in-vitro-method-to-study-sex-based-differences-conjunctival-goblet

<em>In Vitro</em> Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin ...

Harvard Medical School

Specialized pro-resolving lipid mediator resolvin (Rv) E1 stimulates secretion including mucins from conjunctival goblet cells. RvE1 can use both its ChemR23 receptor and the LTB receptor BLT1 to increase [Ca]. The purpose of this study was to determine the expression of ChemR23 and BLT1 and receptors on conjunctival goblet cells and the respective roles these two receptors play in goblet cell responses to RvE1.

RvE1 uses the LTB receptor BLT1 to increase [Ca] and stimulate mucin secretion in cultured rat and human conjunctival goblet cells.

Leukotriene B4 (LTB4) is a major proinflammatory mediator important in host defense, whereas resolvins (Rvs) are produced during the resolution phase of inflammation. The authors determined the actions of both RvE1 and RvD1 on LTB4-induced responses of goblet cells cultured from rat conjunctiva. The responses measured were an increase in the intracellular [Ca] ([Ca]) and high-molecular-weight glycoprotein secretion. Treatment with RvE1 or RvD1 for 30 minutes significantly blocked the LTB4-induced [Ca] increase. The actions of RvE1 on LTB4-induced [Ca] increase were reversed by siRNA for the RvE1 receptor, and the actions of RvD1 were reversed by an RvD1 receptor inhibitor. The RvE1 and RvD1 block of LTB4-stimulated increase in [Ca] was also reversed by an inhibitory peptide to β-adrenergic receptor kinase. LTB4 and block of the LTB4-stimulated increase in [Ca] by RvE1 and RvD1 were partially mediated by the depletion of intracellular Ca stores. RvE1, but not RvD1, counterregulated the LTB4-induced high-molecular-weight glycoprotein secretion. Thus, both RvE1 and RvD1 receptors directly inhibit LTB4 by phosphorylating the LTB4 receptor using β adrenergic receptor kinase. RvE1 receptor counterregulates the LTB4-induced increase in [Ca] and secretion, whereas RvD1 receptor only counterregulates LTB4-induced [Ca] increase.

Resolvin E1 Reduces Leukotriene B4-Induced Intracellular Calcium Increase and Mucin Secretion in Rat Conjunctival Goblet Cells.

To determine the effect of povidone iodine (PI), an antiseptic commonly used prior to ocular surgery, on viability of mixed populations of conjunctival stratified squamous and goblet cells, purified conjunctival goblet cells and purified conjunctival stromal fibroblasts in primary culture.

Povidone iodine treatment is deleterious to human ocular surface conjunctival cells in culture.

The cornea is an avascular, transparent tissue that is essential for visual function. Any disturbance to the corneal transparency will result in a severe vision loss. Due to the avascular nature, the cornea acquires most of the oxygen supply directly or indirectly from the atmosphere. Corneal tissue hypoxia has been noticed to influence the structure and function of the cornea for decades. The etiology of hypoxia of the cornea is distinct from the rest of the body, mainly due to the separation of cornea from the atmosphere, such as prolonged contact lens wearing or closed eyes. Corneal hypoxia can also be found in corneal inflammation and injury when a higher oxygen requirement exceeds the oxygen supply. Systemic hypoxic state during lung diseases or high altitude also leads to corneal hypoxia when a second oxygen consumption route from aqueous humor gets blocked. Hypoxia affects the cornea in multiple aspects, including disturbance of the epithelium barrier function, corneal edema due to endothelial dysfunction and metabolism changes in the stroma, and thinning of corneal stroma. Cornea has also evolved mechanisms to adapt to the hypoxic state initiated by the activation of hypoxia inducible factor (HIF). The aim of this review is to introduce the pathology of cornea under hypoxia and the mechanism of hypoxia adaptation, to discuss the current animal models used in this field, and future research directions.

Hypoxia adaptation in the cornea: Current animal models and underlying mechanisms.

