Real-time imaging of asthmatic epithelial cells identifies migratory deficiencies under type-2 conditions

Abstract

Background: The epithelium is increasingly recognized as a pathologic contributor to asthma and its phenotypes. Although delayed wound closure by asthmatic epithelial cells is consistently observed, underlying mechanisms remain poorly understood, partly due to difficulties in studying dynamic physiologic processes involving polarized multilayered cell systems. Although type-2 immunity has been suggested to play a role, the mechanisms by which repair is diminished are unclear.

Objectives: This study sought to develop and utilize primary multilayered polarized epithelial cell systems, derived from patients with asthma, to evaluate cell migration in response to wounding under type-2 and untreated conditions.

Methods: A novel wounding device for multilayered polarized cells, along with time-lapse live cell/real-time confocal imaging were evaluated under IL-13 and untreated conditions. The influence of inhibition of 15 lipoxygenase (15LO1), a type-2 enzyme, on the process was also addressed. Cell migration patterns were analyzed by high-dimensional frequency modulated Möbius for statistical comparisons.

Results: IL-13 stimulation negatively impacts wound healing by altering the total speed, directionality, and acceleration of individual cells. Inhibition 15LO1 partially improved the wound repair through improving total speed.

Conclusions: Migration abnormalities contributed to markedly slower wound closure of IL-13 treated cells, which was modestly reversed by 15LO1 inhibition, suggesting its potential as an asthma therapeutic target. These novel methodologies offer new ways to dynamically study cell movements and identify contributing pathologic processes.

Keywords: Asthma; IL-13; airway epithelial cells; cell migration; type 2; wound repair.

FIG 1.

IL-13 stimulation decreases wound closure compared to untreated cells. 2mm×10mm images were compared between untreated and IL-13 treated AECs. A, Representative images (10X objective) of IL-13 (10ng/ml) treated or untreated ALI cells. B, Higher magnification images from (A). C, IL-13 stimulation decreased overall wound closure compared to untreated cells by paired-AUC testing (p=0.028, n=6). At both 8 and 16 hrs, the area of the wound under IL-13 conditions was larger than that of the untreated cells (p=0.017, p=0.017 separately, n=6). There were no statistical differences at other time points.

FIG 2.

AEC migration speed and patterns are altered by IL-13 as compared to untreated cells. A, AEC migration was tracked from the same culture regions as shown in Figure E4 in the Online Repository. Nuclei were automatically detected using IMARIS software. Colored lines represent the migration paths of AEC nuclei over the previous 3 hrs. Colors of lines identify specific time intervals after wounding. Blue represents 5–8 hrs; Light blue represents 13–16 hrs; Green represents 21–24 hrs; Light green represents 29–32 hrs; Yellow represents 37–40 hrs; Red represents 45–48 hrs. B, Higher magnification migration images (3 times magnified, 0.7mm×0.7mm) of (A).

FIG 3.

Statistical analyses identify specific differences in the complex migratory patterns of IL-13 stimulated AECs after wounding as compared to untreated cells. (See also Tables 1 and 2). A, FMM for Total speed of untreated vs IL-13 treated AECs. The fitted FMM curve for untreated AECs is shown as the solid blue line. The fitted FMM curve for IL-13 treated AECs is shown as the solid red line. B, FMM for X-axis speed of untreated vs IL-13 treated AECs. The fitted FMM curve for untreated AECs is shown as the solid blue line. The fitted FMM curve for IL-13 treated AECs is shown as the solid red line. C, Total migratory speed (mean with SEM scale bars) D, X-axis migratory speed [net speed toward wound (mean with SEM scale bars)]. E, Acceleration curves for untreated and IL-13 treated AECs in 24 hrs. The acceleration of IL-13 treated AECs rapidly decreased to ~0 μm/h² by 6 hrs, whereas untreated AECs maintained acceleration until 9 hrs before decelerating. After 12 hrs, untreated AECs decelerated rapidly at first and then went back from lowest negative value at 15 hrs to ~0 μm/h² at 21–24 hrs. IL-13 treated AECs continued decelerating even after 12 hrs. Statistically significant differences are shown on the figure, where the acceleration is higher or lower than 0 μm/h².

FIG 4.

Chemical inhibition of 15LO1 under IL-13 conditions improves wound closure compared to IL-13 alone. A, Confirmation of 15LO1 protein increases by IL-13 in ALI-cultured AECs. B, Representative images (10× objective) of wound closure between IL-13+DMSO vs IL-13+BLX treated AECs. C, Magnified 2mm×2mm images of those in (B). D, 15LO1 inhibition with BLX2477 improved wound closure as compared to IL-13+DMSO AECs by paired-AUC testing (p=0.034, n=6). At 16 hrs, the area of the wound was smaller under IL-13+BLX treated conditions, as compared to IL-13+DMSO conditions was larger than that of the untreated cells (p=0.042, n=6). There were no statistical differences at other time points.

FIG 5.

15LO1 inhibition marginally improves AEC migratory patterns under IL-13 conditions towards that of IL-13+DMSO AECs. A, AECs migration was tracked from the same culture regions as those shown in Figure E7 in the Online Repository. Nuclei were automatically detected using IMARIS software. Colored lines represent the migration paths of AEC nuclei over the previous 3 hrs. Colors of lines identify specific time intervals after wounding. Blue represents 5–8 hrs; Light blue represents 13–16 hrs; Green represents 21–24 hrs; Light green represents 29–32 hrs; Yellow represents 37–40 hrs; Red represents 45–48 hrs. B, Higher magnification migration images (3 times magnified, 0.7mm×0.7mm) of (A).

FIG 6.

Statistical analyses identify specific differences in the complex migratory patterns of IL-13 stimulated AECs after wounding as compared to IL-13 stimulated cells in the presence of 15LO1 inhibition (See also Tables E8 and E9 in the Online Repository). A, FMM for Total speed of IL-13+BLX vs IL-13+DMSO treated AECs. The fitted FMM curve for IL-13+BLX treated AECs is shown as the solid green line. The fitted FMM curve for IL-13+DMSO treated AECs is shown as the solid brown line. B, FMM for X-axis speed of IL-13+BLX vs IL-13+DMSO treated AECs. The fitted FMM curve for IL-13+BLX treated AECs is shown as the solid green line. The fitted FMM curve for IL-13+DMSO treated AECs is shown as the solid brown line. C, Total migratory speed (mean with SEM scale bars). D, X-axis migratory speed [net speed toward wound (mean with SEM scale bars)]