Relationships between ocular surface sphingomyelinases, Meibum and Tear Sphingolipids, and clinical parameters of meibomian gland dysfunction

Abstract

Purpose:

Sphingolipids (SPL) are a class of lipid molecules that play important functional and structural roles in our body and are a component of meibum. Sphingomyelinases (SMases) are key enzymes in sphingolipid metabolism that hydrolyze sphingomyelin (SM) and generate ceramide (Cer). The purpose of this study was to examine relationships between ocular surface SMases, SPL composition, and parameters of Meibomian gland dysfunction (MGD).

Methods:

Individuals were grouped by meibum quality (n = 25 with poor-quality, MGD, and n = 25 with good-quality, control). Meibum and tears were analyzed with LC-MS to quantify SPL classes: Cer, Hexosyl-Ceramide (Hex-Cer), SM, Sphingosine (Sph), and sphingosine 1-phosphate (S1P). SMase activity in tears were quantified using a commercially available ‘SMase assay’. Statistical analysis included multiple linear regression analyses to assess the impact of SMase activity on lipid composition, as well as ocular surface symptoms and signs of MGD.

Results:

Demographic characteristics were similar between the two groups. nSMase and aSMase levels were lower in the poor vs good quality group. aSMase activity in tears negatively correlated with SM in meibum and tears and positively with Sph in meibum and S1P in tears. Lower SMase activity were associated with signs of MGD, most notably Meibomian gland dropout.

Conclusion:

This study suggests that individuals with MGD have reduced enzymatic activity of SMases in tears. Specifically, individuals with poor vs good meibum quality were noted to have alterations in SMase activity and SPL composition of meibum and tears which may reflect deviations from normal lipid metabolism in individuals with MGD.

1. Introduction

Meibomian glands (MG) are responsible for the secretion of meibum, and are thought to contribute to maintenance and stabilization of a healthy tear film lipid layer (TFLL) [1]. MG dysfunction (MGD) describes a group of diseases resulting in abnormal properties and secretion of meibum [2]. MGD is common, affecting 32%–78% of the population and is believed to contribute to dry eye (DE) through disruption of the TFLL [3–7]. Specifically, deficiencies in the quantity and quality of meibum contribute to increased evaporation, inflammation, tear hyperosmolarity, and TFLL destabilization, which can contribute to ocular symptoms of pain and discomfort [8].

Several clinical abnormalities in meibomian glands (MG) and their secreted product meibum have been described in individuals with MGD including MG dropout (typically viewed with retro illumination or infrared imaging), plugging of the MG orifices, decreased gland expressibility, and alterations of meibum from a “motor oil” to a “toothpaste” like consistency [8–12]. Various pathophysiologic mechanisms are thought to underlie these clinical changes including obstruction of the gland orifices (due to keratinization or fibrosis) [13], inflammation [14], altered expression of peroxisome proliferator-activated receptor gamma (PPARγ, a regulator of cell differentiation, function, and lipid synthesis), and meibocyte stem cell depletion [15,16], to name a few. Despite the various clinical manifestations and potential contributors, a unifying feature of MGD across studies is an alteration in meibum lipid composition.

Meibum is composed of non-polar lipids (including wax and sterol esters, triglycerides, and hydrocarbons), polar lipids (cerebrosides, polar lipids and sphingomyelin) and free fatty acids yielded in bacterial degradation [10]. Various factors, such as aging, and disease states, such as MGD, have been associated with changes in meibum composition. For example, a prior study which used high performance liquid chromatography to asses meibum composition found differential expression of polar and neutral lipid ions in younger versus older patients [17]. Recent studies suggest, individuals with MGD (defined by the presence of any chronic ocular symptoms and one or more lid margin abnormality: vascularity, displacement of the mucocutaneous junction, irregularity, obstruction, reduced expression) had decreases in non-polar lipids and increases in polar lipids, compared to healthy controls [18]; and alterations in cholesteryol esters do not contribute to tear film dynamics in MGD [19]. Within polar lipids, changes in sphingolipid (SPL) composition in MGD [20], are of significant interest, given their potential structural and functional implications on the ocular surface.

