Comment[ArrayExpressAccession] E-GEOD-25095 Public Release Date 2011-10-18 Investigation Title Protective Role of IL-10 in Ozone-induced Pulmonary Inflammation Comment[Submitted Name] Protective Role of IL-10 in Ozone-induced Pulmonary Inflammation Experiment Description Background: The mechanisms underlying ozone (O3)-induced pulmonary inflammation remain unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine that is known to inhibit inflammatory mediators. Objectives: The current study investigated the molecular mechanisms underlying IL-10-mediated attenuation of O3-induced pulmonary inflammation in mice. Methods: Il10-deficient (Il10-/-) and wild type (Il10+/+) mice were exposed to 0.3-ppm O3 or filtered air for 24, 48 or 72 hr. Immediately following exposure, differential cell counts, and total protein (a marker of lung permeability) were assessed from bronchoalveolar lavage fluid (BALF). mRNA and protein levels of cellular mediators were determined from lung homogenates. We also utilized global mRNA expression analyses of lung tissue with Ingenuity Pathway Analyses (IPA) to identify patterns of gene expression through which IL-10 modifies O3-induced inflammation. Results: Mean numbers of BALF polymorphonuclear leukocytes (PMNs) were significantly greater in Il10-/- mice than in Il10+/+ mice after exposure to O3 at all time points tested. O3-enhanced nuclear NF-kB translocation was elevated in the lungs of Il10-/- compared to Il10+/+ mice. Gene expression analyses revealed several key IL-10 and O3-dependent mediators, including IL-6, MIP-2, IL-1 and CD86. Conclusions: Results indicated that IL-10 protects against O3-induced pulmonary neutrophilic inflammation and cell proliferation. Moreover, gene expression analyses identified three response pathways and several novel genetic targets (e.g. Ccr1, Socs3, Il33, Hat1, and Gale) through which IL10 may modulate the innate and adaptive immune response. These novel mechanisms of protection against the pathogenesis of O3-induced pulmonary inflammation may also provide potential therapeutic targets to protect susceptible individuals. PARALLEL study design with 26 samples. Biological replicates: 2 to 3 replicates per group with wild type air exposed animals as controls for each time point (24, 48, 72 hours). Time-Course, Dose-Response, Strain comparison Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name Kleeberger Backus Howden Fostel Bauer Cho Marzec Peden Kleeberger Person First Name Steven Gillian R Jennifer A Hye-Youn Jacqueline D Steve Person Mid Initials R S K B R Person Email kleeber1@niehs.nih.gov Person Affiliation National Institute of Environmental Health Sciences, NIH Person Phone Person Fax Person Address National Institute of Environmental Health Sciences, NIH, 111 TW Alexander Dr., Building 101, MD D-201, Research Triangle Park, NC, USA Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE25095-1 P-GSE25095-6 P-GSE25095-3 P-GSE25095-8 P-GSE25095-7 P-GSE25095-2 P-GSE25095-4 P-GSE25095-5 Protocol Description ID_REF =
VALUE = RMA data A fragmentation buffer (250mM Tris acetate pH 8.1, 150 mM MgOAc, 500mM KOAc) was used to fragment 15µg of cRNA. The fragmented cRNA was then hybridized to GeneChips (Affymetrix Mouse Genome 430A 2.0). We provided mice with water and pelleted open-formula rodent diet NIH-31 (Zeigler Brothers, Gardners, PA) ad libitum. All experimental procedures were conducted in accordance with approved National Institutes of Health Humane Care and Use of Laboratory Animals guidelines and the American Physiological Society’s “Guiding Principles in the Care and Use of Animals.” Animals were treated humanely and with regard for alleviation of suffering. RMA Express, Visual data mining and statistical analysis Scanned on an Affymetrix scanner. Mice were placed in individual stainless-steel wire cages within a Hazelton 1000 chamber (Lab Products, Maywood, NJ) equipped with a charcoal and high-efficiency particulate air–filtered air supply. Mice were exposed to 0.3 ppm O3 or filtered air for 24, 48 or 72 hr (23.5 hr/day) as described previously (Cho et al, 2007). Left lobes of mouse lung tissue were harvested, flash frozen in liquid nitrogen, and stored at -80oC. For RNA extraction, the lung tissue was homogenized in 2ml Trizol (Invitrogen, Calsbad, CA) with a Tekmar Tissumizer and saw-tooth generator. One ml of homogenate was subsequently processed according to the manufacturer’s (Invitrogen) protocol with the following minor modifications. Two µl of 5 mg/ml glycogen was used as a carrier for the isopropanol precipitation, duration of the isopropanol precipitation was increased to overnight, and all centrifugation times were increased to 15 minutes. RNA pellets were resuspended in nuclease-free water. RNA quality was tested using a Beckman DU680 spectrophotometer, and quality assessment was determined by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. A Qiagen RNeasy total RNA cleanup protocol was subsequently performed followed by re-quantitation by spectophotometry. Double stranded cDNA was synthesized from 4µg of total RNA using the GeneChip Expression 3’ amplification reagents one-cycle cDNA synthesis kit (Affymetrix). The resultant double-stranded cDNA was column-purified using the GeneChip Sample Cleanup Module. Biotinylated cRNA was synthesized from the double-stranded cDNA by in vitro transcription (IVT) using the GeneChip Expression 3’ amplification reagents for IVT labeling (Affymetrix), and was subsequently purified by column purification with the GeneChip Sample Cleanup Module (Affymetrix), and quantified. Quality of biotinylated cRNAs was assessed by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation hybridization grow feature_extraction image_aquisition specified_biomaterial_action nucleic_acid_extraction labeling Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name GENOTYPE VARIATION EXPOSURE DOSE STRAIN EXPOSURE TIME AGENT Experimental Factor Type genotype variation exposure dose strain exposure time agent Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Protective role of interleukin-10 in ozone-induced pulmonary inflammation. Publication Author List Backus GS, Howden R, Fostel J, Bauer AK, Cho HY, Marzec J, Peden DB, Kleeberger SR PubMed ID 20826374 Publication DOI 10.1289/ehp.1002182 Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE25095 Comment[GEOLastUpdateDate] 2011-10-18 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2011-10-17 Comment[ArrayExpressSubmissionDate] 2010-11-03 SDRF File E-GEOD-25095.sdrf.txt