Innate Inflammation Induced by the 8-Oxoguanine DNA Glycosylase-1−KRAS− NF-κB Pathway This information is current as of September 29, 2014.

Leopoldo Aguilera-Aguirre, Attila Bacsi, Zsolt Radak, Tapas K. Hazra, Sankar Mitra, Sanjiv Sur, Allan R. Brasier, Xueqing Ba and Istvan Boldogh

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol published online 29 September 2014 http://www.jimmunol.org/content/early/2014/09/28/jimmun ol.1401625

Published September 29, 2014, doi:10.4049/jimmunol.1401625 The Journal of Immunology

Innate Inflammation Induced by the 8-Oxoguanine DNA Glycosylase-1–KRAS–NF-kB Pathway Leopoldo Aguilera-Aguirre,* Attila Bacsi,*,1 Zsolt Radak,*,2 Tapas K. Hazra,†,‡ Sankar Mitra,†,3 Sanjiv Sur,‡,x Allan R. Brasier,‡,x Xueqing Ba,*,4 and Istvan Boldogh*,x

eactive oxygen species (ROS) generated by biological, physical, and chemical agents, ligand-receptor interactions, and as a byproduct of metabolic processes induce oxidative modifications to lipids, proteins, and DNA, and generate cell activation signals—all of which are thought to be associated with inflammatory disease processes (1). The primary target of ROS in the DNA is guanine, because it has the lowest redox potential of the four nucleobases (2, 3). Although guanine base lesions vary according to the nature of the oxidants (4), 7,8dihydro-8-oxoguanine (8-oxoG) is the most frequent oxidation product in both DNA and RNA. Moreover, the accumulation of 8oxoG along with other oxidatively modified bases in the mammalian genome has been associated with a loss of cellular/tissue homeostasis, and is believed to contribute to various inflammatory processes and aging-related diseases (5, 6). In mammalian cells,

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oxidatively modified base lesions are repaired by the base-specific DNA glycosylases OGG1, endonuclease III–like protein 1 (NTH1), and Nei-like (NEIL1, NEIL2, and NEIL3) (7, 8). OGG1 and NTH1 incise the DNA strand via b-lyase activity, thereby generating 39-phospho-a,b-unsaturated aldehyde terminus (39dRP) and 59phosphate, whereas NEIL1, 2, and 3 have bd-lyase activity, thus generating 39- and 59 phosphate (39P and 59P) at the strand gaps. The 39dRP and 59P are then removed by AP endonuclease 1 (APE1) and polynucleotide kinase 39-phosphatase, respectively, to generate a polymerase-ready 39OH residue. Gap filling can involve 1-nt incorporation by DNA polymerase b (Polb) in the short-patch repair subpathway, or displacement synthesis of 2–8 nt by either Polb or replicative DNA polymerase (Pold) in the long-patch repair subpathway (7–9).

*Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555; †Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555; ‡Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555; and xSealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, TX 77555

The data sets presented in this article have been submitted to the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE60711) under accession number GSE60711.

1 Current address: Department of Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary. 2

Current address: Research Institute of Sport Science, Faculty of Physical Education and Sport Science, Semmelweis University, Budapest, Hungary. 3 Current address: Radiation Oncology, Houston Methodist Research Institute, Houston, TX. 4 Current address: Key Laboratory of Molecular Epigenetics, Institute of Genetics and Cytology, Northeast Normal University, Changchun, China.

Received for publication June 26, 2014. Accepted for publication August 29, 2014. This work was supported by National Institute on Environmental Health Sciences Grant RO1 ES018948 (to I.B.), National Institute of Allergy and Infectious Diseases Grant AI062885 (to I.B., S.S., and A.R.B.), National Institute on Environmental Health Sciences Grant T32 ES007254 (to L.A.-A.), National Heart, Lung, and Blood Institute Proteomic Center Grant N01HV00245 (to I.B., S.S., and A.R.B.), and European Union and European Social Fund Theoretical Atomic, Molecular, and Optical Physics Project TAMOP 4.2.2.A-11/1/KONV-2012-2023 (to A.B.). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401625

Address correspondence and reprint requests to Dr. Istvan Boldogh, Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1070. E-mail address: [email protected] Abbreviations used in this article: AE, airway epithelium; AEC, AE cell; APE1, AP endonuclease 1; BALF, bronchoalveolar lavage fluid; CA, comet assay; CXCL-1,-2, C-X-C motif ligand-1 and -2 chemokines; FapyG, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; FLARE CA, fragment length analysis by repair enzyme CA; FTS, transfarnesylthiosalicylic acid; GSH, reduced glutathione; GSSG, oxidized glutathione; hNBEC, human normal bronchial epithelial cell; HRAS and KRAS, mammalian homolog of Harvey and Kirsten sarcoma virus oncogen; IHC, immunohistochemistry; IIR, innate immune response; i.n., intranasal; MEF, murine embryonic fibroblast; MLE-12, mouse lung epithelial-12; MSK1, mitogen-stress related kinase-1; NEIL1 and 2, Nei (endonuclease VIII)-like DNA glycosylase-1 and -2; NRAS, neuroblastoma RAS viral oncogene homolog; OGG1, 8-oxoguanine DNA glycosylase-1; OGG1-BER, OGG1-initiated DNA base excision repair; 8-oxodG, 8-oxo-29-deoxyguanosine; 8-oxoG, 7,8-dihydro-8-oxoguanine; p, phosphorylated; qRT-PCR, quantitative real-time RT-PCR; RNAi, RNA interference; ROS, reactive oxygen species; WB, Western blotting. Copyright
2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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8-Oxoguanine-DNA glycosylase-1 (OGG1) is the primary enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG) via the DNA base excision repair pathway (OGG1-BER). Accumulation of 8-oxoG in the genomic DNA leads to genetic instability and carcinogenesis and is thought to contribute to the worsening of various inflammatory and disease processes. However, the disease mechanism is unknown. In this study, we proposed that the mechanistic link between OGG1-BER and proinflammatory gene expression is OGG1’s guanine nucleotide exchange factor activity, acquired after interaction with the 8-oxoG base and consequent activation of the small GTPase RAS. To test this hypothesis, we used BALB/c mice expressing or deficient in OGG1 in their airway epithelium and various molecular biological approaches, including active RAS pulldown, reporter and Comet assays, small interfering RNA–mediated depletion of gene expression, quantitative RT-PCR, and immunoblotting. We report that the OGG1-intiated repair of oxidatively damaged DNA is a prerequisite for GDP→GTP exchange, KRAS-GTP–driven signaling via MAP kinases and PI3 kinases and mitogen-stress–related kinase-1 for NF-kB activation, proinflammatory chemokine/cytokine expression, and inflammatory cell recruitment to the airways. Mice deficient in OGG1-BER showed significantly decreased immune responses, whereas a lack of other Nei-like DNA glycosylases (i.e., NEIL1 and NEIL2) had no significant effect. These data unveil a previously unidentified role of OGG1-driven DNA BER in the generation of endogenous signals for inflammation in the innate signaling pathway. The Journal of Immunology, 2014, 193: 000–000.

