Physiological Effects of Soat1 Inactivation on Homeostasis of the Mouse Ocular Surface.

Purpose: Soat1/SOAT1 have been previously reported to be critical for the biosynthesis of cholesteryl esters (CEs) in the mouse Meibomian glands (MGs) as the loss of function led to an arrest of CE production and a substantial accumulation of nonesterified cholesterol in the meibum, causing an increase in its melting temperature. The purpose of this study was to further investigate the role of Soat1 in meibogenesis and ocular surface physiology. Methods: The mouse ocular features of knockout Soat1-/- and wild type (WT) mice were studied using various ophthalmic and histological techniques, mouse lipidomes were monitored using liquid chromatography/mass spectrometry, whereas their transcriptomes were compared to characterize the effects of the mutation on the gene expression profiles in the MG and cornea. Results: Soat1-/- mice displayed increased tear production and severe corneal abnormalities, such as corneal thinning, (neo)vascularization, ulceration, and opacification that progressed with aging. Transcriptomic analyses led to identification of a range of significantly disrupted pathways, which included general and specific lipid metabolism-related pathways, keratinization, angiogenesis/(neo)vascularization, muscle contraction, and several other pathways. In addition, histological and histochemical experiments revealed morphological changes in the MG, cornea, and conjunctiva in Soat1-/- mice. Notably, the mRNA microarray expression level of Soat1 in WT MGs (log2 17.5) was 1000 × of that in the mouse cornea (log2 7.5). Conclusions: These findings suggest a direct involvement of Soat1/SOAT1 in MGs in maintaining ocular surface homeostasis, in general, and corneal health, specifically.

Dysregulation of Lipid Metabolism in Aging Meibomian Glands and Its Molecular Markers.

The main function of exocrine Meibomian glands (MGs) is to produce a lipid-rich secretion called meibum which plays a critical role in maintaining the ocular surface homeostasis of humans and most mammals. The chemical composition of meibum, and its quantity produced by MGs, largely determine whether it can fulfill its role successfully. Aging was frequently associated with the onset of various MG-related pathologies. The goal of this study was to determine how aging affects the chemical composition and quantity of meibum in mice, and identify possible molecular markers of aging. Unbiased, untargeted and targeted lipidomic evaluation of mouse MG lipids was conducted using liquid chromatography-high-resolution mass spectrometry, and the results were analyzed using Principal Component, Orthogonal Projections to Latent Structures Discriminant, and Partial Least Square Discriminant Analyses. We found that aging leads to dysregulation of lipid metabolism in MGs, changing the ratios of major classes of MG lipids (such as wax esters, triacylglycerols, and phospholipids) in a progressive manner. Several lipid species that belong to these groups of MG lipids are proposed as clear markers of aging in a mouse model.

Comparative Biophysical Study of Meibomian Lipids of Wild Type and Soat1-Null Mice: Implications to Meibomian Gland Dysfunction and Dry Eye Disease.

Purpose: The biophysical roles of Meibomian lipids (MLs) played in health and meibomian gland dysfunction (MGD) are still unclear. The purpose of this research is to establish the composition-structure-functional correlations of the ML film (MLF) using Soat1-null mice and comprehensive in vitro biophysical simulations. Methods: MLs were extracted from tarsal plates of wild type (WT) and Soat1 knockout (KO) mice. The chemical composition of ML samples was characterized using liquid chromatography - mass spectrometry. Comprehensive biophysical studies of the MLFs, including their dynamic surface activity, interfacial rheology, evaporation resistance, and ultrastructure and topography, were performed with a novel experimental methodology called the constrained drop surfactometry. Results: Soat1 inactivation caused multiple alternations in the ML profile. Compared to their WT siblings, the MLs of KO mice were completely devoid of cholesteryl esters (CEs) longer than C18 to C20, but contained 7 times more free cholesterol (Chl). Biophysical assays consistently suggested that the KO-MLF became stiffer than that of WT mice, revealed by reduced film compressibility, increased elastic modulus, and decreased loss tangent, thus causing more energy loss per blinking cycle of the MLF. Moreover, the KO mice showed thinning of their MLF, and reduced evaporation resistance. Conclusions: These findings delineated the composition-structure-functional correlations of the MLF and suggested a potential biophysical function of long-chain CEs in optimizing the surface activity, interfacial rheology, and evaporation resistance of the MLF. This study may provide novel implications to pathophysiological and translational understanding of MGD and dry eye disease.

