adhesion of macrophages to matrix (16). Another basic feature of the acute response to CS was the epithelial thickening and goblet cell metaplasia which peaked after 5 d of exposures. Subsequent to this acute phase, components of the innate, host defence response significantly decreased, but did not completely resolve. Conversely, tissue macrophage numbers began to increase. The mechanisms underlying this transition to the second, more chronic phase in the response to smoke is unknown; however, we propose two hypotheses to explain these observations that may occur independently or collectively. Firstly, alveolar macrophages, which migrate into the lung to resolve inflammation, are damaged by oxidants and may become overloaded with particulate matter (PM) with continual exposure to CS. These changes can result in reduced migration and enhanced activation of macrophages (25, 26). This may trigger the

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18 chronic inflammatory response that is observed following 2 w of exposures. This hypothesis is consistent with the observations made in this model, with increased 4HNE staining in alveolar macrophages that are particle laden and accumulating in large aggregates in the alveoli and alveolar ducts by 2 w. Secondly, ultrafine particles may localize to the macrophage mitochondria inducing structural damage (18) thereby reducing the metabolic capacity needed for protection against CS exposure (33). This damage to the metabolic machinery leaves the cell no longer capable of adapting to the meet the added demands of further CS exposures. This may account, at least in part, for the “transcriptional shift” that occurs in gene expression patterns after 2 w of CS exposures and we propose that these changes are responsible for initiating the chronic response to smoke. In summary, we hypothesize that eventually the system becomes overwhelmed with repeated oxidant and particulate challenges, resulting in metabolic insufficiency and subsequently a greater oxidant burden leading to greater inflammation. The second chronic wave of inflammation, oxidant damage, and mucus production was progressive and greater than the initial acute response. As shown previously, the inflammation shifted from a primarily neutrophilic response to one that that had greater macrophage involvement (12, 27). Lymphocytes were present in these aggregates and markers of the adaptive immune response (eg., B cell-mediated immunity pathway, Ly6-C antigen and IgG chains) were increased in the chronic phase; however the presence of lymphocytes were not as apparent as in smoking models reported in other species such as the mouse (7). Associated with this second wave of inflammation were the start of structural changes in the lung consistent with those observed in smokers and COPD patients including collagen deposition around the airways and macrophage aggregation. There

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19 has previously only been one other report that we know of demonstrating a change in airway collagen in rats in response to CS-exposure (30). The increase in airway collagen was no longer significant at 34 w due to the increase level of collagen in the air-exposed controls. After 34 w of exposure there was a significant increase in the mean linear intercept of the alveolar spaces in CS-exposed rats. The failure to see greater structural damage to the lung and subsequent changes to lung function is possibly related to a lack of any viral and/or bacterial infection in this model system. In the smoking cessation group, the changes induced by CS exposure were resolving; however, many of the inflammatory and antioxidant genes remained significantly elevated and were associated with increased numbers of inflammatory cells as has been documented in COPD patients (13). Despite this, other phenotypic changes (eg., mucus production) had returned to control levels. One limitation to this study is that the microarray experiments were conducted using RNA generated from whole lung rather than single cell types. Therefore, changes in expression profiles may reflect changes in cellular constituency of the lung at each time-point. Ideally, it would be useful to analyse the expression changes occurring in a more homogenous cell population using laser capture microdissection. However, this study is strengthened by the fact that samples from individual rats were used for each time-point in contrast to previously published work (9) where pooled samples were used. The use of individual samples allowed us to differentiate CS- and air-exposed groups in a more significant and comprehensive fashion. In addition, for the first time in a rodent smoking model, we used both pathway- and gene-centric analysis of the microarray data in combination with pathological observations. This approach placed these changes in a broader biological context and led to the

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20 identification of changes in biochemical processes previously not associated with the response to CS inhalation or COPD. In summary, this study has provided new insights into the molecular pathways activated in response to CS and possibly involved in the pathogenesis of COPD. After a transient acute response where metabolic pathways appeared most significantly changed, a transcriptional shift in the gene expression pattern after 2 w appears to initiate the chronic phase of the response to smoke inhalation. This shift may be due to changes in metabolic capacity and/or macrophage function; similar to the situation found in man (10, 12). Chronic CS inhalation leads to greater activation of immunity and defence pathways and recapitulated many of the phenotypic changes observed in COPD patients. These include oxidative damage to macrophages, a chronic, low-grade inflammation consisting of neutrophils, macrophages, and lymphocytes, epithelial damage, mucus hypersecretion, airway fibrosis, and emphysema. These data suggest that the rat model may provide insights into the molecular mechanisms underlying the response to chronic smoking that lead to the destructive lung pathologies observed in COPD patients. In addition, using “OMICs” technologies to characterize both animal models and clinical samples may help identify the gaps and parallels in these systems, as well as uncover prospective biomarkers, both of which are vital for identifying and measuring the efficacy of prospective drug candidates for COPD.

