Zulianello and colleagues (5) reported recently that invasion of Pseudomonas aeruginosa into primary human airway (but, more accurately, nasal) epithelium was dependent on bacterial secretion of rhamnolipids to disrupt tight junctions, which promoted paracellular bacterial invasion of the epithelium. They questioned the work of myself and colleagues reporting that the cystic fibrosis transmembrane conductance regulator (CFTR) was a receptor for P. aeruginosa that mediated intracellular endocytosis of airway epithelial cells. While their results support their conclusions, the polarized nasal epithelial monolayers they used are completely unrepresentative of the actual airway epithelial layer in the lung, where transepithelial resistances (TER) in freshly excised human airways are <100 Ω/cm2, typically 60 Ω/cm2, as reported by Boucher et al. and Coyne et al. (2, 3) from the University of North Carolina. In fetal lamb airway epithelia the TER was also much lower (1) than the initial TER of the monolayers used by Zulianello et al. (5). The use of polarized monolayers with TER > 100 Ω/cm2 represents an in vitro artifact when trying to mimic the TER of the human airway epithelium. Consistent with the contention that low TER, as is normally found in the airway, permits P. aeruginosa invasion, Zulianello et al. (5) found that when the TER was reduced after prolonged exposure to rhamnolipids, P. aeruginosa invasion occurred. It was at this point that the TER of their monolayers truly represented that of the intact lung, and this should have been their starting point, where CFTR-dependent interactions could take place (4). In addition, Zulianello et al. (5) neither cited nor commented on our in vivo demonstration in mouse and monkey tracheas that P. aeruginosa is readily internalized into the apical tracheal epithelium 4 h postinfection, as visualized by scanning electron microscopy, whereas no such interaction is seen in transgenic cystic fibrosis knockout mice (4). I would ask Zulianello and colleagues to explain why their hyperpolarized nasal epithelial monolayers should be considered representative of the human airway epithelium that has a normal TER of <100 Ω/cm2, what problems they see with the data of Boucher's group in their reports that the TER of freshly excised airways is low, what contravening data they have regarding these TER measurements on excised airway epithelia, and why their in vitro system, lacking all accessory factors present in the normal airway and based on polarized nasal epithelial cells, is informative and in contrast to our in vivo demonstration of clear, CFTR-dependent, apical invasion of P. aeruginosa into the epithelium in an intact tissue in a living animal, i.e., both mouse and monkey tracheas.
- Copyright © 2006 American Society for Microbiology
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Author's Reply
Our objective is to define the mechanisms involved in the early interaction between Pseudomonas aeruginosa and human epithelial respiratory cells. Although cystic fibrosis mouse models have been developed and contribute significantly to a better understanding of many aspects of the disease (5), we have developed an alternative in vitro model of reconstituted human airway epithelia from primary nasal cells in order to identify virulence factors secreted by the bacterium that might disrupt the epithelial barrier. This model will be suitable to conduct drug delivery studies to strengthen human epithelial resistance towards bacterial infiltration.
Reconstituted human airway epithelia allowed us to identify rhamnolipids as essential virulence factors to enhance the infiltration of Pseudomonas aeruginosa between epithelial cells without favoring their internalization. Rhamnolipids also lead to an improper assembly and interactions of the tight junction strands, which, as a result, functionally release the barrier that normally prevents the access of P. aeruginosa to the paracellular space. The bacteria thus have free access to this space and passively invade the epithelium.
The use of our reconstituted epithelia and unique model system of infection, with Pseudomonas expressing rhlA promoter fusions, will enable us to screen for chemical compounds that might prevent the production of rhamnolipids by infecting bacteria and/or their interaction with the host membrane. Our infection model system allows the simultaneous monitoring of the expression of rhamnolipids and of the cytotoxicity of these compounds on the human airway epithelia.