Sdr16c5 and Sdr16c6 control a dormant pathway at a bifurcation point between meibogenesis and sebogenesis.

Genes Sdr16c5 and Sdr16c6 encode proteins that belong to a superfamily of short-chain dehydrogenases/reductases (SDR16C5 and SDR16C6). Simultaneous inactivation of these genes in double-KO (DKO) mice was previously shown to result in a marked enlargement of the mouse Meibomian glands (MGs) and sebaceous glands, respectively. However, the exact roles of SDRs in physiology and biochemistry of MGs and sebaceous glands have not been established yet. Therefore, we characterized, for the first time, meibum and sebum of Sdr16c5/Sdr16c6-null (DKO) mice using high-resolution MS and LC. In this study, we demonstrated that the mutation upregulated the overall production of MG secretions (also known as meibogenesis) and noticeably altered their lipidomic profile, but had a more subtle effect on sebogenesis. The major changes in meibum of DKO mice included abnormal accumulation of shorter chain, sebaceous-type cholesteryl esters and wax esters (WEs), and a marked increase in the biosynthesis of monounsaturated and diunsaturated Meibomian-type WEs. Importantly, the MGs of DKO mice maintained their ability to produce typical extremely long chain Meibomian-type lipids at seemingly normal levels. These observations indicated preferential activation of a previously dormant biosynthetic pathway that produce shorter chain, and more unsaturated, sebaceous-type WEs in the MGs of DKO mice, without altering the elongation patterns of their extremely long chain Meibomian-type counterparts. We conclude that the Sdr16c5/Sdr16c6 pair may control a point of bifurcation in one of the meibogenesis subpathways at which biosynthesis of lipids can be redirected toward either abnormal sebaceous-type lipidome or normal Meibomian-type lipidome in WT mice.

Primary cilia control cellular patterning of Meibomian glands during morphogenesis but not lipid composition.

Meibomian glands (MGs) are modified sebaceous glands producing the tear film's lipids. Despite their critical role in maintaining clear vision, the mechanisms underlying MG morphogenesis in development and disease remain obscure. Cilia-mediate signals are critical for the development of skin adnexa, including sebaceous glands. Thus, we investigated the role of cilia in MG morphogenesis during development. Most cells were ciliated during early MG development, followed by cilia disassembly during differentiation. In mature glands, ciliated cells were primarily restricted to the basal layer of the proximal gland central duct. Cilia ablation in keratine14-expressing tissue disrupted the accumulation of proliferative cells at the distal tip but did not affect the overall rate of proliferation or apoptosis. Moreover, impaired cellular patterning during elongation resulted in hypertrophy of mature MGs with increased meibum volume without altering its lipid composition. Thus, cilia signaling networks provide a new platform to design therapeutic treatments for MG dysfunction.

Probing dietary triacylglycerol metabolism and meibogenesis in mice: A stable isotope-labeled tracer liquid chromatography-tandem mass spectrometry study.

Exocrine meibomian glands (MGs) play a central role in the ocular physiology and biochemistry by producing in situ and, mostly, de novo a secretion (meibum), which is composed of a complex mixture of homologous lipids of various classes, in a metabolic pathway termed meibogenesis. Recent in vivo experiments with a number of mouse models demonstrated that inactivation of any of the major genes of meibogenesis led to alterations in the lipid composition of meibum and severe ocular and MG abnormalities that replicated various human ocular pathologies. However, the role of dietary lipids in meibogenesis, and in the onset and/or alleviation of these diseases, remains controversial. To uncover the role of dietary lipids, the metabolic transformations of a dietary lipid tracer-stable isotope-labeled glyceryl tri(oleate-1,2,3,7,8-13C5) (13C15-TO)-were investigated using liquid chromatography-high-resolution mass spectrometry. We demonstrated that major metabolic transformations of the tracer occurred in the stomach and small intestines where 13C15-TO underwent immediate and extensive transesterification into 13C5- and 13C10-substituted triacylglycerols of various lengths, giving a mixture of 13C-labeled compounds that remain virtually unchanged in the mouse plasma, liver, and white adipose tissue but were almost undetectable in the feces. Importantly, the tracer and its metabolites were virtually undetectable in MGs, even after 4 weeks of daily supplementation. Notably, unbiased principal component analysis of the data revealed no measurable changes in the overall chemical composition of meibum after the treatment, which implies no direct effect of dietary triacylglycerols on meibogenesis, and left their systemic effects as the most likely mechanism.

