Single cell analysis of short-term dry eye induced changes in cornea immune cell populations.

Background: Dry eye causes corneal inflammation, epitheliopathy and sensorineural changes. This study evaluates the hypothesis that dry eye alters the percentages and transcriptional profiles of immune cell populations in the cornea. Methods: Desiccating stress (DS) induced dry eye was created by pharmacologic suppression of tear secretion and exposure to drafty low humidity environment. Expression profiling of corneal immune cells was performed by single-cell RNA sequencing (scRNA-seq). Cell differentiation trajectories and cell fate were modeled through RNA velocity analysis. Confocal microscopy was used to immunodetect corneal immune cells. Irritation response to topical neurostimulants was assessed. Results: Twelve corneal immune cell populations based on their transcriptional profiles were identified at baseline and consist of monocytes, resident (rMP) and MMP12/13 high macrophages, dendritic cells (cDC2), neutrophils, mast cells, pre T/B cells, and innate (γDT, ILC2, NK) and conventional T and B lymphocytes. T cells and resident macrophages (rMP) were the largest populations in the normal cornea comprising 18.6 and 18.2 percent, respectively. rMP increased to 55.2% of cells after 5 days of DS. Significant changes in expression of 1,365 genes (adj p < 0.0001) were noted in rMP with increases in cytokines and chemokines (Tnf, Cxcl1, Ccl12, Il1rn), inflammatory markers (Vcam, Adam17, Junb), the TAM receptor (Mertk), and decreases in complement and MHCII genes. A differentiation trajectory from monocytes to terminal state rMP was found. Phagocytosis, C-type lectin receptor signaling, NF-kappa B signaling and Toll-like receptor signaling were among the pathways with enhanced activity in these cells. The percentage of MRC1+ rMPs increased in the cornea and they were observed in the basal epithelium adjacent to epithelial nerve plexus. Concentration of the chemokine CXCL1 increased in the cornea and it heightened irritation/pain responses to topically applied hypertonic saline. Conclusion: These findings indicate that DS recruits monocytes that differentiate to macrophages with increased expression of inflammation associated genes. The proximity of these macrophages to cornea nerves and their expression of neurosensitizers suggests they contribute to the corneal sensorineural changes in dry eye.

Ectoine protects corneal epithelial survival and barrier from hyperosmotic stress by promoting anti-inflammatory cytokine IL-37.

PURPOSE: To explore novel role and molecular mechanism of a natural osmoprotectant ectoine in protecting corneal epithelial cell survival and barrier from hyperosmotic stress. METHODS: Primary human corneal epithelial cells (HCECs) were established from donor limbus. The confluent cultures in isosmolar medium were switched to hyperosmotic media (400-500 mOsM), with or without ectoine or rhIL-37 for different time periods. Cell viability and proliferation were evaluated by MTT or WST assay. The integrity of barrier proteins and the expression of cytokines and cathepsin S were evaluated by RT-qPCR, ELISA, and immunostaining with confocal microscopy. RESULTS: HCECs survived well in 450mOsM but partially damaged in 500mOsM medium. Ectoine well protected HCEC survival and proliferation at 500mOsM. The integrity of epithelial barrier was significantly disrupted in HCECs exposed to 450mOsM, as shown by 2D and 3D confocal immunofluorescent images of tight junction proteins ZO-1 and occludin. Ectoine at 5-20 mM well protected these barrier proteins under hyperosmotic stress. The expression of TNF-α, IL-1ß, IL-6 and IL-8 were dramatically stimulated by hyperosmolarity but significantly suppressed by Ectoine at 5-40 mM. Cathepsin S, which was stimulated by hyperosmolarity, directly disrupted epithelial barrier. Interestingly, anti-inflammatory cytokine IL-37 was suppressed by hyperosmolarity, but restored by ectoine at mRNA and protein levels. Furthermore, rhIL-37 suppressed cathepsin S and rescued cell survival and barrier in HCECs exposed to hyperosmolarity. CONCLUSION: Our findings demonstrate that ectoine protects HCEC survival and barrier from hyperosmotic stress by promoting IL-37. This provides new insight into pathogenesis and therapeutic potential for dry eye disease.

Ectoine, from a Natural Bacteria Protectant to a New Treatment of Dry Eye Disease.