Acknowledgements The authors would like to thank Dr. Gabriele Pohlmeyer-Esch and Dr. Horst Posthaus for their help in the pathological characterization of this model and Prof. Ian Adcock

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21 for his careful review of this manuscript. The authors would also like to thank Prof. Jody Wright and Prof. Malcolm Madden for their help in the measurement of the mean linear intercept data and Ms. Danielle Fletcher for her aid in preparing the microarray data.

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22 References 1. Amdur MO and Mead J. Mechanics of respiration in unanesthetized guinea pigs. Am J Physiol 192: 364-368, 1958.

2. Battram C, Charlton SJ, Cuenoud B, Dowling MR, Fairhurst RA, Farr D, Fozard JR, Leighton–Davis JR, Lewis CA, McEvoy L, Turner RJ, and Trifilieff A. In vitro and in-vivo pharmacological characterisation of indacaterol (5-[(R)-2(5,6-Diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one), a novel inhaled {beta}2 adrenoceptor agonist with a 24-hour duration of action. J Pharmacol Exp Ther 317: 762-770, 2007.

3. Beckmann N, Tigani B, Sugar R, Jackson AD, Jones G, Mazzoni L, and Fozard JR. Noninvasive detection of endotoxin-induced mucus hypersecretion in rat lung by MRI. Am J Physiol Lung Cell Mol Physiol. 283: L22-L30, 2002.

4. Birrell MA, Wong S, Hele DJ, McCluskie K, Hardaker E, and Belvisi MG. Steroid-resistant inflammation in a rat model of chronic obstructive pulmonary disease is associated with a lack of nuclear factor-kappaB pathway activation. Am J Respir Crit Care Med 172: 74-84, 2005.

5. Chong BTY, Agrawal DK, Romero FA, and Townley RG. Measurement of bronchoconstriction using whole-body plethysmograph: comparison of freely moving versus restrained guinea pigs. J Pharmacol Toxicol Methods 39: 163-168, 1998.

Page 23 of 41

23 6. Cho HY, Reddy SP, and Kleeberger SR. Nrf2 defends the lung from oxidative stress. Antioxid Redox Signal 8: 76-87, 2006.

7. D'hulst AI, Vermaelen KY, Brusselle GG, Joos GF, and Pauwels RA. Time course of cigarette smoke-induced pulmonary inflammation in mice. Eur Respir J 26: 204-213, 2005.

8. Hautamaki RD, Kobayashi DK, Senior RM, and Shapiro SD. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277: 2002-4, 1997.

9. Gebel S, Gerstmayer B, Bosio A, Haussmann HJ, Van Miert E, and Muller T. Gene expression profiling in respiratory tissues from rats exposed to mainstream cigarette smoke. Carcinogenesis. 25:169-78, 2004.

10. Golpon HA, Coldren CD, Zamora MR, Cosgrove GP, Moore MD, Tuder RM, Geraci MW, Voelkel NF. Emphysema lung tissue gene expression profiling. Am J Respir Cell Mol Biol 31: 595-600, 2004.

11. Heckman CA, and Dalbey WE. Pathogenesis of lesions induced in rat lung by chronic tobacco smoke inhalation. J Natl Cancer Inst 69: 117-29, 1982.

12. Heguy A, O'connor TP, Luettich K, Worgall S, Cieciuch A, Harvey BG, Hackett NR, and Crystal RG. Gene expression profiling of human alveolar macrophages of phenotypically normal smokers and nonsmokers reveals a previously

Page 24 of 41

24 unrecognized subset of genes modulated by cigarette smoking. J Mol Med 84: 31828, 2006.

