Effect of Withdrawing Chronic Topical Immune Modulating Treatment on Schirmer Tear Test Values in Dogs with Dry Eye Disease: Relevance to Dry Eye Studies.

Purpose: To determine the effect of discontinuing chronic topical immune modulating (IM) treatment on Schirmer tear test (STT) values in dogs with dry eye disease (DED). Methods: Serial measurements of STTs from 14 dogs (16 eyes) previously diagnosed with DED were obtained before and after discontinuation of topical IM agents. Dogs with moderate to severe DED that had been well controlled with a topical IM treatment were included. After initial assessment topical IM treatment was discontinued, but topical lubricant was continued, and STT values were obtained sequentially. A mixed-effects regression model was used to evaluate the effects of age, gender, breed, clinical score, frequency of treatment, baseline STT value, and drug type on final STT values after IM withdrawal. P < 0.05 was considered statistically significant. Results: During the follow-up period after the IM treatment had been discontinued (136 ± 29 days), 50% of the eyes (n = 8) exhibited STT values that never decreased to <10 mm/min. In the other 50% (n = 8), STT values decreased from 15.9 ± 4.7 mm/min to 6.1 ± 0.9 mm/min. In this group, the time it took to decrease the STT to <10 mm/min was 21.1 ± 9.5 days. Severe clinical signs of DED and low baseline STT pre-IM treatment significantly affected STT post-IM treatment withdrawal (P < 0.05). Conclusions: The duration that a residual effect of topical IM treatment persists needs to be taken into consideration when studies are designed utilizing dogs with previous IM treatment for DED.

Lack of HPV in pterygium with no evidence of autoinoculation and the role of cytokines in pterygium with dry eye.

This study evaluated human papillomavirus's (HPV) role in pterygium pathogenesis, its autoinoculation from genitalia to ocular surface, potential cytokines involved, and crosstalk cytokines between pterygium and dry eye (DE). This cross-sectional study enrolled 25 healthy controls (HCs) and 116 pterygium patients. Four subgroups of pterygium and DE were used in cytokine evaluations. Conjunctival and pterygium swabs and first-void urine samples (i.e., genitalia samples) were collected for HPV DNA detection using real-time polymerase chain reaction. Tear cytokines interleukin (IL)-6, IL-18, and vascular endothelial growth factor (VEGF) in tears were evaluated. No HPV DNA was detected in conjunctival or pterygium swabs. No association was found between HPV DNA in urine samples and that from conjunctival or pterygium swabs. Tear VEGF levels were significantly higher in pterygium patients than in HCs, with no markedly different levels between primary and recurrent pterygia. Tear IL-6, IL-18, and tear VEGF were significantly higher in participants with DE, regardless of pterygium status. In conclusion, HPV infection was not a pathogenic factor of pterygia. The hypothesis of HPV transmitting from the genitals to ocular surfaces was nullified. Tear VEGF was involved in both pterygia and DE, whereas tear IL-6 and IL-18 played roles only in DE.

Systemic and mucosal antibody responses specific to SARS-CoV-2 during mild versus severe COVID-19.

BACKGROUND: Whereas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody tests are increasingly being used to estimate the prevalence of SARS-CoV-2 infection, the determinants of these antibody responses remain unclear. OBJECTIVES: Our aim was to evaluate systemic and mucosal antibody responses toward SARS-CoV-2 in mild versus severe coronavirus disease 2019 (COVID-19) cases. METHODS: Using immunoassays specific for SARS-CoV-2 spike proteins, we determined SARS-CoV-2-specific IgA and IgG in sera and mucosal fluids of 2 cohorts, including SARS-CoV-2 PCR-positive patients (n = 64) and PCR-positive and PCR-negtive health care workers (n = 109). RESULTS: SARS-CoV-2-specific serum IgA titers in patients with mild COVID-19 were often transiently positive, whereas serum IgG titers remained negative or became positive 12 to 14 days after symptom onset. Conversely, patients with severe COVID-19 showed a highly significant increase of SARS-CoV-2-specific serum IgA and IgG titers after symptom onset. Very high titers of SARS-CoV-2-specific serum IgA were correlated with severe acute respiratory distress syndrome. Interestingly, some health care workers with negative SARS-CoV-2-specific serum antibody titers showed SARS-CoV-2-specific IgA in mucosal fluids with virus-neutralizing capacity in some cases. SARS-CoV-2-specific IgA titers in nasal fluids were inversely correlated with age. CONCLUSIONS: Systemic antibody production against SARS-CoV-2 develops mainly in patients with severe COVID-19, with very high IgA titers seen in patients with severe acute respiratory distress syndrome, whereas mild disease may be associated with transient production of SARS-CoV-2-specific antibodies but may stimulate mucosal SARS-CoV-2-specific IgA secretion.

Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation.

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.

Chemokines in allergic conjunctivitis.

PURPOSE OF REVIEW: Chemokines are a large group of low molecular weight cytokines that attract and activate leukocytes throughout the body and therefore have a key role in the framework of late-phase allergic responses. The purpose of this article is to provide an overview of the main chemokines involved in allergic conjunctivitis, their primary functions and their physiological roles, and therapies targeted at chemokines and their receptors for ocular allergic diseases. RECENT FINDINGS: In recent years, there have been considerable advances in the understanding of ocular pathophysiology of ocular surface inflammatory diseases including both allergic eye diseases and dry eye syndrome. Several therapies being developed for dry eye inflammation are recognized as possible therapies for ocular allergic diseases as there are often common chemokines involved in both disease spectra. SUMMARY: Chemokines represent an integral part of the late-phase cascade of ocular allergic inflammation. A deep understanding of specific chemokines and their interactions will help in targeting therapies to effectively manage ocular clinical findings and symptoms of allergic eye disease.

Dry Eye Disease and Tear Cytokine Levels-A Meta-Analysis.

Background-It is recognized that inflammation is an underlying cause of dry eye disease (DED), with cytokine release involved. We systematically reviewed literature with meta-analyses to quantitatively summarize the levels of tear cytokines in DED. Methods-The PubMed, Embase, Web of Science, Ovid, Cochrane, and Scopus databases were reviewed until September 2019, and original articles investigating tear cytokines in DED patients were included. Differences of cytokines levels of DED patients and controls were summarized by standardized mean differences (SMD) using a random effects model. Study quality was assessed by applying Newcastle-Ottawa-Scale and the GRADE quality score. Methods of analytical procedures were included as covariate. Results-Thirteen articles investigating 342 DED patients and 205 healthy controls were included in the meta-analysis. The overall methodological quality of these studies was moderate. Systematic review of the selected articles revealed that DED patients had higher tear levels of interleukin (IL)-1ß, IL-6, chemokine IL-8, IL-10, interferon-γ, IFN-γ, and tumor necrosis factor-α, TNF-α as compared to controls. Evidence was less strong for IL-2 and IL-17A. Conclusions-Data show that levels of tear cytokines in DED and control display a great variability, and further studies of higher quality enrolling a higher number of subjects are needed, to define a cut-off value.

The Immunological Basis of Dry Eye Disease and Current Topical Treatment Options.

Homeostasis of the lacrimal functional unit is needed to ensure a well-regulated ocular immune response comprising innate and adaptive phases. When the ocular immune system is excessively stimulated and/or immunoregulatory mechanisms are disrupted, the balance between innate and adaptive phases is dysregulated and chronic ocular surface inflammation can result, leading to chronic dry eye disease (DED). According to the Tear Film and Ocular Surface Society Dry Eye Workshop II definition, DED is a multifactorial disorder of the ocular surface characterized by impairment and loss of tear homeostasis (hyperosmolarity), ocular discomfort or pain, and neurosensory abnormalities. Dysregulated ocular immune responses result in ocular surface damage, which is a further contributing factor to DED pathology. Several therapeutics are available to break the vicious circle of DED and prevent chronic disease and progression, including immunosuppressive agents (steroids) and immunomodulators (cyclosporine and lifitegrast). Given the chronic inflammatory nature of DED, each of these agents is commonly used in clinical practice. In this study, we review the immunopathology of DED and the molecular and cellular actions of current topical DED therapeutics to inform clinical decision making.

Cytokine tear film profile determination in eyes of healthy dogs and those with inflammatory periocular and skin disorders.