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OGG1 DNA REPAIR/8-oxoG–INDUCED INNATE IMMUNE RESPONSE

Materials and Methods Reagents Inhibitors. Transfarnesylthiosalicylic acid (FTS, Cayman Chemical, Ann Arbor, MI); GW5074 (5-iodo-3-[(3,5-dibromo-4-hydroxyphenyl)methylene]2-indolinone (Sigma-Aldrich, St. Louis, MO); PD98059 (2-(2-Amino-3methoxyphenyl)-4H-1-benzopyran-4-one (EMD Millipore Chemicals, Gibbstown, NJ); LY294002 (2-morpholin-4-yl-8-phenylchromen-4-one; EMD Millipore Chemicals, Gibbstown, NJ), and DAPI (Sigma-Aldrich). Nucleotides and nucleosides. 8-oxoguanine (Cayman Chemical). 2,6Diamino-4-hydroxy-5-formamidopyrimidine (FapyG) was provided by M. Dizdaroglu (National Institute of Standards and Technology, Gaithersburg, MD). 7,8-Dihydro-8-oxoadenine was purchased from BioLog Life Science Institute (Axxora, San Diego, CA). Guanine and 8-oxo-29-deoxyguanosine (8-oxodG) were from Sigma-Aldrich (St. Louis, MO). 8-OxoG was provided as a hydroacetate salt and dissolved as recommended by the manufacturer (Cayman Chemical). Briefly, 8-oxoG was dissolved in 10 mM NaOH (pH 12). Stock solution was diluted to 0.001– 10 mM concentrations in saline (without Ca2+, Mg2+ [pH 7.4]) and used immediately. For experiments, filter-sterilized, pH-balanced 8-oxoG or solutions of control reagents were used. All reagents were free of LPSs as determined using an LAL Chromogenic Endotoxin Quantitation Kit (catalog number 88282; Pierce Biotechnology (Rockford, IL). Glucose oxidase (catalog number G7141) was purchased from Sigma-Aldrich. The dose of GO was set (in preliminary studies) to 1 mU, which caused an ∼2-fold increase in ROS levels in primary airway epithelial cells, resulting in a 2.5-fold increase in 8-oxoG levels in OGG1expressing airways. Antibodies. Primary Abs–OGG1 Ab (catalog number ab124741) and subtype-specific HRAS Ab (catalog number 1521-1) were purchased from Epitomics (Burlingame, CA). pan-RAS Ab (catalog number 05-1072) was

from Millipore (Billerica, MA) and KRAS (catalog number sc-30), neuroblastoma RAS viral oncogene homolog (NRAS) (catalog number sc3), RelA (catalog number sc-372), and p-RelA (Ser276, catalog number sc101749) Abs from Santa Cruz Biotechnology (Dallas, TX). The following Abs were purchased from Cell Signaling Technologies (Danvers MA): RAF-1 (catalog number 9422), phosphorylated (p)-RAF-1 (Ser338; catalog number 9427), ERK 1,2 (catalog number 4695), p-ERK 1,2 (Thr202/Tyr204, catalog number 9101), MEK 1,2 (catalog number 9122), p-MEK1,2 (Ser217/221; catalog number 9154), mitogen-stress related kinase-1 (MSK1) (catalog number 3489), p-MSK1 (Ser376; catalog number 9591), PI3K (p85; catalog number4292), p-PI3K (p85/Tyr458, p55/Tyr199; catalog number 4228), AKT (catalog number 9272), p-AKT (Ser473; catalog number 4058), IKKa (catalog number 2682), IKKb (catalog number 2370), p-IKKa/b (Ser176/180; catalog number 2697), IkBa (catalog number 4814), and p-IkBa (Ser32/36; catalog number 9246). Secondary Abs (e.g., goat anti-rabbit, rabbit anti mouse and donkey anti-mouse) were from GE Healthcare (Pittsburgh, PA).

Cell cultures Human diploid fibroblasts (MRC-5), murine embryonic fibroblasts (Ogg12/2 MEF, and Ogg1+/+ MEF), and the mouse lung epithelial cell line (MLE-12) were maintained, respectively, in Eagle’s MEM, Dulbecco’s modified Eagle’s low glucose, IMDM, and RPMI 1640, supplemented with 10% FBS. Ogg12/2 and Ogg1+/+ MEF cells were provided by Dr. D.E. Barnes (Clare Hall Laboratories, South Mims, Hertfordshire, U.K.). All media contained glutamine, penicillin, and streptomycin, and cells were grown at 37˚C, in a 5% CO2 atmosphere. Primary mouse lung fibroblasts (catalog number C57-6013) and primary mouse tracheal epithelial cells (catalog number BALB-5033) were obtained from Cell Biologics (Chicago, IL), and cells were maintained as recommended by the provider (in Complete Fibroblast Medium, catalog number M2267 and Complete Epithelial Cell Medium; catalog number M6621). A549 cells expressing GFP-RelA fusion protein were established by Dr. A.R. Brasier’s laboratory and maintained in DMEM containing 10% FBS, penicillin (100 U/ml), streptomycin (100 mg/ml), and hygromycin B (100 mg/ml) at 37˚C in a 5% CO2 incubator. The human telomerase and cyclin-dependent kinase-4–immortalized human normal bronchial epithelial cells (hNBECs) were provided by Dr. J.D. Minna (Hamon Center for Therapeutic Oncology Research, Department of Internal Medicine Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX) and were grown in bronchial airway epithelial cell growth medium (Lonza, Walkersville, MD) in a humidified atmosphere of 5% CO2 (19).

Animals and treatments Animal experiments were performed according to the National Institutes of Health’s Guide for Care and Use of Experimental Animals and approved by the University of Texas Medical Branch Animal Care and Use Committee (approval number 0807044A). Eight- to 10-wk-old female BALB/c mice (The Jackson Laboratory) were used for these studies. Mice (n = 5–6) were challenged via the nasal route with 60 ml pHbalanced saline containing 8-oxoG (pH 7.4; 1 mM), glucose oxidase (1 mU) or other nucleotides or nucleosides under mild anesthesia. Animals were sacrificed at various time points, and their lungs subjected to lavage to obtain bronchoalveolar lavage fluid (BALF). The lungs were then excised and portions sectioned or homogenized for RNA extraction and preparation of lung extracts for Western blotting (WB) or to determine changes in Ras-GTP levels (16). Stealth RNA interference (RNAi) for depletion of target proteins was undertaken as we described under “Depletion of gene expression in lung.” Intraperitoneal inhibitor treatments 2 mg/kg i.p. FTS (RAS inhibitor), 10 mg/kg i.p. GW5074 (Raf1 inhibitor), 10 mg/kg i.p. PD98059 (MEK1/2 inhibitor), or 5 mg/kg i.p. LY294002 (pan-PI3K inhibitor) per mice were added as described in previous studies (20–22).

Evaluation of airway inflammation BALFs were collected, total cell numbers were determined, and cytospin preparations were made for cell typing by Abs using immunohistochemistry or staining with Wright–Giemsa for differential cell counts as we described previously (23, 24). Randomly selected fields were photographed, and differential cell counts were evaluated using ImageJ version 1.47 (National Institutes of Health) with the built-in cell counter plugin. Immunofluorescence staining was carried out (see “Immunohistochemistry”) on nonpermeabilized cells using an Ab to neutrophil elastase (catalog number SC-9518; Santa Cruz Biotechnology). Fluorescent images were captured at a magnification of 3144 using a CoolSNAP HQ2 camera mounted on a Nikon Eclipse Ti microscope. The microscope was operated via NISElements AR Software version 3.22.09 for 64 bit.

