Pollen extracts induce intracellular and mitochondrial reactive oxygen species (ROS) generation in epithelial cells.1Boldogh I. Bacsi A. Choudhury B.K. Dharajiya N. Alam R. Hazra T.K. et al.ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation.J Clin Invest. 2005; 115: 2169-2179Crossref PubMed Scopus (224) Google Scholar, 2Aguilera-Aguirre L. Bacsi A. Saavedra-Molina A. Kurosky A. Sur S. Boldogh I. Mitochondrial dysfunction increases allergic airway inflammation.J Immunol. 2009; 183: 5379-5387Crossref PubMed Scopus (192) Google Scholar Ragweed pollen extract (RWPE) challenge induces 8-oxoguanine DNA glycosylase–mediated DNA-base excitation repair, and suppression of 8-oxoguanine DNA glycosylase inhibits RWPE-induced allergic airway inflammation and TH2 cytokine production.3Bacsi A. Aguilera-Aguirre L. Szczesny B. Radak Z. Hazra T.K. Sur S. et al.Down-regulation of 8-oxoguanine DNA glycosylase 1 expression in the airway epithelium ameliorates allergic lung inflammation.DNA Repair (Amst). 2013; 12: 18-26Crossref PubMed Scopus (63) Google Scholar, 4Li G. Yuan K. Yan C. Fox 3rd, J. Gaid M. Breitwieser W. et al.8-Oxoguanine-DNA glycosylase 1 deficiency modifies allergic airway inflammation by regulating STAT6 and IL-4 in cells and in mice.Free Radic Biol Med. 2012; 52: 392-401Crossref PubMed Scopus (71) Google Scholar We reported that pollens stimulate a Toll-like receptor 4 (TLR4)–dependent innate neutrophil recruitment that in turn stimulates allergic airway inflammation.5Hosoki K. Aguilera-Aguirre L. Brasier A.R. Kurosky A. Boldogh I. Sur S. Facilitation of allergic sensitization and allergic airway inflammation by pollen-induced innate neutrophil recruitment.Am J Respir Cell Mol Biol. 2016; 54: 81-90Crossref PubMed Scopus (7) Google Scholar We identified myeloid differentiation protein 2 (MD2) as a critical TLR4 coreceptor used by pollen and cat dander extract (CDE) to stimulate innate neutrophil recruitment and allergic airway inflammation.6Hosoki K. Boldogh I. Aguilera-Aguirre L. Sun Q. Itazawa T. Hazra T. et al.Myeloid differentiation protein 2 facilitates pollen- and cat dander-induced innate and allergic airway inflammation.J Allergy Clin Immunol. 2016; 137: 1506-1513.e2Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar Like RWPE and CDE, house dust mite signals through TLR4/MD2 agonist, and administration of a functional TLR4 antagonist reduces house dust mite–induced airway lymphocytosis and eosinophilia.7Hammad H. Chieppa M. Perros F. Willart M.A. Germain R.N. Lambrecht B.N. House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells.Nat Med. 2009; 15: 410-416Crossref PubMed Scopus (869) Google Scholar These data indicate a broad-based role of TLR4 in allergen-induced innate and allergic inflammation. However, the role of TLR4 in allergen-induced ROS generation and oxidative DNA damage has not been reported. To test this hypothesis, we first investigated whether allergenic extracts belonging to diverse families generate TLR4-dependent intracellular ROS. Compared with HEK 293 cells that do not express TLR4 (TLR4Null cells), RWPE rapidly induced greater intracellular ROS in cells that overexpress TLR4 (TLR4Hi cells; Fig 1, A). Likewise, firebush, pigweed, timothy, walnut, cottonwood, mountain cedar, cat dander, and cockroach extracts all induced significantly higher intracellular ROS generation in TLR4Hi cells (Fig 1, A). By contrast, Russian thistle, rye, BSA, or LPS at 1 μg/mL (Fig 1, A and B) did not induce ROS generation in TLR4Hi cells, demonstrating specificity of TLR4-dependent ROS generation by certain allergenic extracts. Because the ROS generation observed in Fig 1, A, was elucidated by using cell lines labeled with a redox-sensitive dye, we sought to validate these results by quantifying another marker of oxidative stress: generation of glutathione disulfide (oxidized glutathione [GSSG]). Both RWPE