Sphingolipids (SPL) are ubiquitous lipids found in every cell of the body, mostly within the cell membrane and intra-cellular organelles [21]. In meibum, SPL, mainly in the form of sphingomyelin (SM), represent ~30% of polar lipids [22]. SM is generally considered an inactive membrane lipid, while ceramide (Cer) and sphingosine (Sph) (pro-apoptotic), and ceramide 1-phosphate (C1P) and sphingosine 1-phosphate (S1P) (anti-apoptotic) act as bioactive signaling lipids that are involved in inflammation. These lipids are easily interconvertible, and their proportions change in response to injury, stress, or inter- or intracellular signaling stimuli. This concept is known as the “SPL rheostat”, where interconversion of SPL classes, particularly between ceramide and S1P, occur within a cell and determine the cell’s fate.

The major family of enzymes involved in conversion between SPL classes are sphingomyelinases (SMases). These enzymes are phosphodiesterases which hydrolyze SM to generate Cer and phosphorylcholine [23]. SMases can be classified into classes that operate at neutral pH optimums (nSMase) and acidic pH optimums (aSMase) [24,25]. Oxidative stress is known to increase nSMase and aSMase activity and alters SPL metabolism. Specifically, nSMase activation has been implicated in a number of diseases including atherosclerosis, cancer, Alzheimer’s disease and cystic fibrosis [26–30]. Likewise, aSMase activation has been associated with cancer and emphysema [31–33]. Interestingly, a deficiency in aSMase is the cause of Neimann-Pick disease, a lysosomal storage disorder [34]. In addition to their intracellular location, SMases have also been recovered from human tears. In one study, aSMase activity was tested in various body fluids of 10 normal adults. Enzymatic assay showed higher levels of aSMase activity in tears compared to saliva and urine (11.30 ± 1.92 vs. 4.29 ± 1.82 and 0.59 ± 0.29 respectively) [35].

Furthermore, SMase levels have been found to change in response to stress. One study exposed human corneal epithelial cells to different stressors (ultraviolet B waves, hyperosmolar solution, and lipopolysaccharide) and noted increased SMase activity in stress conditions compared to non-exposed controls [36]. Given the presence of SPL in the tear film [20] and the potential contributions of inflammation and hyperosmolar stress to MGD pathology, research is needed to examine the role of ocular surface SMases in MGD. As such, the aim of this study was to evaluate relationships between human tear SMases with meibum and tear SPL and clinical parameters of MGD.

2. Methods

2.1. Study population

Patients were prospectively recruited from the Miami Veterans Administration Medical Center between October 2013 and August 2016. Exclusion criteria included anatomic abnormalities of the eyelids, conjunctivae, or cornea (e.g., ectropion, pterygium), contact lens use, history of refractive, glaucoma, or retina surgery, use of ocular medications other than artificial tears, active ocular surface process, or cataract surgery within the last 6 months. Patients with human immunodeficiency virus, sarcoidosis, graft-versus-host disease, or a collagen vascular disease (rheumatoid arthritis, lupus, Sjögrens) were also excluded. The Miami Veterans Administration Medical Center approved the prospective study, informed consent was obtained from all patients, and the study was adherent with the principles of the Declaration of Helsinki.

2.2. Data collection

Demographics, past medical history and medication information were collected via self-report and verified using medical records.

2.3. Primary grouping

Individuals were primary grouped based on their meibum quality. For this assessment, meibum was forcefully expressed and graded on a scale of 0–4 (0, clear; 1, cloudy; 2, granular; 3, toothpaste; 4, no meibum extracted) [37]. Individuals were divided into those with good quality meibum (controls), defined as grade 0 or 1 or poor quality meibum (cases), defined as a grade of 2, 3, or 4.

2.4. Ocular surface evaluation

All individuals underwent a standardized tear film assessment of both eyes including measurement of (1) tear osmolarity (TearLAB, San Diego, CA), (2) tear breakup time (TBUT), evaluated after 5 μl of fluorescein was placed on the superior conjunctivae, (3) corneal staining with fluorescein using the National Eye Institute scale [38], (4) Schirmer’s strips with anesthesia, and (5) meibomian gland (MG) assessment including characterization of meibum quality, inferior meibomian gland plugging (MG plugging), and meibomian gland dropout (MG atrophy) using retro-illumination with scoring using the meiboscale. MG plugging was graded on a scale of 0–3, defined as: 0, none; 1, less than 1/3 lid involvement; 2, between 1/3 and 2/3 lid involvement; 3, greater than 2/3 lid involvement [4,39]. MG atrophy was assessed by retroillumination and quantified using a five-point scale (0, area of loss 0%; 1, area of loss <25%; 2, area of loss 25%–50%; 3, area of loss 51–75%; 4, area of loss >75%) [40]. The quality of forcefully expressed meibum was graded as noted above [37].