Resolvin (Rv) D2 and RvD1 are biosynthesized from docosahexaenoic acid (DHA) and promote resolution of inflammation in multiple organs and tissues, including the conjunctiva. Histamine is a mediator produced by mast cells in the conjunctiva during the allergic response. We determined the interaction of RvD2 with histamine and its receptor subtypes in cultured conjunctival goblet cells and compared them with RvD1 by measuring intracellular [Ca] and mucous secretion. Treatment with RvD2 significantly blocked the histamine-induced [Ca] increase as well as secretion. RvD2 and RvD1 counter-regulate different histamine receptor subtypes. RvD2 inhibited the increase in [Ca] induced by the activation of H1, H3, or H4 receptors, whereas RvD1 inhibited H1 and H3 receptors. RvD2 and RvD1 also activate distinct receptor-specific protein kinases to counter-regulate the histamine receptors, probably by phosphorylation. Thus, our data suggest that the counter-regulation of H receptor subtypes by RvD2 and RvD1 to inhibit mucin secretion are separately regulated.

Resolvin D2 and Resolvin D1 Differentially Activate Protein Kinases to Counter-Regulate Histamine-Induced [Ca] Increase and Mucin Secretion in Conjunctival Goblet Cells.

Retinal ischemia is a common cause of many retinal diseases, leading to irreversible vision impairment and blindness. Excessive neuroinflammation, including microglial activation and T-cell responses, has been identified as a critical factor associated with neurodegeneration in retinal ischemia. Baicalein is a natural flavonoid reported to have broad anti-inflammatory and neuroprotective bioactivities. Herein, the effects of baicalein on microglia activation  and  were investigated. We found that baicalein exhibited robust anti-inflammatory effect on cultured human and mouse microglia, as demonstrated by decreased induction of pro-inflammatory cytokines and the phosphorylation of phosphoinositide 3-kinase (PI3K) and nuclear factor kappa B (NFκB). Proteomic analysis further unraveled baicalein's effect on modulating IL-17 signaling pathways and its upstream regulator IL-1β. Intravitreal administration of baicalein in the mouse model of retinal ischemia/reperfusion (I/R) injury attenuated microglial activation and retinal T-cell infiltration, particularly the T helper 17 cells. Additionally, baicalein was shown to exert neuroprotective effects by significantly reducing the retinal ganglion cell (RGC) loss after I/R injury, leading to an improved retinal function and spatial vision. These results suggest that baicalein, a natural flavonoid, acts as a negative regulator of activated microglia and immune responses both  and , effectively alleviating neurodegeneration in retinal I/R injury. This finding indicates that baicalein could be a potential therapeutic agent against currently incurable degenerative retinal diseases.

Baicalein-A Potent Pro-Homeostatic Regulator of Microglia in Retinal Ischemic Injury.

Hyperreflective foci (HRF) features in macular edema associated with different etiologies may indicate the disease pathogenesis and help to choose proper treatment. The goal of this study is to investigate the retinal microstructural features of macular edema (ME) secondary to multiple etiologies with spectral-domain optical coherence tomography (SD-OCT) and analyze the origin of HRF in ME.

Comparison of hyperreflective foci in macular edema secondary to multiple etiologies with spectral-domain optical coherence tomography: An observational study.

The mucin layer of the tear film is produced by goblet cells in the conjunctiva to protect the ocular surface and maintain homeostasis. The pro-resolving lipid mediator resolvin D2 (RvD2) biosynthesized from an omega 3 fatty acid actively terminates inflammation and regulates mucin secretion from conjunctival goblet cells. Our objective was to determine which Ca -dependent signaling pathways RvD2 uses to stimulate conjunctival goblet cell function (CGC). We hypothesize that RvD2 activates multiple intracellular Ca signaling pathways to stimulate CGC secretion. Rat and human CGCs were cultured from conjunctival explants. The amount of RvD2 receptor GPR18/DRV2 message and protein were determined. The intracellular concentration of Ca ([Ca ] ) was measured in CGCs using a fluorescent Ca dye and mucin secretion was determined by measuring protein secretion enzymatically with a lectin. Goblet cells were incubated with signaling pathway inhibitors before stimulation with RvD2 and [Ca ] or secretion was measured. In rat and human CGCs RvD2 receptor and in rat CGCs IP (a molecule that releases Ca from intracellular organelles) receptors 1-3 were detected. In both species of CGC RvD2 increased [Ca ] similarly to RvD1. In rat CGCs, the increase in [Ca ] and secretion stimulated by RvD2 was significantly blocked by inhibitors to phospholipase (PL-) C and IP -receptor, but not protein kinase C. Increase in [Ca ] was blocked by the PLD inhibitor, but not the PLA inhibitor. Secretion was blocked by PLA inhibitor, but not the PLD inhibitor. An inhibitor of the epidermal growth factor receptor blocked the increase in [Ca ] by RvD2 in both species of CGCs. In CGCs RvD2 activates multiple intracellular signaling pathways that are Ca -dependent, along with one Ca -independent and one cAMP/protein kinase A-dependent pathway. Activation of these pathways stimulate mucin secretion from rat and human CGCs into the tear film contributing to ocular surface homeostasis and health.