and CDE induced a greater increase in the concentration of GSSG in cell supernatants of TLR4Hi cells than TLR4Null cells within 30 minutes (Fig 1, C). These data indicate that diverse allergenic extracts stimulate TLR4-dependent and independent ROS and GSSG generation. Because allergenic extracts generate reduced levels of ROS in TLR4Null cells (Fig 1, A and C), it is likely that innate receptors other than TLR4 also contribute to allergen-induced innate ROS generation. Recently, we reported that diverse allergenic extracts, such as RWPE, Bermuda, rye, timothy, pigweed, Russian thistle, eastern cottonwood, black walnut, and CDE, but not firebush and mountain cedar, induced TLR4- and MD2-dependent CXCL secretion from cultured human bronchial epithelial cells.6Hosoki K. Boldogh I. Aguilera-Aguirre L. Sun Q. Itazawa T. Hazra T. et al.Myeloid differentiation protein 2 facilitates pollen- and cat dander-induced innate and allergic airway inflammation.J Allergy Clin Immunol. 2016; 137: 1506-1513.e2Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar Taken together with the data in the present study, the TLR4-mediated ROS-generating ability and CXCL chemokine–inducing ability of different allergens are not always concordant and might reflect differential innate mechanisms of these distinct biological events. To determine the in vivo relevance of these observations, we performed RWPE/CDE nasal challenge in naive mice. A RWPE (Fig 2, A) or CDE (Fig 2, B) single challenge in wild-type (WT) but not Tlr4 knockout mice increased cell-free BALF concentrations of GSSG 30 minutes after challenge. Administration of antioxidants in WT mice before RWPE (Fig 2, A) or CDE (Fig 2, B) challenge inhibited this increase in GSSG levels, confirming that TLR4 contributes to allergen-induced oxidative stress in vivo. Because RWPE stimulated TLR4-dependent GSSG generation, we reasoned that RWPE should also reduce levels of intracellular glutathione, an intracellular antioxidant, in a TLR4-dependent manner. Indeed, RWPE induced a greater reduction of intracellular glutathione levels in TLR4Hi cells than in TLR4Null cells (Fig 2, C), confirming that diverse allergenic extracts induce TLR4-dependent oxidative stress. Previously, we reported that RWPE induces mitochondrial damage and allergic inflammation.2Aguilera-Aguirre L. Bacsi A. Saavedra-Molina A. Kurosky A. Sur S. Boldogh I. Mitochondrial dysfunction increases allergic airway inflammation.J Immunol. 2009; 183: 5379-5387Crossref PubMed Scopus (192) Google Scholar We next assessed the role of TLR4 in RWPE-induced mitochondrial ROS generation. RWPE stimulated mitochondrial ROS generation in TLR4Hi but not TLR4Null cells (Fig 2, D), indicating RWPE induces TLR4-dependent mitochondrial ROS generation. Because the TLR4 ligand LPS stimulates TNF receptor–associated factor 6 (TRAF6)–mediated mitochondrial ROS generation8West A.P. Brodsky I.E. Rahner C. Woo D.K. Erdjument-Bromage H. Tempst P. et al.TLR signalling augments macrophage bactericidal activity through mitochondrial ROS.Nature. 2011; 472: 476-480Crossref PubMed Scopus (421) Google Scholar and induces rapid protein-protein interaction between TLR4 and TRAF6,9Tseng P.H. Matsuzawa A. Zhang W. Mino T. Vignali D.A. Karin M. Different modes of ubiquitination of the adaptor TRAF3 selectively activate the expression of type I interferons and proinflammatory cytokines.Nat Immunol. 