Online Data Supplement

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1 Modulatory role for retinoid-related orphan receptor α (RORα) in allergen-induced lung inflammation Maisa Jaradat, Cliona Stapleton, Stephen L. Tilley, Darlene Dixon, Christopher J. Erikson, Joshua G. McCaskill, Hong Soon Kang, Martin Angers, Grace Liao, Jennifer Collins, Sherry Grissom, Anton M. Jetten Online Data Supplement 39

2 Histopathological Analysis of the Lung Following BAL, the right lobe of the lung was inflated and fixed with 4% neutral buffered paraformaldehyde, processed, and embedded in paraffin as described previously (E1). Serial sections (5 to 6 µm) were stained with hematoxylin and eosin (H&E). Two different types of infiltration were noticed; the first was peribronchiolar/perivascular infiltration and the second was the alveolar sac infiltration. A semi-quantitative score system was followed by a pathologist for the degree of inflammation. Sections were scored in a blinded fashion from 0 to 4. For the peribronchiolar/perivascular infiltration a score of 0 indicated the absence of inflammation; 1, few inflammatory infiltrates of primarily lymphocytic cells mixed with a few neutrophils/eosinophils within the tissue surrounding the bronchioles and vasculature; 2, mild perivascular, intravascular and peribronchiolar infiltrates of lymphocytes mixed with fewer neutrophils/eosinophils within the connective tissue surrounding the bronchioles and vasculature; 3, moderate perivascular, intravascular and peribronchiolar infiltrates of predominantly neutrophils/eosinophils mixed with fewer lymphocytes within the tissue surrounding the bronchioles and vasculature; and 4, severe perivascular, intravascular and peribronchiolar infiltrates of eosinophils and neutrophils mixed with lymphocytes within the tissue surrounding the bronchioles and vasculature. For the alveolar sac infiltration the scoring was as follows; 0 indicated the absence of inflammation; 1, minimal alveolar sac infiltrates of neotrophils/eosinophils, few lymphocytes and increased alveolar macrophages; 2, mild alveolar sac infiltrates of neutrophils/eosinophils, few lymphocytes, increased alveolar macrophages, and a few multinucleate giant cells; 3, moderate alveolar 40

3 sac infiltrates of neutrophils/eosinophils, lymphocytes, alveolar macrophages and numerous multinucleate giant cells; 4, severe alveolar sac infiltrates of neutrophils/eosinophils, lymphocytes and increased alveolar macrophages, and numerous multinucleate giant cells. The scoring was carried out in a blinded fashion. Histological scores were obtained by examining one lung section from each of mice in each group. An entire lung section was examined and then scored (1, 2, 3, or 4). The average score (sum of all scores divided by the number of mice) calculated. To examine mucin production, serial sections of formalin-fixed, paraffin-embedded lung samples were stained with Alcian Blue-Periodic Acid-Schiff (AB-PAS). A semiquantitative score system was used by a pathologist to determine the intensity of AB-PAS stained sections were scored in an unbiased fashion and the level of AB-PAS staining estimated as 0=negative, 1=minimal, 1.5=mild, 2.0=moderate, 2.5=intense, 3.0=very intense. Airway Physiology Lung resistance (R L ) was measured directly in tracheostomized, mechanically ventilated mice. Animals were anesthetized with sodium pentobarbital (70 mg/kg i.p.) and placed on a 37 C heating pad to maintain body temperature throughout the experiment. A tracheostomy was performed using a blunt 19-gauge canula following which mice were connected to a computer-controlled small animal ventilator (Flexivent, SCIREQ Montreal Canada), and ventilated with the following settings: frequency 350 breaths/min, tidal volume 6 ml/kg (avg. 150 µl), PEEP of 3 cm H 2 0. After paralysis with pancuronium (0.5 mg/kg i.p.), baseline R L for each animal was determined by measuring changes in 41

4 pressure at the airway opening during a 6 ml/kg, 1 Hz breath, and calculated by the computer software using the equation of motion (E2). Mice were then exposed to increasing concentrations of methacholine (MCh) (3, 6, 12, 25, 50, and 100 mg/ml) by nebulization via a side-port in the ventilatory circuit. Each MCh dose was delivered at 200 breaths/min for 20 s with the ventilator piston delivering a tidal volume of 0.15 ml. After each dose of aerosol challenge, serial measurements of R L were obtained at 20 s intervals for 3 min, as described above. BAL Fluid Processing and Analysis The chest was opened and lungs were lavaged by instillation as described previously (E1). BAL fluid was kept on ice and centrifuged at 360 xg for 10 min at 4 o C. The supernatants were aliquoted, 10% fetal bovine serum (FBS) was added to each aliquot, and subsequently frozen at -70 o C for chemokine and cytokine analysis. Cell pellets were resuspended in 1 ml of Hanks Balanced Salt Solution (HBSS) and counted in a Coulter counter (Coulter Electronics Ltd., Hialeah, FL). Slides of BAL fluid were prepared using a Cytospin-3 centrifuge (Shandon, Pittsburgh, PA) and stained with Wright-Giemsa (Fisher Scientific, Pittsburgh, PA). Cells were differentially counted using a Hema-Tek II Slide Stainer (Bayer Corp, Tarrytown, NY) in an unbiased manner. Since RORα sg/sg mice are about 20% smaller in weight, cell numbers were adjusted for the weight of the mice. Cytokine and Chemokine Analysis Levels of interleukin 13 (IL-13), thymus and activation- regulated chemokine (TARC or CCL17), and eotaxin (CCL11) in BAL fluid were measured using commercially available 42