Dynamic Changes in the Gene Expression Patterns and Lipid Profiles in the Developing and Maturing Meibomian Glands.

Meibomian glands (MGs) and their holocrine secretion-meibum-play crucial roles in the physiology of the eye, providing protection from environmental factors and desiccation, among other functions. Importantly, aging was implicated in the deterioration of the morphology and functions of MGs, and the quantity and quality of meibum they produce, leading to a loss of its protective properties, while the meibum of young individuals and experimental animals provide ample protection to the eye. Currently, the molecular mechanisms of meibum biosynthesis (termed meibogenesis) are not fully understood. To characterize the physiological changes in developing and maturing MGs, we studied the lipidomes and transcriptomes of mouse MGs ranging from newborns to adults. The results revealed a gradual increase in the critical genes of meibogenesis (such as Elovl3, Elovl4, Awat2, and Soat1, among others) that positively correlated with the biosynthesis of their respective lipid products. The MG transcriptomes of young and adult mice were also analyzed using single-cell RNA sequencing. These experiments revealed the existence of multiple unique populations of MG cells (meibocytes, epithelial cells, and others) with specific combinations of genes that encode meibogenesis-related proteins, and identified clusters and subclusters of cells that were tentatively classified as meibocytes at different stages of differentiation/maturation, or their progenitor cells. A hypothesis was formulated that these cells may produce different types of lipids, and contribute differentially to the Meibomian lipidome.

Differential effects of dietary cholesterol and triglycerides on the lipid homeostasis in Meibomian glands.

Exocrine Meibomian glands (MG) play a central role in the ocular surface physiology by producing meibum - a lipid secretion composed of cholesteryl esters (CE), cholesterol (Chl), triacylgycerols (TAG), waxes and other types of lipids. MG were previously shown to synthesize Meibomian lipids (ML) in situ via a complex array of reactions termed meibogenesis. However, questions remain about the role of dietary lipids in meibogenesis. To establish if dietary Chl (DC) and TAG (DT) can participate in meibogenesis, we studied mice whose diet was supplemented with trace amounts of deuterated Chl (2H-Chl) and 13C-labeled triolein (13C-TO), and the products of their biosynthetic transformations were analyzed using LC/MS. We demonstrated that 2H-Chl, but not 13C-TO, could be directly incorporated into meibum. Furthermore, 2H-Chl was esterified into MG-specific ultra long 2H-CE, which were vastly different from plasma CE and 2H-CE. The measured 2H-Chl/Chl and 2H-CE/CE ratios in meibum increased in a time-dependent manner reaching ∼5% and ∼1.2 %, respectively. The 2H-Chl/2H-CE ratio was about 3.5x higher than that for endogenous unlabeled Chl and CE, indicating accumulation of 2H-Chl in meibum. The elongation pattern of Meibomian 2H-CE closely replicated that of unlabeled CE. On the other hand, 13C-TO was not detected in any of the ML samples as an intact lipid or its metabolized/hydrolyzed products. We conclude that DC can be directly esterified into MG-specific CE, while DT undergo extensive catabolic transformations before reaching MG. These findings demonstrate that DC can have a direct impact on MG and ocular surface lipid homeostasis and pathophysiology.

Depletion of Cholesteryl Esters Causes Meibomian Gland Dysfunction-Like Symptoms in a Soat1-Null Mouse Model.

