Abstract
Purpose.
We discovered that dihydrotestosterone (DHT) decreases the ability of lipopolysaccharide, a bacterial toxin, to stimulate the secretion of leukotriene B4, a potent proinflammatory mediator, by immortalized human meibomian gland epithelial cells (IHMGECs). We hypothesize that this hormone action reflects an androgen suppression of proinflammatory gene activity in these cells. Our goal was to test this hypothesis. For comparison, we also examined whether DHT treatment elicits the same effect in immortalized human corneal (IHC) and conjuntival (IHConj) ECs.
Methods.
Differentiated cells were cultured in media containing vehicle or 10 nM DHT. Cells (n = 3 wells/treatment group) were then processed for RNA isolation and the analysis of gene expression by using Illumina BeadChips, background subtraction, cubic spline normalization and Geospiza software.
Results.
Our results demonstrate that DHT significantly suppressed the expression of numerous immune-related genes in HMGECs, such as those associated with antigen processing and presentation, innate and adaptive immune responses, chemotaxis, and cytokine production. DHT also enhanced the expression of genes for defensin β1, IL-1 receptor antagonist, and the anti-inflammatory serine peptidase inhibitor, Kazal type 5. In contrast, DHT had no effect on proinflammatory gene expression in HCECs, and significantly increased 33 gene ontologies linked to the immune system in HConjECs.
Conclusions.
Our findings support our hypothesis that androgens suppress proinflammatory gene expression in IHMGECs. This hormone effect may contribute to the typical absence of inflammation within the human meibomian gland.
Introduction
A remarkable property of the human meibomian gland (HMG) is its apparent resistance to inflammation.1,2 This does not mean that the HMG cannot become inflamed, such as in a single blocked gland with a chalazion. But rather that the HMG appears to resist inflammatory processes. For example, there is no evidence of overt inflammation within the glandular tissue in obstructive MG dysfunction (MGD), which affects multiple glands and may lead to conjunctival inflammation (i.e. posterior blepharitis).1–5 Moreover, although exposure to the bacterial lipopolysaccharide (LPS) and ligand binding protein (LBP) increases the secretion of the chemoattractant leukotriene B4 (LTB4),6 this endotoxin complex does not elicit the upregulation of proinflammatory response gene ontologies in HMG epithelial cells (ECs).7 This lack of HMGEC response is in striking contrast to those of human corneal (HC) and conjunctival (HConj) ECs. In these cells LPS+LBP stimulates a significant up-regulation of genes associated with inflammatory and immune responses, including those related to chemokine and Toll-like receptor signaling pathways, cytokine-cytokine receptor interactions, and chemotaxis.7
We hypothesize that the transcription of innate anti-inflammatory genes contributes to this resistance to inflammation within the HMG, and that the generation of these transcripts is, at least in part, stimulated by androgens. In support of this hypothesis, we have found that the most highly expressed gene in the HMG encodes for the anti-inflammatory protein, leukocyte-associated immunoglobulin-like receptor-1.8 Further, we have identified HMGEC genes that are upregulated during differentiation and are associated with the suppression of immune cell activation, pro-inflammatory cytokine production and inflammation.9 We have also discovered that androgen administration attenuates the secretion of LTB4 by HMGECs,6 decreases the expression of immune-related genes in proliferating HMGECs,10 and reduces the transcription of immune-response genes in male and female mouse meibomian glands.11,12
The purpose of our present investigation was to continue to test our hypothesis, and determine whether androgen exposure decreases the expression of proinflammatory genes in differentiated HMGECs. For comparison, we also examined whether androgen treatment elicits the same effect in HCECs and HConjECs.
Material and Methods
Cell cultures
Immortalized HMGECs,13 HCECs (from Dr. James Jester, Irvine, CA)14and HConjECs (from Dr. Ilene Gipson, Boston, MA)15 were cultured, as previously described.6,7 Briefly, HMGEC were cultured in keratinocyte serum free medium (KSFM) supplemented with 50 μg/ml bovine pituitary extract (BPE), 5 ng/ml epidermal growth factor (EGF), penicillin (100 IU/ml), and streptomycin (100 μg/ml). HCEC and HConjECs were cultured in KSFM supplemented with BPE (25 μg/ml), EGF, penicillin and streptomycin. Cells were kept in 75 cm2 flasks and then plated for studies in 6-well culture dishes (Corning, Lowell, MA). Once cells reached confluence, their numbers ranged from approximately 4 to 5 × 105 cells/well. The actual counts depended upon the cell type. All cell culture reagents were obtained from Invitrogen Corp. (Carlsbad, CA), except for Dulbecco’s modified Eagle’s medium (DMEM)/F12, which was purchased from Mediatech, Inc. (Manassas, VA).