2.5. DE symptoms

All participants filled out standardized dry eye (DE) questionnaires, the Ocular Surface Disease Index (OSDI, range 0–100) [41] and the 5-Item Dry Eye Questionnaire (DEQ-5, range 0–22) [42].

2.6. Meibum and tear sample collection

For meibum collection, a drop of proparacaine was placed on the ocular surface and on two cotton tip applicators. One applicator was placed behind and one in front of the interior tarsal plate and pressure was applied to forcefully express meibum from the inferior orifices. We placed the two cotton applicators centrally and applied pressure while moving the tips back and forth. On average, we aimed to express meibum from at least five glands from each eye and collect all expressed meibum. The expressed meibum was collected by swiping the cotton tip applicator across the inferior lid margin. The procedure was first performed in the right eye followed by the left eye, using the same applicator. The cotton tip applicator was then broken and the tip placed in an Eppendorf tube and immediately placed in −80 °C. Given this technique, our ‘meibum samples’ likely consist not only of expressed meibum, but also of meibum plugs and other components including marginal epithelial cells, cellular debris, tears, and/or microbes. For tears, Schirmer strips were placed in Eppendorf tubes after clinical testing (performed prior to meibum collection). Samples were frozen at −80 °C until further testing.

2.7. Analysis of SMases

Schirmer’s strips from one eye were used for the tear SPL assay and from the other eye for the SMase assay. The individual frozen Schirmer’s containing tear fluid were extracted in 200 μL of phosphate buffer saline (PBS, pH 7.4) with a protease inhibitor cocktail, which has broad specificity to inhibit serine, cysteine, and metalloproteases, and the protein contents was measured by BCA assay (ThermoFisher Scientific, Waltham, MA) [43,44]. Acidic and neutral sphingomyelinase (aSMase and nSMase, respectively) activity were measured from the extracted tears in triplicate using the Amplex^® Red Sphingomyelinase Assay kit (Invitrogen, Carlsbad, CA) following a previously published protocol from our group [45]. Briefly, in this method, SMase activity was measured in vitro indirectly in an enzyme-coupled assay using a fluorescence microplate reader. The SMase present in the sample first hydrolyzes the sphingomyelin (supplied in the reaction) to yield ceramide and phosphorylcholine. After the action of alkaline phosphatase, which hydrolyses phosphorylcholine, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, H₂O₂, in the presence of horseradish peroxidase, reacts with Amplex^® Red reagent in a 1:1 stoichiometry to generate the highly fluorescent product, resorufin, which is measured at absorption and emission maxima of ~571 nm and 585 nm, respectively. Thus, we present the SMase activity in Relative Fluorescence Unit (RFU); higher RFU associates with higher SMase activity and lower RFU with lower activity. This methodology can be used to continuously assay SMase enzymes with near-neutral pH optima (pH ~ 7.4) i.e. nSMase. Whereas aSMase activity can be measured in two-steps where aSMase reaction is performed at a lower pH, (in 50 mM sodium acetate, pH 5.0) and then the pH is raised to 7.0–8.0 (by adding an equal volume of 100 mM Tris-HCl, pH 8.0) to allow detection with the Amplex^® Red reagent. nSMase and aSMase RFUs were normalized with their protein content and these normalized values were used for statistical analysis. In individuals with nondetectable levels of SMases in tears, we set the values to 0 so that descriptive data could be presented.

2.8. Analysis of sphingolipids

Lipids from the entire cotton bud (meibum), and from the entire Schirmer’s strip (tears) were extracted using a modified Bligh & Dryer method described previously [46]. Further, analysis of SPL was carried out using a liquid chromatography/mass spectrometry, as discussed previously [20].

2.9. Statistical analysis

Descriptive statistics were performed to summarize patient baseline demographics and clinical information. Differences in SMase activity between groups were assessed via non-parametric tests, as given that data was not normally distributed. Correlations between variables were examined via Spearman’s rho. The more severe value from either eye was used when examining DE signs, and in the case of tear osmolarity, the differences between eyes was also correlated with SMase activity. In this paper, there is information given on all variables being compared as opposed to correcting the p-value (e.g., Bonferroni) since the latter methodology has its own limitations [47]. All statistical analyses were performed using SPSS 26.0 (SPSS, Inc., Chicago, IL).