Previous studies on ablation of several key genes of meibogenesis related to fatty acid elongation, omega oxidation, and esterification into wax esters have demonstrated that inactivation of any of them led to predicted changes in the meibum lipid profiles and caused severe abnormalities in the ocular surface and Meibomian gland (MG) physiology and morphology. In this study, we evaluated the effects of Soat1 ablation that were expected to cause depletion of the second largest class of Meibomian lipids (ML)-cholesteryl esters (CE)-in a mouse model. ML of the Soat1-null mice were examined using liquid chromatography high-resolution mass spectrometry and compared with those of Soat1+/- and wild-type mice. Complete suppression of CE biosynthesis and simultaneous accumulation of free cholesterol (Chl) were observed in Soat1-null mice, while Soat1+/- mutants had normal Chl and CE profiles. The total arrest of the CE biosynthesis in response to Soat1 ablation transformed Chl into the dominant lipid in meibum accounting for at least 30% of all ML. The Soat1-null mice had clear manifestations of dry eye and MG dysfunction. Enrichment of meibum with Chl and depletion of CE caused plugging of MG orifices, increased meibum rigidity and melting temperature, and led to a massive accumulation of lipid deposits around the eyes of Soat1-null mice. These findings illustrate the role of Soat1/SOAT1 in the lipid homeostasis and pathophysiology of MG.

Physiological effects of inactivation and the roles of Elovl3/ELOVL3 in maintaining ocular homeostasis.

Recently, elongase of very long chain fatty acids-3 (ELOVL3) was demonstrated to play a pivotal role in physiology and biochemistry of the ocular surface by maintaining a proper balance in the lipid composition of meibum. The goal of this study was to further investigate the effects of ELOVL3 ablation in homozygous Elovl3-knockout mice (E3hom) in comparison with age and sex matched wild-type controls (E3wt). Slit lamp examination of the ocular surface of mice, and histological examination of their ocular tissues, highlighted a severe negative impact of Elovl3 inactivating mutation on the Meibomian glands (MG) and conjunctiva of mice. MG transcriptomes of the E3hom and E3wt mice were assessed and revealed a range of up- and downregulated genes related to lipid biosynthesis, inflammation, and stress response, compared with E3wt mice. Heat stage polarized light microscopy was used to assess melting characteristics of normal and abnormal meibum. The loss of Elovl3 led to a 8°C drop in the melting temperature of meibum in E3hom mice, and increased its fluidity. Also noted were the excessive accumulation of lipid material and tears around the eye and severe ocular inflammation, among other abnormalities.

On the pivotal role of Elovl3/ELOVL3 in meibogenesis and ocular physiology of mice.

The purpose of this study was to examine the role of Elovl3 gene in meibogenesis and the impact of ELOVL3 protein ablation on the physiology of the mouse ocular surface and Meibomian glands (MGs). Elovl3 knockout, ELOVL3-ablated (E3hom) mice and their wild type littermates (E3wt) were studied side by side. E3hom mice had abnormal ocular phenotypes such as delayed eye opening, weeping eyes, crusty eyelids, eyelid edema, highly vascularized cornea and tarsal plates (TPs), slit eye, and increased tearing that resemble symptoms observed in human subjects with various forms of dry eye, MG dysfunction and blepharitis. Lipid profiling of E3hom TPs was conducted using chromatography and mass spectrometry. The analyses revealed that the lipid composition of E3hom TPs was strikingly different from that of their E3wt littermates. The mutation affected major classes of meibomian lipids - cholesteryl esters, wax esters, and cholesteryl esters of (O)-acylated w-hydroxy fatty acids. The studies illuminated the central role of ELOVL3 in producing C21:0-C29:0 fatty acids, including odd-chain and branched ones. Ablation of ELOVL3 leads to selective changes in the lipid composition of meibum, making E3hom mice instrumental in studying the mechanisms of the biosynthesis of meibum and modeling various pathologies of human ocular surface and adnexa.-Butovich, I. A., Wilkerson, A., Bhat, N., McMahon, A., Yuksel, S. On the pivotal role of Elovl3/ELOVL3 in meibogenesis and ocular physiology of mice.

Defective FasL expression is associated with increased resistance to melanoma liver metastases and enhanced natural killer cell activity.

