Search

Herpes simplex keratitis (HSK) is a blinding disease caused by herpes simplex virus type 1 (HSV-1) infection, and rapid eradication of the virus from the affected cornea is imperative. Nod-like receptors (NLRs) are intracellular innate immune sensors closely associated with cell death, inflammation and immune responses. In this study, we investigated the role of NLRP12 in the antiviral immunology in HSK and the underlying mechanisms. We found that NLRP12 expression was significantly decreased in HSV-1-infected human corneal epithelial cells (HCE-Ts) and HSK mouse corneas. Overexpression of NLRP12 significantly reduced viral replication in infected HCE-Ts and functioned through inflammasome-mediated pyroptosis and downstream IL-18-IFN-γ axis. In HSK mouse models, overexpression of NLRP12 reduced viral replication in the cornea and alleviated HSK symptoms. This resulted from enhanced antiviral immune responses including the activation of specific immune cells in both the cornea and the draining lymph nodes. Specifically, the NLRP12-IL-18-IFN-γ axis regulated the interaction between infected corneal epithelial cells and macrophages. In conclusion, our study identified a role of NLRP12 in mediating pyroptosis and regulating antiviral immune responses. This novel finding opens the possibilities of NLRP12 as a viable target in the therapeutic strategies for HSV-1 infection., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mast cells (MCs), traditionally viewed as key players in IgE-mediated allergic responses, are increasingly recognized for their versatile roles. Situated at critical barrier sites such as the ocular surface, these sentinel cells participate in a broad array of physiological and pathological processes. This review presents a comprehensive update on the immune pathophysiology of MCs, with a particular focus on the mechanisms underlying innate immunity. It highlights their roles at the ocular surface, emphasizing their participation in allergic reactions, maintenance of corneal homeostasis, neovascularization, wound healing, and immune responses in corneal grafts. The review also explores the potential of MCs as therapeutic targets, given their significant contributions to disease pathogenesis and their capacity to modulate immunity. Through a thorough examination of current literature, we aim to elucidate the immune pathophysiology and multifaceted roles of MCs in ocular surface health and disease, suggesting directions for future research and therapeutic innovation., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: This study aims to reveal the immunopathogenesis of the high-risk corneal transplantation using a comparative proteomic approach., Methods: The immunological properties of ocular tissues (including corneal grafts, aqueous humour, and iris-ciliary body) were analysed using a high-risk rabbit corneal transplantation model employing a comparative proteomic approach., Results: The corneal grafts revealed a dramatic increase in the immune response both at the early (postoperative day 7) and rejection stages, along with the appearance of transplantation stress-induced cellular senescence in the early stage. The aqueous humour (AH) displayed persistent pathological alterations, indicated by the significant enrichment of complement and coagulation cascades pathway in the early stage and interleukin (IL)-17 signalling pathway in the rejection stage. More surprisingly, the pronounced elevation of immune response was also observed in the iris-ciliary body (I-CB) tissues at the early and rejection stages. The enriched immune-related pathways were associated with antigen processing and presentation, complement and coagulation cascades, and IL-17 signalling pathway. Furthermore, proteomic analysis revealed that the implantation of Cyclosporine A drug delivery system (CsA-DDS) into the anterior chamber obviously mitigated corneal transplantation rejection by inhibiting immunoreaction both in the corneal grafts and I-CB tissues., Conclusion: The results highlighted the involvement of intraocular immunity both in the grafts and I-CB tissues during corneal transplantation rejection, further suggesting the anterior chamber as an optimal drug-delivery site for its treatment., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: Herpes simplex keratitis (HSK) is a blinding disease caused by corneal infection of Herpes simplex virus type 1 (HSV-1). Effective clearance of HSV-1 from the infected cornea is crucial for HSK management. Macrophages play an important part in the innate immune defense against viral infections. This study investigates the immunomodulatory role of NLRP12 in macrophage immune response during HSV-1 infection., Methods: NLRP12 expression post-infection was assessed in various macrophage cell lines. Overexpression of NLRP12 was achieved by lentiviral transfection, and its effect on HSV-1 replication and immune responses were examined. Mechanistic insights into the role of NLRP12 were explored using immunofluorescence and Western Blot. For in vivo studies, ocular adoptive transfer of NLRP12-overexpressing bone marrow derived macrophages (BMDMs) was performed. HSV-1 viral loads, HSK symptoms, and macrophage-mediated immune responses were investigated., Results: A significant decrease in NLRP12 expression post-infection was observed in various macrophage cell lines. Overexpression of NLRP12 in macrophages reduced HSV-1 replication. Mechanistically, overexpression of NLRP12 triggered early and robust pyroptosis in response to HSV-1 infection, inducing interleukin (IL)-18 production and activating downstream antiviral responses through the JAK-STAT signaling pathway. In vivo, ocular adoptive transfer of NLRP12-overexpressing BMDMs to mouse corneas alleviated HSK damage and reduced HSV-1 viral loads. NLRP12-overexpressing BMDMs improved antiviral responses in the cornea and promoted the maturation of corneal-infiltrating macrophages and dendritic cells. Additionally, NLRP12-overexpressing BMDMs amplified the adaptive immune response in the submandibular draining lymph nodes., Discussion: These findings highlight the role of NLRP12 in macrophage-mediated immune response against HSV-1 infection and suggest its potential for possible immunotherapy for HSK., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jiang, Zhang, Liu, Yang, Yang, Liu and Hu.)

Several blinding diseases affecting the retina and optic nerve are exacerbated by or caused by dysregulated inflammation and oxidative stress. These diseases include uveitis, age related macular degeneration, diabetic retinopathy and glaucoma. Consequently, despite their divergent symptoms, treatments that reduce oxidative stress and suppress inflammation may be therapeutic. The production of inflammatory cytokines and their activities are regulated by a class of proteins termed Suppressors of Cytokine Signaling (SOCS). SOCS1 and SOCS3 are known to dampen signaling via pathways employing Janus kinases and signal transducer and activator of transcription proteins (JAK/STAT), Toll-like Receptors (TLR), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen activated kinase (MAPK) and NLR family pyrin domain containing 3 (NLRP3). We have developed cell-penetrating peptides from the kinase inhibitory region of the SOCS1 and SOCS3 (denoted as R9-SOCS1-KIR and R9-SOCS3-KIR) and tested them in retinal pigment epithelium (RPE) cells and in macrophage cell lines. SOCS-KIR peptides exhibited anti-inflammatory, anti-oxidant and anti-angiogenic properties. In cell culture, both Th1 and Th17 cells were suppressed together with the inhibition of other inflammatory markers. We also observed a decrease in oxidants and a simultaneous rise in neuroprotective and anti-oxidant effectors. In addition, treatment prevented the loss of gap junction proteins and the ensuing drop in transepithelial electrical resistance in RPE cells. When tested in mouse models by eye drop instillation, they showed protection against autoimmune uveitis, as a prophylactic as well as a therapeutic. Mice with endotoxin-induced uveitis were protected by eye drop administration as well. R9-SOCS3-KIR was particularly effective against the pathways acting through STAT3, e.g. IL-6 and VEGF-A mediated responses that lead to macular degeneration. Eye drop administration of R9-SOCS3-KIR stimulated production of antioxidant effectors and reduced clinical symptoms in mouse model of oxidative stress that replicates the RPE injury occurring in AMD. Because these peptides suppress multiple pathogenic stimuli and because they can be delivered topically to the cornea, they are attractive candidates for therapeutics for uveitis, macular degeneration, diabetic retinopathy and glaucoma., Competing Interests: CA and HJ are listed as inventors in a patent held by the University of Florida for the technology involving R9-SOCS3-KIR peptide. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ahmed, Johnson and Lewin.)