Alterations in serum cytokine levels and profiles have been reported in association with a variety of disease conditions (e.g., allergic, immune-mediated, etc.) in both humans and animals. In comparison to serum cytokine measurements, tear cytokine measurements might be expected to more accurately reflect the inflammatory milieu associated with periocular disease. The purpose of this study was to use a multiplexed assay to compare the cytokine profile of tears in healthy dogs to those with inflammatory skin and periocular disease. We were able to detect IL-2, IL-6, IL-8, and TNF-α in >47 % of tear samples from both healthy canine patients and those with inflammatory dermatologic disease (with or without concurrent periocular involvement). In contrast, IL-7, IL-10 and IFN-γ were rarely detected. Dogs with both dermatologic and periocular disease (but not dermatologic disease alone) had higher levels of IL-8 (P < 0.001, P > 0.05, respectively) relative to healthy dogs. Patients with concurrent dermatologic and periocular disease also demonstrated significantly greater variability in IL-8 concentrations between eyes than did healthy dogs (P < 0.0001). Our findings suggest that tear cytokine analysis may prove to be a useful tool to investigate the role and interactions of the local ocular immune response in patients with inflammatory periocular disease.

IgE Ratio in Tears: A Predictive Tool of Ocular Allergic Inflammation.

PURPOSE: To demonstrate the tear IgE (measured/exuded) ratio (R) as a useful biological marker of ocular allergy in order to distinguish severe from less severe inflammatory status. METHODS: Tear samples and sera from 78 ocular allergy patients and 19 control subjects were analyzed. Total IgE and albumin were measured for calculating the tear IgE-R defining two subgroups (SG) of samples: R ≥ 4-SG and R < 4-SG. Eosinophil cationic protein, Th1 and Th2 cytokines (IFN-γ, IL-4, -5, -6, -8 and -10) and protein electrophoretic profiles were also investigated in tears. RESULTS: The R < 4-SG compared to the R ≥ 4-SG shows higher levels of tear albumin, eosinophil cationic protein, and Th1 and Th2 cytokines. Moreover, each subgroup presents a specific protein profile. CONCLUSION: This study showed that an IgE-R lower than four must be carefully interpreted as a warning sign of a severe inflammatory context and should be also associated with an exploration of immunological profile.

Differential effects of specific cathepsin S inhibition in biocompartments from patients with primary Sjögren syndrome.

OBJECTIVE: Primary Sjögren syndrome (pSS) is characterized by T and B cell infiltration of exocrine glands. The cysteine protease cathepsin S (CatS) is crucially involved in MHCII processing and T cell stimulation, and elevated levels have been found in patients with RA, psoriasis and pSS. However, little is known about the functional characteristics and mechanisms of SS-A- and SS-B-specific T cells in pSS patients. We herein investigated the inhibition of CatS activity in different biocompartments of pSS patients including antigen-specific T cell responses. METHODS: Ex vivo CatS activity was assessed in tears, plasma and saliva of 15 pSS patients and 13 healthy controls (HC) and in the presence or absence of the specific CatS inhibitor RO5459072. In addition, antigen (SS-A (60kD), SS-B, influenza H3N2, tetanus toxoid and SEB)-specific T cell responses were examined using ex vivo IFN-γ/IL-17 Dual ELISPOT and Bromdesoxyuridin (BrdU) proliferation assays in the presence or absence of RO5459072. Supernatants were analysed for IL-1ß, IL-6, IL-10, TNF-α, IL-21, IL-22 and IL-23, using conventional ELISA. RESULTS: CatS activity was significantly elevated in tear fluid, but not other biocompartments, was inversely associated with exocrinic function in pSS patients and could significantly be suppressed by RO5459072. Moreover, CatS inhibition by RO5459072 led to strong and dose-dependent suppression of SS-A/SS-B-specific T cell effector functions and cytokine secretion by CD14+ monocytes. However, RO5459072 was incapable of suppressing SS-A/SS-B-induced secretion of cytokines in CD14+ monocytes when T cells were absent, confirming a CatS/MHCII-mediated mechanism of suppression. CONCLUSION: CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. Conversely, CatS inhibition diminishes T cell and associated monokine responses towards relevant autoantigens in pSS. Thus, CatS inhibition may represent a promising novel treatment strategy in pSS.

Neurotrophic factors and nerve growth factor in ocular allergy.

PURPOSE OF REVIEW: During allergic reaction, nervous and immune systems mutually interact through release of mediators, including neurotrophic factors and nerve growth factor (NGF). These mediators modulate allergic reaction through binding their receptors expressed by immune and structural cells and by stimulating neuropeptide release by nerves. The role of neuropeptides and NGF has been demonstrated in allergic asthma and rhinitis, and, to a lesser extent, in allergic conjunctivitis. The aim of this review are to elucidate the evidence of the role of NGF and neuropeptides in the pathogenesis of allergic conjunctivitis. RECENT FINDINGS: NGF modulates allergic reaction by stimulating release of cytokines, inflammatory mediators and neuropeptides by immune and structural cells and nerve endings at the site of inflammation. Evidence showed that local and systemic NGF levels increase in patients with allergic conjunctivitis, including allergic rhinoconjuncivitis, vernal keratoconjunctivitis and atopic keratoconjunctivitis. We recently described an increase of conjunctival p75NTR expression in patients with allergic rhinoconjuncivitis, and an increase of tear levels of NGF after conjunctival provocation test with allergen. SUMMARY: NGF modulates ocular allergic reaction. Increasing understanding of the role of neuropeptides in allergic conjunctivitis may pave the way to the development of novel therapeutic approaches and improvement of patients' management.

