Iron induced HIF destabilization and altered Cellular Homeostatic Mechanisms in Cystic Fibrosis Cell Lines

Introduction: Cystic fibrosis (CF) is commonly associated with bacterial infection of the airways usually with P. aeruginosa and S. aureus. The chronic endobronchial inflammation due to persistent bacterial infection leads to progressive lung disease and is the main cause of mortality and morbidity in CF. Toll like receptors (TLR) act as sensors for the bacterial infections through conserved pathogen assisted molecular patterns (PAMPS), which mediate inflammation and innate immune responses of the lung via TLR signaling. LPS induced TLR4 signaling not only recruits inflammatory cytokines but also induces anti-inflammatory pathways which include hemeoxygenase-I (HO-1) thereby maintaining homeostasis of the cell. We recently reported decreased TLR-4 surface expression in the bronchial epithelium of patients with CF compared with healthy control subjects and confirmed our results in a cell culture model with CFBE41o- comparing to their corrected isotypes. We investigated hypoxia, autophagy and anti-inflammatory pathways to determine their involvement during reduced TLR-4 surface expression in CF.

Methods: Immunofluorescence and immunoblotting were performed using the defective CFTR (Cystic Fibrosis Transmembrane Regulator) cell line (CFBE41o-), its corrected counterpart (corr-CFBE41o-) and normal human bronchial epithelial cell line (16HBE14o-) as control to examine the expression of TLR-4, unfolded protein response (UPR), autophagy and anti-inflammatory pathways upon LPS stimulation.

Results: We observed an altered HIF1α stabilization in response to increased iron concentrations in the CFBE41o- cells lines when compared with its corrected counterpart and 16HBE14o- cell line. Upon sequestering of iron using iron chelators (2,2 bipyridyl), HIF1α was stabilized in CFBE41o-. Additionally stabilization of HIF1α resulted in stabilization of HO-1 expression in CFBE41o-.

Conclusion: Our results hint towards a role of iron and HIF in controlling the cellular stress mechanisms and regulating TLR-4 surface expression in cystic fibrosis. Further studies are required to understand the complex mechanisms involved.