Biomarkers for Progenitor and Differentiated Epithelial Cells in the Human Meibomian Gland.

The meibomian gland (MG) is a sebaceous gland that secretes through a holocrine process. Because such secretion requires the destruction of MG acinar epithelial cells, they need constant renewal and differentiation. The processes that promote these regenerative events in the human MG are unknown, nor is it known how to distinguish MG progenitor and differentiated cells. We discovered that Lrig1 and DNase2 serve as biomarkers for human MG progenitor and differentiated cells, respectively. Lrig1 is expressed in MG basal epithelial cells in the acinar periphery, a location where progenitor cells originate in sebaceous glands. DNase2 is expressed in the differentiated epithelial cells of the MG central acinus. Furthermore, proliferation stimulates, and differentiation suppresses, Lrig1 expression in human MG epithelial cells. The opposite is true for DNase2 expression. Our biomarker identification may have significant value in clinical efforts to restore MG function and to regenerate MGs after disease-induced dropout. Stem Cells Translational Medicine 2018;7:887-892.

Meibomian glands (MGs) are eyelid sebaceous glands that secrete through a holocrine mechanism. Because such secretion requires the destruction of MG acinar epithelial cells, they need constant renewal and differentiation. The processes that promote these regenerative events in the human MG are not known, nor is it known how to distinguish MG progenitor and differentiated cells. The authors have discovered biomarkers for human MG progenitor and differentiated cells and have also learned that expression of these biomarkers is plastic and can be induced or suppressed repeatedly by changing the proliferation and differentiation status of these epithelial cells. These biomarker findings may have significant value in clinical efforts to restore MG function and to regenerate MGs after disease‐induced dropout.

Introduction

Meibomian glands (MGs) are large sebaceous glands (SGs) located in the eyelids. The MGs are composed primarily of three types of epithelial cells: progenitor, differentiated, and ductal cells 1. Progenitor epithelial cells (i.e., meibocytes) in these glands, as in skin SGs, appear to emerge from the acinar periphery and differentiate as they migrate across the acinus to the lateral duct 1. Following terminal differentiation, meibocytes release their lipid‐laden contents by holocrine secretion into the ductal system 1. These secretions (i.e., meibum) spread onto the ocular surface from the orifices of the central duct at both the upper and lower eyelid margins and form the outermost lipid layer of the tear film. Meibum provides stabilization to, and prevents evaporation of, the tear film, which is critically important in maintaining ocular surface health 1, 2, 3.

Because holocrine secretion requires continual meibocyte destruction, these epithelial cells need continuous renewal and differentiation 1. However, the processes that promote these regenerative cellular events in the human MG are unknown. Indeed, it is not even known how to distinguish progenitor and differentiated cells in this tissue. Such distinguishing ability, as might be realized with cellular biomarkers, would have tremendous value in clinical efforts to restore MG function and to regenerate these glands after disease‐induced dropout (e.g., through tissue engineering, gene transfer, or cell therapy). The reason is that MG dysfunction (MGD), which is often paralleled by glandular dropout, afflicts hundreds of millions of people, is one of the most frequent causes for patient visits to eye care practitioners, and is the primary cause of evaporative dry eye disease 1, 2, 4, 5, 6. There is no cure for MGD.

We hypothesize that leucine‐rich repeats and immunoglobulin‐like domains protein 1 (Lrig1) and DNase2 will serve as biomarkers for progenitor and differentiated cell populations in the human MG. Our rationale is that Lrig1 is a biomarker for proliferating progenitor cells in the pilosebacous unit and that these precursor cells give rise to differentiated SG epithelial cells (sebocytes) 7, 8, 9. Furthermore, lysosomal DNase2 is known to initiate the nuclear degeneration and holocrine secretion of sebocytes 10, 11.

