Inducible reactive oxygen species (ROS) play a critical regulatory role in the innate immune response (MR) by controlling the phosphorylation of both 1RF3 and NF-KB/ROIA. RelA phosphorylation is required for its inducible acetylation at Lys and binding to the positive elongation factor-b (PTEF-b) complex. The PTEF-b complex of cyclin-dependent kinase (CDK)-9 and bromodomain-4 (Brd4) proteins induces RNA Pol 11 Ser2 phosphorylation to induce transcriptional elongation in downstream genes. P1 will test the hypothesis that ROS modulate innate inflammatory and antiviral activities by modulating the activation states of NFKappaB/ RelA and iRF3 in the airway epithelium in three specific aims.
Aim 1 will examine how ROS modify NF-KappaB/RelA to induce recruitment of the Brd4-CDK9 complex mediating inflammatory cytokine production. The requirement of RelA Lys310 for ROS-induced PTEF-b recruitment will be tested by chromatin immunoprecipitation (ChIP) assays in RelA'''mouse embryonic fibroblasts reconstituted with WT RelA or its non-acetylated Lys310 Arg mutant in response to TNFa, RSV and ragweed pollen particle NADPH oxidase. The role of ROS in inducing promoter-associated inactive CDK9 clearance and replacement with activated CDK9 will be determined in small airway epithelial cells (SAEs). The role of Brd4 as an adapter protein will be examined using siRNA knockdown or in vivo competition using Lys-acetylated histone H4 peptides.
Aim 2 will examine how ROS mediate IRF3 pathway signaling. We will modulate inducible ROS to examine the kinetics of 1RF3 translocation in SAEs, using our novel selected reaction monitoring (SRM) assay. The requirement of ROS for 1RF3 enhanceosome formation will be tested by ChIP assay on IFNp and interferon-stimulated gene (ISG) promoters ? antioxidant modulation. We will determine whether IRF3 induces antiviral transcriptional initiation or elongation by siRNA depletion of CDK-7 and -9.
Aim 3 will examine how transcriptional elongation mediates the effect of the ilR in RSV infection in vivo. How inhibiting NF-KB-PTEF-b recruitment and CDK9 affects cellular inflammation, anti-viral immunity, airway pathology and airway hyper-reactivity (AHR) will be tested in a BALB/c mouse model using an NF-KB inhibitory IKKy/NEMO binding domain (NBD) peptide and small molecule CDK9 inhibitor. These studies will establish the role of the ROS-initiated transcriptional elongation pathway in the MR and so lay the foundation for novel approaches to selectively modulate inflammation in airway mucosa and RSV bronchiolitis.

Public Health Relevance

Asthma is a breathing disorder affecting about 10% of Americans, and is frequently worsened by cold viruses. This project seeks to understand how the lung tissues respond to signals produced by cold viruses that worsen the disease. This knowledge should lead to novel approaches to treat asthma, based on blocking the signals elucidated in this project.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program Projects (P01)
Project #
5P01AI062885-08
Application #
8715677
Study Section
Transplantation Biology &Immunology-2 (AITC)
Project Start
Project End
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
8
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Type
DUNS #
City
Galveston
State
TX
Country
United States
Zip Code
77555
Belanger, KarryAnne K; Ameredes, Bill T; Boldogh, Istvan et al. (2016) The Potential Role of 8-Oxoguanine DNA Glycosylase-Driven DNA Base Excision Repair in Exercise-Induced Asthma. Mediators Inflamm 2016:3762561
Hosoki, Koa; Itazawa, Toshiko; Boldogh, Istvan et al. (2016) Neutrophil recruitment by allergens contribute to allergic sensitization and allergic inflammation. Curr Opin Allergy Clin Immunol 16:45-50
Choudhary, Sanjeev; Boldogh, Istvan; Brasier, Allan R (2016) Inside-Out Signaling Pathways from Nuclear Reactive Oxygen Species Control Pulmonary Innate Immunity. J Innate Immun 8:143-55
Nicholson, Erin G; Schlegel, Chelsea; Garofalo, Roberto P et al. (2016) Robust Cytokine and Chemokine Response in Nasopharyngeal Secretions: Association With Decreased Severity in Children With Physician Diagnosed Bronchiolitis. J Infect Dis 214:649-55
German, Peter; Saenz, David; Szaniszlo, Peter et al. (2016) 8-Oxoguanine DNA glycosylase1-driven DNA repair-A paradoxical role in lung aging. Mech Ageing Dev :
Hosoki, Koa; Aguilera-Aguirre, Leopoldo; Brasier, Allan R et al. (2016) Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment. Am J Respir Cell Mol Biol 54:81-90
Ivanciuc, Teodora; Sbrana, Elena; Ansar, Maria et al. (2016) Hydrogen Sulfide Is an Antiviral and Antiinflammatory Endogenous Gasotransmitter in the Airways. Role in Respiratory Syncytial Virus Infection. Am J Respir Cell Mol Biol 55:684-696
Bacsi, Attila; Pan, Lang; Ba, Xueqing et al. (2016) Pathophysiology of bronchoconstriction: role of oxidatively damaged DNA repair. Curr Opin Allergy Clin Immunol 16:59-67
Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana et al. (2016) Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes. Nat Commun 7:13049
Hosoki, Koa; Boldogh, Istvan; Aguilera-Aguirre, Leopoldo et al. (2016) Myeloid differentiation protein 2 facilitates pollen- and cat dander-induced innate and allergic airway inflammation. J Allergy Clin Immunol 137:1506-1513.e2

Showing the most recent 10 out of 126 publications