After achieving confluence, cells (n = 3 wells/cell type/treatment) were rinsed with PBS and then placed in a differentiation medium containing DMEM/F12, 10% charcoal/dextran-treated fetal bovine serum (FBS; Invitrogen), 10 ng/ml EGF, penicillin and streptomycin in the presence or absence of ethanol or DHT (10 nM; Steraloids, Wilton, NH) for 2 days. Following this time period, cells were incubated with or without androgen (10 nM) for 6 more hours in serum-free DMEM/F12 containing 1% bovine serum albumin (Mediatech, Inc., Manassas, VA), as previously reported6 We used 10 nM DHT because this is the physiological concentration of DHT within tissues in vivo, and should occupy > 90% of cellular androgen receptors.16
RNA extraction and gene microarray analysis
Cellular RNA samples were processed for microarray analyses, as described.17 In brief, total RNA was purified using RNeasy Mini Kit (Qiagen, Inc., Valencia, CA), by following the manufacturer’s instructions. The RNA concentrations and 260/280 nm ratios were obtained by using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA). RNA integrity was examined by using a RNA Nano 6000 Series II Chip with a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). The RNA samples were then additionally processed by Asuragen (Austin, TX) for quantitation of mRNA levels using microarray expression analyses (HumanHT-12 v.4 Expression BeadChips; Illumina, San Diego, CA), as outlined6,8
Data that were non-log-transformed, background subtracted and cubic spline normalized were analyzed with commercial software (GeneSifter.net; Geospiza, Seattle, WA). This comprehensive program also produced Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways’, gene ontology and z-score reports. Standardized hybridization intensities were all adjusted by adding a constant, such that the lowest intensity value for a sample equaled 16.18,19 Illumina data were analyzed with Student’s t-test (two-tailed, unpaired). All data are accessible through the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) via series accession number GSE111496.
Results
Effect of DHT treatment on proinflammatory gene expression in HMGECs
To determine whether androgen treatment suppresses proinflammatory gene expression in differentiated HMGECs, we exposed cells (n = 3 wells/treatment) to vehicle or DHT and then processed cell samples for Illumina BeadChip and Geospiza software analyses.
Our results show that androgen exposure induced a significant influence on the expression of over 2,000 genes in HMGECs (Table 1). The most striking effect was the DHT suppression of numerous immune-related gene ontologies in HMGECs, such as those associated with innate and adaptive immune responses, chemokine activity and receptor binding, and cytokine production (Table 2). For example, DHT decreased the expression of 67 immune response genes (e.g. IL-1β and IL-8), including those involved with antigen processing and presentation, chemotaxis, T cell mediated immunity, and the production, binding, activity and signaling of cytokines and/or chemokines (Table 3). In contrast, DHT enhanced the expression of genes for defensin β1 (1.6-fold; p < 0.0005), IL-1 receptor antagonist (1.4-fold; p < 0.0005), and the anti-inflammatory serine peptidase inhibitor, Kazal type 5 (1.3-fold; p < 0.05) in IHMGECs.
Table 1.
DHT influence on gene expression in differentiated IHMGECs, IHCECs and IHConjECs
| Epithelial cells | Genes ↑ | Genes ↓ | Total genes |
|---|---|---|---|
| Meibomian gland | 1,130 | 1,158 | 2,288 |
| Cornea | 555 | 555 | 1,110 |
| Conjunctiva | 516 | 622 | 1,138 |
Data were evaluated without log transformation. The expression of listed genes was significantly (p < 0.05) up (↑)- or down (↓)-regulated by DHT treatment.
Table 2.
