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Infection of Tracheal Epithelial Cells with RSV or Pseudomonas Aeruginosa Decreases Hyaluronan Levels
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A3879 - Infection of Tracheal Epithelial Cells with RSV or Pseudomonas Aeruginosa Decreases Hyaluronan Levels
Author Block: C. C. Smallcombe1, G. Altawallbeh1, T. L. Bonfield2, F. Rezaee1, G. Piedimonte1; 1Pathobiology, Cleveland Clinic, Lerner Research Institute, Cleveland, OH, United States, 2Case Western Reserve Univ, Cleveland, OH, United States.
Rationale: Cystic Fibrosis (CF) is a genetic disease that causes accumulation of thick mucus in the lungs, pancreas and other organs. In the lungs, the mucus obstructs the airways and traps bacteria leading to infections, extensive lung damage and eventually, respiratory failure. Previously, our murine model demonstrated an increased production of heavy chain-hyaluronic acid (HC-HA) deposition in CF in comparison to WT mice in chronic model of Pseudomonas aeruginosa (PA) infection. We undertook the current using both PA and Respiratory Syncytial Virus (RSV) infection study to examine whether increased HA levels in CF lung tissue are infection-dependent, or as a result of CFTR dysfunction.
Methods: Murine tracheal epithelial cells (mTEC) from WT and delta-F508 CF mice, and primary human bronchial epithelial cells from healthy and CF patients, were grown on semipermeable membrane filters differentiated to air-liquid interface (ALI) conditions. Cells were infected with RSV at a multiplicity of infection (MOI) of 0.5 for 24 hours, then P. aeruginosa at MOI of 0.1, 1, and 10 for 18 hours. HA was quantified by fluorophore-assisted carbohydrate-electrophoresis (FACE). The distributions and colocalization of HA with inter-alpha-inhibitor (IαI), which is an indicative of the covalent HC modification of HA, were examined by immunofluorescence microscopy.
Results: Differentiated epithelial cultures showed ciliary movement, mucus production and possessed a transepithelial electrical resistance (TEER) between 350 and 810 Ω*cm2, with CF mTEC having higher resistance values. FACE analysis showed HA to be present in the apical media and cell lysate of both CF and non-CF cells; with HA concentration higher in non-CF compared to CF. HA levels in healthy controls dropped significantly upon infection with RSV and PA, but no additive affect is seen when in combination. CF HA production was reduced only upon infection with RSV.
Conclusion: CF cells produce less HA than healthy, non-CF cells. These data also demonstrate that RSV infection significantly alters hyaluronan production in both healthy and cystic fibrosis cells; but that Pseudomonas reduces levels in only the former. The effect of HA transport across the lung epithelia is yet to be understood, and future studies will be required to elucidate the mechanisms by which CFTR dysfunction occurs as a result of infection.
Author Block: C. C. Smallcombe1, G. Altawallbeh1, T. L. Bonfield2, F. Rezaee1, G. Piedimonte1; 1Pathobiology, Cleveland Clinic, Lerner Research Institute, Cleveland, OH, United States, 2Case Western Reserve Univ, Cleveland, OH, United States.
Rationale: Cystic Fibrosis (CF) is a genetic disease that causes accumulation of thick mucus in the lungs, pancreas and other organs. In the lungs, the mucus obstructs the airways and traps bacteria leading to infections, extensive lung damage and eventually, respiratory failure. Previously, our murine model demonstrated an increased production of heavy chain-hyaluronic acid (HC-HA) deposition in CF in comparison to WT mice in chronic model of Pseudomonas aeruginosa (PA) infection. We undertook the current using both PA and Respiratory Syncytial Virus (RSV) infection study to examine whether increased HA levels in CF lung tissue are infection-dependent, or as a result of CFTR dysfunction.
Methods: Murine tracheal epithelial cells (mTEC) from WT and delta-F508 CF mice, and primary human bronchial epithelial cells from healthy and CF patients, were grown on semipermeable membrane filters differentiated to air-liquid interface (ALI) conditions. Cells were infected with RSV at a multiplicity of infection (MOI) of 0.5 for 24 hours, then P. aeruginosa at MOI of 0.1, 1, and 10 for 18 hours. HA was quantified by fluorophore-assisted carbohydrate-electrophoresis (FACE). The distributions and colocalization of HA with inter-alpha-inhibitor (IαI), which is an indicative of the covalent HC modification of HA, were examined by immunofluorescence microscopy.
Results: Differentiated epithelial cultures showed ciliary movement, mucus production and possessed a transepithelial electrical resistance (TEER) between 350 and 810 Ω*cm2, with CF mTEC having higher resistance values. FACE analysis showed HA to be present in the apical media and cell lysate of both CF and non-CF cells; with HA concentration higher in non-CF compared to CF. HA levels in healthy controls dropped significantly upon infection with RSV and PA, but no additive affect is seen when in combination. CF HA production was reduced only upon infection with RSV.
Conclusion: CF cells produce less HA than healthy, non-CF cells. These data also demonstrate that RSV infection significantly alters hyaluronan production in both healthy and cystic fibrosis cells; but that Pseudomonas reduces levels in only the former. The effect of HA transport across the lung epithelia is yet to be understood, and future studies will be required to elucidate the mechanisms by which CFTR dysfunction occurs as a result of infection.
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