Effects of Terpinen-4-ol on meibomian gland epithelial cells in vitro

Abstract

Purpose:

Infestation with Demodex mites has been linked to the development of chalazion, meibomian gland dysfunction and blepharitis. An effective treatment is the eyelid application of terpinen-4-ol (T4O), a tea tree oil component. However, T4O is also known to be toxic to non-ocular epithelial cells. We hypothesize that T4O toxicity also extends to human meibomian gland epithelial cells (HMGECs).

Methods:

Immortalized (I) HMGECs were cultured with varying concentrations (1.0 to 0.001%) of T4O under proliferating or differentiating conditions up to 5 days. Experimental procedures included analyses of cell appearance, survival, P-Akt signaling, lysosome accumulation, and neutral lipid content.

Results:

Our findings show that T4O causes a dose- and time-dependent decrease in cell survival of IHMGECs. After 15 minutes of exposure to 1% T4O, IHMGECs exhibited rounding, atrophy and poor adherence. Within 90 minutes of such treatment, almost all cells had died. Reducing the T4O concentration to 0.1% also led to a marked decrease in P-Akt signaling and cell survival of IHMGECs. Decreasing the T4O amount to 0.01% caused a slight, but significant, reduction in IHMGEC number after 5 days of culture, and did not influence the ability of these cells to differentiate.

Conclusions:

T4O, even at levels 10- to 100-fold lower than demodicidal concentrations, is toxic to HMGECs in vitro.

Introduction

Demodex mites are commonly found in human skin, including the eyelid margins¹. Only two subspecies of Demodex have been found to infest human: the larger Demodex folliculorum which congregates in the hair follicle and the smaller Dermodex brevis which resides in the sebaceous gland². Demodex infestation has been implicated with various ocular surface diseases, such as meibomian gland dysfunction, chalazion and blepharitis^3–6. It is suggested that D. brevis, which can burrow deep into the meibomian gland, is a risk factor for multiple or recurrent chalazia⁶.

Tea tree oil (TTO), a natural essential oil extracted from the Australian native plant Melaleuca alternifolia, has been reported to be effective in reducing Demodex and the associated ocular surface inflammation^2,7,8. Of the nearly 100 components in the TTO, terpinen-4-ol (T4O) is found to be the most effective component to kill Demodex⁹. A study in vitro showed Demodex mites could be killed within 88 minutes exposure in 1% T4O and 40 minutes in 4% T4O^9,10. Eyelid hygiene products containing T4O (ranging from 2% to 4%) are now widely suggested in daily practice when ocular demodicosis is confirmed^10–12. However, as well as being cytotoxic to microorganisms, TTO and T4O have long been known to have toxicity against a variety of human cells including epithelial cells and fibroblasts^13,14. While T4O has widely been applied on the patients’ eyelids, the effects of T4O on ocular surface and adnexal cells have not been reported in peer-reviewed literature.

To kill Demodex, especially the D. brevis which may reside deep in the meibomian gland, T4O must reach a certain concentration in the tissue where the mites hide. We hypothesize that in such demodicidal concentrations, T4O would also cause damage to human meibomian gland epithelial cells (HMGECs). To test our hypothesis, we investigated the effects of T4O on the morphology, survival, signaling ability and differentiation of immortalized (I) HMGECs.

Materials and Methods

Short tandem repeat profiling of IHMGECs

We confirmed the identity of our IHMGECs by short tandem repeat (STR) profiling, as previously reported¹⁵. In brief, DNA was extracted from cells in our laboratory at passages 4, 16, 33 and 53 and sent to University of Arizona Genetics Core (Tucson, AZ) for STR profiling by using their standard methodology (https://uagc.arl.arizona.edu/services/complete-solutions/cell-line-authentication). Their technique utilizes the Promega PowerPlex 16HS assay to study 15 autosomal loci and amelogenin, and their data were compared with the DSMZ database (https://www.dsmz.de/services/services-human-and-animal-cell-lines/online-str-analysis.html) with 80% or greater identity at 8 core loci (TH01, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, TPOX) and amelogenin being considered a match. Identical profiles for our IHMGEC cells were obtained for the four passages from our laboratory, as well as three more passages from two other laboratories¹⁵.

Cell culture

IHMGECs¹⁶ were cultured under proliferating or differentiating conditions, as previously described¹⁷. In brief, cells were cultured in keratinocyte serum free medium (KSFM), supplemented with 5 ng/ml epidermal growth factor (EGF) and 50 μg/ml bovine pituitary extract (BPE) (Life Technologies, Grand Island, NY). After reaching confluence, cells were rinsed with PBS and seeded in 12-well culture dishes (Corning, Lowell, MA) for the experiments.

