STAT-1 Attenuates Murine Allergen-Induced Airway Remodeling and Exacerbation by

Transcription

1 Online Supplement STAT-1 Attenuates Murine Allergen-Induced Airway Remodeling and Exacerbation by Carbon Nanotubes Elizabeth A. Thompson, Brian C. Sayers, Ellen E. Glista-Baker, Kelly A. Shipkowski, Mark D. Ihrie, Katherine S. Duke, Alexia J. Taylor, and James C. Bonner

2 Supplementary Methods and Materials Animals. Male wild type (WT) and Stat1 -/- mice bred on a 129SV background were a whole-body knock-out and purchased from Taconic Laboratories (Germantown, NY). Mice were housed in a pathogen-free, climate controlled animal care facility and given food and water ad libitum. All aspects of animal care and experimentation described in this study were conducted according to NIH guidelines and approved by the North Carolina State University Institutional IACUC committee. All mice were 6-8 weeks old upon arrival from the vendor and allowed to acclimate for 1 to 2 weeks prior to experimental procedures. Mice were divided into the following four groups for each time point (Control, OVA, MWCNT, and OVA/MWCNT). The experimental design is illustrated in Fig. 1A. An N=4 animals was used for control or OVA groups and an N=6 was used for MWCNT or OVA/MWCNT groups for each genotype at each time point post-mwcnt treatment. OVA Sensitization of Mice. Mice were sensitized to OVA as previously published (26). In brief, 20 mg OVA (98% pure, Grade V; Sigma-Aldrich, Saint Louis, MO) was adsorbed onto 2 mg of alum (Accurate Chemical and Scientific Corporation, Westbury, NY) in 0.5 ml sterile saline and administered by intraperitoneal (ip) injection on study days 0 and 12. Control mice received 0.5 ml sterile saline via i.p. injection. Mice were challenged intranasally with 20 mg of OVA in 0.1 ml sterile saline while under isoflurane anesthesia on study days 26, 28 and 32. Control mice were administered 0.1 ml of sterile saline intranasally. MWCNT Exposure in Mice. MWCNTs synthesized using carbon vapor deposition with nickel and lanthanum catalysts were obtained from Helix Material Solutions Incorporated (Richardson, TX). These MWCNTs have been previously characterized and reported on by our laboratory (21, 25, 27). Administration of MWCNTs and vehicle control was performed as previously published (28-30). Briefly, on study day 34, mice were exposed to 4 mg/kg MWCNTs in 0.1% Pluronic surfactant solution (Sigma-Aldrich, Saint Louis, MO) diluted with sterile saline by oropharyngeal

3 aspiration (OPA) while under isoflurane anesthesia. Control mice were administered 0.1% Pluronic surfactant solution in sterile saline via OPA. Morphometric Analysis of Airway Fibrosis. Paraffin embedded lung sections were stained with Masson s trichrome blue stain and analyzed under the microscope at 10 X magnification. Photomicrographs of small and medium-sized round and/or oval airways (~500 m diameter) were captured and analyzed for airway collagen deposition using the area/perimeter ratio as described previously (30, 32). Briefly, using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA), we measured around the collagen deposit of the airway or trichrome stain to determine the outer area. We then made a second measurement around the surrounding basement membrane of the same airway to determine the inner area and the airway circumference or perimeter. The difference between the outer and inner area was defined as the area and divided by the perimeter to determine the area/perimeter ratio. A minimum of three measurements was made per animal, and performed on an N=4 animals for control and OVA groups or an N=6 animals for MWCNT and OVA/MWCNT groups, and values were expressed as mean ± SEM for each group. Morphometric Analysis of Mucus Cell Metaplasia. Photomicrographs of paraffin embedded lung sections stained with Alcian Blue Periodic Acid-Schiff (AB-PAS) were taken and images were imported into ImageJ version (National Institutes of Health). Small to medium-sized round and/or oval airways (~500 m diameter) were captured and analyzed for mucous cell metaplasia by Image J as described previously (30). Within ImageJ, cells staining positively for AB-PAS were identified using the de-convolution module and the threshold method, and images were then calibrated for measurements in microns. Data was then expressed as the percentage of epithelial area positively stained for AB-PAS divided by the total epithelial area. These counts were performed on an N=4 animals for control and OVA groups and N=6 animals for MWCNT and OVA/MWCNT groups, and values were expressed as mean ± SEM for each group.

4 ELISA. Quantikine ELISA kits (R & D Systems, Minneapolis, MN) were used to assay protein levels of various mediators of lung disease (IL-13, IL-1, TGF- 1, TNF- and osteopontin) in BALF. Absorbance values were determined using a Thermo Scientific Multiskan FC spectrophotometer microplate reader (ThermoFisher Scientific, Waltham, MA). Protein levels were measured in duplicate from an N=4 animals for control and OVA groups or an N=6 animals for MWCNT and OVA/MWCNT groups. Values were expressed as mean ± SEM for each group. Cell Counts. A Thermo Scientific Cytospin 4 (Thermo Electron Corporation, Waltham, MA) was used to isolate cells from the BALF onto glass slides. Cells were fixed and stained with the Diff- Quik Stain Set (Dade Behring Inc, Newark, DE) and differential cell counts as well as total cell counts were performed for each sample. Differential cell counts were determined by counting all of the cells present in multiple fields of view at 20 X magnification until a minimum of 500 cells was counted per sample. Total cell counts were performed by counting the total number of cells present in three different fields of view at 20 X magnification. These counts were performed on an N=4 animals for control and OVA groups and an N=6 animals for MWCNT and OVA/MWCNT groups, and values were expressed as mean ± SEM for each group. RNA Extraction and Taqman Real-Time PCR. RNA was collected from mouse lung tissue or primary fibroblasts using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer s instructions. RNA was quantitated and diluted to 25 ng/ l. Taqman quantitative real-time PCR was used to determine the expression of Collagen 1A1 and 1A2 in primary fibroblasts treated with TGF- or PBS. 50 ng/ml RNA was used per reaction. Taqman Gene Expression assays for collagen 1A1 (Mm _G1), collagen 1A2 (Mm _M1), IL-10 (Mm m1), and Foxp3 (Mm m1) were performed on an Applied Biosystems StepOnePlus Real- Time PCR System (Applied Biosystems, Foster City, CA). Gene expression was measured relative to the endogenous control, -2 Microglobulin (Mm _M1). All samples were run in duplicate, and relative quantitation values were expressed as fold-change over controls.