Resolvin D2 uses multiple Ca -dependent signaling pathways to stimulate mucin secretion in rat and human conjunctival goblet cells.

Non-invasive electric stimulation (ES) employing a low-intensity electric current presents a potential therapeutic modality that can be applied for treating retinal and brain neurodegenerative disorders. As neurons are known to respond directly to ES, the effects of ES on glia cells are poorly studied. A key question is if ES directly mediates microglial function or modulates their activity merely via neuron-glial signaling. Here, we demonstrated the direct effects of ES on microglia in the BV-2 cells-an immortalized murine microglial cell line. The low current ES in a biphasic ramp waveform, but not that of rectangular or sine waveforms, significantly suppressed the motility and migration of BV-2 microglia in culture without causing cytotoxicity. This was associated with diminished cytoskeleton reorganization and microvilli formation in BV-2 cultures, as demonstrated by immunostaining of cytoskeletal proteins, F-actin and β-tubulin, and scanning electron microscopy. Moreover, ES of a ramp waveform reduced microglial phagocytosis of fluorescent zymosan particles and suppressed lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression in BV-2 cells as shown by Proteome Profiler Mouse Cytokine Array. The results of quantitative PCR and immunostaining for cyclooxygenase-2, Interleukin 6, and Tumor Necrosis Factor-α corroborated the direct suppression of LPS-induced microglial responses by a ramp ES. Transcriptome profiling further demonstrated that ramp ES effectively suppressed nearly half of the LPS-induced genes, primarily relating to cellular motility, energy metabolism, and calcium signaling. Our results reveal a direct modulatory effect of ES on previously thought electrically "non-responsive" microglia and suggest a new avenue of employing ES for anti-inflammatory therapy.

Direct modulation of microglial function by electrical field.

Many conjunctival inflammatory diseases differ between the sexes and altered conjunctival goblet cells (CGCs) response is often involved. Inflammation is initiated by the release of pro-inflammatory mediators and terminated by the biosynthesis of specialized pro-resolution mediators (SPMs). Herein, we determined the sex-based difference in the responses of CGCs to inflammatory stimuli or pro-resolving lipid SPMs and their interaction with sex hormones. GCs were cultured from pieces of human conjunctiva in RPMI media. CGCs were transferred 24 h before the start of experiments to phenol red-free and FBS-free media to minimize exogenous hormones. RT-PCR, immunofluorescence microscopy (IF), and Western Blot (WB) were performed to determine the presence of sex hormone receptors. Cellular response to pro-inflammatory stimuli or SPMs was studied by measuring the increase in intracellular [Ca] ([Ca]) using fura 2/AM microscopy. Use of RT-PCR demonstrated estrogen receptor (ER) α in 4/5 males and 3/3 females; ERβ in 2/4 males and 2/3 females; and androgen receptors (AR) in 3/3 male and 3/3 female CGCs. Positive immunoreactivity by IF and protein expression by WB was detected using antibodies for the ERα and ERβ in 3/3 males and 3/3 females, while AR were only present in males. Significantly different Ca responses between sexes were found with carbachol only at 10 M, but not with histamine or leukotriene (LT) B at any concentration used. Incubation with dihydrotestosterone (DHT), estrone (E1), or estradiol (E2) at 10 M for 30 min significantly inhibited the LTB-stimulated [Ca] increase in male and female CGCs. Incubation with DHT, E1, and E2 overnight significantly inhibited the LTB response in females, while DHT and E2 significantly inhibited the LTB response in males. The SPM lipoxin A (LXA) (10-10 M), but not the resolvins D1 or D2, induced an [Ca] increase that was significantly higher in males compared to females. We conclude that male and female CGCs showed differences in the expression of sex hormone receptors. Treatment with sex hormones altered pro-inflammatory mediator LTB-induced response. Males compared to females have a higher response to the ω-6-fatty acid derived SPM LXA, indicating males may terminate inflammation in conjunctival goblet cells faster than females.