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To address the role of 8-oxoG in the genome, knockout mice lacking OGG1 activity have been generated (10, 11). Surprisingly, Ogg1-null mice did not show impaired embryonic development or phenotypic changes, including altered lifespan, despite supraphysiological levels of 8-oxoG in their genomes. In other reports, Ogg1-null mice were shown to be found resistant to innate and allergic inflammation (12), and when placed on a high-fat diet, showed increased susceptibility to obesity and metabolic dysfunctions (13). Mabley and colleagues (14) showed that that Ogg1-null mice are resistant to endotoxin (LPS)-induced organ dysfunction, neutrophil infiltration, and oxidative stress compared with the response seen in wild-type controls. In another study, sensitized Ogg1-null mice exhibited lower IL-4, IL-6, IL-10, and IL-17 production and decreased levels of inflammatory cell infiltration and oxidative stress in the lungs after OVA challenge compared with levels in wild-type controls (15). Bacsi and colleagues showed that supraphysiological levels of 8-oxoG in the genome of the airway epithelium (AE) evoked a lower inflammatory response in challenged-sensitized mice, as determined by expression of Th2 cytokines, eosinophilia, epithelial metaplasia, and airway hyperresponsiveness (12). Taken together, these observations strongly imply the presence of yet-to-be-defined function(s) of OGG1 protein and/or its repair product, 8-oxoG base, in inflammatory processes. Intriguing recent studies have also shown that OGG1 binds the 8-oxoG base with a high affinity and that the resulting complex (OGG1•8-oxoG) acts as a guanine nucleotide exchange factor that activates RAS family GTPases (16–18). In the current study, we test the hypothesis that the repair of 8oxoG and not the accumulation of 8-oxoG in the genome is etiologically associated with an innate immune response. Using BALB/c mice expressing or lacking OGG1 in the airways, we demonstrated that the repair of oxidatively damaged DNA by OGG1, but not by other lesion-specific DNA glycosylases (NEIL1 and NEIL2), induces the activation of wild-type KRAS, MAP-, PI3-, and MS-kinases and NF-kB, resulting in proinflammatory gene expression and inflammatory cell accumulation.

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Depletion of gene expression in lung

Assessment of RAS-GTP levels

Stealth RNAi was obtained from Invitrogen Life Technologies. Under mild anesthesia, Stealth RNAi to target gene (or control RNAi) in transfection reagent (catalog number 201-10G; Polyplus-transfection, New York, NY) was introduced into the airways i.n. at 0 and 24 h (12, 25). Stealth RNAi duplex for Ogg1 (catalog number MSS237431) was as follows: sense, 59GAUGUCACUUAUCAUGGCUUCCCAA-39; antisense, 59-UUGGGAAGCCAUGAUAAGUGACAUC-39; Stealth RNAi duplex for Kras (catalog number MSS236996_F1N), sense, 59-CACUUUGUGGAUGAGUACGACCCUA-39, antisense, 59-UAGGGUCGUACUCAUCCACAAAGUG-39; and Stealth RNAi duplex for Nras (catalog number NM_010937.2_stealth_244), sense, 59-GAACCACUUUGUGGAUGAAUAUGAU-39, antisense, 59-AUCAUAUUCAUCCACAAAGUGGUUC-39; and Stealth RNAi duplex for Hras (Cat# NM_008284.2_stealth_279), sense, 59-CAGAACCACUUUGUGGACGAGUAUG-39, antisense, 59-CAUACUCGUCCACAAAGUGGUUCUG-39. Neil1 Stealth RNAi (catalog number 1320001; ID number MSS231767), Neil2 Stealth RNAi (catalog number 1320001; ID number MSS283982).

Changes in RAS-GTP levels were quantified using an Active RAS PullDown and Detection Kit (Pierce Biotechnology, Thermo Fisher Scientific) as described previously (16, 17). In brief, RAS-GTP from lung extracts and cell lysates (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 60 mM MgCl2, 1% Nonidet P-40, and 5% glycerol) was captured by the RASbinding domain of Raf1 (RBD) (29) immobilized on Glutathione Resin GST-RAF1-RBD. After washing with binding buffer, captured RAS was eluted in Laemmli buffer and detected by WB. Total RAS proteins were detected using pan-RAS Ab (Millipore). RAS family proteins were identified using subtype-specific Abs for HRAS (Epitomics), KRAS, and NRAS Abs (Santa Cruz Biotechnology).

siRNAs (20–40 nM; optimal final concentrations were determined in preliminary studies) in transfection reagent (INTERFERin; Polyplustransfection) were added to dishes along with 5 3 106 cells in serumfree media for 3 h. Cells were further incubated in growth media for 48 h. Control siRNA and siRNAs to target genes were obtained from Dharmacon, Thermo Fisher Scientific (Rockford, IL) siGENOME Smart pool; Hras catalog number M-004142, Kras catalog number M-005069, and Nras catalog number M-003919). siRNA to deplete mouse OGG1 (catalog number M-048121-01-005) and human OGG1 (catalog number M-005147-03-0005) were purchased from Dharmacon (Pittsburgh, PA). OGG1 was depleted via a simultaneous siRNA transfection and plating method (12, 18). siRNAs to PI3K (catalog number M-041079-01-0005), MEK1-2 (MAP2K1 catalog number M-040605-01-0005, MAP2K2 catalog number M-040606-01-0005), and MSK1 (catalog number M-040751-010005) were from Dharmacon. Depletion of the target at the mRNA/protein levels was determined by quantitative real-time RT-PCR (qRT-PCR) and WB analysis, respectively.

Assessment of genomic 8-oxoG levels Genomic 8-oxoG levels were determined by assessing OGG1-sensitive sites using a FLARE comet assay kit (Trevigen, Gaithersburg, MD) (12, 26, 27). Briefly, freshly isolated, exfoliated airway epithelial cells were suspended in 320 ml 0.6% low melting-point agarose and placed onto precoated microscope slides (Trevigen). Slides with solidified agarose were placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Trish-HCl [pH 10], 1% sodium sarcosinate, and 1% Triton X-100) for 24 h. Slides were gently rinsed three times for 10 min with an enzyme reaction buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 1 mM DTT, and 0.2 mg ml BSA). Slides were than immersed in enzyme reaction buffer 6 10 mg/ml OGG1 [generated locally (28)]. DNA was digested for 180 min at 37˚C. Slides were placed in a precooled (4˚C) electrophoresis tank, and electrophoresis was carried out at pH 11.5 in a buffer provided by the manufacturer (Trevigen). Before electrophoresis, DNA was allowed to unwind for 10 min. After electrophoresis at 1.25 V/cm at 4˚C, preparations were neutralized (0.4 M Tris-HCl [pH 7.4]) three times (15 min). The agarose was air-dried, and the DNA was stained with SYBRGreen. Comets were analyzed using the Comet Assay IV version 4.2 system image analysis software (Perceptive Instruments, Suffolk, U.K.) (12). Depending on cell availability, 120–160 comets were scored. The fold changes in tail intensities are expressed as the mean (6 SD) from three to five mice.

Immunohistochemistry Cells on microscope coverslips were fixed in 4% paraformaldehyde at 4˚C and then permeabilized with Triton X-100 for 30 min at 37˚C. The cells were then incubated for overnight at 4˚C with primary Ab to OGG1 (1:1000), 8-oxodezoxyguanosine (1:400). After washing (PBS-Tween 20: PBS-T) cells were incubated for 1 h at room temperature with Alexa Fluor 488–conjugated secondary Abs. Nuclei of cells were stained for 15 min with DAPI (10 ng/ml; blue). Cells were then mounted in anti-fade medium (DakoCytomation, Carpinteria, CA) on a microscope slide. Microscopy was performed on a NIKON Eclipse Ti System operated via NIS-Elements Software AR version 3.22.09 for 64 bit (NIKON Instruments, Tokyo, Japan).

