IKKα Causes Chromatin Modification on Pro-Inflammatory Genes by Cigarette Smoke in Mouse Lung

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1 IKKα Causes Chromatin Modification on Pro-Inflammatory Genes by Cigarette Smoke in Mouse Lung Se-Ran Yang, Samantha Valvo, Hongwei Yao, Aruna Kode, Saravanan Rajendrasozhan, Indika Edirisinghe, Samuel Caito, David Adenuga, Ryan Henry, George Fromm, Sanjay Maggirwar, Jian-Dong Li, Michael Bulger and Irfan Rahman Online Data Supplement

2 Methods Hematoxylin & Eosin (H&E). Mouse lungs (which had not been lavaged) were inflated with 1% low-melting agarose at 56 C at the pressure of 25 cm H 2 O, and then fixed by 4% paraformaldehyde. Tissues were embedded in paraffin, and sectioned (4 μm). After deparaffinization, the sections were stained with H&E. Macrophage immunohistochemistry. The formalin-fixed, paraffin-embedded lung sections were deparaffinized and hydrated by passing through a series of xylene and graded alcohol. Endogenous peroxidase activity was quenched by exposure to 3% H 2 O 2 in methanol for 3 min. Nonspecific binding of antibodies to the tissues was blocked by incubating the tissue with 5% normal goat serum containing.5% BSA in PBS for 3 min. For the detection of macrophages, rat anti-mouse Mac3 monoclonal antibody (catalog no , BD Pharmingen, San Jose, CA) at a titer of 1:5 was used. The immunodetection was performed with avidin-biotin-peroxidase complex (ABC kit, DAKO Crop., Carpinteria, CA) and DAB reagent (Vector Laboratories Inc, Burlingame, CA) as instructed by the manufacturer. The nuclei were counterstained with Cole s hematoxylin solution. The number of Mac-3 positive cells in the lung sections (5 random microscopic fields per lung section in 3 different sections) were counted manually in a blinded manner under a magnification of 2X and the numbers were averaged (E1-E3). Analysis of pro-inflammatory mediators in BAL fluid. Mice BAL fluid (15 μl) were analyzed for the cytokine levels using the sensitive mouse Multi- Analyte Profile (version 1.6) screening by Luminex (Rules Based Medicine, co-marketed with 2

3 Charles River Laboratories, Austin, TX). The assays permit simultaneous cytometric quantification of multiple chemokines/cytokines with minimal sample volume. 3

4 Results CS induced inflammatory cell influx in mouse lung. To evaluate the CS-induced lung inflammatory response, C57BL/6J mice were exposed to CS for 3 days (acute exposure) or 8 weeks (subchronic exposure), and the number of inflammatory cells were analyzed in BAL fluid and lung sections of air- and CS-exposed mice. In response to CS-exposure, there was a significant accumulation of neutrophils, but not macrophages in BAL fluid compared to airexposed mice (Figures E1 and E2). To assess the infiltration of neutrophils and macrophages in the lung, H&E and Mac-3 staining for macrophages was performed in mid-sagittal sections. H&E staining showed an increased interstitial inflammation with prominent neutrophils and macrophages in lung tissue of CS-exposed mice as compared to air-exposed mice (Fig. E1). In addition, both acute and subchronic CS exposure led to significant macrophage interstitial accumulation, which was determined by immunohistochemistry using Mac-3 antibody in lung tissue (Fig. E2). These results suggested the CS-induced accumulation of inflammatory cells in lung tissue of mice. CS increased the levels of NF-κB-dependent pro-inflammatory mediators in mouse lung. In order to determine whether CS induces inflammatory lung response, C57BL/6J mice were exposed to CS for 3 days and 8 weeks. NF-κB-dependent pro-inflammatory cytokines was analyzed by Luminex-based multiplex assay in BAL fluid of mice exposed to CS for 3 days or 8 weeks after the mice were sacrificed at 24 h post-last exposure. The levels of NF-κB-dependent pro-inflammatory mediators, such as IL-2, IL-1, IP-1, LIF, MCP-1, MCP-3, MCP-5, MIP-3β and MMP-9 were significantly increased after 3 days in lungs of mice exposed to CS exposure (Figure E3). These data confirmed the pro-inflammatory effect of CS in mouse lung. 4