Sex-based differences in conjunctival goblet cell responses to pro-inflammatory and pro-resolving mediators.

Transcorneal electrical stimulation (TcES) is increasingly applied as a therapy for preserving and improving vision in retinal neurodegenerative and ischemic disorders. However, a common complaint about TcES is its induction of eye pain and dryness in the clinic, while the mechanisms remain unknown.

Transcorneal but not transpalpebral electrical stimulation disrupts mucin homeostasis of the ocular surface.

Redox homeostasis is a delicate balancing act of maintaining appropriate levels of antioxidant defense mechanisms and reactive oxidizing oxygen and nitrogen species. Any disruption of this balance leads to oxidative stress, which is a key pathogenic factor in several ocular diseases. In this review, we present the current evidence for oxidative stress and mitochondrial dysfunction in conditions affecting both the anterior segment (e.g., dry eye disease, keratoconus, cataract) and posterior segment (age-related macular degeneration, proliferative vitreoretinopathy, diabetic retinopathy, glaucoma) of the human eye. We posit that further development of therapeutic interventions to promote pro-regenerative responses and maintenance of the redox balance may delay or prevent the progression of these major ocular pathologies. Continued efforts in this field will not only yield a better understanding of the molecular mechanisms underlying the pathogenesis of ocular diseases but also enable the identification of novel druggable redox targets and antioxidant therapies.

Role of Oxidative Stress in Ocular Diseases: A Balancing Act.

Lacrimal gland inflammation triggers dry eye disease through impaired tear secretion by the epithelium. As aberrant inflammasome activation occurs in autoimmune disorders including Sjögren's syndrome, we analyzed the inflammasome pathway during acute and chronic inflammation and investigated its potential regulators. Bacterial infection was mimicked by the intraglandular injection of lipopolysaccharide (LPS) and nigericin, known to activate the NLRP3 inflammasome. Acute injury of the lacrimal gland was induced by interleukin (IL)-1α injection. Chronic inflammation was studied using two Sjögren's syndrome models: diseased  compared to healthy  mice and Thrombospondin-1-null (TSP-1) compared to TSP-1 mice. Inflammasome activation was investigated by immunostaining using the  reporter mouse, by Western blotting, and by RNAseq. LPS/Nigericin, IL-1α and chronic inflammation induced inflammasomes in lacrimal gland epithelial cells. Acute and chronic inflammation of the lacrimal gland upregulated multiple inflammasome sensors, caspases 1/4, and interleukins  and . We also found increased IL-1β maturation in Sjögren's syndrome models compared with healthy control lacrimal glands. Using RNA-seq data of regenerating lacrimal glands, we found that lipogenic genes were upregulated during the resolution of inflammation following acute injury. In chronically inflamed  lacrimal glands, an altered lipid metabolism was associated with disease progression: genes for cholesterol metabolism were upregulated, while genes involved in mitochondrial metabolism and fatty acid synthesis were downregulated, including peroxisome proliferator-activated receptor alpha (PPARα)/sterol regulatory element-binding 1 (SREBP-1)-dependent signaling. We conclude that epithelial cells can promote immune responses by forming inflammasomes, and that sustained inflammasome activation, together with an altered lipid metabolism, are key players of Sjögren's syndrome-like pathogenesis in the  mouse lacrimal gland by promoting epithelial dysfunction and inflammation.

Lacrimal Gland Epithelial Cells Shape Immune Responses through the Modulation of Inflammasomes and Lipid Metabolism.

