Mitotic asynchrony induces transforming growth factor-β1 secretion from airway epithelium

Abstract

We recently proposed that mitotic asynchrony in repairing tissue may underlie chronic inflammation and fibrosis, where immune cell infiltration is secondary to proinflammatory cross-talk among asynchronously repairing adjacent tissues. Building on our previous finding that mitotic asynchrony is associated with proinflammatory/fibrotic cytokine secretion (e.g., transforming growth factor [TGF]-β1), here we provide evidence supporting cause-and-effect. Under normal conditions, primary airway epithelial basal cell populations undergo mitosis synchronously and do not secrete proinflammatory or profibrotic cytokines. However, when pairs of nonasthmatic cultures were mitotically synchronized at 12 hours off-set and then combined, the mixed cell populations secreted elevated levels of TGF-β1. This shows that mitotic asynchrony is not only associated with but is also causative of TGF-β1 secretion. The secreted cytokines and other mediators from asthmatic cells were not the cause of asynchronous regeneration; synchronously mitotic nonasthmatic epithelia exposed to conditioned media from asthmatic cells did not show changes in mitotic synchrony. We also tested if resynchronization of regenerating asthmatic airway epithelia reduces TGF-β1 secretion and found that pulse-dosed dexamethasone, simvastatin, and aphidicolin were all effective. We therefore propose a new model for chronic inflammatory and fibrotic conditions where an underlying factor is mitotic asynchrony.

Keywords: asthma; fibrosis; mitosis; transforming growth factor-β1.

Figure 1.

Induced mitotic asynchrony in proliferating nonasthmatic airway epithelial basal cells stimulates transforming growth factor (TGF)-β1 secretion. (A) Experimental design for inducing mitotic asynchrony in nonasthmatic primary airway epithelial basal cells. Mitotic asynchrony was induced in parallel submerged cultures of nonasthmatic proliferating airway epithelial basal cells via transient exposure to basic media only (for 12 h) in a staggered fashion. At 0 hours, cells from one flask were labeled with the fluorescent membrane dye PKH67. Aliquots of each were reserved as controls before mixing the cultures. Cultures were continuously exposed to bromodeoxyuridine (BrdU). Cells and media were collected at 0, +6, +12, and +24 hours. (B) Cell cycle phase was analyzed by flow cytometry for 7-AAD DNA staining in BrdU⁺ cells. At +6 hours, the mixed cultures showed asynchronous mitosis (G1 [mean ± SEM], S, G2/M: 47 ± 7, 22 ± 2, 31 ± 8%, respectively). Data are shown as mean ± SEM (n = 7 nonasthmatic donors). (C) BrdU⁺ cells as a percentage of the total number of cells in the culture at each time point according to condition. PKH67⁻ cells (i.e., basic before complete medium) proliferate more rapidly in isolation than in mixed conditions. (D) Supernatants were analyzed for TGF-β1 at each time point and compared according to condition. The cells grown in mixed conditions show elevated TGF-β1 secretion between +6 and +12 hours compared with either cell population in isolation. Data are shown as mean ± SEM (n = 5 nonasthmatic donors). *P < 0.05.

Figure 2.

Secreted mediators do not induce mitotic asynchrony in regenerating nonasthmatic airway epithelia. (A) Experimental schema for wounded nonasthmatic (non-) epithelium exposed to conditioned media from regenerating nonasthmatic (non-) or asthmatic (asthma) airway epithelia. Nonasthmatic and asthmatic airway epithelia were cultured, and apical secretions and basolateral conditioned media were collected. New nonasthmatic epithelium was exposed apically to these secretions and basolaterally to these conditioned media as shown for the corresponding times. For example, secretions and media collected from +22 to +24 hours was used for +0 to +22 hours in the new epithelial cultures. (B) Mitotic synchrony/asynchrony according to secretion exposure. Data are shown as mean ± SEM (n = 3 asthmatic and 3 nonasthmatic epithelia). BrdU, bromodeoxyuridine.

Figure 3.

Asthmatic epithelial mitotic asynchrony and transforming growth factor (TGF)-β1 secretion are improved by dexamethasone (DEX). (A) Mean mitotic synchrony/asynchrony in in vitro airway epithelia at air–liquid interface after exposure to PBS or DEX. (B) Basolateral TGF-β1 secretion according to compound and time after wounding. (C) Correlation between DEX-induced improvement in mitotic synchrony (% of mitotically active [BrdU⁺] cells in G₁) and reduction (%) in basolateral TGF-β1 secretion at 24 hours. Data are shown as boxplots with median, interquartile range, and 95% confidence intervals. (n = 8 asthma and 3 nonasthmatic donors). ^○represents ≥ 1 standard deviation from the median; *represents ≥ 2 standard deviations from the median. ^#P < 0.01.

Figure 4.

Asthmatic epithelial mitotic asynchrony and transforming growth factor (TGF)-β1 secretion are improved by simvastatin (SIM) and aphidicolin (APH). (A) Mitotic synchrony/asynchrony in in vitro airway epithelia at air–liquid interface after exposure to PBS, SIM, or APH. (B) Basolateral TGF-β1 secretion according to compound and time after wounding. Data are shown as boxplots with median, interquartile range, and 95% confidence intervals (n = 8 asthma and 3 nonasthmatic donors). ^• represents ≥ 1 standard deviation from the median; ^*represents ≥ 2 standard deviations from the median. ^#P < 0.05 versus PBS.