DHT down-regulation of immune-related gene ontologies in HMGECs
| Ontology | Genes ↓ | Z-score |
|---|---|---|
| Chemokine receptor binding | 10 | 4.45 |
| Positive regulation of adaptive immune response | 8 | 4.16 |
| Positive regulation of positive chemotaxis | 5 | 4.11 |
| Response to cytokine stimulus | 33 | 3.98 |
| Positive regulation of T cell mediated cytotoxicity | 4 | 3.96 |
| Regulation of positive chemotaxis | 5 | 3.94 |
| I-κB kinase/NF-κB cascade | 22 | 3.94 |
| T cell mediated immunity | 7 | 3.72 |
| Regulation of T cell mediated cytotoxicity | 4 | 3.56 |
| Chemokine activity | 8 | 3.56 |
| Regulation of adaptive immune response | 10 | 3.51 |
| Regulation of I-κB kinase/NF-κB cascade | 17 | 3.47 |
| Regulation of transforming growth factor- β production | 4 | 3.39 |
| Transforming growth factor-β production | 4 | 3.39 |
| Positive regulation of leukocyte mediated immunity | 7 | 3.37 |
| Positive regulation of lymphocyte mediated immunity | 7 | 3.37 |
| Cellular response to type I interferon | 10 | 3.18 |
| Type I interferon-mediated signaling pathway | 10 | 3.18 |
| Response to type I interferon | 10 | 3.13 |
| T cell mediated cytotoxicity | 4 | 3.08 |
| Regulation of immune effector process | 17 | 3.06 |
| Regulation of adaptive immune response based on somatic recombination of immune receptors built from | 9 | 3.02 |
| Response to interferon-γ | 11 | 2.97 |
| Antigen processing and presentation of peptide antigen via MHC class I | 4 | 2.94 |
| Regulation of chemotaxis | 9 | 2.91 |
| Regulation of cytokine production | 25 | 2.87 |
| Negative regulation of immune effector process | 5 | 2.84 |
| Cytokine receptor binding | 19 | 2.69 |
| Cytokine production | 26 | 2.56 |
| Chemotaxis | 42 | 2.52 |
| Innate immune response | 33 | 2.47 |
| Cellular response to cytokine stimulus | 21 | 2.41 |
| Positive regulation of leukocyte mediated cytotoxicity | 4 | 2.37 |
| Positive regulation of immune effector process | 9 | 2.34 |
| Regulation of defense response | 26 | 2.27 |
| Regulation of interleukin-2 production | 5 | 2.25 |
| Positive regulation of chemotaxis | 6 | 2.21 |
| Positive regulation of cytokine production | 13 | 2.18 |
| Regulation of lymphocyte mediated immunity | 7 | 2.16 |
| Defense response | 62 | 2.11 |
| Immune system process | 90 | 2.1 |
| Adaptive immune response | 13 | 2.09 |
| Regulation of type I interferon production | 6 | 2.09 |
| Negative regulation of defense response | 6 | 2.04 |
| Immune response | 61 | 2.03 |
Biological process and molecular function immune ontologies (≥ 5 genes/ontology) were identified following the analysis of non-transformed Illumina data. All of the immune ontologies listed in this Table were downregulated in HMGECs following DHT treatment. A z-score is a statistical rating of the relative expression of genes, and demonstrates how greatly they are over- or under-represented in a specific gene list.19 Positive z- scores reflect a greater number of genes meeting the criterion than is anticipated by chance, and values > 2.0 are significant. Terms: Genes ↓ - number of genes down-regulated in a given ontology; z-score - specific score for the down-regulated genes in a given onotology.
Table 3.
DHT downregulation of immune-related gene expression in HMGECs
| Accession # | Gene | Ratio | p value | Ontology |
|---|---|---|---|---|
| NM_004591 | Chemokine (C-C motif) ligand 20 | 2.05 | 0.0042 | chemotaxis |
| NM_002993 | Chemokine (C-X-C motif) ligand 6 | 1.99 | 0.0367 | chemotaxis |
| NM_001511 | Chemokine (C-X-C motif) ligand 1 | 1.99 | 0.0028 | chemotaxis |
| NM_000584 | Interleukin 8 | 1.93 | 0.0019 | angiogenesis |
| NM_001565 | Chemokine (C-X-C motif) ligand 10 | 1.67 | 0.0013 | chemotaxis |
| NM_002994 | Chemokine (C-X-C motif) ligand 5 | 1.61 | 0.0002 | chemotaxis |
| NM_002994 | Chemokine (C-X-C motif) ligand 5 | 1.54 | 0.0159 | chemotaxis |
| NM_022873 | Interferon, alpha-inducible protein 6 | 1.39 | 0.0049 | immune response |
| NM_006290 | Tumor necrosis factor, alpha-induced protein 3 | 1.39 | 0.0304 | B-1 B cell homeostasis |
| NM_000576 | Interleukin 1, beta | 1.33 | 0.0017 | chronic inflammatory response to antigenic stimulus |
| NM_006332 | Interferon, gamma-inducible protein 30 | 1.29 | 0.0293 | cytokine-mediated signaling pathway |
| AB074172 | Interleukin 6 signal transducer | 1.29 | 0.0060 | positive regulation of acute inflammatory response |
| NM_006147 | Interferon regulatory factor 6 | 1.28 | 0.0065 | cytokine-mediated signaling pathway |
| NM_021649 | Toll-like receptor adaptor molecule 2 | 1.28 | 0.0074 | toll-like receptor signaling pathway |
| NM_001004196 | CD200 molecule | 1.27 | 0.0246 | regulation of immune response |
| NM_138806 | CD200 receptor 1 | 1.26 | 0.0239 | regulation of immune response |
| NM_003921 | B-cell CLL/lymphoma 10 | 1.26 | 0.0112 | toll-like receptor signaling pathway |
| NM_003238 | Transforming growth factor, β2 | 1.25 | 0.0109 | immune effector process |
| NM_002089 | Chemokine (C-X-C motif) ligand 2 | 1.19 | 0.0425 | chemotaxis |
| NM_022059 | Chemokine (C-X-C motif) ligand 16 | 1.13 | 0.0199 | chemotaxis |
| NM_004048 | Beta-2-microglobulin | 1.1 | 0.0073 | positive regulation of T cell mediated cytotoxicity |
| NM_002198 | Interferon regulatory factor 1 | 1.09 | 0.0206 | cytokine-mediated signaling pathway |
Accession numbers are the sequence identities of gene fragments expressed on the Illumina BeadChips. These sequences are listed in the nucleotide database of the National Center for Biotechnology Information (NCBI). Relative ratios were calculated by comparing the degree of gene expression in HMGECs after vehicle and DHT treatment. Ratios were determined by using non-transformed data.