Background: The aim of this study was to assess the impact of the ratio between the graft and host corneal size (RGH) on postoperative complications, such as immune reactions, re-bubbling rate and endothelial cell loss (ECL) after Descemet membrane endothelial keratoplasty (DMEK)., Patients and Methods: Retrospectively, 457 patient eyes were included which had undergone surgery between 2016 and 2019 in the Department of Ophthalmology, Saarland University Medical Center in Homburg/Saar using DMEK or triple DMEK, diagnosed as Fuchs' endothelial dystrophy (n = 431), pseudophakic bullous keratopathy (n = 9) and others (n = 17). The follow-up period extended until the end of 2020. Main outcome measures included immune reaction (IR), re-bubbling rate and the postoperative endothelial cell loss (ECL) at 6 weeks, 6 months and 12 months and whether these measures depended on the RGH., Results: The RGH in this study ranged from 0.35 to 0.62 (0.46 ± 0.04). There were 33 (7.2%) postoperative IRs (DMEK n = 25; triple DMEK n = 8). The average RGH without IR (0.46 ± 0.04) was significantly (p = 0.038) smaller than in the group with IR (0.47 ± 0.05). Re-bubbling was necessary in 159 of 457 (34.8%) patient eyes. The RGH in patient eyes with re-bubbling (0.47 ± 0.04) was significantly (p = 0.014) higher than that in eyes without re-bubbling (0.45 ± 0.04). The mean preoperative endothelial cell count (ECD) was 2603 ± 251 cells/mm 2 (min: 2161, max: 3500 cells/mm 2 ). It was shown that a larger RGH had no positive influence on endothelial cell loss (r = 0.001; p = 0.974)., Conclusion: Our results suggest that a larger graft diameter compared to host corneal size is associated with an increased rate of immune reactions and a higher re-bubbling rate after DMEK. Otherwise, a larger RGH had no positive influence on endothelial cell loss after DMEK. Accordingly, the graft size for DMEK should not be unnecessarily large, especially in eyes with Fuchs' endothelial dystrophy., (© 2024. The Author(s).)

Purpose of Review: Cancer immunotherapy is one of the most emerging and rapidly growing fields.Ocular side effects associated with these therapies are common and can be present in up to 70% of patients.The cornea may be involved in different pathogenic mechanisms triggered by different immunotherapeutic agents, and corneal disease varies from mild symptoms to severe corneal ulceration and melting with visual loss.We aimed to review the incidence, mechanism, and management of ocular surface side effects in cancer patients receiving immunotherapy., Recent Findings: With the recent use of immunotherapeutic agents in cancer patients, in particular immune checkpoint inhibitors (ICIs) and epidermal growth factor receptor (EGFR) inhibitors, ocular surface and corneal involvement are common side effects.These patients can be at risk of sight threatening complications that warrant prompt diagnosis and careful monitoring and management., Summary: Immunotherapy- related corneal complications in cancer patients are associated with a decreased quality of life. Prompt recognition and an interdisciplinary approach between ophthalmologists and oncologists are crucial to handle immune related ocular adverse events in these patients, in order to maintain ocular surface integrity and avoid a vision threatening complication., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Sleep deprivation (SD) has a wide range of adverse health effects. However, the mechanisms by which SD influences corneal pathophysiology and its post-wound healing remain unclear. This study aimed to examine the basic physiological characteristics of the cornea in mice subjected to SD and determine the pathophysiological response to injury after corneal abrasion. Using a multi-platform water environment method as an SD model, we found that SD leads to disturbances of corneal proliferative, sensory, and immune homeostasis as well as excessive inflammatory response and delayed repair after corneal abrasion by inducing hyperactivation of the sympathetic nervous system and hypothalamic-pituitary-adrenal axis. Pathophysiological changes in the cornea mainly occurred through the activation of the IL-17 signaling pathway. Blocking both adrenergic and glucocorticoid synthesis and locally neutralizing IL-17A significantly improved corneal homeostasis and the excessive inflammatory response and delay in wound repair following corneal injury in SD-treated mice. These results indicate that optimal sleep quality is essential for the physiological homeostasis of the cornea and its well-established repair process after injury. Additionally, these observations provide potential therapeutic targets to ameliorate SD-induced delays in corneal wound repair by inhibiting or blocking the activation of the stress system and its associated IL-17 signaling pathway., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears., Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1β, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay., Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance., Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Alenezi, Parnell, Schibeci, Ozkan, Willcox, White and Carnt.)

The ocular surface microenvironment, containing the cornea, conjunctiva, and lacrimal gland, constitutes the mucosal frontline of the eye and houses a myriad of immune cells. As a part of unconventional T cells, gamma delta (γδ) T cells differ in the development and functions from canonical alpha beta (αβ) T cells. They are predominantly situated in mucosal sites throughout the body, including ocular surface tissues. Recent research has elucidated that γδ T cells serve as the primary interleukin-17A (IL-17A) source in the conjunctiva. They play a pivotal role in preserving ocular surface homeostasis and exhibit both protective and pathogenic roles in ocular surface diseases. This review delves into the general profiles of γδ T cells, their distribution in ocular surface tissues, and consolidates current insights into their functions in different conditions including dry eye disease, infectious keratitis, corneal wound healing, anterior chamber-associated immune deviation, allergic conjunctival disease, and diabetic ocular surface disease. The aim is to provide a systemic perspective on γδ T cells in the ocular surface microenvironment and outline potential directions for future studies., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Herpetic stromal keratitis (HSK) is a painful and vision-impairing disease caused by recurrent HSV-1 infection of the cornea. The virus replication in the corneal epithelium and associated inflammation play a dominant role in HSK progression. Current HSK treatments targeting inflammation or virus replication are partially effective and promote HSV-1 latency, and long-term use can cause side effects. Thus, understanding molecular and cellular events that control HSV-1 replication and inflammation is crucial for developing novel HSK therapies. In this study, we report that ocular HSV-1 infection induces the expression of IL-27, a pleiotropic immunoregulatory cytokine. Our data indicate that HSV-1 infection stimulates IL-27 production by macrophages. Using a primary corneal HSV-1 infection mouse model and IL-27 receptor knockout mice, we show that IL-27 plays a critical role in controlling HSV-1 shedding from the cornea, the optimum induction of effector CD4+ T cell responses, and limiting HSK progression. Using in vitro bone marrow-derived macrophages, we show that IL-27 plays an antiviral role by regulating macrophage-mediated HSV-1 killing, IFN-β production, and IFN-stimulated gene expression after HSV-1 infection. Furthermore, we report that IL-27 is critical for macrophage survival, Ag uptake, and the expression of costimulatory molecules involved in the optimum induction of effector T cell responses. Our results indicate that IL-27 promotes endogenous antiviral and anti-inflammatory responses and represents a promising target for suppressing HSK progression., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Manually quantifying immune cells (ICs), commonly considered dendritic cells, in the corneal epithelium from in vivo confocal microscopy (IVCM) images can be influenced by observer bias. This study sought to evaluate the repeatability of manual IC quantification. Cell counts were first performed for 184 non-overlapping IVCM images by a single observer. Quantifications were undertaken to establish the total cell numbers per image, and the numbers of three cell morphological subtypes: mature ICs (with elongated dendrites), immature ICs (with short- or non-discernible dendrites) and globular cells (with large bodies and no visible dendrites). Cell counts were then repeated by the same observer, and independently undertaken by a second observer. Prior to these counts, both observers undertook an agreement 'training' process to define IC appearance and delineate the morphological subtypes. Total IC counts demonstrated excellent intra- and inter-observer reliability (intraclass correlation coefficients (ICC) > 0.90). Bland-Altman plots showed that interobserver measurement bias increased as a function of the total IC number in the image prior to consensus training. For total IC counts after the observer training process, there was no significant interobserver measurement bias. For IC morphological subtypes, there was a positive relationship between the mean inter-observer difference and average cell count for mature ICs and globular cells, but not immature ICs. In conclusion, higher variability in manual corneal IC counts exists when more cells are present in an IVCM image. Implementing an observer training process reduced inter-observer variability and minimised systematic measurement error., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Corneal transplantation is the most common form of tissue transplantation. The success of corneal transplantation mainly relies on the integrity of corneal endothelial cells (CEnCs), which maintain tissue transparency by pumping out excess water from the cornea. After transplantation, the rate of CEnC loss far exceeds that seen with normal aging, which can threaten sight. The underlying mechanisms are poorly understood. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide that is constitutively found in the aqueous humor with both cytoprotective and immunomodulatory effects. The curent study found high expression of melanocortin 1 receptor (MC1R), the receptor for α-MSH, on CEnCs. The effect of α-MSH/MC1R signaling on endothelial function and allograft survival in vitro and in vivo was investigated using MC1R signaling-deficient mice (Mc1re/e mice with a nonfunctional MC1R). Herein, the results indicate that in addition to its well-known immunomodulatory effect, α-MSH has cytoprotective effects on CEnCs after corneal transplantation, and the loss of MC1R signaling significantly decreases long-term graft survival in vivo. In conclusion, α-MSH/MC1R signaling is critical for CEnC function and graft survival after corneal transplantation., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Keratitis induced by bacterial toxins, including lipopolysaccharide (LPS), is a major cause of corneal opacity and vision loss. Our previous study demonstrates hepatocyte growth factor (HGF) promotes epithelial wound healing following mechanical corneal injury. Here, we investigated whether HGF has the capacity to suppress infectious inflammatory corneal opacity using a new model of LPS-induced keratitis. Keratitis, induced by two intrastromal injections of LPS on day 1 and 4 in C57BL/6 mice, resulted in significant corneal opacity for up to day 10. Following keratitis induction, corneas were topically treated with 0.1% HGF or PBS thrice daily for 5 days. HGF-treated mice showed a significantly smaller area of corneal opacity compared to PBS-treated mice, thus improving corneal transparency. Moreover, HGF treatment resulted in suppression of α-SMA expression, compared to PBS treatment. HGF-treated corneas showed normalized corneal structure and reduced expression of pro-inflammatory cytokine, demonstrating that HGF restores corneal architecture and immune quiescence in corneas with LPS-induced keratitis. These findings offer novel insight into the potential application of HGF-based therapies for the prevention and treatment of infection-induced corneal opacity., (© 2022. The Author(s).)