2010; 11: 70-75Crossref PubMed Scopus (313) Google Scholar we hypothesized that RWPE might stimulate interaction of TRAF6 with TLR4. Consistent with our hypothesis, stimulation of TLR4Hi cells with RWPE induced rapid association of TRAF6 with TLR4 within 10 minutes (see Fig E1 in this article's Online Repository at www.jacionline.org). Next, we hypothesized that RWPE can induce TLR4-dependent oxidative DNA damage. Consistent with our hypothesis, RWPE induced a greater increase in comet tail moments (a measure of oxidative DNA damage within cells visualized as a comet tail) in TLR4Hi than in TLR4Null cells (Fig 2, E) and greater DNA damage lesions in lung genomic DNA of WT mice than Tlr4 knockout mice within 30 minutes (Fig 2, F) of challenge, validating a critical role of TLR4 in allergen-induced oxidative DNA damage. We next determined whether challenge with physiologically relevant doses of RWPE that induce allergic symptoms in human subjects with allergic rhinitis and a positive skin prick test response to RWPE (see Table E1 in this article's Online Repository at www.jacionline.org) are associated with an increase in oxidative DNA damage. Compared with saline challenge on a previous day, RWPE challenge rapidly increased nasal symptom scores (see Fig E2, A, in this article's Online Repository at www.jacionline.org). This symptom-eliciting dose of RWPE challenge was associated with intranasal levels of 8-hydroxydeoxyguanosine (8-OHdG), a well-defined marker of oxidative DNA damage, at 30 minutes (see Fig E2, B), suggesting that exposure of human subjects to a concentration of ragweed pollen that increases nasal symptoms can also induce local oxidative DNA damage in the nasal airways. Because RWPE induces oxidative stress in the airways of mice independent of mast cells, B cells, and T cells,1Boldogh I. Bacsi A. Choudhury B.K. Dharajiya N. Alam R. Hazra T.K. et al.ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation.J Clin Invest. 2005; 115: 2169-2179Crossref PubMed Scopus (224) Google Scholar our human study suggests an innate mechanism of RWPE-induced oxidative DNA damage. Future human studies involving challenge with a non–cross-reactive allergenic extract (non-IgE mediated) in allergic subjects might provide additional evidence for our hypothesis. Our observations indicate that allergenic extract–induced ROS generation, and oxidative DNA damage is not limited to a stochastic oxidative insult induced by allergen–epithelial cell interaction1Boldogh I. Bacsi A. Choudhury B.K. Dharajiya N. Alam R. Hazra T.K. et al.ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation.J Clin Invest. 2005; 115: 2169-2179Crossref PubMed Scopus (224) Google Scholar but a process that is initiated by innate immune receptors, such as TLR4. It would thus be useful to examine the efficacy of such TLR4 antagonists as the anti-TLR4 mAb NI-0101,10Hennessy E.J. Parker A.E. O'Neill L.A. Targeting Toll-like receptors: emerging therapeutics?.Nat Rev Drug Discov. 2010; 9: 293-307Crossref PubMed Scopus (637) Google Scholar the cell-permeable small-molecule inhibitor of TLR4 signaling TAK-24210Hennessy E.J. Parker A.E. O'Neill L.A. Targeting Toll-like receptors: emerging therapeutics?.Nat Rev Drug Discov. 2010; 9: 293-307Crossref PubMed Scopus (637) Google Scholar and the MD2-TLR4 antagonist Eritoran10Hennessy E.J. Parker A.E. O'Neill L.A. Targeting Toll-like receptors: emerging therapeutics?.Nat Rev Drug Discov. 2010; 9: 293-307Crossref PubMed Scopus (637) Google Scholar in suppressing pollen- and cat dander-induced oxidative stress and DNA damage and attenuating innate and allergic airway inflammation. We thank Dr Robert Esch, PhD, Greer Laboratory, Lenoir, NC, for his scientific input on endotoxin levels in allergenic extracts used in this study. We thank Prof. David Konkel for proofreading this manuscript and providing a critique that substantially improved the quality of the manuscript. Eight- to 12-week-old male WT C57BL/10SNJ mice and Tlr4 knockout mice (C57BL/10ScNJ) were purchased from the Jackson Laboratory (Bar Harbor, Me) and used for these studies. All mice were kept in a pathogen-free environment throughout the experiments at the University of Texas Medical Branch (Galveston, Tex). Animal experiments were performed according to the National Institutes of Health's "Guide for care and use of experimental animals" and