The serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells

Abstract

Background: Serine proteases act through the proteolytic cleavage of the hemagglutinin (HA) of influenza viruses for the entry of influenza virus into cells, resulting in infection. However, the inhibitory effects of serine protease inhibitors on influenza virus infection of human airway epithelial cells, and on their production of inflammatory cytokines are unclear.

Methods: Primary cultures of human tracheal epithelial cells were treated with four types of serine protease inhibitors, including camostat, and infected with A/Sendai-H/108/2009/(H1N1) pdm09 or A/New York/55/2004(H3N2).

Results: Camostat reduced the amounts of influenza viruses in the supernatants and viral RNA in the cells. It reduced the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. Camostat also reduced the concentrations of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the supernatants. Gabexate and aprotinin reduced the viral titers and RNA levels in the cells, and aprotinin reduced the concentrations of TNF-α in the supernatants. The proteases transmembrane protease serine S1 member (TMPRSS) 2 and HAT (human trypsin-like protease: TMPRSS11D), which are known to cleave HA0 and to activate the virus, were detected at the cell membrane and in the cytoplasm. mRNA encoding TMPRSS2, TMPRSS4 and TMPRSS11D was detectable in the cells, and the expression levels were not affected by camostat.

Conclusions: These findings suggest that human airway epithelial cells express these serine proteases and that serine protease inhibitors, especially camostat, may reduce influenza viral replication and the resultant production of inflammatory cytokines possibly through inhibition of activities of these proteases.

Keywords: Airway epithelial cell; Camostat; Cell culture; Influenza; Interleukin; Serine protease.

Fig. 1

A and B: The time course of virus release into the supernatants of primary cultures of human tracheal epithelial (HTE) cells that were obtained at different time-points after exposure to the influenza A/H1N1 pdm 2009 virus (A) or the seasonal influenza A/H3N2 virus (B) in the presence of camostat (10 μg/mL) (closed circles and open triangles) or the vehicle control (1% water) (Control, open circles) from 30 min prior to infection (open and closed circles) or from just after infection (open triangles) until the end of the experiments. The results are expressed as the mean ± SEM (n = 5). Significant differences compared to viral infection alone are indicated by *p < 0.05, **p < 0.01 and ***p < 0.001. C and D: Concentration-dependent effects of the serine protease inhibitor camostat on viral release into supernatants 5 days after infection with the influenza A/H1N1 pdm 2009 virus (C) or the influenza A/H3N2 virus (D). The results are expressed as the mean ± SEM (n = 5). Significant differences compared to vehicle alone (Vehicle) are indicated by *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 2

Western blot analysis of proteins in the supernatants of primary cultures of HTE cells 72 h post infection with the A/H1N1 pdm 2009 virus in the presence of camostat (0.1, 1 or 3 μg/mL) or vehicle (0), showing inhibition of HA0 cleavage. HA0: a hemagglutinin precursor protein, HA1: hemagglutinin subunit, MOCK: without infection.

Fig. 3

A and B: Indirect immunofluorescence staining of TMPRSS2 (A) and TMPRSS11D (B) in primary cultures of HTE cells. TMPRSS2 and TMPRSS11D are stained orange at the cell membrane and in the cytoplasm. Nuclei are stained blue. Magnification: × 630. C: Expression of TMPRSS2 mRNA, TMPRSS4 mRNA and TMPRSS11D mRNA in HTE cells treated with camostat (10 μg/mL) or the vehicle control (1% water). The results are expressed as the ratio of TMPRSSs (TMPRSS2, TMPRSS4 or TMPRSS11D) mRNA expression compared with β-actin mRNA and are reported as the mean ± SEM (n = 3). Significant differences compared to the values of TMPRSS2 in the cells treated with vehicle alone (Vehicle) are indicated by **p < 0.01. Significant differences compared to the values of TMPRSS4 in the cells treated with vehicle alone (Vehicle) are indicated by ++p < 0.01. Camostat did not affect the expression of mRNA of three types of TMPRSSs.