Annexin A1 (AnxA1) is the main mediator of the anti-inflammatory actions of glucocorticoids. AnxA1 functions as a pro-resolving mediator in cultured rat conjunctival goblet cells to ensure tissue homeostasis through stimulation of intracellular [Ca] ([Ca]) and mucin secretion. AnxA1 has several N-terminal peptides with anti-inflammatory properties of their own, including Ac2-26, Ac2-12, and Ac9-25. The increase in [Ca] caused by AnxA1 and its N-terminal peptides in goblet cells was measured to determine which formyl peptide receptors the compounds use and the action of the peptides on histamine stimulation. Changes in [Ca] were determined by using a fluorescent Ca indicator. AnxA1 and its peptides each activated formyl peptide receptors in goblet cells. AnxA1 and Ac2-26 at 10 mol/L and Ac2-12 at 10 M inhibited the histamine-stimulated increase in [Ca], as did resolvin D1 and lipoxin A at 10 mol/L, whereas Ac9-25 did not. AnxA1 and Ac2-26 counter-regulated the H receptor through the p42/p44 mitogen-activated protein kinase/extracellular regulated kinase 1/2, β-adrenergic receptor kinase, and protein kinase C pathways, whereas Ac2-12 counter-regulated only through β-adrenergic receptor kinase. In conclusion, the N-terminal peptides Ac2-26 and Ac2-12, but not Ac9-25, share multiple functions with the full-length AnxA1 in goblet cells, including inhibition of histamine-stimulated increase in [Ca] and counter-regulation of the H receptor. These actions suggest a potential pharmaceutical application of the AnxA1 N-terminal peptides Ac2-26 and Ac2-12 in homeostasis and ocular inflammatory diseases.

Article	Year
RvE1 uses the LTB receptor BLT1 to increase [Ca] and stimulate mucin secretion in cultured rat and human conjunctival goblet cells. The ocular surface\| PubMed ID: 32361084	2020
Resolvin E1 Reduces Leukotriene B4-Induced Intracellular Calcium Increase and Mucin Secretion in Rat Conjunctival Goblet Cells. The American journal of pathology\| PubMed ID: 32561135	2020
Povidone iodine treatment is deleterious to human ocular surface conjunctival cells in culture. BMJ open ophthalmology\| PubMed ID: 32995498	2020
Hypoxia adaptation in the cornea: Current animal models and underlying mechanisms. Animal models and experimental medicine\| PubMed ID: 34977481	2021
Resolvin D2 and Resolvin D1 Differentially Activate Protein Kinases to Counter-Regulate Histamine-Induced [Ca] Increase and Mucin Secretion in Conjunctival Goblet Cells. International journal of molecular sciences\| PubMed ID: 35008563	2021
Baicalein-A Potent Pro-Homeostatic Regulator of Microglia in Retinal Ischemic Injury. Frontiers in immunology\| PubMed ID: 35265083	2022
Comparison of hyperreflective foci in macular edema secondary to multiple etiologies with spectral-domain optical coherence tomography: An observational study. BMC ophthalmology\| PubMed ID: 36038824	2022
Resolvin D2 uses multiple Ca -dependent signaling pathways to stimulate mucin secretion in rat and human conjunctival goblet cells. Journal of cellular physiology\| PubMed ID: 36066128	2022
Direct modulation of microglial function by electrical field. Frontiers in cell and developmental biology\| PubMed ID: 36158207	2022
Sex-based differences in conjunctival goblet cell responses to pro-inflammatory and pro-resolving mediators. Scientific reports\| PubMed ID: 36175572	2022
Transcorneal but not transpalpebral electrical stimulation disrupts mucin homeostasis of the ocular surface. BMC ophthalmology\| PubMed ID: 36522696	2022
Role of Oxidative Stress in Ocular Diseases: A Balancing Act. Metabolites\| PubMed ID: 36837806	2023
Lacrimal Gland Epithelial Cells Shape Immune Responses through the Modulation of Inflammasomes and Lipid Metabolism. International journal of molecular sciences\| PubMed ID: 36901740	2023
Anti-Inflammatory and Pro-resolving Actions of the N-Terminal Peptides Ac2-26, Ac2-12, and Ac9-25 of Annexin A1 on Conjunctival Goblet Cell Function. The American journal of pathology\| PubMed ID: 37423551	2023

Menglu Yang

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