3. Results

Fifty individuals participated in the study (mean age 57 ± 9 years; 84% men; 38% white, 60% black; 18% Hispanic). Comorbidities and systemic drug use were similar between the groups (poor vs good meibum quality), except for NSAID use which was significantly greater in the good meibum quality group (Table 1).

Table 1.

Demographics grouped by meibum quality.

	Good Meibum Quality (N = 25)	Poor Meibum Quality (N = 25)	P-Value
Age, mean ± SD	55 ± 11 years	59 ± 7 years	0.16
Sex, male % (n)	88% (22)	80% (20)	0.44
Race, White % (n)	48% (12)	28% (7)	0.24
Black % (n)	52% (13)	68% (17)
Ethnicity, Hispanic % (n)	16% (4)	20% (5)	0.71
Co-morbidities
PTSD, % (n)	32% (8)	16% (4)	0.19
Depression, % (n)	72% (18)	80% (20)	0.51
Arthritis, % (n)	52% (13)	46% (11)	0.67
Sleep Apnea, % (n)	20% (5)	20% (5)	1.00
Hypercholesterolemia, % (n)	44% (11)	52% (13)	0.57
Hypertension, % (n)	56% (14)	56% (14)	1.00
Rosacea, % (n)	0% (0)	0% (0)	0.00
Benign Prostastic	16% (4)	16% (4)	1.00
Hyperplasia, % (n)
Medication Use
NSAID, % (n)	44% (11)	16% (4)	0.03*
Anti-Depressant, % (n)	52% (13)	72% (18)	0.15
Anti-Anxiety, % (n)	60% (15)	72% (18)	0.37
Antihistamine, % (n)	24% (6)	16% (4)	0.48
Gabapentin, % (n)	36% (9)	20% (5)	0.21
Multivitamin, % (n)	64% (16)	56% (14)	0.56
Beta Blocker, % (n)	20% (5)	8% (2)	0.22
Statin, % (n)	48% (12)	40% (10)	0.57
Aspirin, % (n)	48% (12)	24% (6)	0.08
Sildenafil, % (n)	12% (3)	28% (7)	0.16

SD = standard deviation, n = number in group, PTSD = post-traumatic stress disorder, NSAID = nonsteroidal anti-inflammatory drug

^{* =}p value < 0.05.

First, total nSMase and aSMase activity (measured by RFU on assay) in tears were quantified. Older individuals had lower SMase activity in tears compared to younger individuals (nSMase ρ = −0.41, p = 0.003; aSMase ρ = −0.25, p = 0.08). However, no differences were noted in SMase activity with respect to gender, race, and ethnicity. Individuals with poor meibum quality more frequently had no nSMase (68% vs 100%, p = 0.004) and aSMase (44% vs 92%, p = 0.001) activity noted in tears compared to individuals with good quality meibum. Overall, nSMases and aSMases activity was lower in individuals with poor meibum quality compared to controls (Table 2).

Table 2.

SMase activity in tears in individuals with poor versus good meibum quality.

SMase	Good Quality (N = 25)		Poor Quality (N = 25)		P-Value
	Median	IQR	Median	IQR
nSMase (RFU)	23,748	26,392	4205	13,341	<0.001
aSMase (RFU)	30,893	51,181	0	6263	<0.001

SMase = Sphingomyelinase; nSMase = neutral sphingomyelinase; aSMases = acid sphingomyelinase; RFU = relative fluorescence unit.

^*In individuals with non-detectible Smases, 0 RFU was inputed to provide the noted descriptives).

Next, relationships between SMase activity and mole percentages of SPL classes (obtained by dividing the pmole of the specific SPL by the total pmole of SPL extracted from the individual) were analyzed. Positive associations were noted between nSMase and aSMase activities and meibum Sph (ρ = 0.51 and 0.45, p < 0.001 and p = 0.001, respectively) (Table 3). Similarly, positive associations were noted between nSMase and aSMase activities and tear S1P (ρ = 0.51 and 0.49, p < 0.001, respectively).