13. Hogg JC. Why does airway inflammation persist after the smoking stops? Thorax 61: 96-7, 2006.

14. Ito K, Ito M, Elliott WM, Cosio B, Caramori G, Kon OM, Barczyk A, Hayashi S, Adcock IM, Hogg JC, and Barnes PJ. Decreased histone deacetylase activity in chronic obstructive pulmonary disease. N Engl J Med 352: 1967-76, 2005

15. Jackson A, Kemp P, Giddings J, and Sugar R. Development and validation of a lectin-based assay for the quantitation of rat respiratory mucin. Novartis Found Symp 248: 94-105, 2002.

16. Kirkham PA, Spooner G, Ffoulkes-Jones C, and Calvez R. Cigarette smoke triggers macrophage adhesion and activation: role of lipid peroxidation products and scavenger receptor. Free Radic Biol Med 35: 697-710, 2003

17. Kirschvink N, Vincke G, Onclinx C, Peck MJ, and Gustin P. Comparison between pulmonary resistance and Penh in anaesthetised rats with tracheal diameter reduction and after carbachol inhalation. J Pharmacol Toxicol Methods 51: 123-8, 2005.

Page 25 of 41

25 18. Li N, Sioutas C, Cho A, Schmitz D, Misra C, Sempf J, Wang M, Oberley T, Froines J, and Nel A. Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ Health Perspect 111: 455-60, 2003.

19. Ma B, Kang MJ, Lee CG, Chapoval S, Liu W, Chen Q, Coyle AJ, Lora JM, Picarella D, Homer RJ, and Elias JA Role of CCR5 in IFN-gamma-induced and cigarette smoke-induced emphysema. J Clin Invest 115: 3460-72, 2005

20. Maestrelli P, El Messlemani AH, De Fina O, Nowicki Y, Saetta M, Mapp C, and Fabbri LM. Increased expression of heme oxygenase (HO)-1 in alveolar spaces and HO-2 in alveolar walls of smokers. Am J Respir Crit Care Med 164:1508-13, 2001.

21. Mahadeva R and Shapiro SD. Chronic obstructive pulmonary disease * 3: Experimental animal models of pulmonary emphysema. Thorax 57: 908-14, 2002.

22. Marwick JA, Kirkham PA, Stevenson CS, Danahay H, Giddings J, Butler K, Donaldson K, Macnee W, and Rahman I. Cigarette smoke alters chromatin remodeling and induces proinflammatory genes in rat lungs. Am J Respir Cell Mol Biol 31: 633-42, 2004.

23. Mathers CD, Lopez AD, and Murray CJL. Global burden of disease and risk factors, edited by Lopez AD, Mathers CD, Ezzati M, Murray CJL, and Jamison DT. New York, USA: Oxford University Press, 2006.

Page 26 of 41

26 24. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, Houstis N, Daly MJ, Patterson N, Mesirov JP, Golub TR, Tamayo P, Spiegelman B, Lander ES, Hirschhorn JN, Altshuler D, Groop LC. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34: 267-73, 2003.

25. Morrow PE. Possible mechanisms to explain dust overloading of the lungs. Fundam Appl Toxicol 10: 369-84, 1988.

26. Nikula KJ, Vallyathan V, Green FH, and Hahn FF. Influence of exposure concentration or dose on the distribution of particulate material in rat and human lungs. Environ Health Perspect 109: 311-8, 2001.

27. Ofulue AF, Ko M, and Abboud RT. Time course of neutrophil and macrophage elastinolytic activities in cigarette smoke-induced emphysema. Am J Physiol 275: L1134-44, 1998.

28. Pauwels, RA Buist S Calverley PMA Jenkins CR and Hurd SS. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 163, 1256-1276. 2001.

29. Rangasamy T, Cho CY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW, Yamamoto M, Petrache I, Tuder RM, and Biswal S. Genetic ablation of Nrf2

Page 27 of 41

27 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 114: 1248-59, 2004.

30. Rubio ML, Sanchez-Cifuentes MV, Ortega M, Peces-Barba G, Escolar JD, Verbanck S, Paiva M, and Gonzalez-Mangado N. N-acetylcysteine prevents cigarette smoke induced small airways alterations in rats. Eur Respir J 15: 505-11, 2000.

31. Shinohara T, Kaneko T, Nagashima Y, Ueda A, Tagawa A, and Ishigatsubo Y. Adenovirus-mediated transfer and overexpression of heme oxygenase 1 cDNA in lungs attenuates elastase-induced pulmonary emphysema in mice. Hum Gene Ther 16: 318-27, 2005.