Purpose: To assess and compare corneal sub-basal nerve plexus morphology with circulating lymphocyte subsets, immunologic status and disease activity in Sjögren syndrome (SjS) patients., Methods: Fifty-five SjS patients, 63 Sicca patients (not fulfilling SjS criteria), 18 rheumatoid arthritis (RA) patients and 20 healthy controls (HC) were included. Systemic disease activity in SjS was assessed with the ESSDAI score. Lymphocyte subpopulations were studied with flow cytometry. Corneal confocal microscopy and ImageJ software were used to characterize corneal sub-basal nerve plexus in terms of nerve density (CNFD), length (CNFL) and tortuosity (CNFT). Conventional dry eye tests were also performed., Results: CNFL and CNFD were lower in SjS, Sicca and RA groups, compared to HC (p < 0.001 for both SjS and Sicca); CNFL p = 0.005, CNFD p = 0.018 in RA). CNFT was higher in SjS, followed by Sicca, RA and HC. A negative correlation was found between ESSDAI score and CNFL (r=-0.735, p = 0.012). CNFL correlated negatively with IL21 + CD8 + T cells (r=-0.279, p = 0.039) and a positively with total memory (r = 0.299, p = 0.027), unswitched memory (r = 0.281, p = 0.038) and CD24 Hi CD27 + (r = 0.278, p = 0.040) B cells. CNFD showed a tendency to significance in its negative correlation with ESSDAI (r=-0.592, p = 0.071) and in its positive correlation with switched memory B cells (r = 0.644, p = 0.068)., Conclusions: This is the first study aiming to correlate ocular findings with lymphocyte subsets in SjS. The associations founded between CNFL and CNFD and disease activity, IL21 + follicular T cells and some B-cell subsets suggest that corneal nerve damage may parallel systemic disease activity and inflammatory cells' dynamics., (© 2021 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

The IL-36 cytokines are known to play various roles in mediating the immune response to infection in a tissue- and pathogen-dependent manner. The present study seeks to investigate the role of IL-36R signaling in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. IL-36α -/- , IL-36γ -/- , and IL-36R -/- mice had significantly more severe keratitis than wild-type mice. At six hours postinfection, IL-36α pretreatment augmented P. aeruginosa -induced expression of IL-1Ra, IL-36γ, LCN2, and S100A8/A9. At one day postinfection, exogenous IL-36α suppressed, whereas IL-36α deficiency promoted, the expression of IL-1β. At three days postinfection, exogenous IL-36α suppressed Th1 but promoted Th2 immune response. IL-36α stimulated the infiltration of IL-22-expressing immune cells, and IL-22 neutralization resulted in more severe keratitis. IL-36α alone stimulated dendritic cell infiltration in B6 mouse corneas. Taken together, our study suggests that IL-36R signaling plays a protective role in the pathogenesis of P. aeruginosa keratitis by promoting the innate immune defense, Th2, and/or Th22/IL-22 immune responses. Exogenous IL-36α might be a potential therapy for improving the outcome of P. aeruginosa keratitis., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Immune cell infiltration has been implicated in neurotoxic chemotherapy for cancer treatment. However, our understanding of immune processes is still incomplete and current methods of observing immune cells are time consuming or invasive. Corneal dendritic cells are potent antigen-presenting cells and can be imaged with in-vivo corneal confocal microscopy. Corneal dendritic cell densities and nerve parameters in patients treated with neurotoxic chemotherapy were investigated. Patients treated for cancer with oxaliplatin (n = 39) or paclitaxel (n = 48), 3 to 24 months prior to assessment were recruited along with 40 healthy controls. Immature (ImDC), mature (MDC) and total dendritic cell densities (TotalDC), and corneal nerve parameters were analyzed from in-vivo corneal confocal microscopy images. ImDC was increased in the oxaliplatin group (Median, Md = 22.7 cells/mm 2 ) compared to healthy controls (Md = 10.1 cells/mm 2 , p = 0.001), but not in the paclitaxel group (Md = 10.6 cells/mm 2 ). ImDC was also associated with higher oxaliplatin cumulative dose (r = 0.33, p = 0.04) and treatment cycles (r = 0.40, p = 0.01). There was no significant difference in MDC between the three groups (p > 0.05). Corneal nerve parameters were reduced in both oxaliplatin and paclitaxel groups compared to healthy controls (p < 0.05). There is evidence of elevation of corneal ImDC in oxaliplatin-treated patients. Further investigation is required to explore this potential link through longitudinal studies and animal or laboratory-based immunohistochemical research., (© 2021. The Author(s).)

Herpes simplex virus type-1 (HSV-1) ocular infection is one of the leading causes of infectious blindness in developed countries. The resultant herpetic keratitis (HK) is caused by an exacerbated reaction of the adaptive immune response that persists beyond virus clearance causing substantial damage to the cornea. Intramuscular immunization of mice with the HSV-1(VC2) live-attenuated vaccine strain has been shown to protect mice against lethal ocular challenge. Herein, we show that following ocular challenge, VC2 vaccinated animals control ocular immunopathogenesis in the absence of neutralizing antibodies on ocular surfaces. Ocular protection is associated with enhanced intracorneal infiltration of γδ T cells compared to mock-vaccinated animals. The observed γδ T cellular infiltration was inversely proportional to the infiltration of neutrophils, the latter associated with exacerbated tissue damage. Inhibition of T cell migration into ocular tissues by the S1P receptors agonist FTY720 produced significant ocular disease in vaccinated mice and marked increase in neutrophil infiltration. These results indicate that ocular challenge of mice immunized with the VC2 vaccine induce a unique ocular mucosal response that leads into the infiltration of γδ T cells resulting in the amelioration of infection-associated immunopathogenesis., Competing Interests: Louisiana State University has licensed the VC2 vaccine for genital herpes to Rational Vaccines. KGK serves as a consultant for Rational Vaccines, Inc., (Copyright © 2021 Nabi, Lewin, Collantes, Chouljenko and Kousoulas.)