Table 3.

Spearman’s Rho (ρ) coefficients between SMase activity and sphingolipid relative composition.

Spearmen rho (ρ)	Cer	Hex-Cer	SM	Sph	Sa	S1P
SPL relative composition in meibum
nSMase	−0.09	0.07	−0.23	0.51 ***	0.35 *	ND
aSMase	0.07	0.22	−0.33 *	0.45 ***	0.23	ND
SPL relative composition in tears
nSMase	−0.12	−0.24	−0.30 *	−0.25	ND	0.51 ***
aSMase	0.06	−0.26	−0.29 *	−0.27	ND	0.49 ***

SMase = Sphingomyelinases; nSMase = neutral sphingomyelinase; aSMases = acid sphingomyelinase; Cer = ceramide, Hex-Cer = hexosyl ceramide, SM = sphingomyelin, Sph = sphingosine, Sa = sphinganine, S1P = sphingosine 1-phosphate, ρ = correlation coefficient

^*Statistically significant difference at p-values <0.05,

^**p-value <0.01,

^***p-value <0.001.

Finally, relationships between SMase activity and ocular surface symptoms and signs of DE and MGD were analyzed. SMases, particularly nSMase activity, were positively associated with DE symptoms (DEQ5: ρ = 0.29; p = 0.045; OSDI ρ = 0.30, p = 0.03). SMase activity was negatively related to MG parameters, most strongly meibum quality (nSMases: ρ = −0.49; aSMase: ρ = −0.48, p < 0.005 for both), followed by MG dropout and plugging (Table 4).

Table 4.

Spearman’s Rho (ρ) coefficients between SMase activity and ocular surface symptoms and MGD/dry eye (DE) signs.

Spearman’s ρ	Ocular surface symptoms		MGD/DE Signs
	DEQ-5	OSDI	Meibum Quality	Eyelid Vascularity	Meibomian gland dropout	Meibomian Plugging	TBUT	Fluorescein Stain	Schirmer Test	MMP	Osmolarity	Osmolarity Difference
nSMase	0.29 *	0.30 *	−0.49 ***	0.08	−0.39 **	−0.21	0.21	−0.18	−0.22	0.14	0.09	0.02
aSMase	0.27	0.23	−0.48 ***	−0.02	−0.35 *	−0.30 *	0.12	−0.03	−0.22	0.11	−0.04	−0.05

Relationship between SMase activity in tears and parameter of DE symptoms and MGD/DE signs. For all MGD/DE signs, the more severe value for either eye was used in the analysis. For osmolarity, both the highest value from either eye and the difference in values between eyes were used in the analysis. DE = Dry Eye, SMase = Sphingomyelinases; nSMase = neutral sphingomyelinase; aSMase = acid sphingomyelinase; SPL = total sphingolipid, Cer = ceramide, Hex-Cer = hexosyl ceramide, SM = sphingomyelin, Sph = sphingosine, Sa = sphinganine, S1P = sphingosine 1-phosphate, TBUT = Tear Break-Up Time, MMP = Matrix Metallopeptidase-9, OSDI=Ocular Surface Disease Index, DEQ-5 = Dry Eye Questionnaire, ρ = correlation coefficient,

^*Statistically significant difference at p-values <0.05,

^**p-value <0.01,

^***p-value <0.001.

4. Discussion

This study examined SMase activity in tears and compared its relationship with different SPL metabolites present in meibum and tears with respect to various clinical parameters of MGD and DE. Overall, individuals with poor meibum quality showed less enzymatic activity for both acidic and neutral SMase in tears compared to individuals with good quality meibum. These results also suggest a relationship between SMase activity in tears and altered SPL metabolites in meibum and tears. In past work, it was noted that individuals with poor quality meibum had an increased relative composition of SM and decreased relative composition of Sph [20]. The decreased enzymatic activities of both aSMase and nSMase noted in this study could explain these previous findings, as SMases convert SM to ceramide. As such, it is not surprising that reduced SMase activity correlated with reduced level of S1P in tear and reduced levels of Sph in meibum. With respect to clinical parameters, decreased nSMase activity was most closely associated with abnormal meibum quality and Meibomian grand dropout, thus linking SPL composition, SMases, and clinical signs of MGD. This study supports the hypothesis that individuals with MGD have changes in SMase activity which contributes to changes in their SPL metabolic profile. While these findings did not indicate that changes in SMase activity were related to DE signs, the well-described relationship between MGD and DE warrants further efforts to examine whether SMase activity contributes to the development or propagation of DE disease.