Cell and lung extracts were fractionated on 4–20% gradient SDS polyacrylamide gels (NuSep, Frenchs Forest, Australia), and proteins were subsequently transferred onto nitrocellulose membranes (Amersham Biosciences). Primary Abs (see “Reagents”) against OGG1, RAF-1, pRAF-1, ERK1/2, pERK1/2, MEK1/2, pMEK1/2, MSK1, pMSK1, IkB, pIkB, IKK, pIKK, RelA, and pRelA were used. Ras proteins were detected using panRAS Ab or subtype-specific HRAS, KRAS, and NRAS Abs. Binding of primary Abs was detected with HRP-conjugated secondary (goat antirabbit and donkey anti-mouse) Ab(s). The chemiluminescence signals were detected using the ECL Plus detection system (GE Life Sciences, Pittsburgh, PA). Band intensities were quantitated by densitometry using ImageJ software (version 1.44, National Institutes of Health). In selected studies, the percentage changes in protein levels were calculated using Microsoft Excel.

Luciferase assays Expression vectors were introduced using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Briefly, cells were seeded on plates for 24 h and incubated in growth medium without antibiotics overnight and then cotransfected with pGL4.32 luc2P/NF-kB-RE/ Hygro (Promega, Madison, WI) and pRL-TK Renilla Luciferase plasmid (Promega). After a 24-h transfection, cells were exposed to 8-oxoG (10 mM), and triplicate samples were lysed with 100 ml lysis buffer. Cellular debris was removed by centrifugation. Aliquots of the supernatants (20 ml) were added to the luciferase substrate solution and evaluated on a Glomax Luminometer (Promega). Firefly luciferase values were normalized against Renilla luciferase activity and expressed as relative light units.

Quantitation of chemokines and cytokines To determine the levels of proinflammatory mediators, we used the Bio-Plex Pro Mouse Cytokine 23-plex Assay (catalog number M60-009RDPD; BioRad Laboratories) (30). Briefly, BALF was centrifuged at 14,000 3 g (at 4˚C) to remove cell debris, and 25 ml of supernatant (diluted when needed) was mixed with magnetic beads covalently bound to capture Abs against cytokines, chemokines, or growth factors. After washing, a biotinylated detection Ab specific to an epitope different from that of the capture Ab was added to the reaction mixture. A streptavidin-PE reporter complex was then added to the biotinylated Abs on the bead surface. Plates were evaluated on a Bio-Plex system using a dual-laser, flow-based microplate reader system. Bio-Plex Manager software (version 4.0) was used to calculate the results.

qRT-PCR analysis RNA was extracted using an RNeasy kit per the manufacturer’s instructions (Qiagen, Valencia, CA). Total pooled (n = 5) RNA (1 mg) was reversetranscribed into cDNA using a SuperScript III First-Strand Synthesis System (Invitrogen) and analyzed using a Mouse Inflammatory Cytokines and Receptors PCR Array (catalog number PAMM-011A; SABiosciences, Valencia, CA). Briefly, cDNA in water was mixed with an equal amount of 23 SYBR Green Supermix (Qiagen), and 20 ml of reaction mixture was added to each well containing forward and reverse gene-specific primers; reactions were run for 40 cycles in an ABI7000 thermal cycler. Data were analyzed using RT2 profiler PCR data analysis template version 2.0. The results are reported as mean fold increases for all experimental groups, and data sets were deposited in the National Center for Biotechnology Information Gene Expression Omnibus accession number GSE60711 (http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60711). To confirm expression levels we used primer pairs purchased from Integrated DNA Technologies: Cxcl-1, forward, 59-GCGAAAAGAAGTGCAGAGAG-39, and reverse, 59-CACAAAATGTCCAAGGGAAG-39; Cxcl-2, forward, 59-CTCCTTTCCAGGTCAGTTAGC-39, and reverse, 59-

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Ablation of gene expression in cultured cells

Immunoblotting

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OGG1 DNA REPAIR/8-oxoG–INDUCED INNATE IMMUNE RESPONSE

CAGAAGTCATAGCCACTCTCAA-39; Hras, forward, 59-TGTCTCTGGCTGGAAGTAGG-39, and reverse, 59-TGCAGCAAACACAAGACAGT39; Kras, forward, 59-TTCTTCCCATCTTTGCTCATCT-39, and reverse, 59-ATTCCGTTCATTGAGACCTCAG-39; Nras, forward, 59-TCCCCATCACCTTGAAACTA-39, and reverse, 59-AGACCAAGTGCTGGTCTCAG-39; and Gapdh, forward, 59-GTGGAGTCATACTGGAACATGTAG-39, and reverse, 59-AATGGTGAAGGTCGGTGTG-39. Quantitation of changes in gene expression was calculated using the DDCt method and unstimulated cells as the calibrator and then normalized to GAPDH (25).

Statistical analysis Statistical analysis was performed using Student t test to analyze changes at the mRNA and protein levels. Data from mouse treatment groups were analyzed using ANOVA, followed by Bonferroni posthoc analyses for least significant difference. The data are presented as the means 6 the SEM. Differences were considered to be statistically significant at p , 0.05.

Results

Given our previously published data showing that when in complex with free 8-oxoG, OGG1 activates RAS GTPases (16) and the role

FIGURE 1. Experimental strategy and characterization of site-specific OGG1 depletion. (A) Experimental design. Stealth RNAi decreased both Ogg1 mRNA (B) and protein levels in airway epithelial cells (C), as shown by qRT-PCR and immunohistochemistry, respectively. Green fluorescence mediated by Alexa Fluor 488 shows subcellular localization of OGG1 (left panels). Nuclei of cells were stained with DAPI (blue, right panels). (D) Changes in genomic 8-oxoG levels in OGG1-expressing (control RNAi; blue columns) and OGG1-depleted (Stealth RNAi) airway epithelial cells (red columns) as shown by FLARE Comet assays (Materials and Methods). (E) Genomic 8-oxoG levels in the AE of OGG1expressing mice at time 0 (upper panel) and 1 h after oxidative challenge (lower panel) as shown by IHC (magnification 3216). ***p , 0.001. RAS, mammalian homolog of viral H-, K-, N-RAS protein; Trf-R, transfection reagent for in vivo use (Materials and Methods).