Cornea is one of the most commonly transplanted tissues worldwide. However, it is usually omitted in the field of transplantology. Transplantation of the cornea is performed to treat many ocular diseases. It restores eyesight significantly improving the quality of life. Advancements in banking of explanted corneas and progressive surgical techniques increased availability and outcomes of transplantation. Despite the vast growth in the field of transplantation laboratory testing, standards for corneal transplantation still do not include HLA typing or alloantibody detection. This standard practice is based on immune privilege dogma that accounts for high success rates of corneal transplantation. However, the increasing need for retransplantation in high-risk patients with markedly higher risk of rejection causes ophthalmology transplantation centers to reevaluate their standard algorithms. In this review we discuss immune privilege mechanisms influencing the allograft acceptance and factors disrupting the natural immunosuppressive environment of the eye. Current developments in testing and immunosuppressive treatments (including cell therapies), when applied in corneal transplantation, may give very good results, decrease the possibility of rejection, and reduce the need for retransplantation, which is fairly frequent nowadays., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Justyna Sakowska et al.)

Previously, we reported that herpes simplex virus type 1 (HSV-1) ICP22 binds to the CD80 promoter and suppresses its expression in vitro and in vivo . To better understand the impact of ICP22 binding to CD80 on HSV-1 infectivity and pathogenicity, we mapped the region of ICP22 required to bind the CD80 promoter to a 40-amino-acid (aa) region of ICP22. We constructed a recombinant HSV-1 expressing a truncated form of ICP22 that lacks these 40 aa, which does not bind to the CD80 promoter (KOS-ICP22Δ40) and retains the ability to replicate efficiently in rabbit skin cells, in contrast to ICP22-null virus. The replication of this recombinant virus in vitro and in vivo was higher than that of the ICP22-null virus, but virus replication kinetics were lower than those of the wild-type (WT) control virus. Similar to ICP22-null virus, the KOS-ICP22Δ40 mutant virus increased CD80 expression in dendritic cells (DCs) and interferon gamma (IFN-γ) expression in CD8 + T cells but not CD4 + T cells in infected mouse corneas. In contrast to the significantly reduced virus replication in the eyes of ocularly infected mice, the levels of latency reactivation were similar between KOS-ICP22Δ40 virus and WT virus. Thus, blocking ICP22 binding to the CD80 promoter using a recombinant virus expressing a truncated ICP22 that lacks CD80 promoter binding appears to reduce virus replication and enhance CD8 + IFN-γ + infiltrates in corneas of infected mice, with no effect on latency reactivation. IMPORTANCE Direct binding of HSV-1 ICP22 to the CD80 promoter downregulates the expression of the costimulatory molecule CD80 but not CD86. In this study, we fine mapped the region of ICP22 required for binding to the CD80 promoter and constructed a recombinant virus containing a deletion in ICP22 that failed to bind to the CD80 promoter. This recombinant virus replicated less efficiently in vitro and in vivo than did the WT control virus, although CD80-expressing CD11c + cells and IFN-γ-expressing CD8 + T cells were increased. Interestingly, the levels of latency and reactivation in the two viruses were similar despite lower virus replication in the eyes of infected mice. Therefore, blocking the interaction of ICP22 with the CD80 promoter could be used to temper the immune response.

The ocular surface is the part of the visual system directly exposed to the environment, and it comprises the cornea, the first refractive tissue layer and its surrounding structures. The ocular surface has evolved to keep the cornea smooth and wet, a prerequisite for proper sight, and also protected. To this aim, the ocular surface is a bona fide mucosal niche with an immune system capable of fighting against dangerous pathogens. However, due to the potential harmful effects of uncontrolled inflammation, the ocular surface has several mechanisms to keep the immune response in check. Specifically, the ocular surface is maintained inflammation-free and functional by a particular form of peripheral tolerance known as mucosal tolerance, markedly different from the immune privilege of intraocular structures. Remarkably, conjunctival tolerance is akin to the oral and respiratory tolerance mechanisms found in the gut and airways, respectively. And also similarly, this form of immunoregulation in the eye is affected by ageing just as it is in the digestive and respiratory tracts. With ageing comes an increased prevalence of immune-based ocular surface disorders, which could be related to an age-related impairment of conjunctival tolerance. The purpose of this review was to summarize the present knowledge of ocular mucosal tolerance and how it is affected by the ageing process in the light of the current literature on mucosal immunoregulation of the gut and airways., (© 2021 John Wiley & Sons Ltd.)

The kinetics of antigen-presenting cells (APCs) vary depending on their resident tissues and the manner of immunization. We investigated the long-term changes in mature APC and T-cell subsets over 4 weeks in the ocular surface in murine models of corneal quiescent or potent sterile inflammation, and allosensitization using partial (PT), syngeneic (Syn), and allogeneic (Allo) corneal transplantation. In PT, CD11b int CD11c hi MHCII hi CD86 hi cells increased until 4 weeks with an increase in IFNγ hi T cells. In Syn, both CD11b int CD11c hi MHCII hi CD86 hi and CD11b hi CD11c hi MHCII hi CD86 hi APC subsets increased until 4 weeks with a brief increase in CD69 hi T cells at 2 weeks. In Allo, CD11b int CD11c hi MHCII hi CD86 hi and CD11b hi CD11c hi MHCII hi CD86 hi APC subsets increased until 4 weeks, and an early increase in CD69 hi T cells was observed at 2 weeks followed by a late increase in IFNγ hi T cells at 4 weeks. The frequency of the IFNγ hi T cell subset was positively correlated with the frequency of the CD11b int CD11c hi MHCII hi CD86 hi subset, indicating the existence of APC-T cell interaction in the ocular surface. Together, the results indicate that allosensitization in mature APCs leads to T-cell activation in the ocular surface, whereas sterile inflammation merely induces a brief and non-specific T-cell activation in the ocular surface.

The assessment of disease activity is fundamental in the management of chronic inflammatory demyelinating polyneuropathy (CIDP). Previous studies with small patient numbers found an increase of corneal immune cell infiltrates as a potential marker of inflammation in patients with CIDP. However, its clinical relevance remained unclear. The present study aimed to determine whether the amount of corneal inflammatory cells (CIC) measured by corneal confocal microscopy (CCM) detects disease activity in CIDP. CIC were measured in 142 CCM-investigations of 97 CIDP-patients. Data on clinical disease activity, disability (INCAT-ODSS) and need for therapy escalation at the timepoint of CCM, 3 and 6 months later were analyzed depending CIC-count. Pathological spontaneous activity during electromyography was examined as another possible biomarker for disease activity in comparison to CIC-count. An increased CIC-count at baseline was found in patients with clinical disease activity and disability progression in the following 3-6 months. An increase to more than 25 CIC/mm 2 had a sensitivity of 0.73 and a specificity of 0.71 to detect clinical disease activity and a sensitivity of 0.77 and a specificity of 0.64 to detect disability progression (increasing INCAT-ODSS) in the following 6 months. An increase to more than 50 CIC/mm 2 had a sensitivity of about 0.51 and a specificity of 0.91 to detect clinical disease activity and a sensitivity of 0.53 and a specificity of 0.80 to detect disability progression. CIC count is a non-invasive biomarker for the detection of disease activity in the following 6 months in CIDP., (© 2021. The Author(s).)

We investigated whether aging-dependent changes in dendritic cell (DC) distributions are distinct in autoimmune dry eye compared with an aging-related murine model. Corneal staining and tear secretion were evaluated in young and aged C57BL/6 (B6) and NOD.B10.H2b mice (NOD). In the corneolimbus, lacrimal gland (LG), and mesenteric lymph node (MLN), CD11b - and CD11b + DCs, CD103 + DCs and MHC-II hi B cells were compared between young and aged B6 and NOD mice. With increased corneal staining, tear secretion decreased in both aged B6 and NOD mice ( p < 0.001). In both aged B6 and NOD mice, the percentages of corneolimbal CD11b + DCs were higher ( p < 0.05) than those in young mice. While, the percentages of lymph nodal CD103 + DCs were higher in aged B6 and NOD mice ( p < 0.05), the percentages of corneolimbal CD103 + DCs were only higher in aged NOD mice ( p < 0.05). In aged NOD mice, the proportions of lacrimal glandial and lymph nodal MHC-II hi B cells were also higher than those in young mice ( p < 0.05). It indicates that corneolimbal or lacrimal glandial distribution of CD103 + DCs or MHC-II hi B cells may be distinct in aged autoimmune dry eye models compared to those in aged immune competent murine models.

