Cigarette smoke induces molecular responses in respiratory tissues of ApoE-/- mice that are progressively deactivated upon cessation 

Stéphanie Bouéa*§, Héctor De Leóna*, Walter K. Schlageb, Michael J. Pecka, Horst Weilerb, An

Bergesc, Grégory Vuillaumea, Florian Martina, Baerbel Friedrichsb, Stefan Lebruna, Kris

Meurrensc, Nadine Schrackeb, Michaela Moehringb, Yvonne Steffenb, Jutta Schuellerb, Patrick

Vanscheeuwijcka,b, Manuel C. Peitscha, Julia Hoenga.

aPhilip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000

Neuchâtel, Switzerland.

bPhilip Morris International R&D, Philip Morris Research Laboratories GmbH, Cologne,

Germany

cPhilip Morris International R&D, Philip Morris Research Laboratories bvba, Leuven, Belgium

*These authors contributed equally to this work

§Corresponding author

Online Data Supplement

Supplemental Methods

Gene Expression Data Analysis. Data processing and quality control. Raw data files were read

by the ReadAffy function of the affy package (Gautier et al. 2004) belonging to Bioconductor

(Gentleman et al. 2004) in R (R Development Core Team 2007), and the quality was controlled

by generating RNA degradation plots (thanks to the AffyRNAdeg function of the affy package),

NUSE and RLE plots (thanks to the function affyPLM) (Brettschneider et al. 2008) and

calculating the MA(RLE) values. Arrays that fell below a set of thresholds on the quality control

checks were excluded. Robust Microarray Analysis (RMA) background correction and quantile

normalization were used to generate microarray expression values (Irizarry et al. 2003). Linear

model to calculate fold changes and statistical significance. An overall linear model was fit to

the data for all sample groups, and specific contrasts of interest (for each time point, exposure

(CS or cessation) was compared with its respective sham group) were evaluated to generate raw

p-values for each probe set on the expression array (Gentleman et al. 2004). The Benjamini-

Hochberg False Discovery Rate (FDR) method was then used to correct for multiple testing

effects. Probe sets were considered to have statistically significantly changed expression levels in

a specific comparison if they had an adjusted p-value of lower than 0.05 and an absolute fold

change greater than 1.3. Generation of figures. Volcano plots showing the fold change vs.

statistical significance were generated in R. Different heatmaps were generated using the

heatmap.2 function of the gplots package. Venn diagrams were used to show overlaps between

different lists of probesets or gene sets using the VennDiagram package in R. Gene Set

Enrichment Analyses. GSEA (Subramanian et al. 2005) was performed using the Confero

platform (Hermida et al. 2012) with further developments that will be described in a manuscript

in preparation (Poussin et al.). For each pairwise contrast described above, probesets were

mapped onto Entrez Gene identifiers and collapsed to remove the multiple probeset-to-gene

mappings using the Confero platform. GSEA was performed by ranking genes by their

respective t-statistic, calculated using the Limma package, on the MSigDB (Subramanian et al.

2005) collection of canonical pathways (Cp collection including KEGG, Reactome and Biocarta

pathways) in the curated gene sets (C2), as source of prior biological knowledge. For each

contrast, only gene sets with a FDR below 0.01 were retained for further analysis. To facilitate

and accelerate the interpretation of GSEA results, gene sets were clustered and visualized in the

form of a network using a similar method to the one used in the Enrichment Map (Merico et al.

2010). Briefly, our approach enabled to group gene sets based on common underlying leading

edge genes corresponding to the core genes contributing to the maximum enrichment scores. For

a given contrast, the Jaccard distance was computed between each significant gene set pair. The

Jaccard distance was converted into similarity matrix (1-Jaccard) used as input for APcluster, a

powerful algorithm enabling to cluster gene sets in rich-club and communities (Frey and Dueck

2007). The results were visualized in the form of a network revealing communities of gene sets

representative of perturbed biological processes/pathways in a specific pairwise contrast. Each

community and rich club was then annotated using DAVID 6.7 (Huang da et al. 2009) functional

clustering function with the union of all leading edge genes of the gene sets present in a

community or a rich club. The network of gene sets was visualized using Cytoscape (Shannon et

al. 2003), with the edges length being proportional to the Jaccard distance between gene sets.