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Repair of oxidatively damaged DNA via OGG1-BER is a prerequisite to neutrophil accumulation in the airways

of RAS in inflammatory signaling (31, 32), we proposed that OGG1-BER in the AE is a regulator of an innate immune response. Our experimental strategy to test this hypothesis is shown in Fig. 1A. We generated mice that were OGG1-deficient in their airway epithelia (Fig. 1A, Materials and Methods), as shown by decreased Ogg1 mRNA levels (Fig. 1B) and the lack of OGG1 protein in the nuclei of airway epithelial cells by immunohistochemistry (IHC; Fig. 1C). OGG1-expressing mice were used as a control. Upon a mild oxidative burst (Materials and Methods), there was a multiple-fold increase in genomic 8-oxoG levels in the OGG1-deficient AE, while OGG1-expressing epithelia showed only a transient change (Fig. 1D), as determined by Flare Comet assays (12) and IHC (Fig. 1E). The multiple-fold differences in genomic 8-oxoG levels between OGG1-deficient and OGG1expressing mice imply the repair of 8-oxoG by OGG1-BER. Our results show that 8-oxoG repair by OGG1-BER resulted in inflammatory cell recruitment into the lungs from 4 h on, reaching a maximum at 16 h (Fig. 2A). Differential staining (Fig. 2B, middle panel) and IHC (Fig. 2C) showed that the recruited cells were neutrophils. By contrast, in the absence of OGG1-BER

The Journal of Immunology

To demonstrate the specificity of the 8-oxoG challenge–induced inflammatory response, we showed that in OGG1-expressing mice uric acid (0.0005 mg/kg), a deaminated form of 8-oxoG, a dangerassociated molecular pattern molecule (35), induced considerably fewer neutrophils than did 8-oxoG, and the uric acid–induced accumulation of inflammatory cells was not OGG1-dependent (Fig. 2G). Moreover, the 8-oxoG base was unique in inducing OGG1-dependent inflammation because 8-oxo-deoxyguanosine, 2,6-diamino-4-hydroxy-5-formo-midopyrimidine (substrates of OGG1 in the genome) (33), and the intact guanine base elicited little or no neutrophil recruitment (Fig. 2H). OGG1-BER and RAS activation are prerequisites for the expression of proinflammatory mediators Next, we determined whether the immune response in airways is coupled to OGG1-BER–associated activation of RAS-GTPases. OGG1-BER was initiated by a mild oxidative burst in the airways of OGG1-expressing mice. Lung extracts were then prepared and active RAS levels determined. GTP-bound RAS levels were increased from 30 min on (Fig. 3A). Use of subtype-specific Abs revealed that nearly all this RAS-GTP was KRAS (a homolog of the Kirsten sarcoma virus oncogene; Fig. 3A, upper and middle panels). Challenging OGG1-expressing airways with 8-oxoG caused rapid (from 15 min on) increases in KRAS-GTP levels

FIGURE 2. OGG1-initiated DNA base excision repair results in the recruitment of neutrophils. (A) Kinetics of neutrophil recruitment to the airways after OGG1-BER (empty columns) or challenge with 8-oxoG base (filled columns, 0.0005 mg/kg). Each time point represents the average number of neutrophils from five to six mice. (B) Representative images of differentially stained cells derived from the BALF of saline (left panel)-, OGG1-BER (middle panel)–, and 8-oxoG (right panel)–challenged airways. (C) Immunohistochemical identification of neutrophils based on neutrophil elastase localized to cell membrane (Materials and Methods). Magnification 3144. (D) OGG1 deficiency in airway epithelia decreases the accumulation of neutrophils. (E) The 8oxoG base challenge–induced recruitment of neutrophils is OGG1 dependent. (F) 8-oxoG base induces dose-dependent increases in the recruitment of neutrophils to airways. (G) OGG1-independent changes in neutrophil numbers upon uric acid (0.0005 mg/kg) challenge. (H) 8-oxoG, but not FapyG, 8oxodG, or guanine induces neutrophil recruitment. In (A), (D), (F), (G), and (H), BALF was obtained at 16 h postchallenge, total cell numbers were determined, and cytospin preparations made for differential staining with Wright–Giemsa to determine neutrophil counts. 8-oxoG, FapyG, 8oxodG, guanine base, or uric acid was introduced via the intranasal route in 60 ml saline. **p , 0.01, ***p , 0.001, ****p , 0.0001. Uric A, uric acid.

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(OGG1-deficient airways), significantly fewer (approximately eight times) inflammatory cells were found in the BALF (Fig. 2D; the 16-h time point is shown). This low level of inflammation occurred despite supraphysiological genomic 8-oxoG levels (Fig. 1D). Oxidative DNA base lesions were generated by identical doses of oxidative stress, so the data imply that the ratelimiting event in inflammatory responses is OGG1-BER and the resulting generation of free 8-oxoG base. To mimic OGG1-mediated generation of 8-oxoG (and avoid ROS-induced signaling), we challenged mice with 8-oxoG base, the specific product of OGG1-BER (33). Intriguingly, 8-oxoG base (at 0.00005 mg/kg, chosen based on preliminary studies) is a potent inducer of neutrophils (Fig. 2A, 2B, right panel) in OGG1-expressing airways. In OGG1-deficient airways, an identical dose of 8-oxoG induced a low level of neutrophils (∼10-fold less; Fig. 2E). We have also established a doseresponse relationship (Fig. 2F). The lowest dose that induced an immune response was 0.000005 mg/kg, which can be generated by OGG1-BER in vivo (34). In controls, depletion of NEIL1 had no effect, whereas the lack of NEIL2 somewhat increased neutrophil accumulation after 8-oxoG (0.00005 mg/kg) challenge to the airways (data not shown). The possible role of NEIL2 in immune responses is currently being investigated and will be the subject of a future article.

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(Fig. 3B; the upper panel shows the time course, and the lower panel shows the response of individual animals). In the absence of OGG1 expression, 8-oxoG challenge failed to activate KRAS (Fig. 3C, upper panel), whereas depletion of NEIL1 or NEIL2 had no effect (Fig. 3C, lower panel). FapyG [repaired via both OGG1and NEIL1-BER (36)], 8-oxodG, uric acid, and the intact guanine base failed to activate RAS (Fig. 3D). To pursue these observations, we examined the levels of H(homolog of the Harvey sarcoma virus oncogene), N- (neuroblastoma RAS viral oncogene homolog), and KRAS in lung extracts and the percentages of their GTP-bound forms. Upon quantifying the band intensities, we unexpectedly observed that 85 6 7% of RAS-GTP was KRAS (Fig. 3F, lower panel), which made up only ∼12 6 5% of total RAS (Fig. 3G, lower panel); NRAS represented 78 6 11% of total RAS (Fig. 3G, lower panel), but only 15 6 3% was GTP bound (Fig. 3F, lower panel). HRAS protein was not detectable in 20 mg lung extracts (Fig. 3G). In freshly isolated alveolar macrophages, there were no increases in RAS-GTP levels after 8-oxoG (or oxidative stress) challenge, although NRAS was abundant. HRAS and KRAS were undetectable in 20 mg macrophage extract (Fig. 3E).

Rapid KRAS activation after 8-oxoG challenge to the airways (Fig. 3B), and its dependence on OGG1, suggested that the AE is the site of GDP→GTP exchange. To support this hypothesis, we used primary cultured mouse bronchial epithelial cells (mPBECs) and hNBEC; KRAS-GTP levels were increased in response to 8-oxoG (10 mM) base in both (Fig. 3H). By contrast, NRAS was activated in primary mouse (primary mouse lung fibroblasts) and diploid human (MRC-5) lung fibroblasts after challenge with an identical dose of 8-oxoG (Fig. 3I). These results, along with those shown in Fig. 3A–D and 3F, strongly imply that KRAS is activated at the site of exposure, the AE. Next, we examined whether KRAS activation and downstream signaling result in the expression of proinflammatory chemokines/ cytokines. The airways were challenged for 0, 30, 60, 120, and 180 min, and the mRNA levels of chemokines/cytokines were examined using Mouse Inflammatory Cytokines and Receptors PCR Arrays. Intriguingly, OGG1-BER (Fig. 4A, left panel), or a challenge with 8-oxoG (Fig. 4B, left panel), induced similar expression profiles of proinflammatory mediators (e.g., Cxcl1, Tnfa, Ccl20, Ccl3, Il1a, and Il1b). The expression of these chemokines/ cytokines was greatly decreased in OGG1-deficient compared