The purpose of our study was to test our hypothesis by examining whether Lrig1 and DNase2 serve as biomarkers for progenitor and differentiated cells in the human MG. We also sought to determine whether these biomarkers are plastic and can be induced or suppressed by changing the proliferation and differentiation status of human MG epithelial cells. To help characterize the anatomical location of cells in the human MG, we used antibodies cytokeratins 6 (K6) and 14 (K14) to identify ductal 12, 13, as well as all 14, 15, epithelial cells, respectively.

Materials and Methods

Human Tissue

Discarded and deidentified human eyelids were obtained within 12 hours after eyelid surgeries (two women, one man; age range, 70–82 years). The use of human tissues was approved by the Institutional Review Board of the Massachusetts Eye and Ear Infirmary and Schepens Eye Research Institute and adhered to the tenets of the Declaration of Helsinki. Tissue samples were immediately frozen in optimal cutting temperature compound (Tissue‐Tek, Sakura USA, Torrance, CA) and later sectioned (15 μm) with a cryostat for immunofluorescence and lipid procedures.

Cell Culture

Immortalized human MG epithelial cells (IHMGECs) 16 were cultured in keratinocyte serum‐free medium (KSFM) containing 5 ng/mL epidermal growth factor (EGF) and 50 μg/mL bovine pituitary extract (BPE; Thermo Fisher Scientific, Grand Island, NY) to stimulate cell proliferation 17 (proliferation medium [PM]). After reaching 60%–70% confluence, cells were placed for varying intervals in one of the following media: KSFM alone, PM, or serum‐containing medium composed of 10% fetal bovine serum (Thermo Fisher Scientific) in equal volumes of Dulbecco's modified Eagle's medium and Ham's F12 (differentiation medium [DM], Mediatech, Inc., a Corning Subsidiary, Manassas, VA) to induce cell differentiation 18. To accelerate differentiation of HMGECs, 10 μg/mL azithromycin (AZM; Santa Cruz Biotechnology, Dallas, TX) was added to the DM 19. In some studies, cell culture media were switched multiple times between PM and DM. At experimental termination, cells were processed for immunofluorescent staining or immunoblotting.

Immunofluorescence and Lipid Staining

Human eyelid sections and HMGECs were fixed with cold methanol for 15 minutes at −20°C. Following three phosphate‐buffered saline (PBS) rinses for 5 minutes each, samples were blocked with 2% bovine serum albumin (BSA, Sigma‐Aldrich Corp., St. Louis, MO) in PBS for 60 minutes, and then incubated overnight at 4°C in a moist chamber with antibodies specific for Lrig1 (ab214102,1:100), cytokeratin 14 (ab181595, 1:500), cytokeratin 6 (ab18586, 1:500), DNase2 (ab8119, 1:100; Abcam, Cambridge, MA), or lysosomal‐associated membrane protein 1 (LAMP‐1;H4A3, 1:15; Developmental Studies Hybridoma Bank, Iowa City, IA), or the BSA diluent. After three additional PBS rinses, donkey anti‐rabbit (ab150075, 1:500, Abcam) or donkey anti‐mouse (2492098, 1:500, EMD Millipore, Temecula, CA) secondary antibodies were applied for 1 hour at room temperature. For neutral lipid staining, some eyelid sections were fixed in 4% paraformaldehyde for 15 minutes. Following additional washes, samples were exposed to LipidTOX Green neutral lipid stain (1:500, Thermo Fisher Scientific) in a humid chamber for 30 minutes. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1 μg/mL, Sigma‐Aldrich) and samples were covered with VectaMount mounting medium (Vector Laboratories, Burlingame, CA), and observed with a confocal microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany).