The comparison of gene set networks obtained for different contrasts allowed gaining a dynamic

and robust picture of the evolution of the perturbed biology across different conditions (e.g.

smoking vs. cessation conditions).

Histopathology. Analysis of slides stained with standard hematoxilin/eosin (H&E), Alcian-Blue

Periodic Acid Schiff (AB-PAS), resorcin-fuchsin or immunohistochemical techniques was

performed using an Axio-Imager-Z1 microscope equipped with a high resolution digital color

camera (AxioCam HRc) linked to a computer running Axiovision software 4.6; all instruments

and software were from Zeiss (Göttingen, Germany). Immunohistomechical staining with

MUC5B was conducted to identify mucus-producing cells. Lung histopathology as defined by

three cellular parameters involving macrophage infiltration was scored by a veterinary

pathologist. Macrophages without pigment were defined as free cells in the alveolar lumen not

showing any cytoplasmatic pigmentation. Macrophages with pigment are free cells within the

alveolar lumen containing fine-granular, brownish to yellow cytoplasmic pigmentation.

Pigmented macrophage nests: consist of multiple macrophages in the alveolar lumen with fine-

granular, brownish to yellow cytoplasmic pigmentation; cells are clustered in small groups

within adjacent alveoli and the alveolar epithelium often appears slightly hyperplastic.

Pigmented and unpigmented macrophages were scored as the percentage of alveoli occupied

containing these cell types (0, isolated alveoli with individual cells; 1, <10% alveoli with

aggregates of cells (AAC); 2, 10% ≤ AAC < 25%; 3, 25% ≤ AAC < 50%; 4, 50% ≤ AAC <

75%; AAC ≥ 75%). Number of macrophage nests were counted and scored (0, 0; 1, 1-3, 2, 4-6;

3, 7-10, 4, 11-15; 5, >15).

Lung Morphometric Analysis. Alveolar emphysema was assessed by mean chord length (Lm),

Bronchiole Attachments (BA) and Destructive Index (DI) measurements. Briefly, one step serial

section per animal representing a cross section along the left main stem bronchus and its

branching bronchioles was selected for morphometric evaluation. Lm, BA and DI measurements

were performed in images captured with an Axio-Imager-Z1 microscope equipped with an 8-

specimen holder, a high resolution digital color camera (Olympus DP70) and VIS-software from

Visiopharm (Horsholm, Denmark). All measurements were conducted according to

Visiopharm’s quantitative digital pathology methods. A sampling fraction of 30% was randomly

chosen by an automated image capture system, which generated 10-15 images per animal. Lm

Measurements. Lm is the mean length of line segments spanning the airspace between

intersections of the line with the alveolar surface. Lm was determined with a test line system by

taking the mean of all chords across all alveoli in a captured image according to the following

formula, 1n∑i=1

n

li, where li is the ith measured chord length. Lm estimates the mean free distance

between gas exchange surfaces within the 3D acinar surface complex (alveoli and ducts) and it is

a quantity similar to the mean intercept length.(Hsia et al. 2010) However, the mean intercept

length includes the thickness of one wall intersecting the line segment. Lm is the mean of all the

measurements. BA Measurements. The number of bronchiolar attachments to the alveolar wall

per unit bronchiolar basement membrane length was determined by dividing the number of

attachments (a) by the length of the perimeter of the bronchiole (b). b is measured along the

basement membrane of the bronchiole using a set of lines placed over the bronchiole profile and

counting the number of intersections between the outer surface of the bronchiole and the lines,

according to the following formula, bi= π2

Id , where I is the number of intersections and d is the

distance between the lines. The total number of BA per bronchiolar basement membrane length

is expressed as BA=1n∑i=1

n aili

. DI Measurements. DI is a measure of the fraction of lung tissue

that is damaged and it is a unitless parameter. Using a grid of points set over an acquired image,

the ratio of points falling on destroyed tissue (d) over the points falling on healthy (h) tissue +

destroyed tissue was obtained, DI= dd+h . An average of all measurements per slide was

computed as done for Lm.

Supplemental Figures

Figure S1. Representative images of lung tissue from CS-exposed (A and C), Sham (B) and

Cessation (D) animals. Tissues were stained with H&E and images captured with a high

resolution digital AxioCam HRc camera. CS-exposed tissues (C) showed emphysematous areas

of alveolar destruction, as well as macrophage infiltration in the alveolar lumen (A and C). The

three categories of macrophages (unpigmented, pigmented and nests) used to score inflammation

were present in lungs of CS-exposed mice (A).