The metabolism of SPL is complex and dependent upon the involvement of several enzymes, and it is compartmentalized in different cellular organelles [48]. The de novo SPL synthesis takes place in the endoplasmic reticulum where a key SPL metabolite Cer is generated [49,50]. Cer is then used to synthesize all the higher order SPLs such as SM, which is the most abundant SPL. Sphingomyelinase enzymes can hydrolyze SM to Cer in a reverse reaction. Ceramidases convert Cer into Sph which can be further converted to its phosphorylated form of S1P by sphingosine kinases [51,52]. Maintaining a balanced composition of SPL is important for any cell or tissue, what is known as the ‘sphingolipid rheostat’, which is maintained by the aforementioned key enzymes (Fig. 1).

Fig. 1. Overview of Bioactive Sphingolipid Metabolism from Sphingomyelins.

Sphingomyelins, hexosylceramides, cerebrosides and gangliosides are complex membrane sphingolipids synthesized from Ceramide, the key molecule of sphingolipid metabolism in a cell. The major catabolic pathway of ceramide generation in a cell is from the hydrolysis of Sphingomyelin by Sphingomyelinases (neutral: nSMase, acidic: aSMase). Ceramide kinase phosphorylates ceramide to Ceramide 1-phosphate, and Ceramidases hydrolyze ceramide to Sphingosine. Sphingosine is phosphorylated to Sphingosine 1-phosphate (S1P) by Sphingosine kinases. Ceramide and sphingosine are overall pro-apoptotic lipids, whereas S1P has overall antiapoptotic effects, and a balance of their activity is called sphingolipid rheostat, important for cellular homeostasis.

The findings of reduced SMase activity in the tears of participants with poor quality meibum with increased SM and decreased S1P suggest that SPL metabolism is altered in the MGD group. Such alterations in the setting of MGD may point to a role in alterations in SPL metabolism playing a role in the development of the disease. However, further studies are needed to more robustly test this hypothesis. Increased SM and reduction in Sph in poor quality meibum may result from the affected metabolic pathway of SMase where SM acts as its substrate and Sph is the downstream metabolic product of this pathway (Fig. 1). Decreased S1P in tear may result from the decrease in its precursor (Sph) in the meibum supply. Decreases in S1P, on the other hand, might represent a shift of SPL rheostat towards higher Cer mediated actions in the ocular surface in MGD patients, which may generate a more inflammatory and cell-death inducing environment. Notably, the changes in SMase seen in these results did not correlate with findings of DE such as TBUT, Schirmer’s, etc. This may be the result of participant grouping methodology. It may also reflect differences in the effects of changes in SMase activity within different DE cohorts, such as an increased importance in individuals with signs of MGD.

Though not many studies are found on the role of SMase and the effect of SM accumulation on the ocular surface, loss of aSMase in Niemann-Pick disease A (NPA) leads to significant accumulation of SM and induces neurodegeneration [53,54]. SM is an essential component of the plasma membrane that maintains membrane homeostasis [55]. In NPA, the excessive accumulation of SM at the neuronal plasma membrane causes a higher intracellular calcium level, which induces oxidative stress and cell death [54]. The homeostasis of SM is necessary to maintain proper axonal polarity and synaptic plasticity in the cell [56]. In addition, the imbalance in SM affects cellular repair mechanisms [57]. Interestingly, NPA patients are prone to infection because loss of SM balance leads to decreased ability to repair transmembrane pores [58], thus SM plays a significant role in maintaining membrane integrity.

On the other hand, studies suggest that elevated levels of SMase and increases in Cer are associated with pathological changes in a number of diseases. For example, patients with sepsis showed increased level of SMase and inflammation in plasma [59]. In diabetes, dysfunctional retinal vasculature and vascular repair process have been associated with increased aSMase levels in human retinal endothelial cells [60]. Increased aSMase activity has also been described in atherosclerosis and cancer [29]. Taken together, the existing literature suggests that an optimal level of SMase activity is needed to maintain homeostasis in the sphingolipid rheostat and that reduced or increased levels may contribute to disease, including MGD.