Ectoine, a novel natural osmoprotectant, protects bacteria living in extreme environments. This study aimed to explore the therapeutic effect of ectoine for dry eye disease. An experimental dry eye model was created in C57BL/6 mice exposed to desiccating stress (DS) with untreated mice as controls (UT). DS mice were dosed topically with 0.5-2.0% of ectoine or a vehicle control. Corneal epithelial defects were detected via corneal smoothness and Oregon Green dextran (OGD) fluorescent staining. Pro-inflammatory cytokines and chemokines were evaluated using RT-qPCR and immunofluorescent staining. Compared with UT mice, corneal epithelial defects were observed as corneal smoothness irregularities and strong punctate OGD fluorescent staining in DS mice with vehicle. Ectoine treatment protected DS mice from corneal damage in a concentration-dependent manner, and ectoine at 1.0 and 2.0% significantly restored the corneal smoothness and reduced OGD staining to near normal levels. Expression of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and chemokines CCL3 and CXCL11 was significantly elevated in the corneas and conjunctivas of DS mice, whereas 1.0 and 2.0% ectoine suppressed these inflammatory mediators to near normal levels. Our findings demonstrate that ectoine can significantly reduce the hallmark pathologies associated with dry eye and may be a promising candidate for treating human disease.

Immunologic basis for development of keratoconjunctivitis sicca in systemic autoimmune diseases: Role of innate immune sensors.

The literature is filled with citations reporting an increased incidence of chronic dry eye disease, also known as keratoconjunctivitis sicca, in patients with systemic autoimmune diseases such as rheumatoid arthritis, Sjögren's Syndrome, systemic sclerosis and lupus. As the most environmentally exposed mucosal surface of the body, the conjunctiva constantly responds to environmental challenges which are typically self limited, but when persistent and unresolved may provoke pathogenic innate and adaptive immune reactions. Our understanding of the pathophysiological mechanisms by which systemic autoimmune diseases cause dry eye inducing ocular surface inflammation continues to evolve. Conjunctival immune tone responds to self or foreign danger signals (including desiccating stress) on the ocular surface with an initial non-specific innate inflammatory response. If unchecked, this can lead to activation of dendritic cells that present antigen and prime T and B cells resulting in an adaptive immune reaction. These reactions generally resolve, but dysfunctional, hyper-responsive immune cells found in systemic autoimmune diseases that are recruited to the ocular surface can amplify inflammatory stress responses in the ocular surface and glandular tissues and result in autoimmune reactions that disrupt tear stability and lead to chronic dry eye disease. We here propose that unique features of the ocular surface immune system and the impact of systemic immune dysregulation in autoimmune diseases, can predispose to development of dry eye disease, and exacerbate severity of existing dry eye.

A Mouse Model for Corneal Neovascularization by Alkali Burn.

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the limbus and adversely affects transparency and vision. Alkali burn is one of the most common forms of ocular trauma that leads to CoNV. In this protocol, CoNV is experimentally induced using sodium hydroxide solution in a controlled manner to ensure reproducibility. The alkali burn model is useful for understanding the pathology of CoNV and can be extended to study angiogenesis in general because of the avascularity, transparency, and accessibility of the cornea. In this work, CoNV was analyzed by direct examination under a dissecting microscope and by immunostaining flat-mount corneas using anti-CD31 mAb. Lymphangiogenesis was detected on flat-mount corneas by immunostaining using anti-LYVE-1 mAb. Corneal edema was visualized and quantified using optical coherence tomography (OCT). In summary, this model will help to advance existing neovascularization assays and discover new treatment strategies for pathologic ocular and extraocular angiogenesis.

Differentially Expressed Tear Proteins in Sjögren's Syndrome Keratoconjunctivitis Sicca.

Purpose: The purpose of this study was to determine if there are significant differences in the concentrations of tear proteins in Sjögren's syndrome keratoconjunctivitis sicca (SS KCS) compared to healthy controls. Methods: Tear samples were collected with unmarked Schirmer strips from 15 patients with SS KCS and 21 healthy controls. Tear protein was eluted and the concentration measured. Inflammatory mediators were assayed with a Raybiotech L-507 glass slide array and normalized by strip wetting length. All patients underwent an ocular surface exam to evaluate tear break-up time (TBUT), corneal fluorescein (CF) staining, and conjunctival (CJ) staining. The symptom assessment questionnaire in dry eye (SANDE) scores were collected for all patients. Results: Two hundred fifty-three of the 507 tear proteins analyzed were significantly different in patients with SS compared to controls. Two hundred forty-one if the proteins were upregulated and 12 were downregulated. One hundred eighty-one differentially expressed proteins were significantly correlated with all four clinical parameters: TBUT, CF staining, CJ staining, and SANDE score. Conclusions: These findings indicate that hundreds of factors can be assayed in tear proteins collected from a Schirmer strip. The results suggest tear protein concentrations are altered in patients with SS KCS compared to controls. The upregulated tear proteins correlated with clinical measures of dry eye symptoms and disease severity. Translational Relevance: Tear proteins could serve as important biomarkers for studying pathogenesis and in clinical diagnosis and management of SS KCS.