Purpose: To explore the anti-inflammatory effect of lipoxin A4 (LXA4) in Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms., Methods: In A. fumigatus keratitis mouse models, enzyme-linked immunosorbent assay (ELISA) was used to detect the level of LXA4. Clinical scores were utilized to evaluate fungal keratitis (FK) severity. Fungal load was assessed by plate count. Immunofluorescence staining, HE staining and myeloperoxidase (MPO) assays were carried out to evaluate the neutrophil infiltration and activity. In A. fumigatus infected mouse corneas and inactivated A. fumigatus-stimulated RAW264.7 cells, quantitative real time polymerase chain reaction (qRT-PCR) and ELISA were applied to assess the expression of pro-inflammatory mediators and anti-inflammatory factors.Reactive oxygen species (ROS) was determined by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in RAW264.7 cells., Results: LXA4 level was significantly increased in mice with A. fumigatus keratitis. In an A. fumigatus keratitis mouse model, LXA4 treatment alleviated FK severity, reduced fungal load and repressed neutrophil infiltration and activity. Additionally, LXA4 inhibited the expression of pro-inflammatory mediators including IL-1β, TNF-α, IL-6, cyclooxygenase-2 (COX-2), TLR-2, TLR-4, Dectin-1 and iNOS, and promoted the expression of anti-inflammatory factors IL-10 and Arg-1. In RAW264.7 cells, LXA4 receptor/formyl peptide receptor 2 (ALX/FPR2) blockade reversed the anti-inflammatory effect of LXA4. LXA4 suppressed inactivated A. fumigatus induced elevated ROS production in RAW264.7 cells, which was abrogated by ALX/FPR2 antagonist Boc-2., Conclusion: LXA4 ameliorated inflammatory response by suppressing neutrophil infiltration, downregulating the expression of pro-inflammatory mediators and ROS production through ALX/FPR2 receptor in A. fumigatus keratitis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: Given the role of corneal sensory nerves during epithelial wound repair, we sought to examine the relationship between immune cells and polymodal nociceptors following corneal injury., Methods: Young C57BL/6J mice received a 2 mm corneal epithelial injury. One week later, corneal wholemounts were immunostained using β-tubulin-488, TRPV1 (transient receptor potential ion channel subfamily V member-1, a nonselective cation channel) and immune cell (MHC-II, CD45 and CD68) antibodies. The sum length of TRPV1+ and TRPV1- nerve fibers, and their spatial association with immune cells, was quantified in intact and injured corneas., Results: TRPV1+ nerves account for ∼40% of the nerve fiber length in the intact corneal epithelium and ∼80% in the stroma. In the superficial epithelial layers, TRPV1+ nerve terminal length was similar in injured and intact corneas. In intact corneas, the density (sum length) of basal epithelial TRPV1+ and TRPV1- nerve fibers was similar, however, in injured corneas, TRPV1+ nerve density was higher compared to TRPV1- nerves. The degree of physical association between TRPV1+ nerves and intraepithelial CD45+ MHC-II+ CD11c+ cells was similar in intact and injured corneas. Stromal leukocytes co-expressed TRPV1, which was partially localized to CD68+ lysosomes, and this expression pattern was lower in injured corneas., Conclusions: TRPV1+ nerves accounted for a higher proportion of corneal nerves after injury, which may provide insights into the pathophysiology of neuropathic pain following corneal trauma. The close interactions of TRPV1+ nerves with intraepithelial immune cells and expression of TRPV1 by stromal macrophages provide evidence of neuroimmune interactions in the cornea.

Objective: Keratoconus (KC) is generally described as a non-inflammatory disease, characterized by thinning in the central region of the cornea with consequent tissue degradation producing impaired visual acuity., Materials and Methods: In our experimental study, we analyzed the presence and implications of several inflammatory cytokines in the corneal tissues of patients suffering from keratoconus by immunohistochemical analysis., Results: The analysis showed increased levels of inflammatory factors in the pathological tissues compared to controls, confirming that KC cannot be considered an entirely non-inflammatory pathology and that its etiopathogenesis includes several chronic inflammatory events., Conclusions: In the light of these results, the classification of KC as an inflammatory pathology or as a pathology related to inflammation might be useful in directing future research aimed at developing effective anti-inflammatory therapies to pharmacologically target the inflammatory mediators which contribute to the development and progression of the disease.

The eye and the brain have limited capacities for regeneration and as such, immune-mediated inflammation can produce devastating consequences in the form of neurodegenerative diseases of the central nervous system or blindness as a result of ocular inflammatory diseases such as uveitis. Accordingly, both the eye and the brain are designed to limit immune responses and inflammation - a condition known as "immune privilege". Immune privilege is sustained by physiological, anatomical, and regulatory processes that conspire to restrict both adaptive and innate immune responses., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Niederkorn.)

The purpose of the study was to uncover the role of tenascin X in modulation of healing in mouse corneas subjected to epithelium debridement. Healing in corneas with an epithelial defect was evaluated at the levels of gene and protein expression. Wound healing-related mediators and inflammatory cell infiltration were detected by histology, immunohistochemistry and real-time RT-PCR. Tenascin X protein was upregulated in the wounded wild-type (WT) corneal epithelium. The lack of tenascin X impaired closure of an epithelial defect and accelerated infiltration of neutrophils into the wound periphery as compared to the response in WT tissue. Expression of wound healing-related proinflammatory and reparative components, i.e., interleukin-6, transforming growth factor β, matrix metalloproteinases, were unaffected by the loss of tenascin X expression. Marked accumulation of malondialdehyde (a lipid peroxidation-derived product) was observed in KO healing epithelia as compared with its WT counterpart. Neutropenia induced by systemic administration of a specific antibody rescued the impairment of epithelial healing in KO corneas, with reduction of malondialdehyde levels in the epithelial cells. Finally, we showed that a chemical scavenging reactive oxygen species reversed the impairment of attenuation of epithelial repair with a reduction of tissue levels of malondialdehyde. In conclusion, loss of tenascin X prolonged corneal epithelial wound healing and increased neutrophilic inflammatory response to debridement in mice. Tenascin X contributes to the control of neutrophil infiltration needed to support the regenerative response to injury and prevent the oxidative stress mediators from rising to cytotoxic levels.

Rapid death of infected cells is an important antiviral strategy. However, fast decisions that are based on limited evidence can be erroneous and cause unnecessary cell death and subsequent tissue damage. How cells optimize their death decision making strategy to maximize both speed and accuracy is unclear. Here, we show that exposure to TNF, which is secreted by macrophages during viral infection, causes cells to change their decision strategy from "slow and accurate" to "fast and error-prone". Mathematical modeling combined with experiments in cell culture and whole organ culture show that the regulation of the cell death decision strategy is critical to prevent HSV-1 spread. These findings demonstrate that immune regulation of cellular cognitive processes dynamically changes a tissues' tolerance for self-damage, which is required to protect against viral spread.