Elevated cytokine levels in tears and saliva of patients with primary Sjögren's syndrome correlate with clinical ocular and oral manifestations.

Investigating cytokines in tear fluid and saliva may offer valuable information for understanding the pathogenesis of primary Sjögren's syndrome (pSS). Cytokine profiles in both tear fluid and saliva of pSS patients, non-Sjögren's syndrome (non-SS) subjects with sicca symptoms, and healthy controls without sicca complaints were analysed. Furthermore, relationships associating the severity of clinical ocular and oral manifestations with the upregulated cytokines were assessed. In tear fluid, pSS patients showed elevated levels of IL-1ra, IL-2, IL-4, IL-8, IL-12p70, IL-17A, IFN-γ, IP-10, MIP-1b, and Rantes compared to non-SS subjects and healthy controls. The increased cytokine levels (except IP-10) correlated significantly with reduced tear production, less stable tear film, and greater ocular surface damage. In saliva, pSS patients had a higher IP-10 level, which correlated with higher candida score; and an elevated MIP-1a level, which correlated significantly with lower unstimulated and stimulated whole saliva secretion rates. The upregulated cytokines identified in tear fluid and saliva of pSS patients show a clear interplay between innate and adaptive immune responses that may contribute to disease pathogenesis. The increase of IP-10 and MIP in both tears and saliva further emphasises the essential role of macrophages and innate immunity in pSS.

Immunization of cats to induce neutralizing antibodies against Fel d 1, the major feline allergen in human subjects.

BACKGROUND: Cat allergy in human subjects is usually caused by the major cat allergen Fel d 1 and is found in approximately 10% of the Western population. Currently, there is no efficient and safe therapy for cat allergy available. Allergic patients usually try to avoid cats or treat their allergy symptoms. OBJECTIVE: We developed a new strategy to treat Fel d 1-induced allergy in human subjects by immunizing cats against their own major allergen, Fel d 1. METHODS: A conjugate vaccine consisting of recombinant Fel d 1 and a virus-like particle derived from the cucumber mosaic virus containing the tetanus toxin-derived universal T-cell epitope tt830-843 (CuMVTT) was used to immunize cats. A first tolerability and immunogenicity study, including a boost injection, was conducted by using the Fel-CuMVTT vaccine alone or in combination with an adjuvant. RESULTS: The vaccine was well tolerated and had no overt toxic effect. All cats induced a strong and sustained specific IgG antibody response. The induced anti-Fel d 1 antibodies were of high affinity and exhibited a strong neutralization ability tested both in vitro and in vivo. A reduction in the endogenous allergen level and a reduced allergenicity of tear samples, were observed. CONCLUSION: Vaccination of cats with Fel-CuMVTT induces neutralizing antibodies and might result in reduced symptoms of allergic cat owners. Both human subjects and animals could profit from this treatment because allergic cat owners would reduce their risk of developing chronic diseases, such as asthma, and become more tolerant of their cats, which therefore could stay in the households and not need to be relinquished to animal shelters.

The expression of interleukin-15 and interleukin-17 in tears and orbital tissues of Graves ophthalmopathy patients.

OBJECTIVE: The objective of this study was to study the levels of interleukin (IL)-15 and IL-17 in tears and orbital tissues of the patients with the Graves ophthalmopathy (GO) (active and inactive stages) and in controls. METHODS: Twenty-four active GO patients (CAS ≥ 3/7) were collected in our research. All patients were treated with corticosteroids during the therapy process (4-15 months) and developed into the inactive phase (CAS ≤ 2/7). The tear samples were gathered from inactive and active stages of patients with GO and sex-matched and age-matched controls (volunteers, n = 24) for the enzyme-linked immunosorbent assay measurement of IL-15 and IL-17. The orbital tissues were collected from inactive stage of patients with GO and healthy volunteers for the analysis of IL-15 and IL-17 positive cells using the immunohistochemical analysis. RESULTS: In comparison with the volunteers, significant upregulation of IL-15 and IL-17 were identified in tears of active (P < 0.05) and inactive patients with GO ( P < 0.01). Compared with inactive patients with GO, the levels of IL-15 and IL-17 in the tears were obviously higher than those in the active patients with GO. In addition, higher expression of IL-15 and IL-17 positive cells were found in the orbital organization of inactive patients with GO compared with volunteers. CONCLUSION: The differences in IL-15 and IL-17 expression between GO patients (active and inactive stages) and controls suggested both IL-15 and IL-17 expression were significantly correlated with thyroid-associated ophthalmopathy pathogenesis and development.

