Asthma Pathogenesis Research Highlights

Title: Discovery of NADPH Oxidases Enzyme in Pollens, which causes Oxidative Stress and Worsens Allergic Diseases, including Asthma
    Background and Advances
    Implications & Public Health Impact
    Center Contribution
    Key Researchers
    Publication(s)
    Grant Support
Title: Signaling in Airway Inflammation
    Background and Advances
    Implications & Public Health Impact
    Center Contribution
    Key Researchers
    Publication(s)
    Grant Support
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Title: Discovery of NADPH Oxidases Enzyme in Pollens, which causes Oxidative Stress and Worsens Allergic Diseases, including Asthma

Background and Advances: Exposure to pollen often induces allergic reactions in the nose, lungs and eyes of certain individuals. The role of antigens in allergic airway inflammation is well characterized, but the contributions of other constituents in pollen grains to this process are unknown.

Drs. Istvan Boldogh and Sanjiv Sur in the Asthma Pathogenesis Research Core noticed that ragweed (Ambrosia artemisiifolia) pollen in solution and cells exposed to that solution produced a highly oxidizing environment, as indicated by a dye that become fluorescent in the presence of reactive oxygen species (ROS). This activity of pollen grains and their extracts was found to be due to the presence of an enzyme termed NADPH oxidases. When the enzyme was introduced into the airways of mice, it induced oxidative stress in the airways and enhanced the allergic response and asthma in mice that were sensitized to the pollen proteins. Removal of pollen NADPH oxidase activity from the pollen extract reduced the severity of the asthmatic response. Based on these findings it was proposed that oxidative stress (signal 1) generated by pollen NADPH oxidases augments allergic airway inflammation and worsens asthma symptoms induced by pollen antigen (signal 2).

Subsequent studies examined the chemical basis for the reactions by instilling ragweed pollen onto the lining of the eyes of mice, which had been made allergic to this pollen. The NADPH oxidases in the hydrated pollen generated a reactive intermediate called superoxide anion, which was converted to hydrogen peroxide by host and pollen enzyme superoxide dismutases. These oxygen radicals diffused from pollen grains and increase intracellular ROS levels the eye lining cells in the mice. The immediate symptoms of the eye to the pollen did not require the mice to be allergic, since reactions occurred whether the mice were sensitized with ragweed or not. On the other hand, development of late-phase symptoms required the pollen allergen and ROS. This finding suggested that pollen-induced oxidative insult to the eye lining cells and the allergen was necessary to cause the robust inflammation, including infiltration of inflammatory blood cells into the eye lining. Inactivation of NADPH oxidase activity in the pollen decreased all aspect of the reactions, indicating the importance of this plant enzyme in the eye responses.

In there most recent studies, these investigators have examined the role of free iron in conversion of the superoxide anion produced by the action of NADPH oxidases. In the presence of free iron, the superoxide anion was converted to more reactive oxygen radicals, such as to H2O2 and/or .OH. To remove the iron they added a protein apolactoferrin that binds two iron molecules very tightly. When apolactoferrin was added to the ragweed pollen extract prior to instilling it in the lungs of mice, it decreased the accumulation of inflammatory and mucin-producing cells in airways. LF also lowered the ragweed-induced increase in ROS levels in bronchial epithelial cells. Amb a 1, the major allergenic ragweed pollen protein, which does not have any NADPH oxidase activity, induced a low-grade airway inflammation. However, when the enzyme glucose oxidase which also produce superoxide, was added to Amb a 1 solution the mouse developed a robust airway inflammation, which was significantly reduced by apolactoferrin. Iron-saturated hololactoferrin had little effect on ragweed-induced cellular ROS levels and lung inflammation. The results of these studies suggest that iron in the airway lining fluids and epithelium increase the formation of highly reactive radicals from superoxide anion thereby augment antigen-induced inflammation. These results also support the therapeutic utility of LF in human allergic inflammatory disorders.

