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Research Letter |

Effect of Azithromycin on Lipid Accumulation in Immortalized Human Meibomian Gland Epithelial Cells FREE

Yang Liu, MD1; Wendy R. Kam, MS1; Juan Ding, PhD1; David A. Sullivan, PhD1
[+] Author Affiliations
1Schepens Eye Research Institute, Massachusetts Eye and Ear, and Harvard Medical School, Boston
JAMA Ophthalmol. 2014;132(2):226-228. doi:10.1001/jamaophthalmol.2013.6030.
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Published online

Meibomian gland dysfunction (MGD) is believed to be the leading cause of dry eye disease (DED), which affects tens of millions of Americans.1 Of particular interest, the most common pharmaceutical treatment for the management of MGD in the United States is the off-label use of topical azithromycin.2 This macrolide antibiotic is presumed to be effective because of its anti-inflammatory and antibacterial actions, which may suppress the MGD-associated posterior blepharitis and growth of bacteria on the eyelid.3 However, to our knowledge, no published, peer-reviewed data demonstrate that azithromycin has the ability to act directly on the human meibomian gland to enhance this tissue’s function and to ameliorate the pathophysiology of MGD.

We hypothesize that azithromycin can act directly on human meibomian gland epithelial cells to stimulate their differentiation, enhance the quality and quantity of their lipid production, and promote their holocrine secretion. Our purpose was to begin to test our hypothesis.

Immortalized human meibomian gland epithelial cells (IHMGECs; passages 20-22) were cultured in the presence or absence of 10% fetal bovine serum as previously reported.4 Cells were treated with the ethanol vehicle or azithromycin (10 µg/mL; Santa Cruz Biotechnology) for varying periods. Cellular morphological appearance was recorded, cells were counted with a hemocytometer, and lipid accumulation was assessed by staining cells with LipidTOX green neutral lipid stain (Invitrogen Corp) according to reported methods.4 Staining fluorescent intensities were quantified using ImageJ software (http://rsbweb.nih.gov/ij/index.html). Statistical analyses were performed with t test (2-tailed, unpaired).

Our results show that azithromycin induces a striking, time-dependent accumulation of lipid in IHMGECs (Figure 1A). Within 3 days of azithromycin exposure, the number, size, and staining intensity of intracellular lipid-containing vesicles had markedly increased as compared with those of vehicle-treated control cells. This azithromycin effect on lipids appeared to become maximal at days 3 to 7 of the study (Figure 1B).

Place holder to copy figure label and caption
Figure 1.
Effect of Azithromycin on the Lipid Accumulation and Morphology of Immortalized Human Meibomian Gland Epithelial Cells

Cells were treated with ethanol vehicle or azithromycin in serum-containing media for 7 days. Results are representative of 3 separate experiments. A, Appearance of cellular lipids. Cells were fixed and stained with LipidTOX green neutral lipid stain and 4’,6-diamidino-2-phenylindole (DAPI; red nuclear stain) (Invitrogen Corp) (original magnification ×400). B, LipidTOX staining intensity. Means are reported as fold-change compared with control values on the same day. Error bars indicate standard error. C, Cellular morphology. Images were taken prior to LipidTOX staining. Azithromycin-induced cellular maturation and vesicle accumulation were often followed by cell disruption and vesicle release (arrowhead, day 7) (original magnification ×200).aSignificantly greater than control (P < .005).

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Evaluation of cellular morphology indicated that azithromycin may promote terminal maturation of IHMGECs given that vesicle accumulation was often followed by a cell break-up and vesicle release (Figure 1C).

In contrast to these effects, azithromycin reduced the proliferation of IHMGECs. As shown in Figure 2, this result was found irrespective of whether IHMGECs were cultured under proliferation or differentiation conditions.

Place holder to copy figure label and caption
Figure 2.
Influence of Azithromycin on Proliferation of Immortalized Human Meibomian Gland Epithelial Cells

Cells were cultured in the absence (A) or presence (B) of serum for up to 7 days. Cell numbers at day 0 represent the baseline, and data are reported as mean ± standard error. Similar results were found in 2 additional studies.aSignificantly less than control (P < .005).

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This study supports our hypothesis that azithromycin can act on human meibomian gland epithelial cells and stimulate their lipid accumulation. This azithromycin effect appears to be paralleled by a cellular maturation, a decreased proliferation, and a holocrine-like secretion.

This azithromycin action is quite notable because MGD is thought to be the most common cause of DED.1 Typically, the meibomian glands produce and release a lipid mixture that promotes the stability and prevents the evaporation of the tear film, thereby playing an essential role in ocular surface health. Conversely, MGD destabilizes the tear film and increases its evaporation. Meibomian gland dysfunction is caused primarily by hyperkeratinization of the terminal duct epithelium and reduced secretion quality, and it leads to cystic dilatation of glandular ducts, acinar cell death, and lipid deficiency.1 The end result is DED, characterized by a cycle of tear film hyperosmolarity and ocular surface stress and leading to increased friction, inflammation, and damage to the eye.5 The effect of moderate to severe DED is analogous to conditions such as dialysis and severe angina and is associated with significant pain, role limitations, low vitality, and poor general health.5

Given our finding that azithromycin stimulates the function and differentiation of IHMGECs in vitro, it is possible that this antibiotic may prove beneficial as a treatment for MGD and its associated DED in vivo.