Herpes simplex virus 1 (HSV-1) infects the cornea and caused blinding ocular disease. In the present study, we evaluated whether and how a novel engineered version of fibroblast growth factor-1 (FGF-1), designated as TTHX1114, would reduce the severity of HSV-1-induced and recurrent ocular herpes in the mouse model. The efficacy of TTHX1114 against corneal keratopathy was assessed in B6 mice following corneal infection with HSV-1, strain McKrae. Starting day one post infection (PI), mice received TTHX1114 for 14 days. The severity of primary stromal keratitis and blepharitis were monitored up to 28 days PI. Inflammatory cell infiltrating infected corneas were characterized up to day 21 PI. The severity of recurrent herpetic disease was quantified in latently infected B6 mice up to 30 days post-UVB corneal exposure. The effect of TTHX1114 on M1 and M2 macrophage polarization was determined in vivo in mice and in vitro on primary human monocytes-derived macrophages. Compared to HSV-1 infected non-treated mice, the infected and TTHX1114 treated mice exhibited significant reduction of primary and recurrent stromal keratitis and blepharitis, without affecting virus corneal replication. The therapeutic effect of TTHX1114 was associated with a significant decrease in the frequency of M1 macrophages infiltrating the cornea, which expressed significantly lower levels of pro-inflammatory cytokines and chemokines. This polarization toward M2 phenotype was confirmed in vitro on human primary macrophages. This pre-clinical finding suggests use of this engineered FGF-1 as a novel immunotherapeutic regimen to reduce primary and recurrent HSV-1-induced corneal disease in the clinic., Competing Interests: ED and DB are employees and hold an equity interest in Trefoil Therapeutics, Inc. ED is an inventor on patents claiming TTHX1114. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dhanushkodi, Srivastava, Coulon, Prakash, Roy, Bagnol, David and BenMohamed.)

The endocrine cells confined in the islets of Langerhans are responsible for the maintenance of blood glucose homeostasis. In particular, beta cells produce and secrete insulin, an essential hormone regulating glucose uptake and metabolism. An insufficient amount of beta cells or defects in the molecular mechanisms leading to glucose-induced insulin secretion trigger the development of diabetes, a severe disease with epidemic spreading throughout the world. A comprehensive appreciation of the diverse adaptive procedures regulating beta cell mass and function is thus of paramount importance for the understanding of diabetes pathogenesis and for the development of effective therapeutic strategies. While significant findings were obtained by the use of islets isolated from the pancreas, in vitro studies are inherently limited since they lack the many factors influencing pancreatic islet cell function in vivo and do not allow for longitudinal monitoring of islet cell plasticity in the living organism. In this respect a number of imaging methodologies have been developed over the years for the study of islets in situ in the pancreas, a challenging task due to the relatively small size of the islets and their location, scattered throughout the organ. To increase imaging resolution and allow for longitudinal studies in individual islets, another strategy is based on the transplantation of islets into other sites that are more accessible for imaging. In this review we present the anterior chamber of the eye as a transplantation and imaging site for the study of pancreatic islet cell plasticity, and summarize the major research outcomes facilitated by this technological platform., Competing Interests: P-OB is founder and CEO of Biocrine AB. EI is consultant for Biocrine AB., (Copyright © 2021 Ilegems and Berggren.)

Purpose: of Review: This review offers an informed and up-to-date insight on the immune profile of the cornea and the factors that govern the regulation of such a unique immune environment., Summary: The cornea is a unique tissue that performs the specialized task of allowing light to penetrate for visual interpretation. To accomplish this, the ocular surface requires a distinct immune environment that is achieved through unique structural, cellular and molecular factors. Not only must the cornea be able to fend off invasive infectious agents but also control the inflammatory response as to avoid collateral, and potentially blinding damage; particularly of post-mitotic cells such as the corneal endothelium. To combat infections, both innate and adaptive arms of the inflammatory immune response are at play in the cornea. Dendritic cells play a critical role in coordinating both these responses in order to fend off infections. On the other side of the spectrum, the ocular surface is also endowed with a variety of anatomic and physiologic components that aid in regulating the immune response to prevent excessive, potentially damaging, inflammation. This attenuation of the immune response is termed immune privilege. The balance between pro and anti-inflammatory reactions is key for preservation of the functional integrity of the cornea., Recent Findings: The understanding of the molecular and cellular factors governing corneal immunology and its response to antigens is a growing field. Dendritic cells in the normal cornea play a crucial role in combating infections and coordinating the inflammatory arms of the immune response, particularly through coordination with T-helper cells. The role of neuropeptides is recently becoming more highlighted with different factors working on both sides of the inflammatory balance., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The cornea is a special interface between the internal ocular tissue and the external environment that provides a powerful chemical, physical, and biological barrier against the invasion of harmful substances and pathogenic microbes. This protective effect is determined by the unique anatomical structure and cellular composition of the cornea, especially its locally resident innate immune cells, such as Langerhans cells (LCs), mast cells (MCs), macrophages, γδ T lymphocytes, and innate lymphoid cells. Recent studies have demonstrated the importance of these immune cells in terms of producing different cytokines and other growth factors in corneal homeostasis and its pathologic conditions. This review paper briefly describes the latest information on these resident immune cells by specifically analyzing research from our laboratory., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu and Li.)

Air pollution is a serious environmental issue worldwide in developing countries' megacities, affecting the population's health, including the ocular surface, by predisposing or exacerbating other ocular diseases. Herpes simplex keratitis (HSK) is caused by the herpes simplex virus type 1 (HSV-1). The primary or recurring infection in the ocular site causes progressive corneal scarring that may result in visual impairment. The present study was designed to study the immunopathological changes of acute HSK under urban polluted air, using the acute HSK model combined with an experimental urban polluted air exposure from Buenos Aires City. We evaluated the corneal clinical outcomes, viral DNA and pro-inflammatory cytokines by RT-PCR and ELISA assays, respectively. Then, we determined the innate and adaptive immune responses in both cornea and local lymph nodes after HSV-1 corneal by immunofluorescence staining and flow cytometry. Our results showed that mice exposed to polluted air develop a severe form of HSK with increased corneal opacity, neovascularization, HSV-1 DNA and production of TNF-α, IL-1β, IFN-γ, and CCL2. A high number of corneal resident immune cells, including activated dendritic cells, was observed in mice exposed to polluted air; with a further significant influx of bone marrow-derived cells including GR1+ cells (neutrophils and inflammatory monocytes), CD11c+ cells (dendritic cells), and CD3+ (T cells) during acute corneal HSK. Moreover, mice exposed to polluted air showed a predominant Th1 type T cell response over Tregs in local lymph nodes during acute HSK with decreased corneal Tregs. These findings provide strong evidence that urban polluted air might trigger a local imbalance of innate and adaptive immune responses that exacerbate HSK severity. Taking this study into account, urban air pollution should be considered a key factor in developing ocular inflammatory diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sendra, Tau, Zapata, Lasagni Vitar, Illian, Chiaradía and Berra.)

This report evaluates a dietary manipulation approach to suppress the severity of ocular infections caused by herpes simplex virus infection. The virus causes chronic damage to the cornea that results from a T-cell-orchestrated inflammatory reaction to the infection. Lesion severity can be limited if cells with regulatory activity predominate over proinflammatory T cells and nonlymphoid inflammatory cells. In this report, we show that this outcome can be achieved by including the short-chain fatty acid (SCFA) salt sodium propionate (SP) in the drinking water. Animals given the SP supplement developed significantly fewer ocular lesions than those receiving no supplement. Corneas and lymphoid organs contained fewer CD4 Th1 and Th17 T cells, neutrophils, and macrophages than those of controls, but a higher frequency of regulatory T cells (Treg) was present. The inclusion of SP in cultures to induce CD4 T cell subsets in vitro reduced the magnitude of Th1 and Th17 responses but expanded Treg induction. Dietary manipulation was an effective approach to limit the severity of viral immuno-inflammatory lesions and may be worth exploring as a means to reduce the impact of herpetic lesions in humans. IMPORTANCE Herpetic lesions are a significant problem, and they are difficult to control with therapeutics. Our studies show that the severity of herpetic lesions in a mouse model can be diminished by changing the diet to include increased levels of SCFA, which act to inhibit the involvement of inflammatory T cells. We suggest that changing the diet to include higher levels of SCFA might be a useful approach to reducing the impact of recurrent herpetic lesions in humans., (Copyright © 2021 American Society for Microbiology.)

The ocular surface has the challenging responsibility of maintaining a clear moist refractive surface while protecting the eye from exogenous pathogens and the environment. Homeostasis of the ocular surface, including its innate immune components, is altered in ocular surface disease states. In this review, we focus on antimicrobial peptides and the role they play in the immune response of the ocular surface during healthy states and dry eye diseases. Antimicrobial peptides are of special interest to the study of the ocular surface because of their various roles that include microbial threat neutralization, wound healing, and immune modulation. This review explores current literature on antimicrobial peptides in ocular surface diseases and discusses their therapeutic potential in ocular surface diseases and dry eye.

