Asthmatic Airway Epithelium Is Intrinsically Inflammatory and Mitotically Dyssynchronous

Abstract

Asthma is an inflammatory condition for which anti-inflammatory glucocorticoids are the standard of care. However, similar efficacy has not been shown for agents targeting inflammatory cells and pathways. This suggests a noninflammatory cell contributor (e.g., epithelium) to asthmatic inflammation. Herein, we sought to define the intrinsic and glucocorticoid-affected properties of asthmatic airway epithelium compared with normal epithelium. Human primary differentiated normal and asthmatic airway epithelia were cultured in glucocorticoid-free medium beginning at 248 hours. They were pulsed with dexamethasone (20 nM) or vehicle for 2 hours at 226, 22, 122, and 146 hours. Cultures were mechanically scrapewounded at 0 hours and exposed continuously to bromodeoxyuridine (BrdU). Cytokine secretions were analyzed using cytometric bead assays. Wound regeneration/mitosis was analyzed by microscopy and flow cytometry. Quiescent normal (n 5 3) and asthmatic (n 5 6) epithelia showed similar minimal inflammatory cytokine secretion and mitotic indices. After wounding, asthmatic epithelia secreted more basolateral TGF-b1, IL-10, IL-13, and IL-1b (P , 0.05) and regenerated less efficiently than normal epithelia (148 h wound area reduction 5 [mean 6 SEM] 50.2 6 7.5% versus 78.6 6 7.7%; P 5 0.02). Asthmatic epithelia showed 40% fewer BrdU 1 cells at 148 hours (0.32 6 0.05% versus 0.56 6 0.07% of total cells; P 5 0.03), and those cells were more dyssynchronously distributed along the cell cycle (52 6 10, 25 6 4, 23 6 7% for G1/G0, S, and G2/M, respectively) than normal epithelia (71 6 1, 12 6 2, and 17 6 2% for G1/G0, S, and G2/M, respectively). Dexamethasone pulses improved asthmatic epithelial inflammation and regeneration/mitosis. In summary, we show that inflammatory/fibrogenic cytokine secretions are correlated with dyssynchronous mitosis upon injury. Intermittent glucocorticoids simultaneously decreased epithelial cytokine secretions and resynchronized mitosis. These data, generated in an airway model lacking inflammatory cells, support the concept that epithelium contributes to asthmatic inflammation.

A Heterotopic Xenograft Model of Human Airways for Investigating Fibrosis in Asthma

“…Both the asthmatic grafted epithelium and the asthmatic human lung biopsy tissue show decreased E-cadherin expression. This is all consistent with our previous work showing that, when differentiated at air-liquid interface culture, asthmatic-derived epithelial cells are less differentiated in comparison to nonasthmatic-derived cells, as demonstrated by increased basal cell number and lack of E-cadherin (5,6,11,19).…”

“…We and others (6,11,41) have shown previously that basolateral secretion of TGF-b1 is increased from asthmatic airway epithelium in vitro. The current study furthers that work by demonstrating increased TGF-b1 in asthmatic xenografts, an in vivo system.…”

“…These repair processes are defective in asthma, as evidenced by a reduction in the cell-cell adhesion proteins involved in barrier function (5), asynchrony in mitotic cell division, and increased production of inflammatory mediators and growth factors, such as transforming growth factor (TGF)-b1 (6)(7)(8)(9).…”

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…After 14-21 days (EpiAirway) or 20-45 days (MucilAir) of culture the epithelia are fully ciliated and electrically tight (TEER≈450 Ω cm 2 ). EpiAirway cultures are available in a number of useful insert formats, and have been successfully utilized for numerous applications including airway drug delivery (Agu et al, 2006;Leonard et al, 2005Leonard et al, , 2007, airway disease modelling (Bai et al, 2015;Freishtat et al, 2011; ALI cultures have provided insights into pathogenetic mechanisms, for example the ability of Th2 cytokines, IL-4 and IL-13, to skew bronchial epithelial cell differentiation towards goblet cell hyperplasia, increased mucin gene expression and mucous hypersecretion (Kanoh et al, 2011;Thavagnanam et al, 2011;Zuyderduyn et al, 2011). Furthermore, use of ALI cultures has allowed for comparisons of barrier properties, mucus production and other responses of epithelial cells from asthmatic and healthy subjects, as well as the demonstration that tight junctions are reduced and mucus production and inflammatory cytokine induction are increased in cultures from asthmatic subjects (as observed in vivo).…”

“…Furthermore, use of ALI cultures has allowed for comparisons of barrier properties, mucus production and other responses of epithelial cells from asthmatic and healthy subjects, as well as the demonstration that tight junctions are reduced and mucus production and inflammatory cytokine induction are increased in cultures from asthmatic subjects (as observed in vivo). Such changes have been seen to correspond to an increase in ionic and macromolecular permeability (Xiao et al, 2011;Bai et al, 2015;Freishtat et al, 2011;Watson et al, 2010). It has also been shown that the permeability of the bronchial epithelial cell layer is only increased in asthmatic subjects after exposure to ozone or nitrogen dioxide (Bayram et al, 2002), and that mediator release is altered after exposure of ALI cultures from asthmatic donors to respiratory syncytial virus (RSV) or particulate matter (Hackett et al, 2011b).…”