To explore the short-term effects of T4O, IHMGECs (n = 3 wells/treatment/experiment) were treated with 1.0% or 0.1% T4O (Sigma-Aldrich Corp., St. Louis, MO) or vehicle (dimethyl sulfoxide, DMSO) for up to 90 minutes in the presence or absence of EGF+BPE. It has been demonstrated that DMSO can dissolve T4O and does not interfere or inhibit the biological effect of T4O¹⁸. Cellular morphology was observed and recorded with a phase-contrast microscope.

To assess the long-term influence of T4O, IHMGECs (n = 3 wells/treatment/experiment) were exposed to various doses of T4O (0.1%, 0.01%, and 0.001%) or the DMSO vehicle for 5 days in the presence or absence of EGF+BPE. Cells were counted using a hemocytometer and cellular morphology was examined with a phase-contrast microscope.

Lipid staining

IHMGECs were cultured under differentiating conditions in a 1:1 mixture of DMEM/F12 (Mediatech, Inc., Manassas, VA), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Rockford, IL). Azithromycin (AZM, 10 μg/ml, Santa Cruz Biotechnology, Dallas, TX) was added to the culture medium as a positive control for differentiation.¹⁹ After exposure to T4O (0.1%, 0.01%, and 0.001%), DMSO or AZM for up to 5 days, cells were stained for lysosome numbers using LysoTracker Red DND-99 (50 nM; Thermo Fisher Scientific) and for neutral lipid accumulation with HCS LipidTOX™ Green neutral lipid stain (1:200, Thermo Fisher Scientific) according to reported methods²⁰. Slides were examined by using an Eclipse E800 fluorescent microscope and images were captured with NIS-Elements Basic Research software, version 4.2 (Nikon Instruments, Melville, NY).

SDS-PAGE and immunoblot

IHMGECs were cultured to 50% confluence under proliferating conditions, then treated with 0.1%, 0.01%, and 0.001% of T4O or vehicle control (DMSO) for 30 min. Cells were lysed in Laemmli buffer (Bio-Rad Laboratories, Hercules, CA) supplemented with 1% protease inhibitor cocktail, 200μM sodium orthovanadate and 5% β-mercaptoethanol (Sigma-Aldrich), denatured at 95 °C for 10 min, separated by SDS-PAGE on 4%–20% Tris-glycine gels (Thermo Fisher Scientific), and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with 5% milk in Tris-buffered saline containing 0.01% Tween-20 (TBS/T), then incubated with primary antibodies specific to phospho-phosphoinositide 3-kinase-protein kinase B (P-Akt) (1:1000, rabbit) or β-actin (1:5000, mouse; both from Cell Signaling Technology, Danvers, MA). Following an overnight incubation at 4 °C, membranes were exposed to horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G (1:5000, Sigma-Aldrich). Proteins were visualized with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) using a G-Box gel documentation station (Syngene, Frederick, MD). Image analysis and densitometry were performed with ImageJ (http://rsbweb.nih.gov/ij/index.html).

Statistical analyses

One-way ANOVA and Student’s t-test were performed using Prism 5 (GraphPad Software, Inc., La Jolla, CA). Each experiment was performed in triplicate under the same conditions and repeated at least three times.

Results

Short-term effect of T4O on the morphology and survival of IHMGECs

To determine the short-term effect of T4O on the appearance and survival of IHMGECs, we treated cells with 1.0% and 0.1% T4O for up to 90 min in KSFM with or without EGF+BPE. 1.0 %T4O is reportedly the minimum concentration necessary to kill Demodex⁹.

After 15 minutes of exposure to 1% T4O, IHMGECs exhibited rounding, atrophy and poor adherence (Figure 1). Within 90 minutes of such treatment, almost all cells were detached, floating in the medium, and dead (Figure 1). Reducing the T4O concentration to 0.1% did not elicit the same adverse effects at the 90-minute time point, but IHMGEC rounding and death did increase by 24 hours (Figure 2).

Figure 1.

Short-term effect of 0.1 and 1% T4O on the morphology and survival of IHMGECs in the presence or absence of growth supplements. T4O induced time-dependent morphological changes in IHMGECs. Images shown are representatives of three separate experiments. All images are 200 X magnification. Scale bar = 50 μm.

Figure 2.

Influence of 0.001%, 0.01% and 0.1% T4O on the morphology of IHMGECs in the presence or absence of growth supplements for 24 hours. Cell rounding and death were observed in the 0.1% T4O treatment group. Images shown are representatives of three separate experiments. All images are 200 X magnification. Scale bar = 50 μm.

