Non-typeable Haemophilus influenzae decreases cilia beating via protein kinase Cε.

Kristina L Bailey,Tricia D Levan,Jane M Devasure,Daniel A Yanov,Joseph H Sisson,Todd A Wyatt,Jaqueline A Pavlik

doi:10.1186/1465-9921-13-49

Abstract

BackgroundHaemophilus influenzae infection of the nasal epithelium has long been associated with observations of decreased nasal ciliary beat frequency (CBF) and injury to the ciliated epithelium. Previously, we have reported that several agents that slow CBF also have the effect of activating protein kinase C epsilon (PKCϵ) activity in bronchial epithelial cells. The subsequent auto-downregulation of PKCϵ or the direct inhibition of PKCϵ leads to the specific detachment of the ciliated cells. METHODS: Primary cultures of ciliated bovine bronchial epithelial cells were exposed to filtered conditioned media supernatants from non-typeable H. influenzae (NTHi) cultures. CBF and motile points were measured and PKCϵ activity assayed.ResultsNTHi supernatant exposure significantly and rapidly decreased CBF in a dose-dependent manner within 10 minutes of exposure. After 3 hours of exposure, the number of motile ciliated cells significantly decreased. Direct measurement of PKCϵ activity revealed a dose-dependent activation of PKCϵ in response to NTHi supernatant exposure. Both CBF and PKCϵ activity changes were only observed in fresh NTHi culture supernatant and not observed in exposures to heat-inactivated or frozen supernatants.ConclusionsOur results suggest that CBF slowing observed in response to NTHi is consistent with the stimulated activation of PKCϵ. Ciliated cell detachment is associated with PKCϵ autodownregulation.

Highlights

Non-typeable Haemophilus influenzae (NTHi) is a nonmotile, pleomorphic, gram-negative rod
Our results suggest that ciliary beat frequency (CBF) slowing observed in response to non-typeable H. influenzae (NTHi) is consistent with the stimulated activation of PKCε
Supernatants from NTHi slow cilia in a concentrationdependent manner To determine the time and concentration dependence of NTHi exposure on CBF, bovine bronchial epithelial cells (BBEC) were exposed to increasing concentrations (0%, 10%, 50%, 100%) of NTHi supernatant at multiple time points

Summary

Introduction

Non-typeable Haemophilus influenzae (NTHi) is a nonmotile, pleomorphic, gram-negative rod. When COPD patients are colonized with H. influenza in the stable state, they have an increased number of COPD exacerbations. They have increased symptoms, and increased sputum purulence during exacerbations [1,2]. Part of the detrimental effect NTHi has in COPD may be related to its effects on mucociliary transport. We have reported that several agents that slow CBF have the effect of activating protein kinase C epsilon (PKCε) activity in bronchial epithelial cells. METHODS: Primary cultures of ciliated bovine bronchial epithelial cells were exposed to filtered conditioned media supernatants from non-typeable H. influenzae (NTHi) cultures.

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