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FIGURE 3. OGG1-BER and its product 8-oxoG induce activation of KRAS. (A) OGG1-BER–induced activation of KRAS in the lungs as a function of time. At indicated times, lung extracts were prepared, and changes in GTP-bound RAS were determined by active RAS pulldown assays. Subtype specific Ab to K-RAS and pan-RAS Ab was used to determine KRAS-GTP and total RAS levels, respectively (Materials and Methods). (B) KRAS activation after 8-oxoG challenge (upper panels, time course) and upon initiation of OGG1-BER (lower panels, 30 min time point is shown) in individual lungs. GTP-bound and total RAS levels were determined as described in (A). (C) Activation of KRAS is OGG1 (upper panel) but not NEIL1 or NEIL2 expression dependent (lower panel) in 8-oxoG–challenged airways. GTP-bound RAS levels were determined at 30 min postchallenge. (D) OGG1-BER and 8-oxoG challenge but not FapyG, guanine, uric acid, or 8-oxodG activates RAS (KRAS) in the lungs. The 30-min time point is shown. GTP-bound RAS was determined as described in (A). (E) Expression of NRAS but not KRAS or HRAS in resident alveolar macrophages. Extracts of macrophages from unchallenged lungs (n = 6) were tested by WB using subtype-specific Abs. (F) GTP-bound RAS protein levels in pooled lung extracts (n = 5) after 8-oxoG challenge, and graphical depiction of the percentages of K-, N-, and HRAS-GTP in total RAS-GTP. (G) Expression of RAS subtypes in lung. K-, N-, and HRAS levels (upper panels) are shown by subtype-specific Abs. Lower panel shows graphical depiction of their percentages. Total RAS level is shown by pan-RAS Ab. In (F) and (G), band intensities were quantified using Image J (version 1.44) software. Expression percentages were calculated using Microsfot Excel. (H) KRAS activation in mouse (mPBEC) and hNBECs upon 8-oxoG exposure. (I) Activation of N-RAS upon 8-oxoG exposure in mouse (mPLF) and human (MRC5) lung fibroblast cells. In (A)–(I), 250 mg extract was used for assessing RAS-GTP levels; 20 mg for total RAS. In (B, upper panel), (C), (D), (F), and (G), lungs were challenged with 60 ml saline containing 0.0005 mg/kg 8-oxoG. In (H) and (I), cells were challenged with 10 mM 8-oxoG solution in medium. In (A) and (B, lower panel), oxidative DNA damage was generated by challenging the airways with glucose oxidase (1 mU in 60 ml saline). mPBEC, mouse primary bronchial epithelial cells; mPLF, mouse primary fibroblasts; MRC5, human diploid lung fibroblast.

The Journal of Immunology with OGG1-expressing airways (the 2-h time point is shown, Fig. 4A, 4B, right panels). We expanded these observations by determining the kinetic changes in the mRNA levels in individual airways for Cxcl1 and Cxcl2, mediators of neutrophil recruitment (37). Cxcl1 and Cxcl2 mRNA levels were increased from 30 min on, peaking between 2 and 3 h (Fig. 4C). Later time points were not investigated, because neutrophils were recruited into the lungs from 4 h on (Fig. 2A). Importantly, the expression of Cxcl1 and 2 was significantly lower in OGG1- (Fig. 4C) and KRAS-deficient airways (Fig. 4D). IHC showed that CXCL1 is not detectable in AE cells of OGG1-deficient mice (Ogg1-RNAi; Fig. 4E), whereas it is highly expressed in OGG1-proficient airways (Cont-RNAi; right panel; Fig. 4E). CXCL1 was present at high levels from 2 h on in the BALF from 8-oxoG–exposed AE (Fig. 4F, left panel) and

7 in those airways repairing DNA via OGG1-BER after an oxidative burst (Fig. 4F, right panel). These data are consistent with OGG1driven activation of KRAS (Fig. 3A–C) and KRAS expression– dependent accumulation of neutrophils (Fig. 4G, 4H). In controls, NRAS depletion had no impact on OGG1-BER and 8-oxoG challenge–induced neutrophil levels (Fig. 4G, 4H). These results imply that OGG1-BER–dependent KRAS activation in the AE is a prerequisite for the expression of proinflammatory mediators and for inflammatory cell recruitment. OGG1-BER induces MAP-, PI3-, and MS-kinases for NF-kB activation To examine activation by posttranslational modifications of OGG1-BER-RAS’s downstream effectors, OGG1-expressing airways

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FIGURE 4. OGG1-BER-dependent expression of proinflammatory chemokines and cytokines. (A and B) Expression of proinflammatory mediators upon OGG1-initiated DNA base excision repair (A) and after 8-oxoG (B) challenge of OGG1-expressing (Cont-RNAi, left panel) or OGG1-deficient (Ogg1RNAi, right panel) lungs. RNAs were isolated at from five lungs and pooled, and the synthesized cDNAs were analyzed by Mouse Inflammatory Cytokines and Receptors PCR Array (2-h time point is shown). (C) Time course of OGG1-dependent expression of Cxcl1 (upper panel) and Cxcl2 (lower panel) mRNAs upon 8-oxoG challenge (n = 5-6; N, Cont-RNAi; n, Ogg1-RNAi). (D) KRAS-dependent expression of Cxcl1 and Cxcl2 after 8-oxoG challenge to the airways. KRas was depleted from the airways by RNAi. RNAs isolated after 8-oxoG challenge were analyzed by qRT-PCR (n = 5–6, 2-h time point is shown). (E) Expression of CXCL1 in the airway epithelia (white arrow) of OGG1-proficient (Cont-RNAi) and OGG1-deficient airways (Ogg1-RNAi), as shown by IHC. Representative lung sections (upper panels) are shown after a 2-h 8-oxoG challenge (DAPI staining of sections is shown on lower panel). (F) Levels of CXCL1 in the BALF of mice (n = 5) challenged with 8-oxoG (left panel) or in response to OGG1-BER (n = 6) as determined by BioPlex assays. (G) KRAS but not NRAS expression–dependent recruitment of neutrophils to airways. RAS was depleted by RNAi to Kras or Nras, and airways (n = 5–6) were challenged with 8-oxoG. Neutrophil numbers were determined 16 h postchallenge. (H) OGG1-BER–induced recruitment of neutrophils to airways is KRAS but not NRAS expression dependent. RAS were depleted in airways (n = 5–6) as in (G), and neutrophil numbers were determined 16 h postchallenge. (B, C, D, F, and G) Lungs were challenged with 60 ml saline containing 0.0005 mg/kg 8-oxoG. (A, F, and H) Oxidative DNA damage was generated by challenging the airways with glucose oxidase (Materials and Methods). ***p , 0.001, ****p , 0.0001. Ccl3, chemokine (C-C motif) ligand 3; Ccl20, C-C motif chemokine 20; Cxcl1, chemokine (C-X-C motif) ligand 1 transcript; Cxcl2, chemokine (C-X-C motif) ligand 2 transcript; Il1a, IL-1a; Il1b, IL-1b; Tnfa, TNF.