Along with changes in bioactive signaling, differences in SPL relative composition may result in structural changes within the TFLL. This is because SPL also contribute tear film integrity through integration in the polar lipid monolayer that interfaces with non-polar lipids [61]. Additionally SPL act as a scaffold in the junction between non-polar bulk wax and the cholesterol esters above it [62]. Previous studies have indicated that increasing Cer concentration in meibum in vitro increases meibum melting temperature and rigidity, and destabilizes the lipid tear films [63]. This suggests that beyond their role in cellular signaling, Cer, and other SPL, have biophysical roles on the ocular surface.

Given the complexity of MGD, it is not surprising that discrepancies have been noted with regards to SPL composition between studies. While this group previously reported increased meibum SM in individuals with poor meibum quality [20], another study reported a lower meibum SM% (7.2% SD 4.8 vs 12.6% SD 5.6, p < 0.05) in individuals with chronic anterior and/or posterior blepharitis split by aqueous tear deficiency status (ATD, n = 47; no ATD, n = 84, defined by corneal staining and a low tear meniscus) [64]. However, since all individuals in the latter study had some form of blepharitis, direct comparisons to this current work are challenging. Furthermore, prior studies did not concomitantly examine SMases and thus it is unknown if a similar pattern would have emerged. Our findings of decreased SMase activity in individuals with poor meibum quality can also be contrasted with Robciuc et al. who found that when human epithelial cells were exposed to stressors in the form of ultraviolet B radiation, hyperosmolarity, and lipopolysaccharide, dose-dependent increases in the secretion of SMases from cells were noted [36]. Significant differences in study design may have contributed to the noted differences, among which are the in vitro nature of the work, the specific insults used, and assessment at multiple time points.

As with all studies, these findings must be considered in light of the study limitations which include a specific population of South Florida veterans and specific collection and extraction techniques (placement of a topical anesthetic prior to forceful expression of meibum). In addition, SMase activity was measured in one eye while tear SPL in the other in order to ensure sufficient material. While concomitantly evaluating of SMase activity and SPL composition from the same Schirmer strip was considered, this was deferred given the risk of inaccurate results due to small sample volumes. It was additionally considered that collection of SPLs from meibum and tears could be derived from cell membranes on the ocular surface rather than free lipids. Our group previously examined the proportion of SPL sub-types and found similar SPLs in meibum and tears suggesting that the major source of SPL in tears was the meibomian gland. However, there were also differences in the relative proportions of some SPL sub-types in tears compared to meibum, suggesting that SPLs may also be generated or modified locally by cells and enzymes present on the ocular surface.

Despite these limitations, this study adds knowledge with regards to relationships between ocular surface SMases, SPL in tears and meibum, and a wide variety of ocular surface symptoms and signs. This paper builds on the growing body of research that suggest that SPL metabolism plays a role in MGD. Understanding the contribution of enzymes involved in SPL metabolism to MGD is important as there are potential therapeutic implications. For example, fingolimod (FTY720) is an inhibitor of S1P receptor function and is an approved treatment for multiple sclerosis. Its method of action involves reduction of S1P dependent egress of lymphocytes from lymph nodes and reductions in the recirculation of auto-aggressive T cells via lymph nodes and blood to the central nervous system [65]. One group studied a topical formulation of fingolimod 0.005% in a non-obese diabetic (NOD) mouse model of DE. Compared to baseline, topical administration, 3 times a day for 4 weeks, resulted in a greater improvement in TBUT (0.005%: 3.89s ± 0.21 vs. 2.18 ± 0.17s at baseline, p < 0.05) and a greater reduction in the inflammatory marker IL-1B, compared to untreated controls [66]. As such, future studies are needed to examine the impact of SMase manipulation on MGD.

Abbreviations

DE

dry eye

ADDE

aqueous deficient dry eye

EDE

evaporative dry eye

ATD

aqueous tear deficiency

TFLL

tear film lipid layer

MG

meibomian glands

MGD

meibomian glands dysfunction

SPL

sphingolipids

Cer

ceramide

Sph

sphingosine

C1P

ceramide 1-phosphate

S1P

sphingosine 1-phosphate

Hex-Cer

hexosyl ceramide

SM

sphingomyelin

Sa

sphinganine

SMase

sphingomyelinase

aSMase

acid sphingomyelinase

nSMase

neutral sphingomyelinase