In addition to these anti-inflammatory effects, DHT exerted a significant impact on many nonimmune ontologies. These included an upregulation of numerous genes associated with lipid dynamics and differentiation/autophagic processes, and a downregulation of a multitude of genes related to the cell cycle and cell proliferation (Table 4). DHT also significantly influenced assorted KEGG pathways, increasing those linked to lysosomes and mammalian target of rapamycin (mTOR) signaling, and decreasing those mediating cell cycle, chemokine signaling and antigen processing and presentation (Table 4).
Table 4.
DHT regulation of non-immune-related gene ontologies in HMGECs
| Ontology / Pathway | DHT genes ↑ | DHT genes ↓ | Z-score ↑ | Z-score ↓ |
|---|---|---|---|---|
| Biological process ontologies | ||||
| Lipids | ||||
| Lipid metabolic process | 89 | 41 | 6.26 | −1.81 |
| Cellular lipid metabolic process | 57 | 28 | 4.44 | −1.47 |
| Phosphatidylcholine biosynthetic process | 4 | 1 | 3.66 | 0.12 |
| Steroid metabolic process | 25 | 12 | 3.55 | −0.6 |
| Lipid biosynthetic process | 36 | 25 | 3.49 | 0.43 |
| Phosphatidylcholine metabolic process | 5 | 1 | 3.43 | −0.34 |
| Phospholipid metabolic process | 19 | 9 | 2.99 | −0.62 |
| Lipid transport | 17 | 8 | 2.86 | −0.58 |
| Regulation of lipid metabolic process | 14 | 6 | 2.86 | −0.58 |
| Glycerophospholipid biosynthetic process | 9 | 5 | 2.77 | 0.41 |
| Lipid localization | 18 | 9 | 2.68 | −0.6 |
| Phospholipid biosynthetic process | 12 | 8 | 2.67 | 0.65 |
| Glycerolipid biosynthetic process | 12 | 9 | 2.63 | 1.03 |
| Lipid catabolic process | 18 | 5 | 2.26 | −2.09 |
| Cellular lipid catabolic process | 11 | 3 | 2.21 | −1.41 |
| Sphingolipid metabolic process | 9 | 3 | 2.17 | −0.93 |
| Glycerophospholipid metabolic process | 11 | 6 | 2.12 | −0.25 |
| Cholesterol efflux | 4 | 1 | 2.09 | −0.54 |
| Differentiation / autophagy | ||||
| Vacuole | 42 | 22 | 6.8 | 1.31 |
| Macroautophagy | 8 | 1 | 6.18 | −0.34 |
| Lysosome | 35 | 19 | 6.08 | 1.3 |
| Lytic vacuole | 35 | 19 | 6.08 | 1.3 |
| Autophagy | 13 | 3 | 5.76 | −0.25 |
| Vacuole organization | 11 | 2 | 5.59 | −0.46 |
| Epidermal cell differentiation | 16 | 5 | 5.56 | −0.01 |
| Autophagic vacuole assembly | 6 | 1 | 5.5 | 0 |
| Keratinocyte differentiation | 13 | 5 | 4.83 | 0.38 |
| Vacuolar membrane | 20 | 10 | 4.81 | 0.81 |
| Autophagic vacuole | 6 | 0 | 4.47 | −1.16 |
| Keratinization | 8 | 2 | 4.25 | −0.19 |
| Epithelial cell differentiation | 20 | 9 | 3.17 | −0.71 |
| Cell cycle | ||||
| Mitotic cell cycle | 22 | 94 | −2.02 | 10 |
| Cell cycle phase | 23 | 97 | −2.34 | 9.42 |
| Cell cycle process | 31 | 110 | −2.08 | 9.31 |
| Cell cycle | 51 | 134 | −1.02 | 9.28 |
| Regulation of cell cycle | 31 | 77 | 0.36 | 8.26 |
| Interphase | 11 | 52 | −1.54 | 7.88 |
| Interphase of mitotic cell cycle | 11 | 50 | −1.46 | 7.6 |
| Mitosis | 12 | 46 | −1.08 | 6.89 |
| Nuclear division | 12 | 46 | −1.08 | 6.89 |
| Cell proliferation | 56 | 118 | −0.52 | 6.84 |
| M phase of mitotic cell cycle | 12 | 46 | −1.2 | 6.63 |
| M phase | 16 | 56 | −1.51 | 6.35 |
| G1/S transition of mitotic cell cycle | 6 | 28 | −0.91 | 6.19 |
| Ribosome biogenesis | 3 | 23 | −1.39 | 6.14 |
| KEGG pathways | ||||