The objective was to determine if the absence of FasL signaling would affect melanoma liver metastases by influencing the antimelanoma properties of liver natural killer (NK) cells. Melanoma liver metastases were induced in wild-type C57BL/6 mice and the gld/gld mutant C57BL/6 mouse strain that expresses a defective form of FasL (CD95L) that fails to engage and signal via the Fas receptor (CD95). Liver metastases were produced by intrasplenic injection of B16LS9 melanoma cells. Liver NK cell activity directed against murine B16LS9 melanoma cells was determined in a 24 h in-vitro cytotoxicity assay. Liver NK cells, NK T cells, and the NK cell surface activation marker, NKG2D, were measured by flow cytometry. Mice expressing defective FasL displayed reduced, rather than enhanced, melanoma liver metastases that coincided with increased liver NK cell-mediated tumor cell cytotoxicity. Enhanced cytotoxicity was not mediated by perforin, tumor necrosis factor-α, or tumor necrosis-associated apoptosis-inducing ligand but was closely associated with elevated interferon-γ in the tumor-bearing liver. FasL-defective gld/gld mice also displayed reduced numbers of liver NK T cells, which have been previously implicated in suppression on liver NK cell activity. The absence of functional FasL in the liver correlates with a heightened, not diminished, resistance to melanoma liver metastases. The resistance to liver metastases coincides with a significant, albeit transient, increase in liver NK cytotoxicity and elevated levels of interferon-γ in the liver.

Effects of sex (or lack thereof) on meibogenesis in mice (Mus musculus): Comparative evaluation of lipidomes and transcriptomes of male and female tarsal plates.

The possible role of sex in the biosynthesis of lipids in the Meibomian glands (termed meibogenesis) remains unclear. To determine if there were any major sex-specific differences in the lipid composition of meibomian gland secretions (meibum) and gene expression patterns (GEP) related to meibogenesis, we conducted a study using healthy, age and diet-matched young adult wild-type C57BL/6J mice (2-2.5 month old). Tarsal plates (TP) were surgically excised from the eyelids of mice and subjected to transcriptomic and lipidomic analyses. The GEP were studied using mRNA microarrays. Lipids were extracted with organic solvents and analyzed using liquid chromatography and mass spectrometry. GEP in the TP of female and male mice demonstrated no statistically significant differences in the expression levels of the main protein-coding genes related to lipid metabolism and storage in general, and meibogenesis specifically (such as Elovl, Scd, Fads, Soat, Far, Awat, Acat, Lss, Dhcr, Hmgcr, Hmgcs, Dgat, Bckdh, Dbt, Fasn, and Plin, among others). The meibomian lipid profiles of female and male mice were virtually indistinguishable: all major lipids such as waxes, cholesteryl esters, cholesterol, (O)-acylated omega-hydroxy fatty acids (OAHFA), cholesteryl esters of OAHFA etc., were present in similar ratios. It seems that the major biosynthetic pathways in the Meibomian glands of male and female mice function in a similar fashion and produce secretions of the same overall chemical composition.

Induction of Contrasuppressor Cells and Loss of Immune Privilege Produced by Corneal Nerve Ablation.

Purpose: Severing of corneal nerves in preparation of corneal transplantation abolishes immune privilege of subsequent corneal transplants placed into either eye: a phenomenon termed sympathetic loss of immune privilege (SLIP). SLIP is due to the disabling of T regulatory cells (Tregs) by CD11c+ contrasuppressor (CS) cells. This study characterized the induction, function, and manipulation of CS cell activity and the effect of these cells on Tregs induced by anterior chamber-associated immune deviation (ACAID). Methods: CS cells were induced using a 2.0-mm trephine to score the corneal epithelium. CD11c+ CS cells were evaluated by adoptive transfer and by their capacity to disable CD8+ ACAID Tregs in local adoptive transfer (LAT) of suppression assays. CD11c+ cells were deleted from the ocular surface by subconjunctival injection of clodronate-containing liposomes. Results: CD11c+ CS cell were radiosenstive and long lived. As few as 1000 CS cells blocked the suppressive activity of previously generated CD8+ ACAID Tregs, indicating that CS cells act at the efferent arm of the immune response. Depletion of resident CD11c+ cells at the ocular surface prevented the generation of CS cells. Conclusions: Corneal nerve injury that occurs during keratoplasty converts ocular surface CD11c+ cells into CS cells that block CD8+ Tregs, which are induced by introducing antigens into the anterior chamber (i.e., ACAID Tregs). Depletion of CD11c+ cells at the ocular surface prevents the generation of CS cells and may be a useful strategy for preventing SLIP and enhancing the survival of second corneal transplants.