Bacterial induction of cytokine signaling in epithelial cells

Our laboratory studies the interaction of bacterial pathogens and the airway epithelium, a component of the mucosal immune system that initiates the host response to infection. We use multi-disciplinary approaches to identify key components of bacterial pathogens that are critical in the pathogenesis of airway infection and the nature of the epithelial signaling cascade that recruits inflammatory cells into the lung. As much of the pathology of pneumonia is due to the host response to infection, we have focused upon the host response to bacterial components, and how this response is regulated. A major area of interest has been the pathogenesis of airway infection in cystic fibrosis and we have focused our research on two common CF pathogens, Pseudomonas aeruginosa and Staphylococcus aureus and how they interact with both CF and normal airway epithelial cells.

A major area of research has been the identification of epithelial receptors that initiate chemokine production (IL-8) in response to bacterial gene products. While few organisms are actually adherent to airway epithelial cells, bacterial components ligate TLR2 exposed on the apical surface of airway cells in a lipid raft complex along with asialoGM1, which provides a ligand for many pulmonary pathogens. TLR signaling, NF-kB and MAPK activation initiates chemokine expression to signal the influx of phagocytes to the site of infection. TLR2 signaling also activates a number of secondary messengers including Ca2+ fluxes. Following TLR2 phosphorylation by cSrc, Ca2+ fluxes are generated that can activate adjacent epithelial cells through gap junctions, a response that is also regulated by cSrc kinases. The Ca2+ fluxes also activate calpains, proteases that are involved in modulating the epithelial barrier to facilitate the transmigration of phagocytes into the airway.

In addition, we study the bacterial virulence factors that target epithelial cells. Having explored some of the immunostimulatory properties of flagella and pili, we are now interested in gene products that affect the integrity of the epithelial barrier. The type 3 toxins of P. aeruginosa, for example, are important in modifying epithelial tight junctions to facilitate invasive infection. Ongoing projects are focusing upon the identification of the epithelial junctional proteins that are altered in response to bacterial ligands.

Staphylococcus aureus and particularly MRSA infections have become a major public health issue. Using the same approach, using wild type and mutant strains of S. aureus and comparing the signaling pathways that they activate in vitro in airway epithelial cells and in vivo in transgenic mice, we have identified protein A (SpA) as a major virulence factor in the pathogenesis of pneumonia. Having demonstrated that protein A stimulates TNF signaling, we are now localizing domains of the protein that independently activate type I interferon signals.

Having identified bacterial factors that are critical in pathogenesis, we have embarked upon a translational project to identify small molecule inhibitors to prevent bacterial colonization of the airway. In collaboration with Liang Tong in Dept. of Biology, the crystal structure(s) of bacterial neuraminidases have been solved, and using a bioinformatics approach, small molecule inhibitors predicted to interact with the active site(s) have been identified. Ongoing studies will assess the in vitro and in vivo efficacy of these inhibitors with the eventual goal of identifying compounds that could be given by aerosol to prevent respiratory tract infection.

These projects are supported by the NIH, the Cystic Fibrosis Foundation, and by a Pilot Project from Columbia University.