Does ocular inflammation play a role in xeroderma pigmentosum with endothelial dysfunction: an immunological study.

We report a case of xeroderma pigmentosum (XP) with endothelial dysfunction where the analysis of tears revealed elevated levels of proinflammatory cytokines, even in the absence of active inflammation and neovascularisation of the ocular surface. Although the role of ultraviolet (UV) radiation-induced inflammation in the occurrence of ocular manifestations of XP is known, little is published on the molecular mechanisms and there are no reports quantifying the presence of inflammatory cytokines in the tears of patients with ocular involvement of XP. Tear analysis demonstrated an increase in inflammatory cytokines and chemokines, especially interleukin-8 (2.38 ng/µg), tumour necrosis factor alpha (0.87 ng/µg) and granulocyte monocyte colony stimulating factor (0.44 ng/µg) as compared with the control eye. Effective management of the underlying UV-induced inflammation and promoting DNA repair may play a vital role in managing ocular manifestations and its sequelae in patients of XP.

Survey of cytokines in normal canine tears by multiplex analysis: A pilot study.

OBJECTIVES: The purpose of this pilot study was to use a multiplexed assay to measure cytokines in normal stimulated canine tears. METHODS: 25 healthy dogs were included in the study. Stimulated tears were collected in capillary tubes from the right (OD) and left (OS) eyes and stored at -80 °C until batch sample analysis was performed. The samples were analyzed utilizing Luminex® canine-validated multiplex beads on a Bio-Rad multiplex analyzer for IL-2, IL-6, IL-7, IL-8, IL-10, TNF-α, and IFN-γ. Based upon previous human studies, tears were initially evaluated at a 1:10 dilution. Eight random samples were later re-analyzed without dilution. RESULTS: Diluting the samples 1:10 rendered all analytes undetectable except IL-8. A repeat analysis of eight randomly selected undiluted samples still demonstrated very low cytokine levels except for IL-8 (16/16 eyes; 2254 ±â€¯1677 pg/ml OD, 1095 ±â€¯786.8 pg/ml OS); and IFN-γ (15/16 eyes; 13.37 ±â€¯13.08 pg/ml OD,16.08 ±â€¯19.4 pg/ml OS). CONCLUSION: This pilot study is the first to analyze cytokines in canine tears. This study demonstrated that IL-8 is consistently detected in both diluted and undiluted samples, but undiluted samples may be superior to 1:10 diluted samples for evaluation of other cytokines in canine tears.

Tear osteopontin level and its relationship with local Th1/Th2/Th17/Treg cytokines in children with allergic conjunctivitis

Background: Allergic conjunctivitis (AC) is one of the most common allergic ocular diseases worldwide. Osteopontin (OPN), as a recently described Th2 inflammation related protein, may play a role in the pathogenesis of AC. The aim of this study was to identify the expression of OPN in children with AC. Methods: Eighty AC children (seasonal and perennial AC) and twenty controls were enrolled in this study. Serum and tears of different time points (during and out of the pollen season) were collected and used for enzyme-linked immunosorbent assay (ELISA) of OPN and T-help cell related cytokines, respectively. The relationship between serum and tears OPN and Th1/2/17Treg related cytokines as well as disease severity were analysed. Results: Our results showed that expression of tear OPN protein by perennial AC patients increased significantly compared with controls or seasonal AC patients out of the pollen season. Tear OPN expression was positively related to local Th2/17 cytokines and negatively related to IL-10 and TGF-Beta expression. The tear OPN expression was also significantly related to disease severity. Conclusion: Tear OPN reflects the local clinical status of ocular allergy and might play an important pathophysiological role in local Th2/17/Treg inflammation in children with AC (AU)

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TH17 cells mediate inflammation in a novel model of spontaneous experimental autoimmune lacrimal keratoconjunctivitis with neural damage.

