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. 2013 Jan 1;12(1):18-26.
doi: 10.1016/j.dnarep.2012.10.002. Epub 2012 Nov 3.

Down-regulation of 8-oxoguanine DNA glycosylase 1 expression in the airway epithelium ameliorates allergic lung inflammation

Attila Bacsi  1 , Leopoldo Aguilera-Aguirre, Bartosz Szczesny, Zsolt Radak, Tapas K Hazra, Sanjiv Sur, Xueqing Ba, Istvan Boldogh
Affiliations

Down-regulation of 8-oxoguanine DNA glycosylase 1 expression in the airway epithelium ameliorates allergic lung inflammation

Attila Bacsi et al. DNA Repair (Amst). .

Abstract

Allergic airway inflammation is characterized by increased expression of pro-inflammatory mediators, inflammatory cell infiltration, mucus hypersecretion, and airway hyperresponsiveness, in parallel with oxidative DNA base and strand damage, whose etiological role is not understood. Our goal was to establish the role of 8-oxoguanine (8-oxoG), a common oxidatively damaged base, and its repair by 8-oxoguanine DNA glycosylase 1 (Ogg1) in allergic airway inflammatory processes. Airway inflammation was induced by intranasally administered ragweed (Ambrosia artemisiifolia) pollen grain extract (RWPE) in sensitized BALB/c mice. We utilized siRNA technology to deplete Ogg1 from airway epithelium; 8-oxoG and DNA strand break levels were quantified by Comet assays. Inflammatory cell infiltration and epithelial methaplasia were determined histologically, mucus and cytokines levels biochemically and enhanced pause was used as the main index of airway hyperresponsiveness. Decreased Ogg1 expression and thereby 8-oxoG repair in the airway epithelium conveyed a lower inflammatory response after RWPE challenge of sensitized mice, as determined by expression of Th2 cytokines, eosinophilia, epithelial methaplasia, and airway hyperresponsiveness. In contrast, 8-oxoG repair in Ogg1-proficient airway epithelium was coupled to an increase in DNA single-strand break (SSB) levels and exacerbation of allergen challenge-dependent inflammation. Decreased expression of the Nei-like glycosylases Neil1 and Neil2 that preferentially excise ring-opened purines and 5-hydroxyuracil, respectively, did not alter the above parameters of allergic immune responses to RWPE. These results show that DNA SSBs formed during Ogg1-mediated repair of 8-oxoG augment antigen-driven allergic immune responses. A transient modulation of OGG1 expression/activity in airway epithelial cells could have clinical benefits.

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Conflict of interest statement