5 ELISA assay kits according to the manufacturer s instructions (R&D Systems, Minneapolis, MN). Levels of interleukins IL-2, IL-4, IL-5, IL-10, IL-12, (GM-CSF), interferon γ (IFNγ), and tumor necrosis factor α (TNFα) were determined using a Bio- Plex cytokine assay according to the manufacturer s protocol (BioRad, Hercules, CA). IgE Assay Blood was obtained from the aorta while mice were deeply anesthetized with pentobarbital following measurements of lung function. Blood was placed in an Eppendorf tube and allowed to coagulate. Samples were spun at 6,000g for 2 min and serum was removed and stored at -80 C. The samples were thawed on ice and total IgE levels were determined by ELISA using rat anti-mouse IgE (R35-92) according to the manufacturer s instructions (BD Biosciences Pharmingen, San Diego, CA). OVAspecific IgE was measured by the same ELISA with the following modification: the 96 well plate was coated with OVA (100 µl of 20 mg/ml OVA in the coating buffer provided by the manufacturer). Samples, standard and controls were incubated overnight at 4 C. Fluorescence-activated Cell Sorter Analysis of BAL Lymphocytes BAL lymphocytes expressing CD3, CD4, CD8, and B220 were stained as previously described (E3). Briefly, cell suspensions (5 X 10 5 cells) were incubated with a combination of FITC- or phycoerythrin-conjugated CD4, CD3, CD8 and B220 antibodies (Pharmingen, San Diego, CA). Cells were sorted using an LSR flow cytometer (Becton 43

6 Dickinson, San Jose, CA). Data were analyzed using CellQuest software (Becton Dickinson). Statistical Analysis of Data Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Boneferroni s Method (Multiple Comparison) when comparing between groups and student t-test for comparison within the group using the Sigmastat 2.0 software (Jandel, CA). All values are expressed as averages + standard error. Microarray Analysis Microarray analyses were carried out by the NIEHS Microarray Group (NMG). Gene expression analysis was conducted using Agilent mouse oligo arrays (Agilent Technologies, Palo Alto, CA) representing about 20,000 genes. Total RNA was isolated from lavaged lungs of saline- or OVA-challenged WT and RORα sg/sg mice using Qiagen RNeasy Mini Kit. For each condition, equal amounts of total RNA from lungs of three individual mice were pooled and amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Analyses were performed in duplicate. Starting with 1 µg of total RNA, Cy3- or Cy5-labeled crna was produced according to manufacturer s protocol. For each two-color comparison, 750 ng of each Cy3- and Cy5- labeled crnas were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 16 h in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v7.1), using defaults for all parameters. 44

7 Images and GEML files, including error and p-values, were exported from the Agilent Feature Extraction software and deposited into Rosetta Resolver (version 3.2, build ) (Rosetta Biosoftware, Kirkland, WA). The resultant ratio profiles were combined into ratio experiments as described (E4). Intensity plots were generated for each ratio experiment and genes were considered signature genes if the p value was less than Real Time QRT-PCR Analyses Real Time QRT-PCR analyses were performed to validate some of the genes identified with the microarray analyses. Equal amounts of total RNA isolated from 3 or four different mice were pooled and 50 ng of pooled RNA used in each Real Time QRT-PCR reaction. The PCR reactions were carried out in triplicate in a 7300 Real Time PCR system (Applied Biosystems) using the TaqMan One-Step RT-PCR mix (Applied Biosystems) and the following cycles: 30 min at 48 0 C, 10 min at 95 0 C, then 40 cycles each at 95 0 C for 15 sec and 60 0 C for 60 sec. Pre-designed Assays-on-Demand primers/probe sets for Ccl17 (Mm _m1), Ccl24 (Mm _m1), Saa3 (Mm _m1), and Igf1 (Mm _m1) were purchased from Applied Biosystems. All results were normalized to an internal control, the 18S transcript. Primers (F: 5 -cggctaccacatccaaggaa-3, R: 5 -gctggaattaccgcggct-3 ) and probe (5 - FAM-TgcTggcAccAgAcTTgcccTc-TAMRA-3 ) for the 18S were designed using the Primer Express 2.0 software and synthesized at Sigma/Genosys. 45

8 References E1. Stapleton CM, Jaradat M, Dixon D, Kang HS, Kim SC, Liao G, Carey MA, Cristiano J, Moorman MP, Jetten AM. Enhanced susceptibility of staggerer (RORasg/sg) mice to lipopolysaccharide-induced lung inflammation. Am J Physiol Lung Cell Mol Physiol 2005;289:L144-L152. E2. Zosky G, Garnier C, PA. S, Holt P, Sly P, Turner D. The pattern of methacoline responsiveness in mice is dependent on antigen challenge dose. Resp Res 2004;5:15. E3. Kurebayashi S, Ueda E, Sakaue M, Patel DD, Medvedev A, Zhang F, Jetten AM. Retinoid-related orphan receptor gamma (RORgamma) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis. Proc Natl Acad Sci USA 2000;97: E4. Stoughton RS, Dai H. Statistical combining of cell expression profiles. US Patent No 6,351,712,

9 Figure E1. OVA-induced mucus cell hyperplasia in airway epithelium of WT and RORα sg/sg mice. Sections of airways from WT and RORα sg/sg mice (n = in each group) were stained by PAS and then scored in an unbiased manner. A representative section of an airway from saline-challenged WT (A), OVA-challenged WT (B), salinechallenged RORα sg/sg (C), and OVA-challenged RORα sg/sg mice (D) is shown. 47

10 A B C D Supplemental Figure E1A-D