Influence of T4O on P-Akt expression of IHMGECs

To examine whether T4O influences the activity of the P-Akt pathway in IHMGECs, we cultured cells in serum-free conditions with 0.1%, 0.01% and 0.001% of T4O or the DMSO vehicle for 30 minutes and then processed them for immunoblotting. Our results show that T4O caused a dose-dependent decrease in P-Akt levels, with 0.1% T4O eliciting a significant reduction in signaling activity (Figure 3).

Figure 3.

T4O alters the P-Akt signaling pathway of IHMGECs. Cells were treated with vehicle control or T4O in serum-free medium for 30 min. Cells treated with 0.1%T4O showed a significant decrease in AKT phosphorylation. Results are reported as mean studies performed under the same conditions. Results are shown as mean ± standard error. ** indicates p< 0.01, NS indicates not significant.

Longer-term impact of T4O on the survival of IHMGECs

To evaluate the longer-term impact of T4O on IHMGECs, cells were exposed to the DMSO vehicle, or 0.1%, 0.01% or 0.001% T4O in media with or without growth supplements for 5 days. IHMGECs were then examined for appearance and enumerated.

As illustrated in Figure 4A, 0.1% T4O induced cell rounding, perinuclear vesicle accumulation, cellular atrophy, poor adherence and detachment by day 5 in the presence or absence of EGF+BPE. Extremely few IHMGECs survived this 0.1% T4O exposure (Figure 4B). Treatment with 0.1% and 0.01%, but not 0.001%, T4O also significantly decreased the number of IHMGECs, as compared to the placebo condition (Figure 4B).

Figure 4.

Longer-term effect of T4O on the survival of IHMGECs. Cells were treated with vehicle or T4O in the presence or absence of growth supplements for 5 days before cell counting. (A) T4O induced dose-dependent morphological changes in IHMGECs. Images shown are representatives of three separate experiments. All images are 200 X magnification. Scale bar = 50 μm. (B) Cell counting results at the fifth day with different dosages of T4O. Data from one experiment are shown as a representative of three studies performed under the same conditions. Results are shown as mean ± standard error. *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05.

Effect of T4O on neutral lipid and lysosome accumulation of IHMGECs

To assess the effect of T4O on IHMGECs differentiation, cells (n = 2 wells/treatment/experiment) were treated for 5 days with 0.01% or 0.001% T4O, vehicle or AZM in serum-containing medium and then processed for neutral lipid and lysosome evaluation. We could not determine the influence of the demodicidal 1.0% T4O concentration, or the dose ten-fold lower (i.e. 0.1%), because these T4O amounts in differentiating medium killed all the IHMGECs.

We found that the 0.01% and 0.001% concentrations of T4O had no effect on the neutral lipid content or lysosome numbers in IHMGECs (Figure 5). In these studies, azithromycin, the positive control, consistently increased these parameters (Figure 5).

Figure 5.

Impact of T4O on neutral lipid and lysosome accumulation in IHMGECs. Cells (n = 2 wells/treatment/experiment) were treated for 5 days with 0.01% or 0.001% T4O, or vehicle or AZM in serum containing medium, then stained for neutral lipids (LipidTox Neutral Green) and lysosomes (LysoTracker Red). Images shown are representative of three separate experiments. All images are 200 X magnification.

Discussion

Our findings show that T4O caused a dose- and time-dependent decrease in the survival of IHMGECs, and also significantly reduced their P-Akt signaling ability. The demodicidal 1.0% T4O concentration killed almost all the IHMGECs within 90 minutes. Reducing the T4O level by 100-fold to 0.01% still decreased the survival of IHMGECs but did not interfere with their capacity to differentiate. Overall, our results support our hypothesis that the demodicidal concentrations of T4O are toxic to IHMGECs.

Tighe et al. revealed that the mean survival time of Demodex in 1% T4O was 87.6 ± 13.5 min⁹. However, our findings show nearly all HMGECs were dead within 90 min in 1% T4O, which means 1% T4O might kill the Demodex and HMGECs at the same time. Moreover, the concentrations of T4O in commercial lid hygiene products are even higher, and the recommended treatment regimen is to apply lid hygiene products twice a day for at least 6 weeks to cover two Demodex life cycles^2,12,,21. Indeed, the American Academy of Ophthalmology website states “Typically, a daily lid scrub with 50 percent tea tree oil and lid massage with 5 percent tea tree oil ointment will take care of ocular Demodex infestation”²². T4O is a small lipophilic molecule that rapidly penetrates the epidermis^23–26 and is known as a penetration enhancer (i.e. an excipient which reversibly alters the barrier function of the stratum corneum leading to increased permeability of the skin)^23,24,27. Following application, approximately 2 to 4% of applied TTO components permeate the skin, and between 0.23 to 0.37% of these products are retained after 24 hours²⁵. If such pharmacodynamics also occur in the eyelid, this would lead to levels of T4O between 0.2% (permeation) and 0.02% (retention), which we demonstrate either kill, or significantly reduce the survival of, IHMGECs, respectively.