Immunoblotting

Cells (n = 3 wells/experiment) were lysed in sodium dodecyl sulfate (SDS) Laemmli buffer (Bio‐Rad Laboratories, Inc., Hercules, CA) supplemented with a 1% protease inhibitor cocktail and 5% β‐mercaptoethanol (Sigma‐Aldrich). Lysates were heated at 95°C for 15 minutes, separated by SDS‐polyacrylamide gel electrophoresis on 4%–20% gradient gels (Thermo Fisher Scientific), and transferred to poly(vinylidene difluoride) membranes (Bio‐Rad). Membranes were blocked with 5% milk or BSA in Tris‐buffered saline containing 0.01% Tween‐20 (TBS/T). Membranes were then incubated with primary antibodies to Lrig1 (ab36707, 1: 500, Abcam), proliferating cell nuclear antigen (PCNA; D3H8P, 1: 1000, Cell Signaling Technology, Danvers, MA), DNase2 (1: 1000, Sigma‐Aldrich), or β‐actin (1: 10000, Cell Signaling Technology) in TBS/T supplemented with 5% nonfat dry milk or BSA. Following an overnight incubation at 4°C, membranes were exposed to goat anti‐rabbit or goat anti‐mouse secondary antibodies coupled to horseradish peroxidase (1: 5000, Sigma‐Aldrich). Staining intensities were quantified with ImageJ software (https://imagej.nih.gov/ij/download.html).

Statistical Analysis

The significance of the differences between groups was determined by using Student's unpaired, two‐tailed t test (Prism 5, GraphPad Software, Inc., La Jolla, CA). Values with p < .05 were considered statistically significant.

Results

Lrig1 identification in the human MG

To determine whether Lrig1 serves as a biomarker for progenitor cells in the human MG, we processed human tissues for the identification of ductal and acinar epithelial cell regions and then stained samples with antibodies to Lrig1. As shown in Figure 1A, K6 expression is limited to MG ductal cells, whereas K14 staining highlights both ductal and acinar epithelial cells. Immunofluorescence analysis of Lrig1 with or without K14 costaining demonstrated that Lrig1‐positive cells are located solely in the acinar basal layer of the human MG (Fig. 1B). This presence of Lrig1 in the acinar periphery, and its absence in the central acinus, is analogous to that of progenitor cell distribution in other SGs.

Figure 1

Identification of K14, K6, and Lrig1 in the human meibomian gland (MG). (A): K14 is present in both ductal and acinar epithelial cells, whereas K6 expression is restricted to ductal cells. (B): Lrig1‐positive cells are located only in the basal layer of the MG acinus. Scale bar = 50 μM. Abbreviations: K6, cytokeratins 6; K14, cytokeratins 14.

Lrig1 expression in proliferating versus differentiating HMGECs

To examine whether Lrig1 expression is unique to proliferating, as compared with differentiating, cells, we cultured HMGECs in PM, DM, or DM + AZM media and processed them for morphological, immunofluorescence, and Western blot analyses. Proliferating HMGECs feature a distinctive, cobblestone morphology (Fig. 2A), which is associated with a pronounced expression of Lrig1 (Fig. 2B). In contrast, culture of HMGECs under differentiating conditions promotes a morphological shift to a large and flattened cellular appearance (Fig. 2A) and a loss of Lrig1 expression (Fig. 2B). The significant decrease in Lrig1 protein expression, which is induced by switching HMGECs from proliferating to differentiating media, was confirmed by Western blotting (Fig. 2C).

Figure 2

Differential appearance of Lrig1 in proliferating versus differentiated human meibomian gland epithelial cells (HMGECs). In parallel with a change in cellular morphology (A), Lrig1 expression in proliferating HMGECs (B) (PM frame) appears to be lost when cells differentiate (B; DM and AZM frames). (C): This decrease in Lrig1 levels was confirmed by Western blot (n = 3 experiments, **p < .01). Scale bar = 50 μM. Abbreviations: AZM, azithromycin; DM, differentiation medium; PM, proliferation medium.

To verify the linkage between Lrig1 expression and cellular proliferation, we grew HMGECs until 70% confluent, and then cultured them in either basal KSFM or PM media for an additional 3 days. After this period, we analyzed cell lysates for both Lrig1 and PCNA, which is an indicator of cell cycle entry 20. As shown in Figure 3, the expression of both Lrig1 and PCNA proteins were significantly enhanced in PM conditions.