were approved by the University of Texas Medical Branch Animal Care and Use Committee (approval no. 9708038A). Lyophilized RWPE (lot no. 169118), firebush (lot no. 23242), pigweed (lot no. 144530), timothy grass (lot no. 210887), rye grass (lot no. 147138), black walnut (lot no. 148412), eastern cottonwood (lot no. 24168), mountain cedar (lot no. 228278), Russian thistle (lot no. 134436), American cockroach (lot no. 172660), and cat dander (lot no. 169124) were purchased from Greer Laboratories (Lenoir, NC). Because LPS and allergens signal through the same TLR4 receptor,E1Hosoki K. Aguilera-Aguirre L. Brasier A.R. Kurosky A. Boldogh I. Sur S. Facilitation of allergic sensitization and allergic airway inflammation by pollen-induced innate neutrophil recruitment.Am J Respir Cell Mol Biol. 2016; 54: 81-90Crossref PubMed Scopus (29) Google Scholar, E2Hosoki K. Boldogh I. Aguilera-Aguirre L. Sun Q. Itazawa T. Hazra T. et al.Myeloid differentiation protein 2 facilitates pollen- and cat dander-induced innate and allergic airway inflammation.J Allergy Clin Immunol. 2016; 137: 1506-1513.e2Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar we examined endotoxin contamination in allergenic extract by using the LAL chromogenic endotoxin quantitation kit (Thermo Scientific, Hudson, NH). All allergenic extracts used in the present study contained endotoxin levels of less than 0.1 ng/1 mg protein of allergen, a level that we have reported cannot account for the TLR4-dependent biological effects in our earlier studies.E1Hosoki K. Aguilera-Aguirre L. Brasier A.R. Kurosky A. Boldogh I. Sur S. Facilitation of allergic sensitization and allergic airway inflammation by pollen-induced innate neutrophil recruitment.Am J Respir Cell Mol Biol. 2016; 54: 81-90Crossref PubMed Scopus (29) Google Scholar, E2Hosoki K. Boldogh I. Aguilera-Aguirre L. Sun Q. Itazawa T. Hazra T. et al.Myeloid differentiation protein 2 facilitates pollen- and cat dander-induced innate and allergic airway inflammation.J Allergy Clin Immunol. 2016; 137: 1506-1513.e2Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Mice were sedated with low-dose intraperitoneal xylazine/ketamine anesthetic mixture for intranasal challenge and killed by means of lethal intraperitoneal xylazine/ketamine overdose.E3Boldogh I. Bacsi A. Choudhury B.K. Dharajiya N. Alam R. Hazra T.K. et al.ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation.J Clin Invest. 2005; 115: 2169-2179Crossref PubMed Scopus (304) Google Scholar WT and Tlr4 knockout mice were challenged intranasally with a single dose of RWPE or CDE (100 μg/60 μL) and killed after 0.5 hours. In some experiments WT or Tlr4 knockout mice were treated with a single intraperitoneal injection of freshly made antioxidants (N-acetyl cysteine; Sigma-Aldrich, St Louis, Mo; 320 mg/kg; ascorbic acid, 400 mg/kg adjusted to pH 7.4 with 12.5 mol/L sodium hydroxide) or PBS control 1 hour before RWPE challenge.E4Dharajiya N. Choudhury B.K. Bacsi A. Boldogh I. Alam R. Sur S. Inhibiting pollen reduced nicotinamide adenine dinucleotide phosphate oxidase-induced signal by intrapulmonary administration of antioxidants blocks allergic airway inflammation.J Allergy Clin Immunol. 2007; 119: 646-653Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar BALF for biochemical examinations was taken, as previously described.E5Sur S. Lam J. Bouchard P. Sigounas A. Holbert D. Metzger W.J. Immunomodulatory effects of IL-12 on allergic lung inflammation depend on timing of doses.J Immunol. 1996; 157: 4173-4180Crossref PubMed Google Scholar Briefly, to collect BALF, after a lethal dose of anesthesia, tracheas of mice were cannulated, and lungs were lavaged with two 0.7-mL aliquots of ice-cold Dulbecco PBS (Corning, Manassas, Va). Lungs were dissected out, and genomic DNA was extracted for DNA damage quantification. Briefly, butylated hydroxytoluene (0.5 mmol/L; Sigma-Aldrich) was added to prevent further lipid oxidation in some aliquots. GSSG levels in BALF were measured spectrophotometrically by using a glutathione/GSSG-412 kit (Bioxytech; OXIS, Portland, Ore).E3Boldogh I. Bacsi A. Choudhury B.K. Dharajiya N. Alam R. Hazra T.K. et al.ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation.J Clin Invest. 2005; 115: 2169-2179Crossref PubMed Scopus (304) Google Scholar Genomic DNA from mouse lungs were isolated by using Qiagen Genomic-tip 500/G and Genomic DNA buffer set (Qiagen, Valencia, Calif), according to vendor's protocol. The LORD-Q assayE6Lehle S. Hildebrand D.G. Merz B. Malak P.N. Becker M.S. Schmezer P. et al.LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis.Nucleic Acids Res. 2014; 42: e41Crossref PubMed Scopus (34) Google Scholar was performed by using KAPA2G Fast Hot-Start ReadyMix (Kapa Biosystems, Wilmington, Mass) and LightCycler 480 High Resolution Melting Dye (Roche, Indianapolis, Ind). Cycling conditions were as follows: a preincubation phase of 5 minutes at 95°C was followed by 50 cycles of 10 seconds at 95°C, 30 seconds at 60°C, and 1 second at 72°C for small amplicons or 2:15 minutes at 72°C for long amplicons (ABI 7500; Applied Biosystems, Foster City, Calif). For analysis of the DNA damage in genomic DNA, amplicons were generated with primers as follows: mTop2A forward 5′-GGGAAAGGAATCCCTGTTGTTG-3′ and reverse 5′-GGAGGTCAGGAGTTGTCCAAA-3′ for small amplicons (81-bp amplicons) and forward 5′-GGGAAAGGAATCCCTGTTGTTG-3′ and reverse 5′-TGCCATGGTATGCACAGAGG-3′ for long amplicons (3478-bp amplicons); these primers were purchased from Integrated DNA Technologies (Coralville, Iowa). HEK 293 cells that do not express TLR4 (TLR4Null) and HEK 293 cells that stably overexpress TLR4, CD14, and MD2 (TLR4Hi; InvivoGen, San Diego, Calif) were used for the experiment described below. We analyzed whether allergenic extract–induced ROS generation was determined by using minor modifications of a previously described protocol.E3Boldogh I. Bacsi A. Choudhury B.K. Dharajiya N. Alam R. Hazra T.K. et al.ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation.J Clin Invest. 2005; 115: 2169-2179Crossref PubMed Scopus (304) Google Scholar Briefly, cells were suspended in PBS at a concentration of 2 × 107 cells/mL. 2′,7′-Dichlorodihydrofluoresceine diacetate (Sigma-Aldrich) was added at a concentration of 50 μmol/L for 30 minutes.E3Boldogh I. Bacsi A. Choudhury B.K. Dharajiya N. Alam R. Hazra T.K. et al.ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation.J Clin Invest. 2005; 115: 2169-2179Crossref PubMed Scopus (304) Google Scholar After 2 washes, cells were plated at 1 × 106 cells per well in a 96-well plate and stimulated with BSA, RWPE, firebush, pigweed, timothy grass, rye grass (rye), black walnut (walnut), eastern cottonwood (cottonwood), mountain cedar, Russian thistle, American cockroach (cockroach), and cat dander at a concentration of 100 μg/mL and LPS (Sigma-Aldrich) at a concentration of 1 μg/mL. The change in DCF fluorescence intensity was assessed in a FLx800 microplate reader (Bio-Tek, Winooski, Vt) at 488-nm excitation and 530-nm emission every 10 minutes for 60 minutes. Cells were loaded with MitoSOX (Invitrogen, San Diego, Calif) at a concentration of 2.5 mmol/L for 30 minutes at 37°C in a 5% CO2 atmosphere.E7West A.P. Brodsky I.E. Rahner C. Woo D.K. Erdjument-Bromage H. Tempst P. et al.TLR signalling augments macrophage bactericidal activity through mitochondrial ROS.Nature. 