The role of Interleukin-33 in the development of Idiopathic Pulmonary Fibrosis

The student will stimulate primary lung fibroblasts with increasing concentrations of recombinant IL-33 and measure markers of fibrosis including pSmad2/3, alphaSMA, collagen gene and protein expression. They will compare the effect of IL-33 on fibroblasts from patients with and without IPF. They will stimulate human macrophages (PMA stimulated THP1 cells, blood derived monocytes and primary alveolar macrophages) with IL-33 and measure
macrophage polarization and profibrotic cytokines, including TGF-beta. The effect of IL-33 function blocking antibodies on markers of fibrosis and macrophage polarization from cells obtained from patients with IPF, and controls, will be assessed.
Aim 2: NHBE cells will be stimulated with poly I:C and recombinant GM-CSF individually, and in combination, to induce IL-33 expression in the presence of intracellular (Caspase 1, 2, and 7) and extracellular proteases (neutrophil elastase and cathepsin) known to cleave IL-33. Levels, and isoforms, of intracellular IL-33 will be assessed by quantitative PCR and immunoblot. Levels of extracellular IL-33 will be measured by ELISA. The effect of extracellular IL-33 on macrophage polarization will be assessed by culturing macrophages in the conditioned media from epithelial cells with anti-IL-33 blocking reagents. Due to the labile nature of IL-33 (Cohen et al, Submitted), co-culture experiments will be performed to confirm results. The effect of epithelial apoptosis on IL-33 expression the effect of staurosporin on IL-33 secretion will be assessed. The cellular co-localization of IL-33 and intracellular proteases in lung tissue from patients, and controls, will be assessed using paraffin sections. Subsequently the levels of IL-33 and its cleavage fragments will be measured by immunoblot from frozen tissue obtained from patients or controls.