Figure 3

Stimulation of Lrig1 expression in immortalized human meibomian gland epithelial cells. (A) Cells were cultured in basal KSFM or PM media. The latter promoted both Lrig1 and PCNA appearance, as shown by Western blot (n = 3 experiments, *p < .05) (B). Abbreviations: KSFM, keratinocyte serum‐free medium; PCNA, proliferating cell nuclear antigen; PM, proliferation medium.

DNase2 expression in the human MG and in HMGECs

To determine whether DNase2 serves as a protein biomarker for differentiated cells in the human MG, we processed human tissues for the identification of neutral lipids and lysosomes in differentiated cells and then stained samples with antibodies to DNase2. As anticipated, the maturing, lipid‐containing cells were situated in the central region, but not the basal layer, of the MG acinus (Fig. 4A, 4D). DNase2 expression was identified in, and restricted to, the lipid‐laden lysosomes in these differentiated cells (Fig. 4B, 4C, 4D). DNase2 could not be detected within the progenitor cell area of the acinar periphery (Fig. 4B).

Figure 4

Cellular location of DNase2 in the human meibomian gland (MG). To identify neutral lipids and lysosomes, cells were stained with LipidTOX 19 and antibodies to LAMP‐1 31. LipidTOX staining of neutral lipids is present in the epithelial cells of the acinus, but not the basal layer (A). DNase2 staining localizes in central cells of the acinus (B). DNase2 is located in neutral‐lipid (D) containing lysosomes (C) in the human MG. Note the cytoplasmic lipid droplets (D), which are also found in sebocytes 32. Scale bar = 50 μM. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; LAMP‐1, lysosomal‐associated membrane protein 1.

To confirm that DNase2 protein is expressed primarily in differentiated, as compared with proliferating, cells, we cultured HMGECs in PM, DM, or DM + AZM media and processed samples for immunofluorescence and Western blot analyses. Our results show that DNase2 is present predominantly in differentiated HMGECs (Fig. 5A, 5B).

Figure 5

Differential expression of DNase2 in proliferating versus differentiated human meibomian gland epithelial cells (HMGECs). DNase2 is manifest in HMGECs when cultured in DM or AZM, but not in PM, as shown by immunofluorescence (A) and Western blot (B) (n = 3 experiments,**p < .01). Scale bar = 50 μM. Abbreviations: AZM, azithromycin; DM, differentiation medium; PM, proliferation medium.

Plasticity of Lrig1 and DNase2 biomarkers in HMGECs

To determine whether the Lrig1 and DNase2 biomarkers are plastic and can be induced or suppressed by changing the proliferation and differentiation status of HMGECs, we performed two types of reversibility studies. First, we cultured HMGECs in PM for 3 days, switched the media to DM for another 3 days, and then returned the cells to PM (Fig. 6A). Second, we altered the media sequence, and began by placing the HMGECs in DM media, then switching to PM, followed by DM (Fig. 6C). As shown in Figures 6B and 6D, we discovered that proliferation stimulates, and differentiation suppresses, Lrig1 expression in HMGECs. The opposite is true for DNase2 expression (Fig. 6B, 6D).

Figure 6

Plasticity of biomarkers in human meibomian gland epithelial cells (HMGECs). Switching the culture media from PM to DM to PM (A, B), or from DM to PM to DM (C, D) induces corresponding shifts in cellular morphology (A, C) and Lrig1 and DNase2 expression (B, D). Proliferation stimulates, and differentiation reduces, Lrig1 appearance in HMGECs (A, B). The opposite response is found for DNase2 (C, D). Scale bar = 100 μM. Abbreviations: DM, differentiation medium; PM, proliferation medium.

Discussion

Our results demonstrate Lrig1 is expressed in the basal epithelial layer of the human MG, and not in the differentiated cells in the central section of the MG acinus. In contrast, DNase2 is expressed in the differentiated epithelial cells of the MG central acinus, and not in peripheral basal cells. Our findings also show that proliferation stimulates, and differentiation suppresses, Lrig1 expression in HMGECs. The opposite is true for DNase2 expression. These results support our hypothesis that Lrig1 and DNase2 serve as biomarkers for progenitor and differentiated cells in the human MG.

