Exposure to pollens is one of the most common causes of respiratory allergies such as allergic rhinitis and allergic asthma worldwide. We have previously shown that pollens contain an enzyme called NADPH oxidase (pollen-NOX), and that this enzyme is critical for the induction of allergic inflammation. However, very little is known about the molecular mechanism by which these oxidases induce allergic diseases. The goal of this project is to address this critical gap in knowledge about how pollen-NOX induces allergic inflammation. The airway epithelium is the first line of defense against the harmful effects of pollens. Epithelial and intraepithelial dendritic (DCs) cells are two types of cells in the airway epithelium that are the key first responder cells to inhaled pollen allergens. We propose that one of the earliest events when pollens land on the airway epithelium is that pollen-NOX binds to a receptor on the surface of DCs called Toll-like receptor (TLR4). This cell-surface receptor is best known for its ability to bind to a bacterial product called LPS, but its ability to bind pollen-NOX has not been described. This binding initiates oxidative stress in the DCs that is sufficiently severe to damage DNA, the cells' genetic material. This damaged DNA is repaired by a DNA repair enzyme called OGGI, and 8-oxoG- the damaged base in the DNA, is released. OGGI and 8- oxoG then form a signaling complex in DCs called OGGI-guanine nucleotide exchange factor, or OGG1gef. This induces a signaling cascade that activates DCs and promotes allergic inflammation. This model will be tested in three Specific Aims. In Aim 1 a cell line called 293 and mouse DCs will be used to test how pollen- NOX binds to TLR4, and determine the importance of this binding in the activation of DCs. Aim 2 will test whether DNA damage and repair induced by pollen-NOX is crucial for activating a signaling cascade that induces allergic lung inflammation in mice. Aim 3 will test the human relevance of the observations made in cell lines and mice in Aims 1 and 2. We will determine whether binding of pollen-NOX to TLR4 activates DNA damage/repair-mediated signaling pathways in DCs from healthy human subjects and those with allergic rhinitis. We will also perform nasal challenge with pollen extract to healthy subjects and subjects with allergic rhinitis, and determine if this challenge induces DNA damage and repair in the nose. Achieving these Aims should identify novel molecular pathways utilized by pollen NADPH oxidases to activate DCs and induce allergic inflammation in humans, and so identify novel molecular targets for new treatments of allergic rhinitis and allergic asthma.