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.

Introduction

Meibomian gland dysfunction (MGD) is the leading cause of dry eye disease (DED)^1–6, which afflicts hundreds of millions of people throughout the world.^7–9 Normally, meibomian glands (MGs) generate abundant lipids that accumulate in lysosomes, are secreted in a holocrine manner into lateral ducts, and are ultimately released onto the ocular surface.^4,10 This glandular secretion, termed meibum, provides a clear optical surface for the cornea, interferes with bacterial colonization, and retards tear overflow.^1,7–11 Meibum also promotes the stability and prevents the evaporation of the tear film, thereby playing a critical role in the health of the ocular surface.^1,10,12–15

However, MGD, and the resulting meibum insufficiency, destabilizes the tear film, increases its osmolarity and evaporation, and leads to DED.^{1–6,12,14,16–19} The most frequent cause of human MGD is excretory duct obstruction, due to reduced meibum quality and hyperkeratinization of the terminal duct epithelium.^1,20–27 This obstruction may lead to cystic dilatation of glandular ducts, and the atrophy and loss of human MG epithelial cells (HMGECs).^1,22,28 MGD may also facilitate bacterial growth on the lid margin^29,30 and promote inflammation in the adjacent conjunctiva (i.e., posterior blepharitis).^1,31,32

The most frequent pharmaceutical treatment in the United States for the management of MGD is the off-label use of azithromycin (AZM).³³ This macrolide antibiotic was thought to be effective due to its anti-bacterial and anti-inflammatory actions, which may suppress the MGD-associated growth of lid bacteria and posterior blepharitis.^34–37 However, we discovered that AZM also has the ability to act directly on HMGECs to stimulate their differentiation, enhance the quality and quantity of their lipid production, and promote their holocrine secretion.³⁸ We hypothesize that this AZM capability is due to its cationic amphiphilic drug (CAD) nature and reflects a phospholipidosis (PLD)-like effect.³⁹ CADs feature a hydrophobic ring structure linked to a hydrophilic domain with a cationic amine group.⁴⁰ Drug-induced PLD, in turn, is characterized by an intracellular accumulation of cholesterol and phospholipids, and the formation of distinct, onion-shaped secretory lysosomes, termed lamellar bodies.⁴⁰

In support of our hypothesis, we discovered that AZM stimulates the accumulation of free and esterified cholesterol, neutral lipids, phospholipids, and lysosomes in immortalized (I) HMGECs. This increase of neutral lipid content occurs predominantly within lysosomes, many of which appear to be onion-shaped lamellar bodies.³⁹ This intracellular accumulation of phospholipids and the concurrent development of lamellar bodies are the hallmark features, and indeed the gold standard, for identifying PLD.^41,42

Given our research findings, we further hypothesize that other CADs should be able to reproduce AZM’s action on IHMGECs and potentially serve as a therapy for human MGD. To begin to test this hypothesis, we sought to determine whether solithromycin (SOL), a CAD, mirrors AZM activity, and stimulates the differentiation of IHMGECs. In addition, because it is possible that PLD-inducing drugs may elicit toxic effects in other tissues⁴³, we assessed the impact of SOL on the survival and proliferation of IHMGECs.

Materials and methods

IHMGECs were cultured under differentiating and proliferating conditions, according to published protocols.^44–46 For differentiation studies IHMGECs were grown on glass coverslips in 6-well plates until ~80% confluent, then cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 (50:50; Thermo Fisher Scientific, Waltham, MA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), SOL (2, 10, or 20 μg/ mL; Cempra), or AZM (10 μg/ml; Santa Cruz Biotechnology, Dallas, TX) for up to 7 days. Cells were also treated with equivalent concentrations of both ethanol (AZM vehicle) and dimethyl sulfoxide (DMSO; SOL vehicle). At experimental termination, total cellular neutral lipid and lysosome accumulations were evaluated by staining cells with LipidTOX green neutral lipid stain (Thermo Fisher Scientific) and LysoTracker® Red DND-99 (Thermo Fisher Scientific), a fluorescent probe designed for labeling acidic organelles (e.g., lysosomes), as previously reported.^38,39,47,48 Coverslips were mounted on slides with ProLong Gold antifade reagent with 4ʹ,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) for DNA staining and allowed to dry overnight. Images were captured with a Nikon fluorescent TS100 phase contrast microscope and staining intensities were quantified with ImageJ.⁴⁹ Data were analyzed by ANOVA and Newman–Keuls multiple comparisons tests by using Prism 5 software (GraphPad Software, Inc., La Jolla, CA). Image quantification was performed using ImageJ (http://rsbweb.nih.gov/ij/index.html). Eight images were analyzed for each condition and background intensity was subtracted.

For proliferation experiments, IHMGECs were seeded in 24-well plates in keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF; 5 μg/mL) and bovine pituitary extract (BPE; 50 μg/mL). Cells were then washed and the media were changed to unsupplemented (basal) KSFM with or without SOL (2, 10, or 20 μg/mL), AZM (10 μg/mL), or fresh KSFM containing EGF and BPE. After 7 days, cell numbers were enumerated using a hemocytometer. The EGF- and BPE-treated cells were positive controls for proliferation in these studies.⁴⁵

Results

Dose-dependent effect of SOL on lipid and lysosome accumulation in IHMGECs

To determine whether SOL stimulates the accumulation of neutral lipids and lysosomes in IHMGECs, we cultured cells in the presence of vehicles (i.e., ethanol and DMSO), SOL (2, 10, or 20 μg/ml), or the positive-control AZM (10 μg/ml) for up to 7 days under differentiating conditions. Our results demonstrate that SOL, like AZM, elicits a marked increase in the neutral lipid content and lysosome appearance in IHMGECs (Figure 1). As with AZM, this SOL-induced rise in lipid levels occurred predominantly within lysosomes (Figure 1).