Figure S2. Gene Set Enrichment Analysis of RNE after Six Months Exposure. - CS vs.

sham. Perturbation of pathways primarily related to cell adhesion (RC1), glutathione metabolic

process (RC2), and lysosomal function (RC6) were shown after CS exposure.

Figure S3. Gene Set Enrichment Analysis of RNE after Six Months Exposure. – Cessation

vs. sham. Smoking cessation still showed perturbation of pathways related to cell adhesion,

wound healing and regulation of immune response. No changes were observed in xenobiotic

metabolism or lysosomal function.

Figure S4. Xenobiotic metabolism pathways in NE and lung parenchyma. (A) A set of six

genes (Adh7, Gsto1, Cyp2b10, Cbr3 Gstp1 and Aldh3b2) showed the largest CS-dependent

upregulation in NE, whereas only one gene (Cyp1a1) showed comparable changes in levels of

expression in lung parenchyma. Three genes (Gsta2, Cyp2s1 and Cyp2f2) were downregulated

by CS exposure in lung parenchyma but remained unchanged in NE. Expression levels of most

genes in NE of cessation group were lower at any time upon cessation, whereas only Cyp1a1 and

Gsta3 exhibited a similar pattern in lung. Genes that were down-regulated in the lung

parenchyma after CS exposure, showed a time-dependent up-regulation upon smoking cessation

(Cyp2s1 and Cyp2f2). Log intensity values are depicted here.

Figure S5. Selected xenobiotic metabolism genes perturbed after 6 months of CS exposure

and smoke exposure discontinuation in NE and lung parenchyma. A set of genes that were

upregulated in NE, including Adh7, Gsto1, Cyp2b10 and Cbr3 were unmodified in lung

parenchyma. Conversely, genes that were upregulated by CS exposure in parenchyma (Cyp1a1)

were not modified in NE. Furthermore, expression of some genes underwent no changes by CS

exposure in NE but were downregulated in lung parenchyma (e.g. Cyp2f2). For all genes,

smoking cessation resulted in the opposite effect triggered by CS exposure. Log fold-change data

for the CS group (6 months) expressed as CS vs. sham; data for the cessation group expressed as

cessation vs. CS.

Figure S6. Expression levels of selected complement and coagulation genes in NE and lung parenchyma after 6 months of CS exposure or smoking cessation. Three complement genes (C1qb, C1qc and C3ar1) were clearly upregulated by CS exposure in lung parenchyma, whereas expression levels remained unmodified in NE. Conversely, F13a1 (coagulation factor XIII, subunit 1) displayed larger expression levels after CS exposure in NE, whereas minimal expression changes were observed in lung parenchyma. Plat (plasminogen activator) was downregulated in response to CS exposure and upregulated in the smoking cessation group in lung parenchyma. Log fold-change data expressed as described in Figure S4.

Figure S7. Expression levels of selected immunomodulatory genes in NE and lung parenchyma after 6 months of CS exposure (3R4F) or smoke exposure discontinuation (cessation). (C) Genes involved in immunomodulatory functions and regulation of IgA transport in epithelial cells (Pigr) were highly responsive to CS exposure and smoke exposure discontinuation. Four genes, Pigr, Cd86, Cxcl12 and H2-Ab1 showed the highest expression levels in lung parenchyma of cessation group. These 4 genes were virtually unaffected by CS exposure in NE, however, Pigr expression in NE was stimulated by smoking cessation. Log fold-change data expressed as described in Figure S4.

Supplemental Tables

Table S1. Number of Replicates for Each Measurement.

Groupsham CS cessation

Nicotine metabolites - 3mo (3-5 mice/cage) 14 16 -Nicotine metabolites - 6mo (3-4 mice/cage) 10 8 9BALF cell count - 6mo 8 8 8BALF MAP - 6mo 8 5 8COHb - 3mo 8 8 -COHb - 6mo 8 8 8Lung histopathology - 6mo 18 20 20Lung histomorphometry - 6mo 16 17 19Lung parenchyma gene expression - 3mo 8 8 -Lung parenchyma gene expression - 4mo 8 8 8Lung parenchyma gene expression - 5mo 7 8 7Lung parenchyma gene expression - 6mo 7 8 8Nasal epithelium gene expression - 3mo 7 8 8Nasal epithelium gene expression - 4mo 8 8 8Nasal epithelium gene expression - 5mo 7 8 6Nasal epithelium gene expression - 6mo 7 8 8

mo: months (time point)

Table S2. Test Atmosphere Characterization.