5 TUNEL Staining. Paraffin embedded lung tissue was examined for the presence of apoptotic cells using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI). Briefly, tissue was de-paraffinized, rehydrated, fixed in 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO), and permeabilized in 20 μg/ml Proteinase K solution. The fragmented DNA characteristic of apoptotic cells was visualized due to the incorporation of fluorescein-12-dutp (a) at 3 -OH DNA ends. Slides were cover-slipped using VECTASHIELD + DAPI (Vector Labs, Burlingame,CA) for nuclei detection. Photomicrographs of airways were taken and images were imported into ImageJ version (National Institutes of Health). Within ImageJ, fluorescent cells were counted by first selecting image, then adjust, then threshold to choose black & white. De-grouping clumps of cells was performed by selecting process, then binary, then convert to mask, then selecting process, binary, and watershed. Finally, counting was performed by selecting analyze and then selecting analyze particles. Data was then expressed as the percentage of TUNEL-positive cells relative to the total number of DAPI-positive cells within a microscopic field. Counts were performed on three airways per lung and from N=4 animals for control or OVA groups and N=6 from MWCNT or OVA/MWCNT groups. Primary Mouse Lung Fibroblasts. Primary mouse lung fibroblasts were isolated from adult male wild type (WT) and Stat1 -/- (KO) mice bred on a 129SV background as previously published (33). Fibroblasts were cultured in Dulbecco s modified Eagle s medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% Fetal Bovine Serum (Life Technologies, Grand Island, NY), 1% antibiotic/antimycotic (Life Technologies, Grand Island, NY) and 0.1% Fungizone (Life Technologies, Grand Island, NY). Second passage primary cells were plated in six-well plates and grown to 100% confluence. They were then placed in serum free medium (SFDM) for 24 hours prior to treatment with 10 ng/ml recombinant mouse TGF- 1 protein (R & D Systems, Minneapolis, MN) or PBS for 48 hours. Sircol Collagen Assay. Soluble collagen secreted by mouse lung fibroblasts in culture was measured by Sircol assay (Biocolor, Carrickfergus, UK). Briefly, WT or STAT-1 -/- mouse lung

6 fibroblasts were grown to confluence in 24-well tissue culture plates in 1 ml 10% FBS-DMEM, then rendered quiescent in 1 ml SFDM for 24 hrs, and treated with recombinant mouse TGF- 1 (10 ng/ml) or SFDM alone (control) for 72 hrs prior to collecting the supernatant. Cell culture supernatants were analyzed for soluble collagen according to the manufacturer s instructions and absorbance of standards and samples was measured on a microplate reader at a wavelength of 540 nm.

7 Supplementary Figure Legends Fig. S1. PDGF-AA protein levels in bronchoalveolar lavage fluid (BALF) of wild-type (WT) and Stat1 -/- mice following OVA sensitization and exposure to MWCNTs. Protein levels PDGF-AA were assessed in BALF using ELISA. Open bars represent wild-type (WT) mice and solid bars represent STAT-1-deficient (Stat1 -/- ) mice. Data are the mean SEM (N=4 animals control and OVA groups, N=6 MWCNT and OVA/MWCNT groups). *p<.0.05, **p<0.01, ***p<0.001 compared to control as determined by one-way ANOVA. # p<0.05, ## p<0.01 between genotypes within the same treatment group as determined by two-way ANOVA. Fig. S2. T-bet mrna levels in lung tissue from wild-type (WT) and Stat1 -/- mice following OVA sensitization and exposure to MWCNTs. Levels of T-bet mrna in lung tissue from WT mice (open bars) or Stat1 -/- mice (solid bars) were measure by Taqman real-time RT-PCR. Data are the mean SEM. Data are the mean SEM (N=3 animals for each group). *p<.0.05, **p<0.01 compared to control as determined by one-way ANOVA. ## p<0.01, ### p<0.001 between genotypes within the same treatment group as determined by two-way ANOVA.

8 Fig. S1 WT Stat1 -/- 1 day 21 day PDGF-AA (pg/ml) *** * *** *** *** ## *** # *** *** 0 Control MWCNT OVA OVA/MWCNT 0 Control MWCNT OVA OVA/MWCNT

9 Fig. S2 WT Stat1 -/- 1 day 21 day T-bet mrna (fold change) Control MWCNT OVA OVA/MWCNT 0 Control MWCNT OVA OVA/MWCNT