Delphi Panel Consensus Regarding Current Clinical Practice Management Options for Demodex blepharitis.

Purpose: To obtain consensus on Demodex blepharitis (DB) treatment using a modified Delphi panel process. Methods: Literature search identified gaps in knowledge surrounding treatment of DB. Twelve ocular surface disease experts comprised the Demodex Expert Panel on Treatment and Eyelid Health (DEPTH). They completed a live roundtable discussion in addition to 3 surveys consisting of scaled, open-ended, true/false, and multiple-choice questions pertaining to the treatment of DB. Consensus for scaled questions using a 1 to 9 Likert scale was predefined as median scores of 7-9 and 1-3. For other question types, consensus was achieved when 8 of 12 panelists agreed. Results: The experts agreed that an effective therapeutic agent for treatment of DB would likely decrease the necessity of mechanical intervention, such as lid scrubs or blepharoexfoliation (Median = 8.5; Range 2-9). When treating DB, panelists believed that collarettes serve as a surrogate for mites, and that eliminating or reducing collarettes should be the main clinical goal of treatment (Median = 8; Range 7-9). The panelists would treat patients with at least 10 collarettes, regardless of other signs or symptoms and agreed that DB can be cured, but there is always the possibility for a reinfestation (n = 12). There was also consensus that collarettes, and therefore mites, are the primary treatment target and the way by which clinicians can monitor patient response to therapy (Median = 8; Range 7-9). Conclusion: Expert panelists achieved consensus on key facets of DB treatment. Specifically, there was consensus that collarettes are pathognomonic for DB, that DB patients with >10 collarettes should be treated even in the absence of symptoms, and that treatment efficacy can be tracked by collarette resolution. By increasing awareness about DB, understanding the goals of and monitoring treatment efficacy, patients will receive better care and, ultimately, better clinical outcomes.

Comparison of Efficacy and Inflammatory Response to Thermoconjunctivoplasty Performed with Cautery or Pulsed 1460 nm Laser.

Conjunctivochalasis is a degenerative condition of the conjunctiva that disrupts tear distribution and causes irritation. Thermoreduction of the redundant conjunctiva is required if symptoms are not relieved with medical therapy. Near-infrared laser treatment is a more controlled method to shrink the conjunctiva than thermocautery. This study compared tissue shrinkage, histology, and postoperative inflammation in thermoconjunctivoplasty performed on the mouse conjunctiva using either thermocautery or pulsed 1460 nm near-infrared laser irradiation. Three sets of experiments were performed on female C57BL/6J mice (n = 72, 26 per treatment group and 20 control) to assess conjunctival shrinkage, wound histology, and inflammation 3 and 10 days after treatment. Both treatments effectively shrunk the conjunctiva, but thermocautery caused greater epithelial damage. Thermocautery caused greater infiltration of neutrophils on day 3 and neutrophils and CD11b+ myeloid cells on day 10. The thermocautery group had significantly higher conjunctival expression of IL-1ß on day 3. Expression of chemokine CCL2 was higher in the conjunctiva on day 3 and tear concentrations were higher on day 7 in the laser group. These results suggest that pulsed laser treatment causes less tissue damage and postoperative inflammation than thermocautery while effectively addressing conjunctivochalasis.

Clinical diagnosis and management of Demodex blepharitis: the Demodex Expert Panel on Treatment and Eyelid Health (DEPTH).