Fully polarized airway epithelial cell monolayers are suitable in vitro models to analyze host-pathogen interactions. Airway epithelial (nasal) cell monolayers used in our studies are representative of and resemble the epithelial nasal tissue of the respiratory tract. Electron microscopy confirms that the monolayer is pseudostratified and highly differentiated, and it shows numerous microvilli and cilia on the apical surface.
While, indeed, the short circuit corresponds to 70 to 100 μA/cm2 (6), as reported by Boucher et al. (2), the transepithelial resistances (TER) of reconstituted human epithelia from nasal biopsies are generally around ≥500 (up to 2,000) Ω.cm2 (4). Techniques of culturing primary human nasal cells described by Yamaya et al. have been shown to have properties similar to those of the original tissue and to our culture system with respect to their ultrastructure and ion transport, and the TER is around 2,000 Ω.cm2 (10). Recently, Boucher and colleagues have been performing epithelial permeability tests on in vitro cultures of primary airway cells from human subjects and selected specifically cell cultures with a TER of ≥600 Ω/cm2 to start with fully polarized cells (3).
The apparent discrepancies between the initial analyses of Boucher (8) and others might be attributed to the difficulties in comparing functions of epithelia freshly excised from human subjects and reconstituted, well-differentiated human airway epithelia cultures on cell support. It cannot also be excluded that surgery, excision, and hypoxia of human airway samples leads to abnormal bioelectric parameters (8) that will not be observable after 15 days of culture. It is also not excluded that bioelectric properties of bronchi are affected due to an induction in expression of specific claudins (or tight junction-associated proteins) that might lead to a more leaky barrier when resection was performed. To end with this point, it should be mentioned that human nasal epithelial cells, fully polarized, differentiated, and with a significant TER (≥500 Ω.cm2), are only observed when collagen support matrices are used (1). It should also be underlined that, indeed, relatively higher TER (1,349 ± 508 Ω.cm2) are seen in polymerized collagen such as Vitrogen gel (11, 12). However, based on mitochondrial dehydrogenase activity, lactate dehydrogenase release, ciliary beat frequency studies, TER, permeation, and electron microscopy studies, this gel is functionally suitable for in vitro reconstitution of nasal epithelia (1, 12).
Upon treatment with 150 μg/ml purified rhamnolipids, a significant drop of TER is observed within 60 min that permits P. aeruginosa infiltration and residency in the paracellular space (for as long as 5 h after treatment with rhamnolipids). This infiltration is also observed after an overnight infection of the human cells with PAO1. As shown by confocal and electron microscopies, Pseudomonas aeruginosa resides in the intercellular spaces and was not internalized by the epithelial cells, although these cells are positive for the cystic fibrosis transmembrane conductance regulator (CFTR).
In our model, shorter exposures to PAO1 (from 1 to 5 h and up to 8 h) in the absence of rhamnolipids do not induce the binding of the bacteria to the cell surface or its internalization by host cells.
Furthermore, rhamnolipids, either purified or expressed by PAO1, alter the tight junction architecture, causing a loss of cell polarity with the displacement of apical markers, such as ezrin, to the basolateral membranes. Therefore, when the TER is lowered by the toxin (≤200 Ω.cm2), apical and basolateral markers are no longer segregated in separated domains which do not support a central role for CFTR as an apical receptor at this stage. Furthermore, polarized epithelial cells lacking CFTR were also infiltrated by PAO1 once the polarity was lost. By scanning electron microscopy studies, Schroeder and colleagues (9) observed that the main location for uptake of bacteria is on the edge of the transition of ciliated to nonciliated epithelia cells. Will electron microscopy be more informative? In a previous study, internalization of the bacteria was evaluated after gentamicin treatment, which has been reported to favor expression of specific virulence factors by Pseudomonas (7). The antibiotic exclusion assay may also account for the different observations made by Schroeder and colleagues (10) and our studies (12) on bacterial internalization.
In conclusion, nasal and bronchial cell culture systems (cystic fibrosis and control) must be developed and validated as alternatives to animal models. This is imperative to conduct nasal drug delivery studies. To this end, primary cultures of human nasal epithelium are the most relevant.