Plasmacytoid dendritic cells (pDCs) are a unique subpopulation of immune cells, distinct from classical dendritic cells. pDCs are generated in the bone marrow and following development, they typically home to secondary lymphoid tissues. While peripheral tissues are generally devoid of pDCs during steady state, few tissues, including the lung, kidney, vagina, and in particular ocular tissues harbor resident pDCs. pDCs were originally appreciated for their potential to produce large quantities of type I interferons in viral immunity. Subsequent studies have now unraveled their pivotal role in mediating immune responses, in particular in the induction of tolerance. In this review, we summarize our current knowledge on pDCs in ocular tissues in both mice and humans, in particular in the cornea, limbus, conjunctiva, choroid, retina, and lacrimal gland. Further, we will review our current understanding on the significance of pDCs in ameliorating inflammatory responses during herpes simplex virus keratitis, sterile inflammation, and corneal transplantation. Moreover, we describe their novel and pivotal neuroprotective role, their key function in preserving corneal angiogenic privilege, as well as their potential application as a cell-based therapy for ocular diseases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The search for alternatives to allotransplants is driven by the shortage of corneal donors and is demanding because of the limitations of the alternatives. Indeed, current progress in genetically engineered (GE) pigs, the introduction of gene-editing technology by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, and advanced immunosuppressants have made xenotransplantation a possible option for a human trial. Porcine corneal xenotransplantation is considered applicable because the eye is regarded as an immune-privileged site. Furthermore, recent non-human primate studies have shown long-term survival of porcine xenotransplants in keratoplasty. Herein, corneal immune privilege is briefly introduced, and xenogeneic reactions are compared with allogeneic reactions in corneal transplantation. This review describes the current knowledge on special issues of xenotransplantation, xenogeneic rejection mechanisms, current immunosuppressive regimens of corneal xenotransplantation, preclinical efficacy and safety data of corneal xenotransplantation, and updates of the regulatory framework to conduct a clinical trial on corneal xenotransplantation. We also discuss barriers that might prevent xenotransplantation from becoming common practice, such as ethical dilemmas, public concerns on xenotransplantation, and the possible risk of xenozoonosis. Given that the legal definition of decellularized porcine cornea (DPC) lies somewhere between a medical device and a xenotransplant, the preclinical efficacy and clinical trial data using DPC are included. The review finally provides perspectives on the current standpoint of corneal xenotransplantation in the fields of regenerative medicine., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: To investigate the clinical effects of IRT5 probiotics in the environmental dry eye model., Methods: Eight week old male C57BL/6 mice were randomly divided into two groups; control group (n = 16) received oral gavage of 300 μL phosphate-buffered saline (PBS) alone once daily, IRT5 group (n = 9) received oral gavage of 1 x 109 CFU IRT5 probiotics powder in 300 μL PBS once daily, both groups for 11 to 12 days. Simultaneously, all mice underwent dry eye induction. Tear secretion, corneal staining and conjunctival goblet cell density were evaluated. Quantative real-time polymerase chain reaction (RT-PCR) for inflammation-related markers was performed. 16S ribosomal RNA of fecal microbiome was analyzed and compositional difference, alpha and beta diversities were assessed., Results: There was no difference in NEI score but significant increase in tear secretion was observed in IRT5 group (p < 0.001). There was no significant difference in goblet cell density between groups. Quantative RT-PCR of cornea and conjunctiva revealed increased TNF-α expression in IRT5 group (p < 0.001) whereas other markers did not significantly differ from control. IRT5 group had significantly increased species diversity by Shannon index (p = 0.041). Beta diversity of genus by UniFrac principle coordinates analysis showed significant distance between groups (p = 0.001). Compositional differences between groups were observed and some were significantly associated with tear secretion. Multivariate linear regression analysis revealed Christensenellaceae (p = 0.009), Lactobacillus Helveticus group (p = 0.002) and PAC001797&#95;s (p = 0.011) to strongly influence tear secretion., Conclusion: In experimental dry eye model, IRT5 probiotics treatment partially improves experimental dry eye by increasing tear secretion which was associated with and influenced by the change in intestinal microbiome. Also, intestinal microbiome may affect the lacrimal gland through a different mechanism other than regulating inflammation., Competing Interests: Sin-Hyeog Im is the CEO of ImmunoBiome Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Herpes simplex virus type 1 (HSV-1)-infected corneas can develop a blinding immunoinflammatory condition called herpes stromal keratitis (HSK), which involves the loss of corneal sensitivity due to retraction of sensory nerves and subsequent hyperinnervation with sympathetic nerves. Increased concentrations of the cytokine VEGF-A in the cornea are associated with HSK severity. Here, we examined the impact of VEGF-A on neurologic changes that underly HSK using a mouse model of HSV-1 corneal infection. Both CD4 + T cells and myeloid cells produced pathogenic levels of VEGF-A within HSV-1-infected corneas, and CD4 + cell depletion promoted reinnervation of HSK corneas with sensory nerves. In vitro, VEGF-A from infected corneas repressed sensory nerve growth and promoted sympathetic nerve growth. Neutralizing VEGF-A in vivo using bevacizumab inhibited sympathetic innervation, promoted sensory nerve regeneration, and alleviated disease. Thus, VEGF-A can shape the sensory and sympathetic nerve landscape within the cornea, with implications for the treatment of blinding corneal disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Here, we report our studies of immune-mediated regulation of Zika virus (ZIKV), herpes simplex virus 1 (HSV-1), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the human cornea. We find that ZIKV can be transmitted via corneal transplantation in mice. However, in human corneal explants, we report that ZIKV does not replicate efficiently and that SARS-CoV-2 does not replicate at all. Additionally, we demonstrate that type III interferon (IFN-λ) and its receptor (IFNλR1) are expressed in the corneal epithelium. Treatment of human corneal explants with IFN-λ, and treatment of mice with IFN-λ eye drops, upregulates antiviral interferon-stimulated genes. In human corneal explants, blockade of IFNλR1 enhances replication of ZIKV and HSV-1 but not SARS-CoV-2. In addition to an antiviral role for IFNλR1 in the cornea, our results suggest that the human cornea does not support SARS-CoV-2 infection despite expression of ACE2, a SARS-CoV-2 receptor, in the human corneal epithelium., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Autoimmune-mediated dry eye disease is a pathological feature of multiple disorders including Sjögren's syndrome, lupus and rheumatoid arthritis that has a life-long, detrimental impact on vision and overall quality of life. Although late stage disease outcomes such as epithelial barrier dysfunction, reduced corneal innervation and chronic inflammation have been well characterized in both human patients and mouse models, there is little to no understanding of early pathological processes. Moreover, the mechanisms underlying the loss of cornea homeostasis and disease progression are unknown. Here, we utilize the autoimmune regulatory (Aire)-deficient mouse model of autoimmune-mediated dry eye disease in combination with genome wide transcriptomics, high-resolution imaging and atomic force microscopy to reveal a potential extracellular matrix (ECM)-biomechanical-based mechanism driving cellular and morphological changes at early disease onset. Early disease in the Aire-deficient mouse model is associated with a mild reduction in tear production and moderate immune cell infiltration, allowing for interrogation of cellular, molecular and biomechanical changes largely independent of chronic inflammation. Using these tools, we demonstrate for the first time that the emergence of autoimmune-mediated dry eye disease is associated with an alteration in the biomechanical properties of the cornea. We reveal a dramatic disruption of the synthesis and organization of the extracellular matrix as well as degradation of the epithelial basement membrane during early disease. Notably, we provide evidence that the nerve supply to the cornea is severely reduced at early disease stages and that this is independent of basement membrane destruction or significant immune cell infiltration. Furthermore, diseased corneas display spatial heterogeneity in mechanical, structural and compositional changes, with the limbal compartment often exhibiting the opposite response compared to the central cornea. Despite these differences, however, epithelial hyperplasia is apparent in both compartments, possibly driven by increased activation of IL-1R1 and YAP signaling pathways. Thus, we reveal novel perturbations in corneal biomechanics, matrix organization and cell behavior during the early phase of dry eye that may underlie disease development and progression, presenting new potential targets for therapeutic intervention., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The protective efficacy of a live-attenuated HSV type 1 (HSV-1) vaccine, HSV-1 0∆ nuclear location signal (NLS), was evaluated in mice prophylactically in response to ocular HSV-1 challenge. Mice vaccinated with the HSV-1 0∆NLS were found to be more resistant to subsequent ocular virus challenge in terms of viral shedding, spread, the inflammatory response, and ocular pathology in a dose-dependent fashion. Specifically, a strong neutralizing Ab profile associated with low virus titers recovered from the cornea and trigeminal ganglia was observed in vaccinated mice in a dose-dependent fashion with doses ranging from 1 × 10 3 to 1 × 10 5 PFU HSV-1 0∆NLS. This correlation also existed in terms of viral latency in the trigeminal ganglia, corneal neovascularization, and leukocyte infiltration and expression of inflammatory cytokines and chemokines in infected tissue with the higher doses (1 × 10 4 -1 × 10 5 PFU) of the HSV-1 0∆NLS-vaccinated mice, displaying reduced viral latency, ocular pathology, or inflammation in comparison with the lowest dose (1 × 10 3 PFU) or vehicle vaccine employed. Fifteen HSV-1-encoded proteins were uniquely recognized by antisera from high-dose (1 × 10 5 PFU)-vaccinated mice in comparison with low-dose (1 × 10 3 PFU)- or vehicle-vaccinated animals. Passive immunization using high-dose-vaccinated, but not low-dose-vaccinated, mouse sera showed significant efficacy against ocular pathology in HSV-1-challenged animals. In summary, we have identified the minimal protective dose of HSV-1 0∆NLS vaccine in mice to prevent HSV-mediated disease and identified candidate proteins that may be useful in the development of a noninfectious prophylactic vaccine against the insidious HSV-1 pathogen., (Copyright © 2020 The Authors.)