2011; 472: 476-480Crossref PubMed Scopus (1056) Google Scholar After 2 washes, cells were stimulated with RWPE at a concentration of 100 μg/mL for 20 minutes and subjected to fluorometric analysis (infinite 200; Tecan, Durham, NC). Mean fluorescence intensities were calculated at excitation of 400 nm and emission of 590 nm. Cells were stimulated with BSA, RWPE, or CDE at a concentration of 100 μg/mL for 30 minutes. Supernatants were collected and subjected to spectrophotometry to measure GSSG levels, as described above. Cells were stimulated with RWPE at a concentration of 100 μg/mL for 6 hours. After centrifugation and removal of cell-culture supernatants, intracellular glutathione levels were assayed by using GSH-Glo glutathione assay (Promega, Madison, Wis), according to the manufacturer's instructions. Cells were stimulated with RWPE at a concentration of 100 μg/mL for 1 hour. The comet assay (Travigen, Gaithersburg, Md) was performed, as previously described.E8Bacsi A. Kannan S. Lee M.S. Hazra T.K. Boldogh I. Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells.Free Radic Biol Med. 2005; 39: 1650-1659Crossref PubMed Scopus (22) Google Scholar, E9Boldogh I. Roy G. Lee M.S. Bacsi A. Hazra T.K. Bhakat K.K. et al.Reduced DNA double strand breaks in chlorambucil resistant cells are related to high DNA-PKcs activity and low oxidative stress.Toxicology. 2003; 193: 137-152Crossref PubMed Scopus (54) Google Scholar, E10German P. Szaniszlo P. Hajas G. Radak Z. Bacsi A. Hazra T.K. et al.Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair.DNA Repair (Amst). 2013; 12: 856-863Crossref PubMed Scopus (55) Google Scholar Briefly, cells were suspended in 0.75% low-melting point agarose (Travigen) and put on microscope slides (Travigen). The slides were then immersed in lysis buffer. Electrophoresis of DNA was performed. After neutralization, the DNA was stained with SYBR Green. Comets were evaluated by using the Comet Assay IV v4.2 system (Perceptive Instruments, Suffolk, United Kingdom). The tail intensity, which was defined as the percentage of DNA migrating from the head of the comet into the tail, was measured. We have reported that RWPE requires MD2 in addition to TLR4 to evoke innate immunologic responses.E2Hosoki K. Boldogh I. Aguilera-Aguirre L. Sun Q. Itazawa T. Hazra T. et al.Myeloid differentiation protein 2 facilitates pollen- and cat dander-induced innate and allergic airway inflammation.J Allergy Clin Immunol. 2016; 137: 1506-1513.e2Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Because high-quality immunoprecipitation antibody against hemagglutinin (HA) exists, coimmunoprecipitation experiments were performed with HEK 293 cells stably overexpressing HA-tagged TLR4 but not MD2 (TLR4HA). To facilitate innate immune recognition of RWPE by TLR4HA cells, cells were transfected with 2 μg of plasmid encoding MD2 (plasmid 13028; Addgene, Cambridge, Mass) by using Lipofectamine 2000 to generate TLR4-MD2 expressing cells (TLR4HA-MD2), as previously described.E2Hosoki K. Boldogh I. Aguilera-Aguirre L. Sun Q. Itazawa T. Hazra T. et al.Myeloid differentiation protein 2 facilitates pollen- and cat dander-induced innate and allergic airway inflammation.J Allergy Clin Immunol. 2016; 137: 1506-1513.e2Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Twenty-four hours after transfection, TLR4HA-MD2 cells were stimulated with RWPE at 100 μg/mL for 10 or 30 minutes at 37°C in a 5% CO2 atmosphere. Coimmunoprecipitation and Western blot analysis were performed by using the methods described previously,E11Cui W. Xiao N. Xiao H. Zhou H. Yu M. Gu J. et al.beta-TrCP-mediated IRAK1 degradation releases TAK1-TRAF6 from the membrane to the cytosol for TAK1-dependent NF-kappaB activation.Mol Cell Biol. 2012; 32: 3990-4000Crossref PubMed Scopus (40) Google Scholar, E12Shi H.X. Liu X. Wang Q. Tang P.P. Liu X.Y. Shan Y.F. et al.Mitochondrial ubiquitin ligase MARCH5 promotes TLR7 signaling by attenuating TANK action.PLoS Pathog. 