32. Stevenson CS, Coote K, Webster R, Johnston H, Atherton HC, Nicholls A, Giddings J, Sugar R, Jackson A, Press NJ, Brown Z, Butler K, and Danahay H. Characterization of cigarette smoke-induced inflammatory and mucus hypersecretory changes in rat lung and the role of CXCR2 ligands in mediating this effect. Am J Physiol Lung Cell Mol Physiol 288: L514-22, 2005.

33. Stevenson CS, Koch LG, and Britton SL. Aerobic capacity, oxidant stress, and chronic obstructive pulmonary disease-A new take on an old hypothesis. Pharmacol Ther 110: 71-82, 2006.

34. Stevenson CS, Winny C, Coote K, Giddings J, Whittaker P, Pohlmeyer-Esch G, Charman C, Danahay H, Butler K. A chronic rat model of smoke-induced lung

Page 28 of 41

28 injury and comparison with an acute 24 h screening model. Am J Respir Crit Care Med 169: A205, 2004.

35. Szustakowski JD, Lee JH, Marrese CA, Kosinski PA, Nirmala NR, Kemp DM. Identification of novel pathway regulation during myogenic differentiation. Genomics 87: 129-138, 2006.

36. Tagawa A, Kaneko T, Shinohara T, Ueda A, Sato T, and Ishigatsubo Y. Heme oxygenase-1 inhibits cigarette smoke-induced increase in the tracheal mucosal permeability in guinea pigs in vivo. Inflamm Res 54: 229-34, 2005.

37. Thurlbeck WM. Christie lecture: Emphysema then and now. Can Respir J 1: 2139, 1994.

38. Wright JL and Churg A. Animal models of cigarette smoke-induced COPD. Chest 122: 301S-306S, 2002.

39. van der Vaart H. Postma DS, Timens W, Hylkema MN, Willemse BW, Boezen HM, Vonk JM, de Reus DM, Kauffman HF, and ten Hacken NH. Acute effects of cigarette smoking on inflammation in healthy intermittent smokers. Respir Res 6: 22, 2005.

40. Yamada N, Yamaya M, Okinaga S, Nakayama K, Sekizawa K, Shibahara S, and Sasaki H. Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to emphysema. Am J Hum Genet 66: 187-95, 2001.

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29 Figure Legend Figure 1. Temporal changes in gene expression in response to cigarette smoke exposure. Heat-maps represent hierarchal clustering of all genes present on the microarray chip from selected time-points over the course of the study. Each timepoint is indicated above its respective heat-map. The red branch-lines indicate individual sham animals and the blue branch-lines represent CS-exposed rats. Blue bars represent low expression levels, red bars represent high expression levels, and yellow bars represent no change relative to normalized median gene expression values. Figure 2. Metabolic pathways are increased in response to smoke exposure. Heat-maps represent selected genes from metabolic functions (B). Blue bars represent low expression levels, red bars represent high expression levels, and yellow bars represent no change relative to normalized median gene expression values. Data shown from selected time-points representing the transition from acute to chronic smoke exposure, with the last column for each group representing the smoking cessation group. Figure 3. Stress response and inflammatory pathways are also increased in response to cigarette smoke exposure. Heat-maps represent selected genes from stress response and inflammatory processes. Blue bars represent low expression levels, red bars represent high expression levels, and yellow bars represent no change relative to normalized median gene expression values. Data shown from selected time-points representing the transition from acute to chronic smoke exposure, with the last column for each group representing the smoking cessation group. Figure 4. Lung 4HNE staining of alveolar macrophages in the rat-smoking model. Representative picture of 4HNE adduct staining in alveolar macrophages in

