The long-term goal of this project is to understand how natural killer T (NKT) cells recognize and respond to microorganisms in the respiratory mucosa. We have previously shown that NKT cells in the lungs play a critical role in the development of airway inflammation and asthma. In addition, we have shown that glycolipids from the bacterial species Sphingomonas can directly activate pulmonary NKT cells in mice and induce airway inflammation and airway hyperreactivity (AHR), a cardinal feature of asthma. These results suggest that NKT cells in the lung mucosa may respond to other microorganisms that enter the lung, and that NKT cells could play a previously unsuspected critical role in regulating pulmonary mucosal immune responses. Although only a few microorganisms are known to activate NKT cells, based on strong preliminary data, we believe that many pulmonary microorganisms, including Streptococcus pneumoniae, Burkholderia cenocepacia, Sphingomonas paucimobilis and Aspergillus fumigatus express glycolipids that can activate NKT cells, resulting in innate and adaptive mucosal immune responses. Furthermore, we hypothesize that microorganisms such as Sphingomonas paucimobilis, are much more common in the airway mucosa of patients with chronic pulmonary inflammatory diseases than previously recognized, and that host responses to these previously unrecognized microorganisms in the endobronchial mucosa can trigger chronic diseases in the airways. We therefore propose to identify glycolipids from S. pneumoniae, B. cenocepacia, and A. fumigatus that can directly activate NKT cells, thus demonstrating a specific mechanism by which these microorganisms can activate NKT cells and induce AHR, inflammation and asthma. Further, to demonstrate the clinical relevance of such mechanisms, we propose to examine the lungs of patients with chronic lung disease (e.g., asthma) for the presence of bacteria, using an extremely sensitive, novel non-culture based 16S rRNA PhyloChip microarray method, which uses approximately 500,000 probes to detect the 16S ribosomal RNA signatures from 9,000 bacteria. Surprisingly, preliminary data indicate that a highly diverse complex bacterial consortia, including S. pneumoniae, and bacteria not previously detected by cultured based techniques, such as Sphingomonas species (expressing glycolipids that can activate NKT cells), are indeed present in the lungs of a large fraction of patients with asthma. We have assembled an outstanding team of investigators, including experts in lung biology, lipid biochemistry, microbiology and immunology. Using very novel techniques, we have generated exciting preliminary data, which suggest that our studies will greatly expand the fundamental understanding of the types of immune responses that develop against microorganisms present in the respiratory mucosa, and how these responses result in the development of chronic lung diseases, such as asthma.