2011; 7: e1002057Crossref PubMed Scopus (50) Google Scholar with minor modification. Cells were lysed in coimmunoprecipitation buffer (50 mmol/L Tris [pH 7.4], 156 mmol/L NaCl, 1% Nonidet P-40, and 1 mmol/L EDTA) freshly supplemented with complete protease inhibitor cocktail (P8340, Sigma-Aldrich) and 1 mmol/L phenylmethylsulfonyl fluoride. One part of total lysates was subjected to Western blotting with monoclonal rat anti-HA (Roche, Indianapolis, Ind) and rabbit anti–β-actin (4967S, Cell Signaling Technology, Beverly, Mass) to validate equal protein levels in cell lysates. The remaining part of each cell lysate was subjected to immunoprecipitation by using mouse anti-HA (12CA5, Roche) to pull down TLR4. Protein-G-agarose (16-201, Millipore; 3 hours, at 4°C) bound immunocomplexes were washed and subjected to Western blot analysis. TLR4 was visualized by means of Western blotting with monoclonal rat anti-HA (Roche), and TRAF6 was visualized with anti-TRAF6 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif). From December 7, 2007, to September 3, 2008, 9 patients with allergic rhinitis were recruited from the University of Texas Medical Branch at Galveston Allergy Clinic for this study. Mean age was 41.7 years, with a range of 26 to 55 years; 8 patients were female (Table E1). Protocols and consent forms were reviewed and approved by the Institutional Review Board at the University of Texas Medical Branch at Galveston (protocol IRB#05-093). Informed consent was obtained from all subjects who participated in the study. All subjects met the following inclusion criteria, presence of seasonal symptoms of allergic rhinitis, including nasal congestion, nasal discharge, sneeze, positive prick skin test response to RWPE, and male and female patients aged 18 to 60 years of all ethnic groups. Exclusion criteria were a history of smoking; moderate-to-severe asthma; systemic disorders, such as diabetes, chronic obstructive pulmonary disease, and lupus; and pregnancy. Intranasal challenge with saline or RWPE solution was performed. All subjects had allergic rhinitis with a positive prick skin test response to RWPE. Nasal lavage fluid on different days in the same subjects were collected from subjects by using the methods described previously,E13Iliopoulos O. Proud D. Adkinson Jr., N.F. Norman P.S. Kagey-Sobotka A. Lichtenstein L.M. et al.Relationship between the early, late, and rechallenge reaction to nasal challenge with antigen: observations on the role of inflammatory mediators and cells.J Allergy Clin Immunol. 1990; 86: 851-861Abstract Full Text PDF PubMed Scopus (102) Google Scholar with minor modifications. Briefly, squirts of saline or RWPE solution were instilled into each nostril. The concentration of RWPE solution was increased until symptoms appeared. Provoking doses of RWPE (Greer Laboratories) with Amb a 1 equivalent doses of 0.05, 0.13, 0.41, 1.21, 3.65, and 10.96 μg were delivered in 0.137 mL per squirt. During the nasal challenge, symptom scores were recorded at 30 minutes and 1, 2, 3, 4, and 5 hours after the nasal challenge by using a modification of a method reported elsewhere.E14Lumry W. Hampel F. LaForce C. Kiechel F. el-Akkad T. Murray J.J. A comparison of once-daily triamcinolone acetonide aqueous and twice-daily beclomethasone dipropionate aqueous nasal sprays in the treatment of seasonal allergic rhinitis.Allergy Asthma Proc. 2003; 24: 203-210PubMed Google Scholar Symptom scoring was as follows: (1) sneezing, 0 to 3 points (0 sneeze = 0 points, 1-5 sneezes = 1 point, 6-10 sneezes = 2 points, an >10 sneezes = 3 points); (2) stuffy nose, 0 to 3 points (0 = none, 1 = mild, 2 = moderate, and 3 = severe symptoms); and (3) runny nose, 0 to 3 points (0 = none, 1 = mild, 2 = moderate, and 3 = severe symptoms). The total symptom score was the sum of scores of sneezing, stuffy nose, and runny nose. Therefore the total score ranged from 0 to 9. A positive clinical challenge result was defined as an increase in greater than 3 total points. At baseline and 30 minutes after saline or RWPE challenge, 5 mL of prewarmed saline was squirted into each nostril with a syringe, kept in the nostril for 10 seconds, and