Sham CS

TPM (µg/l) < LOQ 593.5 ± 18.4

CO (µg/l) < LOQ 695.4 ± 20.6

Particle size, MMAD (m) - 0.78 ± 0.01

Nicotine (µg/l) < LOQ 33.5 ± 2.8

Formaldehyde (µg/l) - 0.67 ± 0.06

Acetaldehyde (µg/l) - 39.4 ± 1.1

Acrolein (µg/l) - 3.9 ± 0.2

CS: mainstream cigarette smoke; TPM: total particulate matter; MMAD: mass median aerodynamic diameter; LOQ: limit of quantification. Data reported as mean ± SD.

Table S3. Difference between groups for inflammatory cell subpopulations and macrophage activation in BALF.

Difference between groups

CS vs.

sham

CS vs.

cessation

cessation vs.

sham

Cell Type

Macrophages210.7

(83.9, 337.5)*128.7

(1.9, 255.5)*82.0

(-44.8, 208.8)

Dendritic Cells31.3

(22.4, 40.3)*23.1

(14.1, 32.1)*8.3

(-0.7, 17.3)

Lymphocytes139.8

(85.5, 194.1)*97.9

(43.6, 152.2)*41.9

(-12.5, 96.2)

Neutrophils143.8

(85.5, 202.0)*137.0

(78.7, 195.3)*6.8

(-51.5, 65.1)

Eosinophils0.76

(0.15, 1.37)*0.64

(0.04, 1.25)*0.12

(-0.49, 0.72)

Macrophage Activation Markers

CD545160

(4031, 6289)*3931

(2802, 5060)*1230

(101, 2359)*

CD86604

(275, 932)*336

(7, 664)*268

(-60, 597)

MHCII-263

(-811, 285)-699

(-1247, -151)*436

(-113, 984)

CD11b796

(316, 1277)*736

(255, 1216)*61

(-420, 542)

Data displayed are differences between groups and related 95%-CI estimated by the Fisher’s Least Significant Difference (LSD) method (* means that confidence interval does not contain 0). Cell numbers are expressed in absolute numbers (x1000).

Table S4. Concentrations of mediators in BALF

Data expressed as means ± SEM. *: different to Sham (p < 0.05). #: different to CS (p < 0.05)

Table S5. Histopathological scores (mean+/- SD and min-max per group).

Sham CS Cessation

Pigmented macrophage nests

Mean SD

Min-max

0.0 ± 0.0

0-0

3.4 ± 0.7

2-4

1.6 ± 1.4

0-5

Macrophages without pigment

Mean SD

Min-max

0.0 ± 0.0

0-0

1.1 ± 0.2

1-2

0.6 ± 0.5

0-1

Pigmented macrophages

Mean SD

Min-max

0.0 ± 0.0

0-0

2.9 ± 0.7

2-4

0.6 ± 0.6

0-2

Table S6. Lung histomorphometric measurements (mean +/- SD)

Sham CS Cessation

Lm (µm) 28.6 ± 0.4 33.5 ± 0.6 30.8 ± 0.4

BA (n/mm) 15.3 ± 0.8 6.9 ± 0.5 12.1 ± 0.6

DI 0.59 ± 0.01 0.8 ± 0.02 0.65 ± 0.01

Table S7. Difference between groups for lung histomorphometric measurements.

Measurement

Difference between groups

CS vs. sham CS vs. cessation cessation vs. sham

Lm (µm) 4.97 (3.53, 6.41)* 2.75 (1.37, 4.14)* 2.22 (0.81, 3.62)*

BA (n/mm) -8.43 (-10.24, -6.62)* -5.25 (-6.98, -3.51)* -3.18 (-4.94, -1.42)*

DI 0.21 (0.17, 0.25)* 0.15 (0.11, 0.19)* 0.06 (0.02, 0.1)*

Lm: mean chord length, BA: bronchiole attachments, DI: destructive index. Data displayed are

differences between groups and related 95%-CI estimated by the Fisher’s Least Significant

Difference (LSD) method (* means that confidence interval does not contain 0).

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