BACKGROUND: Twelve ocular surface disease experts convened to achieve consensus about Demodex blepharitis (DB) using a modified Delphi panel process. METHODS: Online surveys were administered using scaled, open-ended, true/false, and multiple-choice questions. Consensus for questions using a 1 to 9 Likert scale was predefined as median scores of 7-9 and 1-3. For other question types, consensus was achieved when 8 of 12 panellists agreed. Questions were randomized, and results of each survey informed the following survey. RESULTS: Twelve practitioners comprised the Demodex Expert Panel on Treatment and Eyelid Health (DEPTH). Following 3 surveys, experts agreed that DB is chronic (n = 11) and recurrent (n = 12) and is often misdiagnosed. Consensus was achieved regarding inflammation driving symptoms (median = 7; range 7-9), collarettes as the most common sign (n = 10) and pathognomonic for DB (median = 9; range 8-9), and itching as the most common symptom (n = 12). Panellists agreed that DB may be diagnosed based on collarettes, mites, and/or patient symptoms (n = 10) and felt that patients unresponsive to typical therapies should be evaluated for DB (n = 12). Consensus about the most effective currently available OTC treatment was not reached. CONCLUSIONS: The Delphi methodology proved effective in establishing consensus about DB, including signs, symptoms, and diagnosis. Consensus was not reached about the best treatment or how to grade severity. With increased awareness, eyecare practitioners can offer DB patients better clinical outcomes. A follow-up Delphi panel is planned to obtain further consensus surrounding DB treatment.

Non-invasive and objective tear film breakup detection on interference color images using convolutional neural networks.

PURPOSE: Dry eye disease affects hundreds of millions of people worldwide and is one of the most common causes for visits to eye care practitioners. The fluorescein tear breakup time test is currently widely used to diagnose dry eye disease, but it is an invasive and subjective method, thus resulting in variability in diagnostic results. This study aimed to develop an objective method to detect tear breakup using the convolutional neural networks on the tear film images taken by the non-invasive device KOWA DR-1α. METHODS: The image classification models for detecting characteristics of tear film images were constructed using transfer learning of the pre-trained ResNet50 model. The models were trained using a total of 9,089 image patches extracted from video data of 350 eyes of 178 subjects taken by the KOWA DR-1α. The trained models were evaluated based on the classification results for each class and overall accuracy of the test data in the six-fold cross validation. The performance of the tear breakup detection method using the models was evaluated by calculating the area under curve (AUC) of receiver operating characteristic, sensitivity, and specificity using the detection results of 13,471 frame images with breakup presence/absence labels. RESULTS: The performance of the trained models was 92.3%, 83.4%, and 95.2% for accuracy, sensitivity, and specificity, respectively in classifying the test data into the tear breakup or non-breakup group. Our method using the trained models achieved an AUC of 0.898, a sensitivity of 84.3%, and a specificity of 83.3% in detecting tear breakup for a frame image. CONCLUSIONS: We were able to develop a method to detect tear breakup on images taken by the KOWA DR-1α. This method could be applied to the clinical use of non-invasive and objective tear breakup time test.

Quantitative assessment of botulinum toxin injection on blink rate in blepharospasm.

PURPOSE: To objectively measure the blink rate in patients with blepharospasm managed by botulinum toxin type-A injections. METHODS: In this prospective, non-interventional case series, the complete blink rates of subjects were measured before incobotulinumtoxina injection and at follow-up within 4 weeks using slow-motion video-taping. Additionally, subjects graded the frequency of blinking, the severity of light-sensitivity, and the severity and frequency of dry eye symptoms on a categorical visual analog scale. The results are reported as median (range). RESULTS: Ten subjects were enrolled, with nine females. The total duration of treatment was 70 (5-116) months with total of 27.5 (2-51) injections. The subjects were grouped as short-time (<52w) or long-time (>52w) treatments. The median age, follow-up time, and injected doses were 73.5 (49-81) years, 21 (14-28) days, and 38 (8-47) units, respectively, with no significant difference between groups. The total complete blinks per minute before incobotulinumtoxina injection was 39 (23-64) which decreased to 18.5 (1-60) at follow-up (p = 0.004). The average change in complete blink rate was -67.4 ± 23.7% in long-time and -45.2 ± 31.2% in short-time groups (mean ± SD, p = 0.01). The total self-graded frequency of blinking and light-sensitivity decreased significantly at follow-up (p = 0.004, p = 0.047, respectively). Similar patterns of subject reported grades were seen in both groups. CONCLUSION: Videotaping is a low-cost method for objective measurement of blink rate in blepharospasm patients after incobotulinumtoxina injection. There was a significant reduction in blink rate after incobotulinumtoxina injections with higher percentage of change in the long-time treatment group. Incobotulinumtoxina injection also significantly improves subjective photophobia.

Imbalanced IL-37/TNF-α/CTSS signaling disrupts corneal epithelial barrier in a dry eye model in vitro.