How pollen-carried allergens contribute to development of inflammation in lower airways of the lungs has remained a puzzle, because pollen grains are too large to penetrate into the lower airways. However, in an additional study Drs. Boldogh and Sur provide evidence that small sub-pollen particles that are released from weed (e.g., ragweed) pollen grains contain both antigenic and oxidative properties. The investigators were able to show that when fresh ragweed pollen grains were mixed with water, they released 0.5 to 4 mm particles, which contain Amb a 1 and other allergenic proteins of ragweed pollen and posses NADPH oxidase activity. Administration of these particles significantly increased the levels of ROS in airway epithelial cells and induced accumulation of eosinophils and formation of mucin producing cells in the lungs of mice. Pre-treatment of the particles with quinacrine, a NADPH oxidase inhibitor, decreased their capacity to induce ROS and subsequent airway inflammation. They concluded that allergenic potency of the sub-micronic particles of pollen contains all of the constituents necessary to cause robust allergic reactions deep within the lungs.

Implications and Public Health Impact: The key factor that makes pollen proteins so allergenic has not been identified. Our findings identify NADPH oxidase(s) generated oxidative stress as key factor that significantly increase the ability of pollen antigens to induce allergic inflammation. As a consequence of this discovery, a patent was issued. Inhibitors of this oxidase may be developed to reduce allergic inflammation and the severity of the asthma.

Center Contribution: The NIEHS Center Cell Biology Facility Core, Biomolecular Resource Facility Core and the NHLBI Proteomics Centers were critical for these breakthrough papers.

Key Researchers:
Istvan Boldogh, Asthma Pathogenesis Research Core and Cell Biology Facility Core, Department of Microbiology and Immunology

Sanjiv Sur, Asthma Pathogenesis Research Core, Department of Internal Medicine

Publication(s):
Boldogh, I., BK. Choudhury, TK. Hazra, R. Alam, R. Goldblum, S. Mitra and S. Sur. 2005 Aug 1; Oxidative stress generated by pollen NADPH oxidases provide a second signal that augments antigen-induced allergic inflammation. J. Clinical Investigations, 115:2169-2179.

Bacsi, A., Choudhury, B.K., Dharajiya, N., Sur, S., and Boldogh, I. 2005 Oct 1; Impact of pollen-mediated oxidative stress on hypersensitivity reactions and lat-phase inflammation in allergic conjunctivitis. J. Allergy Clinical Immunology, 116:836-843.

Bacsi, A., B.K. Choudhury, N. Dharajiya, S. Sur, I. Boldogh, Sub-pollen particles: carriers of allergens and oxidases. J. Allergy Clinical Immunology, 2006.

Kruzel, M.L. A. Bacsi, B. Choudhury, S. Sur, and I. Boldogh, Lactoferrin decreases pollen antigen-induced allergic airway inflammation in a murine model of asthma. Immunology 2006.

Patent(s):
Boldogh, I., et al., and S. Sur. Methods for Inhibiting Allergic Inflammation and Other Responses Initiated By Pollens, Molds, And Other Non-Animal Derived Allergens. U.S. Patent Application No.: 026.00711, 2003.

Grant Support:
NIH/NHLBI R01 HL071163
Role of oxidative stress in asthma initiation

NIH/NIAID AI062885
Signaling in airway epithelium
(Dr. Sur is Principal Investigator of project 4 entitled, “Effect of Pollen-induced ROS on allergic asthma”.)
(Dr. Boldogh is Principal Investigator of project 5 entitled, “Mitochondria: antigen-induced airway inflammation”.)

NIH/NHLBI N01 HB28184
Proteomic Technologies to Study Airway Inflammation

Title: Signaling in Airway Inflammation

Background and Advances: Five of the long-standing members of the Asthma Pathogenesis Research Core have used their history of fruitful collaboration within that Core to acquire Program Project (P01 AI062885-01) funding from NIAID to support and extend investigations largely initiated within the NIEHS Core. Dr. Allan Brasier, the principal investigator of this project, has served as the Director to the Asthma Pathogenesis core for the last two years. The term of the program project extends from September 1, 2005 to March 31, 2010. This funding represents a major advance for this Core, since it will bring in approximately $4.2M total direct cost in new grant funding dedicated to investigating mechanisms by which airway inflammation causes asthma. The five interdigitating and synergistic research projects will investigate the hypothesis that cytokines, viruses and allergens induce specific intracellular signaling pathways in airway epithelial cells that induce genetic programs important in the inflammatory response. Reactive Oxygen Species (ROS) generated during viral replication or from exposure to pollen-associated NADPH oxidases induce mitochrondrial dysfunction, further enhancing ROS stress, and activate signaling pathways controlling chemokine expression. Epithelial derived chemokines, in turn, induce leukocytic recruitment into the airways that induce lung damage, and airway hyper-reactivity. A brief description of each of the individual projects is listed below:

Project 1. Gene Networks in Stimulated Epithelium
Project Leader: A.R. Brasier, M.D.
Dr. Brasier will pursue hypothesis that the nuclear factor-κB (NF-κB) transcription factor is a central regulator of airway inflammation in the stimulated epithelial cell. NF-kB activation of target promoters requires chromatin remodeling, is redox-sensitive, and requires formation of a macromolecular complex with the NF-κB inducing kinase, IκB kinase alpha, and the p300 coactivator.

Project 2. Role of epithelial chemokines in the pathogenesis of virus-induced wheezing
Project Leader, R.P. Garofalo, M.D.
Dr. Garofalo will pursue the hypothesis that MIP-1a, by virtue of its activities on natural killer (NK) cells and cytotoxic T lymphocytes (CTL), may function as a bridge between innate and adaptive immune responses to RSV.

Project 3. Redox-sensitive pathways mediating RSV pathogenesis
Project Leader, A. Casola, M.D.
Dr. Casola will pursue the hypothesis that ROS generated during RSV infection controls two critical intracellular pathways, one leading to inhibition of tyrosine phosphatases, which results in STAT activation, and the second regulating IKKe induction and subsequent IRF activation and NF-kB-transcriptional activation.

Project 4. Effect of Pollen-Induced ROS on Lung Gene Expression
Project Leader, Sanjiv Sur, M.D.
Dr. Sur will pursue the hypothesis is that oxidized glutathione (GSSG) and 4-hydroxynonenal (4-HNE) generated by the interaction of intrinsic NADPH oxidases in pollens with substrate in airway lining fluid provides a critical second signal that activates MAP kinases and induces expression of genes in the lungs that augment mucin production, airway inflammation and AHR in allergic asthma.

Project 5. Impact of Intrinsic Pollen Oxidases on Mitochondria in Asthma
Project Leader, Istvan Boldogh, Ph.D.
Dr. Boldogh will pursue the hypothesis that by their intrinsic oxidant activity, plant/mold aero-allergenic particles cause mitochondrial dysfunction and the released ROS are critically important in inflammatory chemokine production and vigorous antigen-driven allergic inflammation.

Implications and Public Health Impact: Asthma remains a leading cause of emergency department and hospitalizations, particularly for children. The most effective medications currently available for asthma management have broad anti-inflammatory activities and consequently multiple adverse effects. A more complete understanding of the cellular signaling patterns that connect the common, environmental triggering factors: allergen inhalation and respiratory viral infection with the inflammatory process in the airways will allow us to design therapies that prevent signal initiation or halt the pathway prior to the pathophysiological changes characteristics of asthma exacerbations.

Center Contribution: The NIEHS Center at UTMB has supported the initiation and growth of the Asthma Pathogenesis Research Core for the last seven years though pilot project grants and access to highly sophisticated technologies of the excellent Service Cores. The administrative structure of the NIEHS Center has also supported the numerous meetings and other interactions that helped to foster the development and integrations of projects, such as those supported by this Program Project grant.

Key Researchers:
Allan R. Brasier, Asthma Pathogenesis Research Core, Department of Internal Medicine

Roberto Garofalo, Asthma Pathogenesis Research Core, Department of Pediatrics and Department of Biochemistry and Molecular Biology

Antonella Casola, Asthma Pathogenesis Research Core, Department of Pediatrics

Sanjiv Sur, Asthma Pathogenesis Research Core, Department of Internal Medicine

Istvan Boldogh, Asthma Pathogenesis Research Core and Cell Biology Facility Core, Department of Microbiology and Immunology

Publication(s):
Tian, B., Nowak, D., and Brasier, A.R A TNF Induced Gene Expression Program Under Oscillatory NF-kB Control. BMC Genomics, 6:137, 2005.

Grant Support:
NIH/NIAID AI062885
Signaling in airway epithelium

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