then collected with gravitational aid. Nasal fluids were analyzed for 8-OHdG by using by an 8-OHdG DNA Damage ELISA kit (Cell Biolabs, San Diego, Calif), according to the manufacturer's instructions. In all figures the continuous outcome data are expressed as means ± SEMs. The paired t test was used for comparing variables before and after the challenge separately for each group. The unpaired t test was used to compare the groups with respect to their mean differences/changes from baseline/before to and after challenge time points. ANOVA was used to compare the mean differences among 3 or more groups. Rejection of the null hypothesis was determined by using the Bonferroni multiple comparisons test. To assess the interaction effects of 2 variables (time vs intensity of symptom score), 2-way ANOVA was used on the outcomes, and rejection of the null hypothesis was performed with the Bonferroni multiple comparisons test. The software package GraphPad Prism 6 (GraphPad Software, San Diego, Calif) was used for all data analyses and preparation of graphs.Fig E2RWPE challenge rapidly increases oxidative DNA damage in the noses of allergic human subjects. Intranasal RWPE challenge in human subjects is shown. Patients with allergic rhinitis and positive skin prick test responses to RWPE were challenged with saline on one day and RWPE on another. A, RWPE challenge increased nasal symptom scores for congestion, drainage, and sneezing recorded on subjective scale scores of 0 to 3, yielding a combined total symptom score for all 3 symptoms of a maximum of 9. B, RWPE challenge increased levels of 8-OHdG in nasal fluids 30 minutes after challenge. *P < .05, **P < .01, and ***P < .001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Subjects' characteristicsAge (y)SexRace55FW39FB23FW43FH50FW52MW26FW38FW49FWB, Black; F, female; H, Hispanic; M, male; W, white. Open table in a new tab B, Black; F, female; H, Hispanic; M, male; W, white.

This paper describes the application of NASA's software that calculates optical aberrations (wavefront errors). The power of this software lies in its ability to use an optical system's existing camera as a sensor to detect its own error, without installing any separate devices. This software is expected to sharpen images from space and improving the image quality of the astronomical telescopes, it could also redefine perfect vision for humans.

The innate immune response of airway epithelial cells to airborne allergens initiates the development of T cell responses that are central to allergic inflammation. Although proteinase allergens induce the expression of interleukin 25, we show here that epithelial matrix metalloproteinase 7 (MMP7) was expressed during asthma and was required for the maximum activity of interleukin 25 in promoting the differentiation of T helper type 2 cells. Allergen-challenged Mmp7(-/-) mice had less airway hyper-reactivity and production of allergic inflammatory cytokines and higher expression of retinal dehydrogenase 1. Inhibition of retinal dehydrogenase 1 restored the asthma phenotype of Mmp7(-/-) mice and inhibited the responses of lung regulatory T cells, whereas exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating interleukin 25 while simultaneously inhibiting retinoid-dependent development of regulatory T cells.

This paper presents an adaptive optics (AO) system for a large IR telescope. The Keck Telescope on Mauna Kea is used as a case study. The system is designed to provide low-order wavefront correction using faint natural guide stars. It is shown that two innovations, namely the use of an AO-optimized CCD detector, and use of a variable control bandwidth, result in correction to a Strehl ratio of about 0.1 using guide stars of visual magnitude 18. Sky coverage is approximately 20% at this level of performance. Sky coverage rises to 100% at a Strehl ratio of 0.05.