Page 30 of 41

30 (A) sham-exposed rat lungs, (B) 3 day smoke-exposed rat lungs (C) 8 week smokeexposed rat lungs and (D) 6 month smoke-exposed rat lungs. Arrows show alveolar macrophages. (E) Alveolar macrophage 4HNE staining is elevated, in smoke-exposed rat lungs compared to sham-exposed animals (n=6). Closed circles represent smokeexposed animals, open circles represent sham-exposed animals and the horizontal lines indicate the mean values with n = 6 per group; * = p < 0.05. Figure 5. Temporal changes in cigarette smoke-induced neutrophil chemokine cytokine production. Plots illustrate changes in the levels of neutrophil chemokines, CINC-1 and CINC-2, after 1 day until 8 months of exposure, including the smoking cessation group. Chemokine levels were assayed for in both lavage fluid (CINC-1, (A) and CINC-2 (C)) and tissue homogenate supernatant (CINC-1 (B); CINC-2, (D)). Data presented as mean values + SEM with n = 8 per group; * = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to sham control. Figure 6. Temporal changes in cigarette smoke-induced inflammatory cell infiltration in the lung. Plots describe changes in BAL fluid neutrophil (A) tissue neutrophil (B), BAL fluid macrophage (C), and tissue macrophage (D) numbers after 1 day until 8 months of exposure, including the smoking cessation group. Data presented as mean values + SEM with n = 8 per group; * = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to sham control. Figure 7. Macrophage aggregates fill alveoli in response to chronic smoke exposure. Pictures show the progression of macrophage infiltration in the alveoli from air-exposed rats (A) to rats exposed to smoke for 3 weeks (B), 8 weeks (C) and 34 weeks (D). Figure 8. Cigarette smoke induces epithelial thickening, goblet cell metaplasia, and increased mucus levels in the BAL fluid. Figure illustrates tracheal epithelial

Page 31 of 41

31 sections stained for mucin using UEA-1 in air-exposed rats (A), rats exposed to smoke for 5 days (B), 8 weeks (C), or 6 months followed by 2 months with no exposures – ie., smoking cessation group (D). The graphs describe changes in mucin levels in the tracheal epithelium (E) and primary bronchus (F) in response to 1 day through 8 months of exposure, including the smoking cessation group. BAL fluid mucin levels were also assessed using an ELLA system (G). Data presented as mean values + SEM with n = 8 per group; * = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to sham control. Figure 9. Temporal effects of cigarette smoke-induced airway remodelling. Plots describe changes in the area of collagen staining (A) and smooth muscle staining (B) around the airways (excluding the primary bronchus) in response to 16 and 34 weeks of smoke exposure. Data presented as mean values + SEM with n = 8 per group; **** = p < 0.0001 compared to sham control. Figure 10. Cigarette smoke exposure cause airspace enlargement after 8 months of exposure. The mean linear intercept of alveolar spaces was significantly increased in rats exposed to smoke for 8 months compared to age-matched air-exposed controls. Data presented as mean values + SEM with n = 8 per group; * = p < 0.05 compared to sham control.

Page 32 of 41

Figure 1.

D5

D14

Page 33 of 41

2

Cess

34W

16W

4W

3D

34W

16W

4W

3D

Figure 2.

Amino acid metabolism Arginase 1 Gamma-glutamyltransferase 1 Glutamine synthetase 1 Phosphoserine aminotransferase 1 Similar to proline dehydrogenase (oxidase) 2

Carbon fixation Aldolase A Malic enzyme 1 Transketolase

Glutamate metabolism Glutamate cysteine ligase, modifier subunit 1 Glutamine synthetase

Glycolysis Enolase 1, alpha GAPDH Hexokinase 2 Lactate dehydrogenase A Phosphofructokinase, platelet

Oxidative Phosphorylation ATPase, H+ transporting, lysosomal 16 kDa ATPase, H+ transporting, lysosomal, beta 56/58 kDa, isoform 2 Cytochrome c oxidase subunit IV isoform 1 Cytochrome c oxidase, subunit Va