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were 8-oxoG–challenged (in these studies an oxidative burst was not used to exclude the role of ROS in the activation of redoxsensitive kinases), and lung extracts were prepared. WB analysis showed increased levels of p-RAF1 (v-raf1 murine leukemia viral oncogene homolog 1), p-MEK1/2, p-ERK1/2, and p-MSK1 (Fig. 5A). Moreover, we observed phosphorylation of another RAS target, phosphatidylinositol-4,5-bisphosphate 3-kinase (p-PI3K(p85), which is an activator of the serine/threonine protein kinase B (AKT; Fig. 5B). To demonstrate the relevance of activated KRAS (shown in Fig. 3A, 3B) and its targets (MAPKs and PI3K) to an immune response, we showed that treating mice with FTS [an inhibitor of RAS membrane interactions (38)], inhibitors of RAF1 (GW5074) and MEK1/2 (PD98059), or the pan-PI3K

inhibitor LY294002 (20-22) decreased the recruitment of neutrophils to the airways (Fig. 5C). Signaling by MAP and PI3 kinases have been linked to activation of the trans-acting factor NF-kB, which is rate-limiting for transcription from proinflammatory genes, including Cxcl1 and Cxcl2 (39, 40). Upon challenging OGG1-expressing airways with 8-oxoG, we observed a rapid increase in the levels of the p-IkB kinase complex (p-IKKa/b), p-IkB, and p-RelA (at Ser 276 ) (Fig. 5D). To validate these observations, we transfected MLE-12 cells and hNBECs with a luciferase reporter construct (luc2P) driven by NF-kB promoter elements. We found that 8-oxoG challenge of these cells increased luc2P activity in control siRNA-, but not Ogg1 siRNA-transfected mouse (MLE-12) and human

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FIGURE 5. OGG1-BER–dependent activation of MAPK, PI3K, and MSK and NF-kB. (A and B) 8-oxoG challenge to the airways increased the phosphorylated levels of RAS targets, MAPKs (RAF1, MEK1/2, ERK1/2, and MSK1) (A) and PI3K and AKT (B). In (A) and B), mice were challenged via the nasal route with 8-oxoG (0.0005 mg/kg), and the lungs were harvested after 0, 15, 30, and 60 min. Lung extracts were prepared from five animals per time points. Then, 40 mg protein extract was fractionated by 5–20% SDS PAGE, and proteins transferred to membranes were identified by phopsphospecific and pan Abs (Materials and Methods). (C) Pharmacological inhibitors of RAS, RAF-1, MEK1/2, and PI3K decreased the recruitment of neutrophils to the airways. BALF was derived at 16 h postchallenge (n = 6), and cell types were determined by differential staining of cells. (D) Phosphorylation of IKK, I-kB, and Rel/p65 in 8-oxoG–challenged airways. Mice were challenged, lung extracts were made, and changes in phosphorylated and total IKK, I-kB, and Rel/p65 were detected by immune blotting using phopspho-specific and pan Abs as in (A) and (B). (E) OGG1-dependent activation from an NF-kB-driven promoter. MLE-12 and hNBEC were transfected with control or siRNA to OGG1, and 36 h later a luc2P reporter driven by NF-kB response elements was introduced and the cells exposed to 8-oxoG (10 mM). Luc activities were assessed 6 h later. (F) Ogg1+/+ and Ogg12/2 murine fibroblasts were transfected with control or siRNA to OGG1, and 36 h later, a luc2P reporter driven by NF-kB response elements was introduced and the cells exposed to 8-oxoG (10 mM). Luc activities were assessed 6 h later. (G) Nuclear translocation of GFP-RelA in A549 cells after exposure to 8-oxoG (10 mM). (H) Ablation of OGG1 or (I) KRAS by siRNA from GFP-RelA–expressing A549 cells decreased the 8-oxoG–induced nuclear accumulation of GFP-RelA. Ablation of H- and NRAS, had no effect. (J) siRNA depletion of PI3K or MAPK from GFP-RelA–expressing cells decreased the nuclear accumulation of GFP-RelA. ***p , 0.01, ****p , 0.001. AKT, serine/threonine kinase (protein kinase B); GW5074, RAF-1 kinase inhibitor; PD98059, MAP kinase kinase (MEK1,2); LY294002, PI3K inhibitor; RAF1, v-raf1 murine leukemia viral oncogene homolog 1.

The Journal of Immunology

Discussion The mechanism by which DNA damage causes inflammation is complex; it often, if not in all documented cases, involves kinases activated by DNA single- or double-strand breaks, or DNA fragments that were recognized by pattern-recognition receptors inducing downstream inflammatory signaling (41, 42). Our data show for the first time, to our knowledge, that the release of the 8-oxoG base from the genome by OGG1-BER is a prerequisite for signaling increased expression of proinflammatory mediators and an innate immune response. Downstream of OGG1-BER, KRAS, PI3K, MAPK, and MSK activate the canonical NF-kB pathway, a central mediator of airway mucosal inflammation, by inducing RelA activation by its phosphorylation at Ser276 and nuclear translocation (Fig. 6). These two events are essential for the full activation of NF-kB–dependent inflammation; Ser276 phosphorylation is a posttranslational modification required for its interactions with histone acetyl transferases and transcriptional elongation complexes for activation of the proinflammatory innate subnetwork, including CXCLs (40). OGG1 is the key enzyme in removing 8-oxoG and FapyG from DNA with equal specificity and kinetics during BER to prevent mutations and maintain genomic integrity (33, 43). Traditionally,

FIGURE 6. Schematic depiction of the role of OGG1-initiated DNA base excision repair in an innate immune response.

accumulation of oxidatively damaged DNA base lesions, including 8-oxoG, has been linked to various inflammatory diseases, as well as aging processes (6, 7, 44). In this study, we document that OGG1-BER or challenging the airways with 8-oxoG base, a product of OGG1-BER, induced proinflammatory gene expression and inflammatory cell recruitment to airways. By contrast, a lack of OGG1 activity in airway epithelia resulted in poor inflammation. In light of earlier studies (12) these results are not entirely surprising, but the involvement of OGG1-BER-dependent signaling in NF-kB activation and proinflammatory gene expression is a novel observation. Oxidative DNA damage was generated by challenging airways with mild oxidative stress using glucose oxidase (Materials and Methods) that we have previously characterized (45). ROS generated in the AE both induce damage to molecules and signal transduction via phosphorylation (at tyrosine or serine residues) or cysteine modification of proteins (46). Moreover, small GTPases, including RAS, have redox-sensitive motifs adjacent to their guanine nucleotide-binding sites, so guanine nucleotide exchange can be activated by reactive species (47), and ROS signaling is also etiologically linked to inflammatory processes (48). OGG1expressing and OGG1-deficient lungs were exposed to identical doses of an oxidative burst, but only the OGG1-expressing ones showed activation of RAS, increased chemokine/cytokine expression and robust inflammation. Although these observations require further investigation, they imply that OGG1-BER appears to override the effect of ROS on RAS activation and downstream signaling. Another possibility is that OGG1 deficiency mediates a resistance to oxidative stress, as suggested previously (14, 15). To exclude this possibility, we determined GSSG levels after oxidative challenge using BALF of OGG1-expressing and deficient mice; there were no differences. An unexpected observation was that both OGG1-BER and 8-oxoG challenge selectively activated KRAS in lungs, although the level of NRAS was approximately six times higher in total lung extracts. These results imply that KRAS activation occurs at the site of exposure. To confirm, we used primary epithelial and fibroblast cells. In epithelial cells, the GTP-bound level of KRAS was increased, whereas in fibroblast cells, NRAS-GTP levels were increased upon 8-oxoG challenge, even though the abundance of KRAS and NRAS was similar in these cells (HRAS was undetectable). We speculate that OGG1, via its 8-oxoG base–dependent GEF activity, increased GTP-bound RAS levels in a cell typespecific manner whose mechanism is being investigated. We also considered the possibility that the proinflammatory response due to OGG1-BER induced single-strand gap/break signaling, as shown in a previous study (12, 41). Via its glycosylase activity, OGG1 generates abasic sites at which it cleaves the DNA backbone via its AP lyase activity, transiently generating single-strand gaps (33, 49, 50). When not repaired in a timely fashion, these could generate signaling for proinflammatory gene expression, as shown previously (12, 41). To exclude the role of these DNA repair intermediates, and also ROS-induced signaling we used OGG1-BER’s product, the 8-oxoG base. 8-OxoG challenge of the airways caused rapid KRAS GDP→GTP exchange, leading to proinflammatory gene expression, and recruitment of neutrophils. We propose that 8-oxoG penetrated the airway lining fluid, was internalized by AE, similarly to those occur in cultured cells (16). The internalized 8-oxoG then mediates the GEF activity of OGG1, which, in turn, activates KRAS in a cell type-dependent manner. Indeed, the innate immune response occurred only in OGG1-expressing airways. Moreover, 8-oxoG was unique in activating RAS in the airways and inducing an immune response, as the FapyG base, like