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.

The influence of SOL on IHMGEC differentiation was dose-dependent. As shown in Figure 2, we observed a dramatic increase in both neutral lipids and lysosomes in the IHMGECs treated with 10 μg/ml and 20 μg/ml SOL concentrations. In contrast, the 2 μg/ml SOL dosage may have induced a slight rise in neutral lipid accumulation within lysosomes, but this was difficult to determine, as the signal intensity was similar to that in vehicle-treated controls.

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.

Time-course of SOL’s influence on lipid and lysosome accumulation in IHMGECs

To examine the time-dependence of the SOL stimulatory effect on IHMGEC differentiation, as well as to assess whether SOL activity occurs in a time-frame analogous to that of AZM, we cultured cells in the presence of vehicles (i.e., ethanol and DMSO), SOL (10 μg/ml) or the positive-control AZM (10 μg/ml) for 1, 3, and 5 days under differentiating conditions.

As demonstrated in Figure 3, SOL induced a rapid and robust increase in lysosome accumulation. This effect, which occurred within 1 day, was unique to SOL and not duplicated by AZM. After 3 (Figure 3) and 5 days of treatment, both SOL and AZM had essentially the same upregulatory effects on the numbers of lysosomes in IHMGECs. With regard to neutral lipid accumulation in IHMGECs, the stimulatory actions of SOL and AZM followed a similar time course (Figure 3).

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.

Impact of SOL on the proliferation of IHMGECs

To investigate whether SOL modulates the survival or proliferation of IHMGECs, we cultured cells in the presence of 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. Our findings demonstrate that cellular exposure to EGF and BPE caused, as anticipated, a significant increase in IHMGEC proliferation in serum-free media (Figure 4). However, neither SOL nor AZM altered the number of cells, as compared to that of the vehicle-treated control (Figure 4).

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.

Discussion

Our results demonstrate that SOL induces a rapid and dose-dependent increase in the accumulation of neutral lipids and lysosomes in IHMGECs. 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 positive 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 in serum-free media during a 7-day time period. Our results support our hypothesis that SOL, a CAD, is able to reproduce AZM’s impact on lysosome and lipid accumulation of HMGECs. Of particular interest, the ability of SOL to act like a CAD on IHMGECs to stimulate their PLD-like maturation³⁹ cannot be duplicated by non-CAD compounds, such as the antibiotics tetracycline, doxycycline and minocycline⁴⁸, or omega 3 and 6 fatty acids.⁵⁰

The mechanism by which SOL promotes the differentiation of IHMGECs most likely involves a PLD-like effect. SOL is a novel fluoroketolide⁵¹ that is known to accumulate in other eukaryotic cells (e.g., macrophages, kidney epithelial cells)^52,53, become sequestered in lysosomes, and induce PLD.⁵³ This PLD process has been associated with enhanced cholesterol synthesis⁴³ and is characterized by an intracellular accumulation of cholesterol and phospholipids, as well as the formation of distinct secretory lysosomes called lamellar bodies.⁴⁰ A lamellar body is a form of lysosome specialized for lipid storage and secretion.^54–58 These concentric vesicle-like lamellar bodies have been identified in IHMGECs following exposure to AZM³⁹, and they appear to accumulate lipids in the meibomian gland.^59,60 SOL, in turn, increases the neutral lipid level and lysosome number in IHMGECs, and this lipid accumulation occurs predominantly within the lysosomes.

It is noteworthy that the pharmaceutical industry has considered PLD a toxic effect that warrants elimination.⁶¹ In fact, the development of various lead compounds has been terminated after PLD was discovered in various tissues in clinical trials.⁶¹ Such termination is understandable when one considers that the PLD induced by certain aminoglycoside and macrolide antibiotics is linked to cellular apoptosis and necrosis.^62,63 However, studies to date indicate that SOL exposure, when used in microbiologically relevant concentrations, shows no evidence of cellular toxicity or impairment of anti-microbial defense.⁵³ Indeed, at the concentrations used in our study, SOL had no toxic impact on IHMGECs. Rather, SOL elicited a positive differentiative effect.

The SOL-induced generation of lysosomes and the accumulation of lipids within these vesicles are similar to events that typically occur during the differentiation of HMGECs.⁴⁶ This cellular process appears to start with small, undifferentiated cells located in the periphery of meibomian gland acini. These cells possess large quantities of free ribosomes and mitochondria, and a poorly developed smooth endoplasmic reticulum (SER) and Golgi apparatus. As cells mature and begin their migration toward the lateral ductules, the SER and Golgi become more apparent, lysosomes are generated, and lipids begin to accumulate. Ultimately, cells terminally differentiate, a process associated with a marked increase in volume, a profusion of lipid-filled vesicles, and nuclear pyknosis. Cells then undergo holocrine secretion, which involves cellular autophagy, apoptosis, disintegration and the release of lipid-laden contents into the lateral ductules, the central duct, and eventually the ocular surface.^1,60,64–67

The capacity of SOL to stimulate this differentiative process of HMGECs may, like AZM, be beneficial for the treatment of MGD. Further, because SOL harbors both anti-bacterial⁶⁸ and anti-inflammatory⁶⁹ activities, it is possible that this antibiotic may also decrease the MGD-linked growth of lid bacteria and development of posterior blepharitis.^34–37 However, whether SOL can duplicate the clinically significant ophthalmic effects of AZM in vivo remains to be determined.