SHAM

SMOKED

Page 34 of 41

3 Figure 3.

Acidic AcidicChitinase Chitinase

Cess

34W

16W

4W

3D

34W

16W

3D

Cess

34W

16W

4W

3D

34W

16W

4W

3D

4W

INFLAMMATION

STRESS RESPONSE

Cathepsin Cathepsin D D

Aldehyde dehydrogenase Aldehyde dehydrogenase

CD36 CD36

Alpha-1 acidglycoprotein glycoprotein Alpha-1 acid

Complement component 1q Complement component 1q

CytochromeP450 P450 4B1 Cytochrome 4B1

Complement component 3 Complement component 3

CytochromeP450 P450 1B1 Cytochrome 1B1

CX3CL1 CX3CL1

γ Glutamate cysteinesynthetase synthetase γ-Glutamate cysteine

CXCR1 CXCR1

γ Glutamyltranspeptidase γ-Glutamyltranspeptidase

Ferritin Light Chain 1 Ferritin Light Chain 1

Glutamate cysteine ligase ligase Glutamate cysteine

GRO GRO

Glutathione transferase A1 Glutathione SS transferase A1

Interleukin-1 receptor Interleukin-1 receptor type II type II

HemeOxygenase Oxygenase Heme

JE responsive element JE responsive element

Lipocalin 22 Lipocalin

Ly6-C antigen Ly6-C antigen

Metallothionein Metallothionein

MHC class II II MHC class

NAD(P)H-menadione oxioreductase NAD(P)H-menadione oxioreductase

MIP1a MIP1a

NF-E2 Factor2 NF-E2 Factor 2

MME MMP-12

SuperoxideDismutase Dismutase 22 Superoxide

NRAMP2 NRAMP

Thioredoxinreductase reductase Thioredoxin

Osteopontin Osteopontin

UDP-glucuronosyltransferase 1A6 UDP-glucuronosyltransferase 1A6

Small Inducible cytokine A20 Small inducible cytokine A20

UDP-glucuronosyltransferase 1A7 UDP-glucuronosyltransferase 1A7

T-Kininogen T-Kininogen

SHAM

SMOKED

SHAM

SMOKED

Page 35 of 41

4 Figure 4.

(b)

(a)

Sham-exposed lungs

3 days smoke-exposed lungs

(c)

(d)

8 weeks smoke-exposed lungs

6 months smoke-exposed lungs

*

(e)

*

Alveolar Macrophage Staining

3

2

1

0 Sham

Exposed 3 Day

Sham

Exposed 8 Week

Cigarette Smoke Exposure

Sham

Exposed 6 Month

Page 36 of 41

5

A.

1200

**

Sham

1000

Smoked

800

400

***

***

600

***

CINC-2 (pg/mL)

3500

**

***

** **

200 0

**

*

1d 3d 5d 2w 3w 4w 6w 8w12w16w26w34wcess

C.

**

3000

***

2500

***

2000

***

1500 1000 500 0

***

**

***

*** ** *

***

***

CINC-1 (pg/mg protein)

CINC-1 (pg/mL)

1400

800

CINC-2 (pg/mg protein)

Figure 5.

600

1d 3d 5d 2w 3w 4w 6w 8w12w16w26w34wcess

700 600 500 400 300

***

B. Sham Smoked

* ***

200

***

n=1

*** ** **

***

*** ***

100 0

1d 3d 5d 2w 3w 4w 6w 8w12w16w26w34wcess

D.

***

500

*** ***

400

**

**

300 200

***

***

*** *** *** *

100 0

1d 3d 5d 2w 3w 4w 6w 8w12w16w26w34wcess

Page 37 of 41

6

A. ***

Macrophages x 10^6

**

** ***

1d 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

*** *** ***

3d

5d

***

100 90 80 70 60 50 40 30 20 10 0

Neutrophils / mm2

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

C.

120

p=0.067 ***

* ***

1d

3d

***

***

5d

**

6w

12w 16w 26w 34 w 26w + 8w rec

smoke

**

D.

100

*

sham

B.

** *

** p=0.057

1d 3d 5d 3w 4w 6w 8w 16w 26w 34w 26w+ 8w rec

6w 12w 16w 26w 34w 26w + 8w rec

Macrophages / mm2

Neutrophils x 10^6

Figure 6.

80

*** ***

***

***

*** ** *** *** **

60 40 20 0 1d 3d 5d 3w 4w 6w 8w 16w 26w 34w 26w+ 8w rec

Page 38 of 41

7 Figure 7.

A.

Macrophage aggregates containing neutrophils filling and distending alveoli. Pneumocyte type II hyperplasia

C.

Macrophage aggregates filling alveoli

B.

Large aggregation and consolidation

D.

Page 39 of 41

8 Figure 8.

A.

B.

Area mucin / micron2

C.

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

D.

***

E.

*** ** **

**

***

**

***

*

1d

3d

5d

3w 4w 6w 8w 16w 26w 34w 26w+ 8w rec

Mucin (units/mL)

25

***

F.

20 **

***

15 *

10 *

5 0

*

1d

*

3d 5d

*

***

*

2w 3w 4w 6w 8w 12w 16w 26w 34w 26 + 8w rec

Page 40 of 41

9 Figure 9.

A.

Thickness (μm)

75

*** ****

Control Smoked

50

25

0

B.

16 Weeks

34 Weeks

16 Weeks

34 Weeks

Thickness (μ m)

6 5 4 3 2 1 0

Page 41 of 41

10 Figure 10.

*

80

Lm

70 60 50 40 30 Sham

Smoke