PURPOSE: To explore novel role and molecular mechanism of a natural anti-inflammatory cytokine interleukin (IL) 37 in preventing corneal epithelial barrier disruption from hyperosmolar stress as can occur in dry eye disease. METHODS: Primary human corneal epithelial cells (HCECs) were cultured from fresh donor limbal explants. An in vitro dry eye model with hyperosmolar stress was established by switching HCECs from isosmolar (312mOsM) to hyperosmolar medium (350-500 mOsM), and some cells were treated with rhIL-37 or rhTNF-α, for different periods (2-48 h). The expression of cytokines and cathepsin S, and barrier protein integrity were evaluated by RT-qPCR, ELISA, and immunofluorescent staining with confocal microscopy. RESULTS: The integrity of epithelial barrier was significantly disrupted in HCECs exposed to hyperosmolar medium, as shown by immunofluorescent images of tight junction (TJ, ZO-1, occludin and claudin-1) and adheren junction (E-cadherin) proteins. TNF-α accentuated hyperosmolar-induced disruption of TJ barrier functional integrity whereas exposure to IL-37 blunted or even reversed these changes. Cathepsin S, encoded by CTSS gene, was found to directly disrupt epithelial barrier integrity. Interestingly, CTSS expression was significantly induced by TNF-α and hyperosmolarity, while exogenous rhIL-37 inhibited TNF-α and CTSS expression at mRNA and protein levels following hyperosmolar stress. Furthermore, rhIL-37 restored barrier protein integrity, observed in 2D and 3D confocal immunofluorescent images, in HCECs under hyperosmolar stress. CONCLUSION: Our findings demonstrate a novel signaling pathway by which anti-inflammatory cytokine IL-37 prevents corneal epithelial barrier disruption under hyperosmotic stress via suppressing TNF-α and CTSS expression. This study provides new insight into mechanisms protecting corneal barrier in dry eye disease.

Nicotinic acetylcholine receptor stimulation: A new approach for stimulating tear secretion in dry eye disease.

Tear secretion is regulated by the lacrimal functional unit consisting of afferent and efferent nerve innervation. The afferent arm consists of trigeminal nociceptors on the ocular surface and nasal mucosa. When stimulated by agonists, nicotinic acetylcholine receptors on nerve endings in the nose initiate a reflex arc resulting in instantaneous tear secretion. Pharmacologic nasal neural stimulation to increase endogenous tear production is a novel approach to treating dry eye disease.

Single-cell transcriptional profiling of murine conjunctival immune cells reveals distinct populations expressing homeostatic and regulatory genes.

Immune cells in the exposed conjunctiva mucosa defend against environmental and microbial stresses. Expression profiling by single-cell RNA sequencing was performed to identify conjunctival immune cell populations expressing homeostatic and regulatory genes. Fourteen distinct clusters were identified, including myeloid cells (neutrophils, monocytes, macrophages), dendritic cells (DC), and lymphoid cells (B, T, γδT, ILC2, and NK) lineages. Novel neutrophil [lipocalin (Lcn2) high and low), and MHCIIlo macrophage (MP) clusters were identified. More than half of the cells map to myeloid and dendritic cell populations with differential expression profiles that include genes with homeostatic and regulatory functions: Serpinb2 (MHCIIlo macrophage), Apoe (monocyte), Cd209a (macrophage), Cst3 (cDC1), and IL4i1 in migratory DC (mDC). ILC2 expresses the goblet cell trophic factor IL-13. Suppressed inflammatory and activated anti-inflammatory/regulatory pathways were observed in certain myeloid and DC populations. Confocal immunolocalization of identity markers showed mDC (CCR7, FASCIN1) located on or within the conjunctival epithelium. Monocyte, macrophage, cDC1 and IL-13/IL-5+ ILC2 were located below the conjunctival epithelium and goblet cells. This study found distinct immune cell populations in the conjunctiva and identified cells expressing genes with known homeostatic and immunoregulatory functions.

IL-17 Producing Lymphocytes Cause Dry Eye and Corneal Disease With Aging in RXRα Mutant Mouse.