BACKGROUND: Dry eye disease (DED) affects one third of the population worldwide. In prior studies, experimental autoimmune lacrimal keratoconjunctivitis (EALK) induced by desiccating stress in mice has been used as a model of DED. This model is complicated by a requirement for exogenous epithelial cell injury and administration of anticholinergic agents with broad immunologic effects. OBJECTIVE: We sought to develop a novel mouse model of EALK and to demonstrate the responsible pathogenic mechanisms. METHODS: CD4+CD45RBhigh naive T cells with and without CD4+CD45RBlow regulatory T cells were adoptively transferred to C57BL/10 recombination-activating gene 2 (Rag2)-/- mice. The eyes, draining lymph nodes, lacrimal glands, and surrounding tissues of mice with and without spontaneous keratoconjunctivitis were evaluated for histopathologic changes, cellular infiltration, and cytokine production in tissues and isolated cells. Furthermore, the integrity of the corneal nerves was evaluated using whole-tissue immunofluorescence imaging. Gene-deficient naive T cells or RAG2-deficient hosts were evaluated to assess the roles of IFN-γ, IL-17A, and IL-23 in disease pathogenesis. Finally, cytokine levels were determined in the tears of patients with DED. RESULTS: EALK developed spontaneously in C57BL/10 Rag2-/- mice after adoptive transfer of CD4+CD45RBhigh naive T cells and was characterized by infiltration of CD4+ T cells, macrophages, and neutrophils. In addition to lacrimal keratoconjunctivitis, mice had damage to the corneal nerve, which connects components of the lacrimal functional unit. Pathogenic T-cell differentiation was dependent on IL-23p40 and controlled by cotransferred CD4+CD45RBlow regulatory T cells. TH17 rather than TH1 CD4+ cells were primarily responsible for EALK, even though levels of both IL-17 and IFN-γ were increased in inflammatory tissues, likely because of their ability to drive expression of CXC chemokines within the cornea and the subsequent influx of myeloid cells. Consistent with the findings of this model, the tears of patients with DED had increased levels of inflammatory cytokines, including IL-17A and TNF-α. CONCLUSION: We describe a novel model of spontaneous EALK that supports a role for TH17 cells in disease pathogenesis and that will contribute to our understanding of autoimmune lacrimal keratoconjunctivitis in many human eye diseases, including DED.

An Ocular Commensal Protects against Corneal Infection by Driving an Interleukin-17 Response from Mucosal γδ T Cells.

Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but proof of a living, resident ocular microbiome remains elusive. Here, we used a mouse model of ocular surface disease to reveal that commensals were present in the ocular mucosa and had functional immunological consequences. We isolated one such candidate commensal, Corynebacterium mastitidis, and showed that this organism elicited a commensal-specific interleukin-17 response from γδ T cells in the ocular mucosa that was central to local immunity. The commensal-specific response drove neutrophil recruitment and the release of antimicrobials into the tears and protected the eye from pathogenic Candida albicans or Pseudomonas aeruginosa infection. Our findings provide direct evidence that a resident commensal microbiome exists on the ocular surface and identify the cellular mechanisms underlying its effects on ocular immune homeostasis and host defense.

Pathological Analysis of Ocular Lesions in a Murine Model of Sjögren's Syndrome.

Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by severe inflammation of exocrine glands such as the salivary and lacrimal glands. When it affects the lacrimal glands, many patients experience keratoconjunctivitis due to severely dry eyes. This study investigated the pathological and immunological characteristics of ocular lesions in a mouse model of SS. Corneal epithelial injury and hyperplasia were confirmed pathologically. The number of conjunctival mucin-producing goblet cells was significantly decreased in the SS model mice compared with control mice. Expression levels of transforming growth factor (TGF)-ß, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and C-X-C motif chemokine (CXCL) 12 were significantly higher in the corneal epithelium of the SS model mice than in control mice. Inflammatory lesions were observed in the Harderian, intraorbital, and extraorbital lacrimal glands in the SS model mice, suggesting that the ocular glands were targeted by an autoimmune response. The lacrimal glands of the SS model mice were infiltrated by cluster of differentiation (CD)4⁺ T cells. Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed significantly increased mRNA expression of TNF-α, TGF-ß, CXCL9, and lysozyme in the extraorbital lacrimal glands of the SS model mice compared with control mice. These results add to the understanding of the complex pathogenesis of SS and may facilitate development of new therapeutic strategies.