| Lysosome | 24 | 5 | 7.28 | −0.85 |
| Metabolic pathways | 81 | 64 | 3.69 | −0.03 |
| mTOR signaling pathway | 8 | 1 | 3.36 | −1.21 |
| Aldosterone-regulated sodium reabsorption | 6 | 1 | 2.71 | −0.95 |
| RNA transport | 5 | 25 | −1.11 | 5.51 |
| Cell cycle | 5 | 19 | −0.59 | 4.51 |
| Chemokine signaling pathway | 9 | 20 | −0.27 | 2.79 |
| Antigen processing and presentation | 6 | 8 | 1.41 | 2.14 |
Biological process ontologies (≥ 5 genes/category) related to lipids, differentiation / autophagy and cell cycle, as well as assorted KEGG pathways, were selected after analyses of non-transformed data. Those ontologies and pathways with a z-score > 2.0 are highlighted in bold. Terms are analogous to those described in the legend to Table 2.
Influence of DHT on proinflammatory gene expression in HCECs and HConjECs
To examine whether androgen exposure also decreases proinflammatory gene expression in differentiated HCECs and HConjECs, we treated these cells (n = 3 wells/treatment) with vehicle or DHT and then processed the samples for molecular biological analyses.
Our findings demonstrate that DHT exposure caused a significant change in the expression of more than a thousand genes in both HCECs and HConjECs and (Table 1). Notably, of the 240 biological process gene ontologies (≥ 5 genes/ontology) significantly altered by DHT in HCECs, none were proinflammatory and only one was immune-related. More specifically, DHT increased the expression of 17 genes in the immune system development ontology (z score [zsc] = 2.32). In contrast, the 14 biological process gene ontologies with the highest z scores following DHT treatment of HCECs, as well as 90% of the top 31 such ontologies, were all related to upregulation of the cell cycle (Table 5)
Table 5.
DHT up-regulation of cell cycle gene ontologies in HCECs
| Ontology | Genes ↑ | Z-score |
|---|---|---|
| Pyrimidine ribonucleotide metabolic process | 4 | 5.62 |
| Cell cycle process | 47 | 5.27 |
| Mitotic cell cycle | 38 | 5.22 |
| Cell cycle phase | 39 | 4.8 |
| Cell cycle | 55 | 4.75 |
| Negative regulation of cell cycle process | 8 | 4.74 |
| Regulation of cell cycle process | 22 | 4.69 |
| DNA-dependent DNA replication initiation | 5 | 4.33 |
| Negative regulation of mitosis | 5 | 4.33 |
| Negative regulation of nuclear division | 5 | 4.33 |
| DNA-dependent DNA replication | 9 | 4.32 |
| Regulation of cell cycle arrest | 16 | 4.3 |
| Base-excision repair | 5 | 4.25 |
| Spindle checkpoint | 5 | 4.25 |
| DNA conformation change | 12 | 4.13 |
| Mitosis | 20 | 4.09 |
| Nuclear division | 20 | 4.09 |
| Cell cycle checkpoint | 15 | 4.06 |
| Cell cycle arrest | 20 | 4 |
| Regulation of cell cycle | 30 | 3.95 |
| Organelle fission | 20 | 3.93 |
| M phase of mitotic cell cycle | 20 | 3.93 |
| Pyrimidine nucleoside metabolic process | 5 | 3.85 |
| Mitotic cell cycle checkpoint | 10 | 3.76 |
| Double-strand break repair | 7 | 3.7 |
| M phase | 24 | 3.61 |
| DNA strand elongation involved in DNA replication | 4 | 3.59 |
| Negative regulation of cell cycle | 21 | 3.58 |
Cell cycle-related ontologies (≥ 5 genes/ontology) were selected after the analysis of biological process data. All of the ontologies listed in this Table were upregulated in HCECs following DHT treatment.