Purpose: To investigate IL-17 related mechanisms for developing dry eye disease in the Pinkie mouse strain with a loss of function RXRα mutation. Methods: Measures of dry eye disease were assessed in the cornea and conjunctiva. Expression profiling was performed by single-cell RNA sequencing (scRNA-seq) to compare gene expression in conjunctival immune cells. Conjunctival immune cells were immunophenotyped by flow cytometry and confocal microscopy. The activity of RXRα ligand 9-cis retinoic acid (RA) was evaluated in cultured monocytes and γδ T cells. Results: Compared to wild type (WT) C57BL/6, Pinkie has increased signs of dry eye disease, including decreased tear volume, corneal barrier disruption, corneal/conjunctival cornification and goblet cell loss, and corneal vascularization, opacification, and ulceration with aging. ScRNA-seq of conjunctival immune cells identified γδ T cells as the predominant IL-17 expressing population in both strains and there is a 4-fold increased percentage of γδ T cells in Pinkie. Compared to WT, IL-17a, and IL-17f significantly increased in Pinkie with conventional T cells and γδ T cells as the major producers. Flow cytometry revealed an increased number of IL-17+ γδ T cells in Pinkie. Tear concentration of the IL-17 inducer IL-23 is significantly higher in Pinkie. 9-cis RA treatment suppresses stimulated IL-17 production by γδ T and stimulatory activity of monocyte supernatant on γδ T cell IL-17 production. Compared to WT bone marrow chimeras, Pinkie chimeras have increased IL-17+ γδ T cells in the conjunctiva after desiccating stress and anti-IL-17 treatment suppresses dry eye induced corneal MMP-9 production/activity and conjunctival goblet cell loss. Conclusion: These findings indicate that RXRα suppresses generation of dry eye disease-inducing IL-17 producing lymphocytes s in the conjunctiva and identifies RXRα as a potential therapeutic target in dry eye.

A multicenter report of the use of plasma rich in growth factors (PRGF) for the treatment of patients with ocular surface diseases in North America.

PURPOSE: To investigate the efficacy and safety of plasma rich in growth factors (PRGF) eyedrops in the management of patients with ocular surface diseases in North America. METHODS: Multicenter interventional case series of patients using PRGF eyedrops for the first time. A cohort of patients was analyzed for corneal staining score at initial visit and at 3 months of therapy with PRGF. Another cohort responded to a 10-item questionnaire that evaluated patients' satisfaction and safety, which included the symptom assessment questionnaire in dry eye (SANDE) score, after 6 months of PRGF treatment. RESULTS: A total of 153 patients were analyzed. Of these, 102 were reviewed for corneal epitheliopathy and 99 patients responded to the questionnaire. The mean (±SD) age of the population was 63.7 ± 17 years and 72.5% were female. The clinical indications for PRGF usage were dry eye (60%), neurotrophic keratopathy (15%), dormant corneal ulcers (12%), limbal stem cell deficiency (10%), and cicatrizing conjunctivitis (4%). At the final visit, 74.3% of patients showed an improvement of their corneal staining. Those who had punctate epithelial erosions or epithelial defects were reduced from 76.5% to 47% and 23.5% to 7.8% respectively (p < 0.0001). Symptoms, measured via SANDE score, significantly decreased from a median of 90 to 34.6 out of 100 points on follow-up (p < 0.0001). Only one patient (0.98%) complained of ocular burning sensation as a side effect. CONCLUSIONS: This multicentric study demonstrates the safety and efficacy of the use of PRGF for treating signs and symptoms in patients with significant ocular surface diseases.

Gut Microbiota From Sjögren syndrome Patients Causes Decreased T Regulatory Cells in the Lymphoid Organs and Desiccation-Induced Corneal Barrier Disruption in Mice.

Sjögren syndrome (SS) is an autoimmune inflammatory disorder characterized by secretory dysfunction in the eye and mouth; in the eye, this results in tear film instability, reduced tear production, and corneal barrier disruption. A growing number of studies show that homeostasis of the ocular surface is impacted by the intestinal microbiome, and several 16S sequencing studies have demonstrated dysbiosis of the intestinal microbiota in SS patients. In this study, we utilized metagenomic sequencing to perform a deeper analysis of the intestinal microbiome using stools collected from sex- and age-matched healthy (n = 20), dry eye (n = 4) and SS (n = 7) subjects. The observed Operational Taxonomic Units (OTUs) and Shannon alpha diversity were significantly decreased in SS compared to healthy controls, and there was a significant inverse correlation between observed OTUs and ocular severity score. We also identified specific bacterial strains that are differentially modulated in SS vs. healthy subjects. To investigate if the differential composition of intestinal microbiome would have an impact on the immune and eye phenotype, we performed functional studies using germ-free mice colonized with human intestinal microbiota from SS patients and healthy controls. Flow cytometry analysis demonstrated reduced frequency of CD4+ FOXP3+ cells in ocular draining cervical lymph nodes (CLN) in mice colonized with SS patient intestinal microbiota 4 weeks post-colonization. We also found that offspring of SS-humanized mice also have fewer CD4+FOXP3+ cells in the CLN as well as spleen, demonstrating vertical transmission. SS-humanized mice subjected to desiccating stress exhibited greater corneal barrier disruption as compared to healthy control humanized mice under the same conditions. Taken together, these data support the hypothesis that the intestinal microbiota can modulate ocular surface health, possibly by influencing development of CD4+ FOXP3+ regulatory T cells (Tregs) in the ocular draining lymph nodes.

