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

Abstract

Background: Haemophilus 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.

Results: NTHi 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.

Conclusions: Our 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.

Figure 1

Supernatants from NTHi slow ciliary beat frequency in a time-and concentration-dependent manner.A) Bovine bronchial epithelial cells (BBECs) were exposed to 0%, 10%, 50% and 100% NTHi supernatant for 10 minutes. Ciliary beat frequency (CBF) was measured each minute. NTHi supernatant exposure rapidly slows CBF at the 50% (*p < 0.05) and 100% (#p < 0.001) concentrations. There is no difference between 10% and media control. (n = 6) B) BBECs were exposed to 50% NTHi supernatant, or 50% NTHi growth medium for up to 48 hours and CBF was measured hourly. The CBF slowing is sustained, with statistically significant CBF slowing at each timepoint compared to control. (*p < 0.05; #p < 0.001 vs. media) (n = 8).

Figure 2

PKCϵ activity is rapidly stimulated by NTHi.A) Bovine bronchial epithelial cells (BBECs) were exposed to 0, 10, 25, 50, and 100% NTHi for 1–120 minutes. The 50% and 100% NTHi rapidly stimulates PKCϵ within 1 minute. PKCϵ activity returns to baseline after 2 hours (p < 0.05 Media vs. 50% from 1–60 min. p < 0.01 Media vs. 100% from 1–60 min) (n = 6). B) PKCϵ activity remains auto-downregulated at 24 hours (*p < 0.001 vs. Media) (n = 6).

Figure 3

NTHi supernatant fails to activatePKCϵif it is heat-inactivated or frozen.A) Heat inactivation of the NTHi for 5 minutes at 95°C blocks the stimulation of PKCϵ at 10 min and 1 hr (*p < 0.05) (n = 6). PMA is included as a positive control for PKCϵ activation. B) Freeze-thawing the NTHi supernatant blocks the stimulation of PKCϵ (*p < 0.05) at 10 min and 1 hr (n = 6).

Figure 4

NTHi supernatant decreases the total number of motile points after 24–48 hours of exposure. BBECs were exposed to 50% NTHi supernatant and 50% NTHi growth media alone (Brain-heart infusion broth) for 1–48 hours. The total number of motile points was measured. At 24 and 48 hrs, a significant decrease in motile cilia was detected (*p < 0.01) in the ciliated cells exposed to NTHi supernatents (n = 8).

Figure 5

NTHi supernatant does not cause cell lysis, but it does result in the preferential detachment of ciliated cells.A) Media supernatants were collected from bovine bronchial cell cultures treated with 50% NTHi supernatants. Supernatants were assayed for lactate dehydrogenase (LDH) activity. As a positive control, cells were intentionally lysed using sonication. The cells that were exposed to 100% NTHi for 1–48 hours showed no elevation in LDH. This indicates that NTHi does not cause cytotoxicity (*p < 0.05 + control vs. NTHi) (n = 6) B) BBECs were exposed to 50% NTHi supernatant for 1–48 hours. The supernatant from these cultures was pelleted to collect any ciliated cells that had detached. Pellets were vacuum transferred and blotted for 13S dynein. Detached ciliated cells were detected at 18–48 hr NTHi supernatant exposure. Positive control is purified bovine tracheal axonemes (0.1 μg) (n = 3).

Figure 6

Schematic for the proposed role of PKC epsilon.