The Effect of Solithromycin, a Cationic Amphiphilic Drug, on the Proliferation and Differentiation of Human Meibomian Gland Epithelial Cells

Abstract

Purpose: We previously discovered that azithromycin (AZM) acts directly on immortalized human meibomian gland epithelial cells (IHMGECs) to stimulate their lipid and lysosome accumulation and overall differentiation. We hypothesize that this phospholipidosis-like effect is due to AZM's cationic amphiphilic drug (CAD) nature. If our hypothesis is correct, then other CADs (e.g., solithromycin [SOL]) should be able to duplicate AZM's action on IHMGECs. Our purpose was to test this hypothesis.

Materials and methods: IHMGECs were cultured in the presence of vehicle or SOL (2, 10, or 20 µg/ml) for up to 7 days under proliferating or differentiating conditions. Positive (epidermal growth factor and bovine pituitary extract for proliferation; AZM for differentiation) and negative (vehicle) controls were included with the experiments. IHMGECs were evaluated for cell number, neutral lipid content, and lysosome accumulation.

Results: Our results demonstrate that SOL induces a rapid and dose-dependent increase in the accumulation of neutral lipids and lysosomes in HMGECs. The lysosomal effects were most prominent with the 10 and 20 µg/ml doses, and occurred earlier (i.e., 1 day) with SOL than with the AZM (10 µg/ml) control. The effects of SOL and AZM on IHMGEC differentiation were essentially the same after 3 days of culture. SOL did not influence the proliferation of HMGECs during a 7-day time period.

Conclusions: Our results support our hypothesis that SOL, a CAD, is able to reproduce AZM's impact on lysosome and lipid accumulation, as well as the differentiation, of HMGECs. The effect of SOL on lysosome appearance was faster than that of AZM.

Keywords: Solithromycin; azithromycin; cationic amphiphilic drug; dry eye disease; meibomian gland dysfunction; phospholipidosis.

Figure 1.

Effect of SOL on neutral lipid and lysosome accumulation in IHMGECs. Cells (n = 3 wells/treatment; n = 2 experiments) were treated with vehicles (i.e. ethanol and DMSO), SOL (10 μg/ml) or AZM (10 μg/ml) for 5 days under differentiating conditions. Neutral lipids were stained with LipidTOX (green), lysosomes with LysoTracker (red), and nuclei with DAPI (blue). A. The appearance of lysosome and neutral lipid staining after 5 days of antibiotic treatment. All images are 400X magnification. B. Fluorescence intensity of the neutral lipid staining was quantified using Image J. C. Fluorescence intensity of the lysosome staining. Data shown are mean fluorescence intensity ± SEM. ** indicates p < 0.01 compared to control.

Figure 2.

Impact of different doses of SOL on neutral lipid and lysosome accumulation in IHMGECs. Cells (n = 3 wells/treatment; n = 4 experiments) were cultured in the presence of vehicles (i.e. ethanol and DMSO), SOL (2, 10, or 20 μg/ml) or AZM (10 μg/ml) for 7 days under differentiating conditions. The targets of the LipidTOX, LysoTracker and DAPI stains are explained in the Methods and the legend for Figure 1. All images are 400X magnification. B. Fluorescence intensity of the neutral lipid staining was quantified using ImageJ. C. Fluorescence intensity of the lysosome staining. Data shown are mean fluorescence intensity ± SEM. ** indicates p < 0.01 compared to control, † indicates p < 0.05 compared to azithromycin-treated cells.

Figure 3.

Time-dependent effect of SOL on neutral lipid and lysosome accumulation in IHMGECs. Cells (n = 3 wells/treatment; n = 2 experiments) were exposed to vehicles (i.e. ethanol and DMSO), SOL (10 μg/ml) or AZM (10 μg/ml) for 1 and 3 days under differentiating conditions. A. The appearance of lysosome and neutral lipid staining after antibiotic treatments. B. Fluorescence intensity of neutral lipid staining on day 1 was measured using ImageJ. C. Fluorescence intensity of the lysosome staining on day 1. D. Fluorescence intensity of neutral lipid staining on day 3. E. Fluorescence intensity of the lysosome staining on day 3. Data shown are mean fluorescence intensity ± SEM. * indicates p < 0.05 compared to control, ** indicates p < 0.01 compared to control, † indicates p < 0.05 compared to azithromycin-treated cells.

Figure 4.

Influence of SOL on IHMGEC proliferation. Cells (n = 3 wells/treatment; n = 4 experiments) were cultured with vehicles, SOL (2, 10 or 20 μg/ml), AZM (10 μg/ml), or the positive-control EGF and BPE for 7 days in KSFM media. Data from one experiment are shown. Columns depict the mean ± standard error. * indicates p < 0.05.