Gut-derived butyrate suppresses ocular surface inflammation.

Dry eye is a common ocular inflammatory disorder characterized by tear film instability and reduced tear production. There is increasing evidence that homeostasis of the ocular surface is impacted by the intestinal microbiome. We are interested in investigating the potential role of microbially produced small molecules in mediating the interaction between the intestinal microbiota and the ocular surface. One such molecule is butyrate, a short-chain fatty acid (SCFA) produced by certain members of the gut microbiota through fermentation of dietary fiber. Here we show that SCFA transporter SLC5A8 is expressed in vivo in murine conjunctival and corneal epithelium. Pre-treatment of in vitro corneal epithelial cultures or bone marrow-derived dendritic cells (BMDCs) with phenylbutyrate (PBA) reduces lipopolysaccharide-induced pro-inflammatory Tnf expression. Corneal epithelial cultures and BMDCs isolated from Slc5a8 knockout mice are unable to respond to PBA pre-treatment, suggesting that SLC5A8 is required for the protective effect of PBA. The treatment of mice undergoing desiccating stress (DS) with oral tributyrin, a prodrug form of butyrate, reduces inflammation at the ocular surface in vivo, and this effect partially requires SLC5A8. Finally, expression analysis on conjunctival tissue isolated from mice subjected to DS with and without tributyrin treatment revealed that treatment downregulated genes involved in Type I interferon signaling. Together these data support our hypothesis that SCFAs produced in the gut participate in the maintenance of ocular surface homeostasis.

IL-36α/IL-36RA/IL-38 signaling mediates inflammation and barrier disruption in human corneal epithelial cells under hyperosmotic stress.

PURPOSE: To explore the distinct expression and diverse roles of IL-36 cytokines in dry eye disease using an in vitro hyperosmolarity model of human corneal epithelial cells (HCECs). METHODS: Primary HCECs were cultured from fresh donor limbal explants. Hyperosmolarity model was established by switching HCECs from isosmotic (312 mOsM) to hyperosmotic medium (350-500 mOsM) alone or with addition of rhIL-36RA or rhIL-38 for 2-48 h. Some cultures were treated with IL-36α (1-10 ng/ml) with or without rhIL-36RA or rhIL-38. Gene expression was detected by RT-qPCR; and protein production and barrier disruption were evaluated by ELISA and/or immunofluorescent staining. RESULTS: IL-36 cytokines were differential expressed in primary HCECs. Among 3 pro-inflammatory agonists, IL-36α, but not IL-36ß and IL-36γ, was distinctly induced at osmolarity-dependent manner while two antagonist IL-36RA and IL-38 were significantly suppressed in HCECs exposed to hyperosmotic stress. IL-36α increased to 4.4-fold in mRNA and 6.9-fold at protein levels (116.0 ± 36.33 pg/ml vs 16.79 ± 6.51 pg/ml in controls) by 450 mOsM, but dramatically inhibited by addition of rhIL-36RA or rhIL-38. Exogenous rhIL-36α stimulated expression of TNF-α and IL-1ß at mRNA and protein levels and disrupted tight junction proteins ZO-1 and occludin. However, rhIL-36RA or rhIL-38 suppressed TNF-α and IL-1ß production and protected HCECs from barrier disruption in response to IL-36α or hyperosmolarity. CONCLUSIONS: Our findings demonstrate that the stimulated pro-inflammatory IL-36α with the suppressed antagonists IL-36RA and IL-38 is a novel mechanism by which hyperosmolarity induces inflammation in dry eye. IL-36RA and IL-38 may have a therapeutic potential in dry eye.