5 References E1. Foronjy RF, Mirochnitchenko O, Propokenko O, Lemaitre V, Jia Y, Inouye M, Okada Y, D'Armiento JM. Superoxide dismutase expression attenuates cigarette smoke- or elastase-generated emphysema in mice. Am J Respir Crit Care Med 26;173: E2. Foronjy RF, Mercer BA, Maxfield MW, Powell CA, Okada Y, D'Armiento J. Structural emphysema does not correlate with lung compliance: lessons from the mouse smoking model. Exp Lung Res 25;31: E3. Rangasamy T, Cho CY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW, Yamamoto M, Petrache I, Tuder RM, Biswal S. Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 24;114:

6 Figure Legends Figure E1. CS exposure increased inflammatory cell influx in mouse lungs. A) Acute CS exposure decreased the number of macrophages in BALF when the animals were sacrificed at 24 h post-last exposure compared to air-exposed mice. However, the number of macrophages in BALF was not altered when the animals were sacrificed at 2 hr and 24 hr post-last sub-chronic CS exposure compared to that of air-exposed mice. B) The number of neutrophils in BAL fluid was significantly increased in response to CS exposure both after acute (3 days) and sub-chronic (8 weeks). Data are shown as mean ± SEM (n=8 per group). p<.5, p<.1, p<.1, significant compared with respective air-exposed mice. Figure E2. CS exposure induced neutrophils and macrophages influx in mouse lung tissue. A) H&E-stained lung sections from 3 days and 8 weeks CS-exposed mice showed increased infiltration of inflammatory cells compared to air-exposed mice. Arrows indicate inflammatory cells. B) Upper panel: Immunohistochemical detection of macrophages (arrows) using rat antimouse Mac-3 antibody in lung at 24 h post-last CS exposure for both 3 days and 8 weeks air- or CS-exposed mice. Lower panel: Figures shown are from a representative experiment with 3-4 mice/group. Histogram showing the number of Mac-3 positive cells in the lung sections counted as described in Materials and Methods. Original magnification, 1X (H&E and Mac3) and 2X (H&E and Mac3 staining). 3 d, 3 days; 8 w, 8 weeks (n=8 per group). p<.1, significant compared with respective air-exposed mice. 6

7 Figure E3. CS exposure increased the levels of pro-inflammatory mediators in BAL. The levels of RelA/p65 NF-κB-dependent pro-inflammatory cytokines were analyzed by Luminex assay in BAL fluid (15 μl) of mice exposed to CS for 3 days. The animals were sacrificed at 24 h of post-last CS exposure. Data are shown as mean ± SEM, n=6/group. p<.5, p<.1, p<.1, significantly different from respective air-exposed mice. White square indicates airexposed mice, and black square indicates CS-exposed mice. a: IL-2 (Interleukin-2), b: IL-1 (Interleukin-1), c: IP-1 (Inducible protein-1), d: LIF (Leukemia inhibitory factor), e: MCP-1 (Monocyte chemoattractant protein-1), f: MCP-3 (Monocyte chemoattractant protein-3), g: MCP-5 (Monocyte chemoattractant protein-5), h: MIP- 3β (Monocyte chemoattractant protein-3beta), i: MMP-9 (Matrix metalloproteinase-9). 7

8 Figure E1 A Macrophages Cell Number x 1 5 / ml 4 Air CS hr 24 hr 2 hr 24 hr 3 days 8 weeks (Cigarette Smoke Exposure) B Cell Number x 1 5 / ml 1.5 Air CS 1..5 Neutrophils 2 hr 24 hr 2 hr 24 hr 3 days 8 weeks (Cigarette Smoke Exposure)

9 Figure E2 Air CS No. of macrophages per field 8 w Mac3 3 d 8 w H&E 3 d Air CS Air CS 3 days 8 weeks

10 Figure E3 a) IL-2 IL-1 IP-1 LIF b) Air CS Air CS Air CS Air CS c) d) e) f) g) MCP-1 MCP-3 MCP-5 MIP-3β ng/ml h) Air CS Air CS Air CS Air CS i) ng/ml MMP-9 Air CS