DHT significantly influenced the expression of 338 biological process gene ontologies (≥ 5 genes/ontology) in HConjECs. Thirty-three of these ontologies were associated with the immune system and all were upregulated by DHT (Table 6). Of interest, only five of the immune ontologies (e.g. cytokine production, regulation of defense response) increased by DHT in HConjECs were the same as those decreased by DHT in HMGECs (Table 2). DHT also had an effect on the expression of several cell cycle ontologies in HConjECs. DHT upregulated two (i.e. cell division, 15 genes increased [↑], zsc = 2.01; regulation of DNA replication, 5 genes ↑, zsc = 2.75) and downregulated four (regulation of cell cycle process, 17 genes decreased [↓], zsc = 2.62; cell cycle checkpoint, 13 genes ↓, zsc = 2.91; regulation of cell cycle arrest, 13 genes ↓, zsc = 2.75; G2/M transition, 5 genes ↓, zsc = 4.94) of these ontologies.
Table 6.
DHT up-regulation of immune-related gene ontologies in HConjECs
| Ontology | Genes ↑ | Z-score |
|---|---|---|
| Myd88-dependent toll-like receptor signaling pathway | 8 | 4.78 |
| Positive regulation of defense response | 13 | 4.75 |
| Toll-like receptor signaling pathway | 9 | 4.72 |
| Pattern recognition receptor signaling pathway | 9 | 4.56 |
| Innate immune response-activating signal transduction | 9 | 4.52 |
| Activation of innate immune response | 9 | 4.44 |
| Positive regulation of innate immune response | 10 | 4.18 |
| Toll signaling pathway | 7 | 3.88 |
| Regulation of interleukin-6 production | 5 | 3.79 |
| Toll-like receptor 4 signaling pathway | 7 | 3.76 |
| Intracellular transport | 35 | 3.74 |
| Cytokine secretion | 5 | 3.73 |
| Interleukin-6 production | 5 | 3.67 |
| Toll-like receptor 1 signaling pathway | 6 | 3.46 |
| Toll-like receptor 2 signaling pathway | 6 | 3.41 |
| Regulation of defense response | 16 | 3.33 |
| Positive regulation of NF-κB transcription factor activity | 6 | 3.29 |
| Immune response-activating signal transduction | 11 | 3.25 |
| Immune response-regulating signaling pathway | 11 | 3.17 |
| Regulation of innate immune response | 10 | 3 |
| Positive regulation of immune system process | 19 | 2.95 |
| Positive regulation of immune response | 14 | 2.87 |
| Myd88-independent toll-like receptor signaling pathway | 5 | 2.82 |
| Leukocyte cell-cell adhesion | 3 | 2.82 |
| Cytokine production | 14 | 2.79 |
| Toll-like receptor 3 signaling pathway | 5 | 2.78 |
| Activation of immune response | 11 | 2.48 |
| Regulation of cytokine production | 12 | 2.4 |
| Positive regulation of response to stimulus | 27 | 2.25 |
| Positive regulation of T cell activation | 7 | 2.18 |
| Positive regulation of lymphocyte activation | 8 | 2.1 |
| Regulation of adaptive immune response based on somatic recombination of immune receptors built from | 4 | 2.08 |
| Regulation of adaptive immune response | 4 | 2.04 |
| Cytokine activity | 10 | 2.59 |
Immune-related ontologies (≥ 5 genes/ontology) were identified after the analysis of biological process and molecular function ontology data. All of the ontologies listed in this Table were upregulated in HConjECs following DHT treatment.
Discussion
The purpose of this study was two-fold. First, to test our hypothesis that DHT suppresses proinflammatory gene expression in IHMGECs. Second, to determine whether DHT elicits analogous effects in IHCECs and IHConjECs. Our results demonstrate that DHT significantly decreased the expression of numerous immune-related genes in IHMGECs, including those related to antigen processing and presentation, innate and adaptive immune responses, chemotaxis, and cytokine production. DHT also enhanced the expression of genes encoding anti-inflammatory proteins in IHMGECs. In contrast, DHT had no influence on proinflammatory gene expression in IHCECs, and significantly enhanced the activity of genes linked to the immune system in IHConjECs. Overall, our data support our hypothesis that androgens reduce proinflammatory gene expression in IHMGECs. This hormone action, which does not occur in IHCECs and IHConjECs, may contribute to the typical lack of inflammation within the HMG.