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1. Changes in 8-oxoG levels in the genomic DNA of airway epithelial cells after RWPE challenge
Sensitized Ogg1Pand O gg1Dmice (proficient and deficient in O gg1 expression in the airway epithelium, respectively) were challenged with RWPE, and the levels of 8 -oxoG in exfoliated airway epithelial cells were examined at various time points thereafter. (A) Representative images of comet moments with and without digestion of DNA with recombinant hOGG1(n = 200). (B) Quantitation of 8-oxoG levels in the DNA of airway epithelial cells by comet assay (n = 200 comets for each time point) from Ogg1P(n = 7) and O gg1Dmice (n = 6); (C) Ogg1 mRNA levels in airway epithelial cells 48 h after instillation of control or Ogg1-specific Stealth siRNA into the lungs (n = 5–11). (D) GSH:GSSG ratios ± SEM in bronchoalveolar lavage fluid (BALF, n = 7–9). Ogg1P, mice proficient in Ogg1 expression in the airway epithelium, Ogg1D, mice deficient in Ogg1 expression in the airway epithelium; hOGG1: recombinant human OGG1 protein; RWPE, ragweed pollen grain extract. **P< 0.01, *** P< 0.001.
Figure 2
Figure 2. DNA strand damage in the airway epithelium after RWPE challenge of sensitized mice
Ogg1P and Ogg1Dmice were RWPE -challenged, and exfoliated AECs were subjected to Comet assays. (A) Kinetics of changes in SSB levels in the airway epithelium from Ogg1Pand O gg1D mice after RWPE challenge. For each time points and treatments, >200 comet moments were evaluated. (B) Representative comet moments under neutral (left images) and alkaline (right images) electrophoretic conditions, representing DNA double-strand breaks and single-strand breaks, respectively, 16 h after RWPE challenge. CA, comet assay; Ogg1P, mice proficient in Ogg1 expression in the airway epithelium; O gg1D, mice deficient in Ogg1 expression in the airway epithelium; RWPE, ragweed pollen grain extract; SSBs, single strand breaks;. *P< 0.05, ** P< 0.01, *** P< 0.001.
Figure 3
Figure 3. The levels of SSBs and intracellular ROS in cultured cells proficient or deficient in Ogg1 expression after RWPE challenge or co-culture with neutrophils
(A) SSB levels in cultured cells. MLE-12 cells and mouse embryonic fibroblast (MEF) cells were Ogg1-depleted using siRNA, and co-incubated with activated neutrophils. In parallel experiments Ogg1−/− MEF cells were co-cultured with activated neutrophils. SSB levels were determined by alkaline Comet assays. For each time points and treatments, >200 comet moments were evaluated. **P < 0.01, ***P < 0.001. (B) RWPE challenge did not induce a detectable increase in SSB levels. MLE-12 and Ogg1+/+MEF cells transfected with control siRNA or O gg1 siRNA, as well as Ogg1−/− MEF cells were treated with RWPE for 2 h. Levels of SSBs were determined by alkaline Comet assays. In controls, MLE-12 cells were co-incubated with activated neutrophils for 2 h. For each treatment, >200 comet moments were evaluated. (C) Changes in ROS levels in MLE-12 cells after addition of RWPE or activated neutrophils. ROS levels in MLE -12 cells were determined using 2′-7′-dihydro-dichlorofluorescein diacetate (n = 3–4) as described in Materials and Methods. (D) Ogg1 depletion had no effect on the neutrophil-induced increase in cellular ROS levels. Ogg1 was depleted via siRNA as in (A). Ogg1−/− MEF cells were used as a control (n = 2–4). (E) Ogg1 protein (upper panel) and mRNA (lower panel) levels in control siRNA and Ogg1 siRNA-transfected MLE-12 and Ogg1+/+MEF cells. Right panel: 8 -oxoG excision by cell extracts from control and Ogg1 siRNA-transfected cells. MEF, mouse embryonic fibroblast; RWPE, ragweed pollen grain extract; SSBs, single strand breaks. **P< 0.01, *** P< 0.001.
Figure 4
Figure 4. Depletion of Ogg1 in the airway epithelium decreases allergen-driven inflammation and the expression of Th2 cytokines in the lungs of sensitized mice after RWPE challenge
Sensitized Ogg1P and Ogg1Dmice were challenged with saline or RWPE, and BALF and the lungs were then analyzed. (A) The number of eosinophils infiltrated into the BALF (n=9). (B) Inflammatory cell infiltration into the peribronchial area (n= 7–9 per group). Magnification: x96. Scale bars represent 100 μm. (C) MUC5A/C levels in BALF as determined by ELISA (n = 6–9 per group). (D), Airway epithelial hyperplasia (PAS staining; magnification: x96). Representative images are shown out of 6–9 mice per group. (E) Airway hyperresponsiveness was assessed at 60 h post-challenge (n = 6–9 per group). *P< 0.05, **P < 0.01.(F) Changes in levels of IL-4 (upper panel), IL-5 (middle panel) and IL-13 (lower panel) in BALF as a function of time after RPWE challenge (n = 6–8 per group). (G) Depletion of Neil1 and Neil2 DNA glycosylases had no effect on the eosinophil numbers in BALF (n = 5–7 per group). Neil1 and Neil2 were depleted as described in Methods. Neil1P: mice proficient in Neil1 expression in the airway epithelium;Neil1D: mice deficient in Neil1 expression in the airway epithelium; Neil2P: mice proficient in Neil2 expression in the airway epithelium; Neil2D: mice deficient in Neil2 expression in the airway epithelium; Ogg1P, mice proficient in Ogg1 expression in the airway epithelium; Ogg1D, mice deficient in Ogg1 expression in the airway epithelium; RWPE, ragweed pollen grain extract; BALF, bronchoalveolar lavage fluid. *P< 0.05, **P< 0.01, *** P< 0.001.
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References

    1. Dharajiya N, Boldogh I, Cardenas V, Sur S. Role of pollen NAD(P)H oxidase in allergic inflammation. Curr Opin Allergy Clin Immunol. 2008;8:57–62. - PMC - PubMed
    1. Vrtala S, Grote M, Duchene M, van Ree R, Kraft D, Scheiner O, Valenta R. Properties of tree and grass pollen allergens: reinvestigation of the linkage between solubility and allergenicity. Int Arch Allergy Immunol. 1993;102:160–169. - PubMed
    1. D’Amato G, Liccardi G, D’Amato M, Cazzola M. Outdoor air pollution, climatic changes and allergic bronchial asthma. Eur Respir J. 2002;20:763–776. - PubMed
    1. Annesi-Maesano I, Rouve S, Desqueyroux H, Jankovski R, Klossek JM, Thibaudon M, Demoly P, Didier A. Grass Pollen Counts, Air Pollution Levels and Allergic Rhinitis Severity. Int Arch Allergy Immunol. 158:397–404. - PubMed
    1. Boldogh I, Bacsi A, Choudhury BK, Dharajiya N, Alam R, Hazra TK, Mitra S, Goldblum RM, Sur S. ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation. J Clin Invest. 2005;115:2169–2179. - PMC - PubMed

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