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(hNBEC) cells (Fig. 5E). Consistent with these data, the luc2P reporter activity was increased in 8-oxoG-challenged Ogg1+/+, but not Ogg1‒/‒ fibroblasts (Fig. 5F). To gain further insights into the role of OGG1-BER-driven MAPK and PI3K pathways in NF-kB/RelA activation, we used human AE cells (A549) stably expressing a RelA-fused GFP (GFP-RelA) (40) localized to the cytoplasm in nonstimulated cells (Fig. 5G, lower panels). In response to 8-oxoG, GFP-RelA was imported into the cell nuclei (Fig. 5G, lower panels). siRNA depletion of OGG1 (Fig. 5H) or KRAS (but not NRAS or HRAS) decreased the percentage of GFP-RelA–positive nuclei (Fig. 5I). Depleting PI3K, MEK1, MEK2, or MSK1 with siRNAs significantly decreased NF-kB/RelA’s nuclear accumulation (Fig. 5J). Taken together, these results show that OGG1-BER–KRAS induces the canonical NF-kB activation pathway for proinflammatory chemokine/cytokine expression and the innate immune response (Fig. 6).

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OGG1 DNA REPAIR/8-oxoG–INDUCED INNATE IMMUNE RESPONSE agreement with previous studies (56–58). Importantly, the evolutionarily conserved 8-oxoguanine DNA glycosylase-1 is not only a key repair enzyme charged with maintaining genomic integrity and preventing mutations, but it also plays an essential role in the host’s innate immune response. Genomic DNA is continuously challenged by ROS, and its integrity should be continually maintained by DNA repair. It is thus plausible to speculate that OGG1-initiated DNA BER is fueling the chronic inflammation that is a major underlying process associated with asthma and COPD. Moreover OGG1-BER could also be etiologically linked to other inflammatory diseases such as atherosclerosis, arthritis, cancer, osteoporosis, dementia, vascular diseases, obesity, the metabolic syndrome, and diabetes. Taken together, our results point to a novel discovery—the role of OGG1-dependent repair of damaged DNA in proinflammatory signaling, which may be one among many mechanistic explanations for OGG1’s previously proposed link to disease and aging processes. These data also imply that modulating OGG1’s guanine nucleotide exchange factor activity and/or cellular 8-oxoG base levels could benefit human health.

Acknowledgments We greatly appreciate the critical editing of the manuscript by Dr. David Konkel (Institute for Translational Sciences, University of Texas Medical Branch) and Mardelle Susman (Department of Microbiology and Immunology, University of Texas Medical Branch). We also thank Drs. Miral Dizdaroglu and Pawel Jaruga (Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD) for providing FapyG.

Disclosures The authors have no financial conflicts of interest.

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other nucleotides and nucleosides, failed to do so. Uric acid, a deaminated form of 8-oxoG and a major alarmin inflammatory molecule (35, 51), induced low levels of inflammation independent of OGG1. 8-oxoG exposure and OGG1-BER (induced by an oxidative burst) increased chemokine and cytokine expression in an identical pattern. The only differences we observed were the precise fold changes. Expression of these proinflammatory mediators was dependent on OGG1 in both 8-oxoG and oxidatively challenged airways. These results are consistent with OGG1-dependent activation of KRAS GTPases after oxidative burst, raising the possibility that ROS-generated signals are transmitted via KRAS. In this scenario, 8-oxoG is first formed in DNA because of guanine’s susceptibility to oxidation. Because it is a highly mutagenic 8-oxoG is promptly repaired via OGG1-BER. The released 8-oxoG base forms OGG1•8-oxoG complex [a GEF (16)] activate KRAS and downstream signaling. In addition to the epithelium, oxidative (and 8-oxoG) challenge impacts other cell types including macrophages in the airways. To address the role of macrophages in an inflammatory response, we showed that freshly isolated alveolar macrophages express NRAS, whereas expression of KRAS and HRAS was not at detectable levels. Interestingly, there were no increases in NRAS-GTP levels after oxidative stress (or 8-oxoG) challenge and downregulation of NRAS (expressed in macrophages and epithelium) had no effect on the inflammatory response. Taken together, these data suggest that proinflammatory cytokines and chemokines are produced primarily by airway epithelial cells, resulting in neutrophil recruitment to the airways. 8-oxoG challenge of OGG1-expressing airways resulted in both MAP (RAF1, MEK1,2, ERK1,2 and MSK-1) and PI3 kinase activation, leading to activation of IkB kinase (IKK). Activated IKK phosphorylates IkB, which then becomes a target for ubiquitination and proteasome-mediated degradation, leading to liberation of NF-kB homo- and heterodimers, including RelA-p65 (52). In addition to its release, RelA-p65 is subject to phosphorylation at multiple sites, including serine residues 210, 276, 529, 536, and others (39). However, among the serine phosphorylation, that at aa 276 appears to be one of the most important, which is mediated by the catalytic subunit of protein kinase A and MSK1 (39, 53–55). These events can be explained by the results from previous studies showing that OGG1 binds the 8-oxoG base at a substrate-independent site, and that 8-oxoG–induced conformational changes in the OGG1 molecule have allowed its physical interaction with and activation of RAS GTPases (16, 17), whose targets are RAF/MAPK and IP3Ks. To further verify the significance of OGG1-KRAS/MAPK/PI3K signaling for nuclear translocation of RelA-p65, a critical step in gene activation, we show that depletion of OGG1, KRAS, MEK1,2, MSK1, or PI3Ks by siRNA decreased RelA-p65 nuclear accumulation. Activation of NF-kB via OGG1-BER–KRAS signaling was further confirmed by reporter assays. Moreover, to support OGG1-KRAS/MAPK/PI3K signaling in the immune response, we showed that inhibitors of RAS membrane anchorage (FTS), as well as RAF1, MEK1/2 or a pan-PI3K inhibitor, decreased neutrophilia in the lungs. Combining the results from cultured cells and those from the lungs strongly support the relevance of OGG1BER signaling to an immune response via KRAS, MAPKs, PI3K, MSK1 and the NF-kB pathway. In conclusion, in this paper, we document a novel mechanism by which the repair of oxidatively damaged DNA via OGG1-inititated DNA BER and KRAS signaling lead to activation of NF-kB (and possibly other transacting factors) in the AE to mount an immune response in the lungs. These data also show that epithelial cells are the primary cell type in orchestrating an inflammatory response, in

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