Although we hypothesized, we also anticipated, that DHT would suppress proinflammatory gene expression in IHMGECs for a number of reasons. Androgens have long been known to exert a significant influence on the development, expression and activity of immunity in both health and disease.20–23 For example, androgens have been shown to control the maturation, proliferation, migration, activation, and/or function of B cell precursors, T cells, monocytes, macrophages and neutrophils; and to regulate the synthesis, secretion and/or expression of antibodies, cytokines, adhesion molecules and autoantigens.22,24,25 In general, the result of these androgen activities appears most often to be protective and immunosuppressive.20,22,23,25 An example is this hormone’s impact on the autoimmune lacrimal gland in Sjögren syndrome, where we have discovered that androgens cause a dramatic suppression of the inflammation in, and a significant increase in the functional activity of, this tissue.26–30
The anti-inflammatory effect in IHMGECs is in stark contrast to that observed in sebocytes, in which androgens have been reported to increase the production of pro-inflammatory cytokines.31 This differential response, though, is not surprising, given that considerable differences appear to exist in the control mechanisms between different types of sebaceous glands.2,32 Further, as we have previously reported, androgen action in sebaceous glands is significantly influenced by steroidogenic enzymes. The activity of these enzymes may vary according to tissue location in the body, cellular position within a pilosebaceous unit, as well as microenvironmental factors.2
In addition to its anti-inflammatory impact, DHT upregulated numerous genes associated with cellular differentiation and lipid dynamics, and downregulated many genes related to the cell cycle and cell proliferation. We also anticipated these effects, because androgens stimulate the differentiation and lipogenesis of the MG.2,23,33 These actions are likely mediated through binding to androgen receptors (ARs) in HMGECs,34,35 because androgen effects appear to depend on the presence of functional ARs.36,37
It was of particular interest that the DHT effects on immune, lipid, cell cycle and mTOR signaling genes in differentiated IHMGECs were similar to those induced by DHT in proliferating IHMGECs.10 Our finding was surprising, given that gene expression in proliferating versus differentiating IHMGECs is typically quite different.38 As we recently discovered, significant differences exist in the expression of more than 9,200 genes in proliferating versus differentiating IHMGECs.38
Our results demonstrate a very potent DHT influence on IHMGEC gene expression, and this finding is quite different than that recently published by other investigators.39 These researchers reported that DHT had minimal or no effect on specific gene expression, as well as on the morphology, proliferation, metabolic activity, and lipid accumulation of IHMGECs. Our apparently ‘opposite’ results can be explained by differences in experimental design. For our differentiation experiments, we used charcoal- and dextran-treated FBS. The charcoal and dextran pretreatment removes all free steroids, a procedure that has been used for over 45 years in endocrine studies.40,41 In contrast, the differentiation studies of Schröder et al. involved culturing IHMGECs in 10% FBS.39 Such FBS, without charcoal-dextran stripping, contains testosterone, at levels that can be converted to 10 nM DHT within cells that harbor steroidogenic enzymes.16 This concentration of DHT results in the occupation of > 90% of the intracellular androgen receptors.16 The human meibomian gland, in turn, which is made up solely of epithelial cells, contains all these steroidogenic enzymes.42 Thus, the Schröder et al. “control” IHMGECs were cultured in androgen-containing FBS, and essentially exposed to 10 nM DHT. The Schröder “DHT” treated cells were treated with another 10 nM DHT. Given that the first 10 nM DHT would occupy almost all of the androgen receptors, it would be anticipated that there would be no differences found between the responses of “control” and “DHT” treated IHMGECs. That is what Schröder et al. reported.39 In effect, Schröder et al. compared the results of DHT-treated control cells versus DHT-treated cells. Our data are based upon results from IHMGECs cultured in the absence (controls), or presence (treated), of 10 nM DHT, an experimental design that would permit the identification of an androgen influence.
The DHT-induced suppression of proinflammatory genes in IHMGECs was not duplicated in IHCECs or IHConjECs. Rather, DHT exerted no influence on proinflammatory gene expression in IHCECs, and significantly increased the activity of genes related to the immune system in IHConjECs. These observations are similar to our previous findings when we compared the DHT effects on the gene activity of proliferating IHMGECs versus proliferating IHConjECs,10 as well as on the protein synthesis of the lacrimal gland versus other mucosal tissues.10,43,44 In brief, the nature of androgen influence is most often cell- and tissue-specific.10,43,44
The most striking effect of DHT on IHCECs was its upregulation of a tremendous number of cell cycle genes. This proliferative response may help to explain why androgen administration has been demonstrated to stimulate mitosis and repair defects in the cornea45–47 In contrast, the most marked effect of DHT on differentiated IHConjECs was an upregulation of immune response genes. Thus, DHT increased the expression of 33 immune-related gene ontologies (e.g. cytokine production, regulation of defense response) in IHConjECs, and the majority of these were different than those suppressed by DHT in IHMGECs. DHT also modulated several cell cycle ontologies in IHConjECs, with the majority being downregulated.