The toxicity of TTO and T4O is not unique to IHMGECs. Investigators have reported that demodicidal concentrations and less of TTO or T4O kill human hepatic, cervical and breast epithelial, T, B, and bone marrow cells, as well as fibroblasts and peripheral blood monocytes in vitro^{13,14,28–31}. TTO is also known to: [a] cause allergic contact dermatitis, with a 0.7% prevalence in patch-tested patients³²; [b] generate secondary organic aerosols containing ultrafine particles, which may elicit inflammation and oxidative stress³³; [c] contribute to the development of antibiotic resistance in human pathogens and commensals, when used repeatedly at sub-lethal bactericidal concentrations (0.1 to 0.25%)³⁴; and [d] induce prepubertal gynecomastia in young boys^29,30.

This gynecomastia response has been attributed to TTO’s role as an endocrine disruptor^29,30. Endocrine disruptors are natural and synthetic chemicals that disrupt the normal functioning of the endocrine system and produce inappropriate effects in hormone-responsive target tissues³⁰. TTO possesses both estrogen (e.g. 0.025% TTO) and antiandrogen (e.g. 0.005% TTO) activities^28–30. Estrogens are well-known suppressors of sebaceous gland function³² and may promote the development of meibomian gland dysfunction (MGD)^35–37. Androgens, in turn, stimulate lipogenesis and suppress keratinization of the meibomian gland^35,36, and antiandrogen use is linked to the induction of MGD and dry eye^38,39. In fact, androgen deficiency is a major risk factor for the development of MGD and dry eye disease in humans^{35,36,40–42}.

Greay et al. found T4O had significant anti-proliferative activity against murine tumor cell lines by eliciting G1 cell cycle arrest⁴³. In our study, T4O reduced Akt phosphorylation in IHMGECs after only 30 min exposure. The P-Akt pathway is an important regulator of cell proliferation and survival⁴⁴. As a large sebaceous gland that secretes through a holocrine mechanism, the human meibomian gland requires constant renewal and differentiation of meibomian gland acinar epithelial cells, which means IHMGECs are metabolically active and more sensitive to cell cycle inhibitors³⁵.

The fact that all IHMGECs were dead in differentiating medium after 5 days of treatment with 0.1% T4O further proved its toxicity in vitro. However, 0.01% and 0.001% T4O in differentiating medium exerted no influence on neutral lipid and lysosome accumulation in IHMGECs. This finding was consistent with our previous study about the toxicity of cosmetic preservatives on human ocular surface and adnexal cells, which also found the toxic preservatives don’t necessarily interfere with cell differentiation¹⁷. This might be explained by the fact that cellular proliferation is quite distinct from differentiation. Toxic compounds that significantly reduce proliferation may have no effect on differentiation⁴⁵. Besides, the differentiation culture media contains serum, which has very high amounts of albumin. Albumin might bind toxic compounds through its amino group, thus neutralizing the biological effect of lower concentrations of T4O⁴⁶.

The European Cosmetic Toiletry and Perfumery Association in 2002 published the following recommendation: “Tea Tree Oil should not be used in cosmetic products in a way that results in a concentration greater than 1% oil being applied to the body”⁴⁷. Given that Manuka honey⁴⁸, microblepharoexfoliation⁴⁹ and intense pulsed light therapy^50–53 have all been shown to be effective against Demodex, is it possible that these treatments could replace TTO and T4O in the future?

Overall, Demodex is a major cause of ocular discomfort and T4O has proved its efficacy in reducing Demodex and the related symptoms^2,3,54,55. Our study discovered that T4O was toxic to HMGECs, but it was limited to cellular exposure in vitro. More clinical studies are necessary to determine the benefits and harms of daily use of T4O on the eyelid margins, as well as the indications and contraindications of T4O application. In addition, as previously suggested²⁹, the medical community should be aware of the possibility of endocrine disruption and should caution patients about repeated exposure to TTO or T4O.

Conclusion

T4O, at demodicidal concentrations, alters the morphology and inhibits the survival of IHMGECs. T4O at lower concentrations also reduces the proliferation and activity of a cell survival mediator in IHMGECs.