Air-liquid interface cultures using murine tracheal respiratory epithelium have revolutionized the in vitro study of airway diseases. However, these cultures often are impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. The ability to study ciliary physiology in vitro is of utmost importance in the research of chronic rhinosinusitis (CRS). Our hypothesis is that the murine nasal septum is a better source of ciliated respiratory epithelium to develop respiratory epithelial air-liquid interface models.
Nasal septa and tracheas were harvested from 10 BALB/c mice. The nasal septa were harvested by using a simple and straightforward novel technique. Scanning electron microscopy was performed on all specimens. Cell counts of ciliated respiratory epithelial cells were performed at one standard magnification (1535x). Comparative analysis of proximal and distal trachea, midanterior and midposterior nasal septal epithelium, was performed.
Independent cell counts revealed highly significant differences in the proportion of cell populations (p < 0.00001). Ciliated cell counts for the trachea (106.9 +/- 28) were an average of 38.7% of the total cell population. Nasal septal ciliated epithelial cells (277.5 +/- 16) comprised 90.1% of the total cell population.
To increase the yield of respiratory epithelial cells harvested from mice, we have found that the nasal septum is a far superior source when compared with the trachea. The greater surface area and increased concentration of ciliated epithelial cells has the potential to provide an eightfold increase in epithelial cells for the development of air-liquid interface cultures.
"Murine Sinonasal Epithelial cells (MNSE) were harvested and differentiated at an air-liquid interface previously described [16,17]. Briefly, tissue was harvested and grown on Costar 6.5-mm-diameter permeable filter supports (Corning Life Sciences, Lowell, MA) submerged in culture medium. "
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is characterized by acute pulmonary exacerbations (APE). The CF nasal airway exhibits a similar ion transport defect as the lung, and colonization, infection, and inflammation within the nasal passages are common among CF patients. Nasal lavage fluid (NLF) is a minimally invasive means to collect upper airway samples.
We collected NLF at the onset and resolution of CF APE and compared a 27-plex cytokine profile to stable CF outpatients and normal controls. We also tested IP-10 levels in the bronchoalveolar lavage fluid (BALF) of CF patients. Well-differentiated murine sinonasal monolayers were exposed to bacterial stimulus, and IP-10 levels were measured to test epithelial secretion.
Subjects hospitalized for APE had elevated IP-10 (2582 pg/mL [95% CL of mean: 818,8165], N=13) which significantly decreased (647 pg/mL [357,1174], P<0.05, N =13) following antimicrobial therapy. Stable CF outpatients exhibited intermediately elevated levels (680 pg/mL [281,1644], N=13) that were less than CF inpatients upon admission (P=0.056) but not significantly different than normal controls (342 pg/mL [110,1061]; P=0.3, N=10). IP-10 was significantly increased in CF BALF (2673 pg/mL [1306,5458], N=10) compared to healthy post-lung transplant patients (8.4 pg/mL [0.03,2172], N=5, P<0.001). IP-10 levels from well-differentiated CF murine nasal epithelial monolayers exposed to Pseudomonas PAO-1 bacteria-free prep or LPS (100 nM) apically for 24 hours were significantly elevated (1159 ± 147, P<0.001 for PAO-1; 1373 ± 191, P<0.001 for LPS vs. 305 ± 68 for vehicle controls). Human sino-nasal epithelial cells derived from CF patients had a similar response to LPS (34% increase, P<0.05, N=6).
IP-10 is elevated in the nasal lavage of CF patients with APE and responds to antimicrobial therapy. IP-10 is induced by airway epithelia following stimulation with bacterial pathogens in a murine model. Additional research regarding IP-10 as a potential biomarker is warranted.
PLoS ONE 08/2013; 8(8):e72398. DOI:10.1371/journal.pone.0072398 · 3.23 Impact Factor
"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 , . 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 , . "
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 03/2013; 8(3):e59925. DOI:10.1371/journal.pone.0059925 · 3.23 Impact Factor
"In addition, cultures were observed with light microscopy following sectioning and staining with toluidine blue (Figure 4e,f). We observed a trend for a greater percentage of ciliated cells in nasal respiratory cell cultures compared with tracheal cell cultures as previously reported26,27; otherwise, there were no striking differences in morphology between the cells cultured from the two regions. In addition, both nasal and tracheal cultures exhibited similar amiloride-sensitive currents and cAMP-activated CFTR-dependent chloride currents (data not shown). "
[Show abstract][Hide abstract] ABSTRACT: Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN) responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV) in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction.Molecular Therapy - Nucleic Acids (2013) 2, e69; doi:10.1038/mtna.2012.60; published online 29 January 2013.
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