This differential distribution of Lrig1 in the basal layer, and not in the central area, of the MG is analogous to that found in SGs 1, 7, 8, 9. The SG basal layer is the source of Lrig1‐positive, proliferating progenitor cells, which give rise to differentiated sebocytes in the central region 7, 8, 9, 21, 22. Thus, it appears that meibocytes are generated from Lrig1‐positive progenitor cells in the MG basal layer 1, 23. This finding is consistent with the observation that MGs display a gradient of meibocyte maturation, with more undifferentiated, immature cells in the basal layer, and more mature, lipid‐containing cells in the center of the acinus 21, 22.

Lrig1 has also been identified as a marker of human corneal 24, epidermal 8 and intestinal 25, 26 stem cells, and the majority of Lrig1‐positive intestinal stem cells are proliferating cells 26. Other studies, though, have reported that Lrig1‐expressing stem cells are predominantly quiescent 8, 25, and that Lrig1 serves as a negative regulator of EGF receptor signaling to suppress stem cell proliferation 8, 27, 28. How, then, do we explain our finding that EGF and BPE promote proliferation, and stimulate the accumulation of Lrig1, in HMGECs? It is possible that Lrig1 may act within a negative feedback loop. As demonstrated by Gur et al. 27, Lrig1 expression increases in response to growth factor induced proliferation 17, in order to attenuate cell proliferation and maintain epithelial quiescence.

We discovered that DNase2 is expressed only in the lipid‐containing differentiated cells of the human MG. This cellular location likely reflects DNase2’s ultimate role, as in sebocytes, to initiate nuclear degeneration and holocrine secretion 10, 11. This autolytic process appears to be driven by endolysosomal DNase2, and results in a unique form of programmed cell death 10, 11. Of interest, we have observed that androgen treatment of HMGECs upregulates the gene expression for DNase2 (unpublished data). This response is associated with an androgen‐induced increase in genes promoting cellular differentiation and a decrease in those driving cell proliferation 29. The nature of this hormone response seems to mimic that of AZM, which decreases cell proliferation 19, stimulates differentiation, and enhances DNase2 accumulation in HMGECs.

We found that expression of the Lrig1 and DNase2 biomarkers is plastic and that both biomarkers can be induced or suppressed repeatedly by changing the proliferation and differentiation status of HMGECs. Such reversibility has also been observed in primary human corneal cells 30, indicating that the immortalized HMGECs share characteristics of primary cells. Indeed, we have previously found that many of the genetic responses of immortalized HMGECs are the same as those of primary HMGECs 18. These findings suggest that this MG cell line, which features a normal karyotype, represents an appropriate model in vitro to study HMGEC dynamics 16. Furthermore, it may be that primary HMGECs have similar biomarker plasticity, which could have implications for MG regeneration in vivo.

Conclusion

In summary, our results support our hypothesis that Lrig1 and DNase2 serve as biomarkers for progenitor and differentiated cells, respectively, in the human MG. Future studies will help determine the precise role of these proteins in HMGECs.

Author Contributions

D.A.S. and Y.L.: conception and design; H.‐T.X., D.C., M.P.H. and Y.L.: data collection; H.‐T.X., D.A.S. and Y.L.: data analysis and interpretation; M.P.H.: provision of study material; H.‐T.X., D.A.S., W.R.K. and Y.L.: manuscript writing; H.‐T.X., D.A.S., D.C., M.P.H., W.R.K. and Y.L.: final approval of manuscript; D.A.S.: funding acquisition.

Disclosure of Potential Conflict of Interest

D.A.S. discloses patent holding, research funding and ownership interest with Singularis/Lμbris Biopharma ‐ Lubricin product in development and has no relationship to stem cells. All other authors indicated no potential conflicts of interest.