Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection

Henneke P (2013) Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection. PLoS ONE 8(3): e59925. doi:10.1371/journal.pone.0059925 Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection Manfred Fliegauf 0 Andreas F.-P. Sonnen 0 Bernhard Kremer 0 Philipp Henneke 0 Samithamby Jeyaseelan, Louisiana State University, United States of America 0 1 Centre of Chronic Immunodeficiency (CCI), University Medical Centre Freiburg and University of Freiburg , Freiburg, Germany , 2 Department of Paediatrics and Adolescent Medicine, University Medical Centre Freiburg , Freiburg , Germany Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance. - Funding: This study was supported by the German Federal Ministry of Education and Research (BMBF 01 EO0803) and by DFG grant HE 3127/5-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Pneumonia can be caused by various pathogens (bacteria, viruses, fungi) and is a leading cause of death due to infectious disease in industrialized countries [1]. Streptococcus pneumoniae (pneumococcus) is the major pathogen of community-acquired pneumonia and causes more than one million infant deaths every year worldwide [2]. Pneumococci usually asymptomatically colonize the upper respiratory tract (nasopharynx) of humans [2]. Accordingly, they mainly exist as commensal bacteria along with other co-resident microorganisms [3]. Colonizing pneumococci can persist for weeks in adults or even months in children without any medical sequelae (colonization stage/carrier stage). On occasions, pneumococci pass to other areas where they can cause severe diseases (pathogenic stage). These include the lower airways/lungs (pneumonia), the middle ear (middle ear infections/ otitis media), the cerebrospinal fluid of the brain (meningitis), and the blood (bacteriaemia or septicemia), respectively [4,5]. Although pneumococci are thought to follow similar strategies to attack ciliated respiratory and ciliated ependymal epithelia, the mechanisms that transform the persistently colonizing phenotype to an invasive pneumococcal disease with high morbidity and mortality are poorly understood. Under normal conditions, the tracheal, bronchial and lung epithelia act as a mechanical barrier and sentinel system against pathogens. The mucus in the tracheo-bronchial tree traps inhaled particles, pathogens and toxins and transports them quickly through the trachea towards the pharynx by means of ciliary beating and cough [6,7]. Each ciliated epithelial cell carries approximately 200 cilia (,710 mm in length), which move the extracellular mucus by constant, orchestrated and vigorous beating [6,8]. This mucociliary clearance mechanism ensures that inhaled particles do not come into direct contact with the epithelial cells and do not reach the alveolar cavities. Only when pathogens have resisted mucociliary clearance and have spread within the lung tissue, are resident alveolar macrophages required to neutralize the pathogenic bacteria and/or to recruit other elements of innate immunity e.g. via Toll-like receptor-mediated signaling [1,9]. The importance of the mucociliary escalator is further emphasized by the consequences of its dysfunction. For instance, patients with impaired motile cilia function (PCD, primary ciliary dyskinesia) and patients with highly viscous mucus (as in CF, Cystic Fibrosis) suffer from recurrent and severe sinopulmonary infections that can result in chronic scarring and bronchiectasis [10]. Based on the observations that reduced mucus flow is advantageous for the pathogens to resist airway clearance, it has been suggested that pneumococci (and analogously other airway pathogens) might tightly adhere to ciliated epithelia and/or slow down the ciliary beat. Other possible mechanisms to escape the powerful forces of mechanical clearance include embedding into biofilms, lytic damage or direct invasion of host epithelial cells, or increase of mucus viscosity [11,12]. However, simplified experimental (murine) models that allow for analysis of the dynamic interaction of pathogenic organisms and the highly specialized ciliated respiratory epithelium were difficult to establish and only recently became available [13,14]. Pneumolysin is a key virulence factor in pneumococcal infections [2,5,1521]. The 53 kDa monomer is released at low concentrations during colonization but at high levels upon bacterial autolysis in later stages of infection. 3050 monomers assemble a large transmembrane channel (,260 A in diameter) that allows for free exchange of ions and small molecules and eventually mediates cell lysis and tissue damage. Purified pneumolysin has been reported to cause rapid, dose-dependent inhibition of the ependymal ciliary beat frequency and simultaneous cell damage. However, a pneumolysin-deficient strain had similar effects when compared to the parental wildtype pneumococcal strain, suggesting additional, (...truncated)