There are several limitations in our research. First, we did not perform RT-PCR confirmations of selected BeadChip data. Our reason was that for RT-PCR verification of Illumina results we try to select genes with intensities ≥ 500, at least in one group, and a mean difference between groups ≥ 2.0. We find it very difficult to detect genes with lower Illumina intensities and lower comparative ratios with RT-PCR. In our current study, there were only three genes that met our intensity and ratio criteria in the IHMGECs (i.e. S100 calcium binding protein A7, TAF15 RNA polymerase II and aspartic peptidase, retroviral-like 1), one gene in the HCECs (heat shock 10kDa protein 1) and one gene in the IHConjECs (activating transcription factor 3). However, we have confirmed DHT’s influence on three of these genes in earlier experiments, which involved IHMGECs cultured in serum-free media (Table 7). The effects of DHT (i.e. significantly up- or down-regulated) were the same as in the current study.
Table 7.
Confirmation of the DHT influence on the expression of specific genes in IHMGECs, IHCECs and IHConjECs
| Current study | Previous study | ||||
|---|---|---|---|---|---|
| Cells | Gene | Ratio | p value | Ratio | p value |
| DHT gene ↑ | |||||
| IHConjECs | Activating transcription factor 3 | 2.50 | 0.0001 | 5.00 | 0.0006 |
| DHT genes ↓ | |||||
| IHMGECs | TAF15 RNA polymerase II | 2.03 | 0.0198 | 1.43 | 0.0003 |
| IHCECs | Heat shock 10kDa protein 1 | 2.17 | 0.0372 | 1.30 | 0.0273 |
The previous study involved culturing cells in the presence or absence of 10 nm DHT in serum-free media for 3 (IHMGECs), 4 (IHConjECs), or 5 (IHCECs) days. The experimental procedures, and methods for determining gene expression, were analogous to those previously reported.10 The IHCECs were a gift from Dr. Ilene Gipson (Boston, MA). Relative ratios were calculated by comparing the degree of gene expression in cells after vehicle and DHT exposure. Abbreviations: ↑- upregulation; ↓- downregulation
We should also note that we have consistently found that differences in gene expression in the MG are typically modest (i.e. ≤ 2.0-fold), irrespective of the comparison (e.g. hormone versus placebo, male versus female, etc). For example, we have found that over 96.5% of the sexually dimorphic genes in the BALB/c mouse MGs display modest differences in gene expression.48 Such differences, though, are far greater than observed in somatic tissues, the vast majority of which are very modest (i.e. ≤ 1.2-fold). As we have previously reported,48 this is true for 71.4% of sexually dimorphic genes in adipose tissue, 81.3% in liver, 82.5% in muscle and 94.4% in brain.49 These differences are biologically valid.50
A second limitation in our research is that our data reflect mRNA levels, and we have yet to determine whether these transcripts are translated into functional proteins. A third limitation is that the IHMGECs, IHCECs and IHConjECs all originated from male tissues.51 Because equivalent cell lines from female tissues do not exist, we could not evaluate the effects of androgens on immortalized “female” cells. It may be that the vast majority of genes regulated by androgens in cells from male and female meibomian glands, for example, are the same, given our findings in mice.52 However, it is also known that there may be distinct, sex-related differences in cellular receptor and signaling pathways, as well as in cellular responses to pharmaceuticals and stress.53–55 Indeed, the understanding that sex-specific cellular characteristics mediate sex-specific responses to a variety of stimuli has led cardiovascular researchers to propose sex-specific treatments for cardiovascular disease.56 The reality is that cells have a “sex,” and cells from males and females can behave differently.57 Future research will have to address whether sex-related differences occur in the behavior of human ocular surface and adnexal cells to androgen exposure.
Lastly, given the apparent property of the HMG to resist inflammation,1,2 might this ability be related to the glandular location? More specifically, given that the MGs are embedded in the tarsus of the eyelids, is it possible that the MGs are poorly accessible to the adaptive immune system with its cell populations (e.g. macrophages, lymphocytes, plasma cells, neutrophils)? Studies would indicate that MGs are not so hidden from the immune system. For example, investigators have reported extensive periglandular inflammation in eyelids of patients with Sjögren syndrome.58 And others have reported that hyperreflective, polymorphous cellular structures are present outside HMG acini in patients with MGD.59 Qazi et al. also suggest that these structures may be immune cells, and that some of them may also be found in intraepithelial locations and within conjunctival ducts (i.e. presumably MG).59 As an additional consideration, proinflammatory cytokines in the tear film may promote hyperkeratinization of the MG terminal duct epithelium and thereby facilitate the development of obstructive MGD.1,2,60,61
Overall, our results support our hypothesis that androgens decrease proinflammatory gene expression in IHMGECs. This hormone action, which is not reproduced in IHCECs or IHConjECs, may contribute to the typical absence of overt inflammation within the HMG.
Supplementary Material
Disclosure of Funding:
Supported by NIH grant EY05612, the Margaret S. Sinon Scholar in Ocular Surface Research fund, ARVO/Pfizer, TUBITAK, and the Guoxing Yao Research Fund
Footnotes
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Disclosure/Conflict of Interest
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