Corresponding Author: Yang Liu, MD, Schepens Eye Research Institute, 20 Staniford St, Boston, MA, 02114 (yang.liu@schepens.harvard.edu).

Published Online: December 19, 2013. doi:10.1001/jamaophthalmol.2013.6030.

Author Contributions: Dr Liu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Liu, Sullivan.

Acquisition of data: Liu.

Analysis and interpretation of data: All authors.

Drafting of the manuscript: Liu, Sullivan.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Liu, Sullivan.

Obtained funding: Liu, Sullivan.

Administrative, technical, or material support: Kam, Ding.

Study supervision: Liu, Sullivan.

Conflict of Interest Disclosures: Schepens Eye Research Institute is planning to submit a provisional patent based, in part, on the data presented in the article. No other disclosures were reported.

Funding/Support: This work was supported by grant EY05612 from the National Institutes of Health, the Margaret S. Sinon Scholar in Ocular Surface Research Fund, and the Guoxing Yao Research Fund.

Role of the Sponsor: The funding organizations had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Knop  E, Knop  N, Millar  T, Obata  H, Sullivan  DA.  The international workshop on meibomian gland dysfunction: report of the subcommittee on anatomy, physiology, and pathophysiology of the meibomian gland. Invest Ophthalmol Vis Sci. 2011;52(4):1938-1978.
PubMed   |  Link to Article
Lemp  MA, Nichols  KK.  Blepharitis in the United States 2009: a survey-based perspective on prevalence and treatment. Ocul Surf. 2009;7(2)(suppl):S1-S14.
PubMed   |  Link to Article
Geerling  G, Tauber  J, Baudouin  C,  et al.  The international workshop on meibomian gland dysfunction: report of the subcommittee on management and treatment of meibomian gland dysfunction. Invest Ophthalmol Vis Sci. 2011;52(4):2050-2064.
PubMed   |  Link to Article
Liu  S, Kam  WR, Ding  J, Hatton  MP, Sullivan  DA.  Effect of growth factors on the proliferation and gene expression of human meibomian gland epithelial cells. Invest Ophthalmol Vis Sci. 2013;54(4):2541-2550.
PubMed   |  Link to Article
Sullivan  DA, Hammitt  KM, Schaumberg  DA,  et al.  Report of the TFOS/ARVO Symposium on global treatments for dry eye disease: an unmet need. Ocul Surf. 2012;10(2):108-116.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.
Effect of Azithromycin on the Lipid Accumulation and Morphology of Immortalized Human Meibomian Gland Epithelial Cells

Cells were treated with ethanol vehicle or azithromycin in serum-containing media for 7 days. Results are representative of 3 separate experiments. A, Appearance of cellular lipids. Cells were fixed and stained with LipidTOX green neutral lipid stain and 4’,6-diamidino-2-phenylindole (DAPI; red nuclear stain) (Invitrogen Corp) (original magnification ×400). B, LipidTOX staining intensity. Means are reported as fold-change compared with control values on the same day. Error bars indicate standard error. C, Cellular morphology. Images were taken prior to LipidTOX staining. Azithromycin-induced cellular maturation and vesicle accumulation were often followed by cell disruption and vesicle release (arrowhead, day 7) (original magnification ×200).aSignificantly greater than control (P < .005).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Influence of Azithromycin on Proliferation of Immortalized Human Meibomian Gland Epithelial Cells

Cells were cultured in the absence (A) or presence (B) of serum for up to 7 days. Cell numbers at day 0 represent the baseline, and data are reported as mean ± standard error. Similar results were found in 2 additional studies.aSignificantly less than control (P < .005).

Graphic Jump Location

Tables

References

Knop  E, Knop  N, Millar  T, Obata  H, Sullivan  DA.  The international workshop on meibomian gland dysfunction: report of the subcommittee on anatomy, physiology, and pathophysiology of the meibomian gland. Invest Ophthalmol Vis Sci. 2011;52(4):1938-1978.
PubMed   |  Link to Article
Lemp  MA, Nichols  KK.  Blepharitis in the United States 2009: a survey-based perspective on prevalence and treatment. Ocul Surf. 2009;7(2)(suppl):S1-S14.
PubMed   |  Link to Article
Geerling  G, Tauber  J, Baudouin  C,  et al.  The international workshop on meibomian gland dysfunction: report of the subcommittee on management and treatment of meibomian gland dysfunction. Invest Ophthalmol Vis Sci. 2011;52(4):2050-2064.
PubMed   |  Link to Article
Liu  S, Kam  WR, Ding  J, Hatton  MP, Sullivan  DA.  Effect of growth factors on the proliferation and gene expression of human meibomian gland epithelial cells. Invest Ophthalmol Vis Sci. 2013;54(4):2541-2550.
PubMed   |  Link to Article
Sullivan  DA, Hammitt  KM, Schaumberg  DA,  et al.  Report of the TFOS/ARVO Symposium on global treatments for dry eye disease: an unmet need. Ocul Surf. 2012;10(2):108-116.
PubMed   |  Link to Article

Correspondence

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