In vivo confocal microscopy (IVCM) allows the evaluation of the living human cornea at the cellular level. The non-invasive nature of this technique longitudinal, repeated examinations of the same tissue over time. Image analysis of two-dimensional time-lapse sequences of presumed immune cells with and without visible dendrites at the corneal sub-basal nerve plexus in the eyes of healthy individuals was performed. We demonstrated evidence that cells without visible dendrites are highly dynamic and move rapidly in the axial directions. A number of dynamic cells were observed and measured from three eyes of different individuals. The total average displacement and trajectory speeds of three cells without visible dendrites (N = 9) was calculated to be 1.12 ± 0.21 and 1.35 ± 0.17 μm per minute, respectively. One cell with visible dendrites per cornea was also analysed. Tracking dendritic cell dynamics in vivo has the potential to significantly advance the understanding of the human immune adaptive and innate systems. The ability to observe and quantify migration rates of immune cells in vivo is likely to reveal previously unknown insights into corneal and general pathophysiology and may serve as an effective indicator of cellular responses to intervention therapies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The presence and potential functions of resident plasmacytoid dendritic cells (pDCs) in peripheral tissues is unclear. We report that pDCs constitutively populate naïve corneas and are increased during sterile injuries or acute herpes simplex virus 1 (HSV-1) keratitis. Their local depletion leads to severe clinical disease, nerve loss, viral dissemination to the trigeminal ganglion and draining lymph nodes, and mortality, while their local adoptive transfer limits disease. pDCs are the main source of HSV-1-induced IFN-α in the corneal stroma through TLR9, and they prevent re-programming of regulatory T cells (Tregs) to effector ex-Tregs. Clinical signs of infection are observed in pDC-depleted corneas, but not in pDC-sufficient corneas, following low-dose HSV-1 inoculation, suggesting their critical role in corneal antiviral immunity. Our findings demonstrate a vital role for corneal pDCs in the control of local viral infections., Competing Interests: Declaration of Interests P.H. and A.J. are co-inventors on two pending patent applications, which are in part related to this work., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rationale: Corneal transplantation is an effective treatment to corneal blindness. However, the immune rejection imperils corneal allograft survival. An interventional modality is urgently needed to inhibit immune rejection and promote allograft survival. In our previous study, subconjunctival injections of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a rat model of corneal allograft rejection extended allograft survival for 2 d. In this study, we sought to generate IL-10-overexpressing BM-MSCs, aiming to boost the survival-promoting effects of BM-MSCs on corneal allografts and explore the molecular and cellular mechanisms underlying augmented protection. Methods: A population of IL-10-overexpressing BM-MSCs (designated as IL-10-BM-MSCs) were generated by lentivirus transduction and FACS purification. The self-renewal, multi-differentiation, and immunoinhibitory capabilities of IL-10-BM-MSCs were examined by conventional assays. The IL-10-BM-MSCs were subconjunctivally injected into the model of corneal allograft rejection, and the allografts were monitored on a daily basis. The expression profiling of long noncoding RNA (lncRNA) in the allografts was revealed by RNA sequencing and verified by quantitative real-time PCR. The infiltrating immune cell type predominantly upregulating the lncRNA expression was identified by RNAscope in situ hybridization. The function of the upregulated lncRNA was proved by loss- and gain-of-function experiments both in vivo and in vitro . Results: The IL-10-BM-MSCs possessed an enhanced immunoinhibitory capability and unabated self-renewal and multi-differentiation potentials as compared to plain BM-MSCs. The subconjunctivally injected IL-10-BM-MSCs reduced immune cell infiltration and doubled allograft survival time (20 d) as compared to IL-10 protein or plain BM-MSCs in the corneal allograft rejection model. Further, IL-10-BM-MSCs significantly upregulated lncRNA 003946 expression in CD68 + macrophages infiltrating corneal allografts. Silencing and overexpressing lncRNA 003946 in macrophage cultures abolished and mimicked the IL-10-BM-MSCs' suppressing effects on the macrophages' antigen presentation, respectively. In parallel, knocking down and overexpressing the lncRNA in vivo abrogated and simulated the survival-promoting effects of IL-10-BM-MSCs on corneal allografts, respectively. Conclusion: The remarkable protective effects of IL-10-BM-MSCs support further developing them into an effective interventional modality against corneal allograft rejection. IL-10-BM-MSCs promote corneal allograft survival mainly through upregulating a novel lncRNA expression in graft-infiltrating CD68 + macrophages. LncRNA, for the first time, is integrated into an IL-10-BM-MSC-driven immunomodulatory axis against the immune rejection to corneal allograft., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The eye is provided with immune protection against pathogens in a manner that greatly reduces the threat of inflammation-induced vision loss. Immune-mediated inflammation and allograft rejection are greatly reduced in the eye, a phenomenon called 'immune privilege'. Corneal tissue has inherent immune privilege properties with underlying three mechanisms: (1) anatomical, cellular, and molecular barriers in the cornea; (2) an immunosuppressive microenvironment; and (3) tolerance related to regulatory T cells and anterior chamber-associated immune deviation. This review describes the molecular mechanisms of the immunosuppressive microenvironment and regulatory T cells in the cornea that have been elucidated from animal models of ocular inflammation, especially those involving corneal transplantation, it also provides an update on immune checkpoint molecules in corneal and systemic immune regulation, and its relevance for dry eye associated with checkpoint inhibitor therapy.

Ocular herpes simplex virus 1 (HSV-1) infection leads to an immunopathogenic disease called herpes stromal keratitis (HSK), in which CD4 + T cell-driven inflammation contributes to irreversible damage to the cornea. Herpesvirus entry mediator (HVEM) is an immune modulator that activates stimulatory and inhibitory cosignals by interacting with its binding partners, LIGHT (TNFSF14), BTLA (B and T lymphocyte attenuator), and CD160. We have previously shown that HVEM exacerbates HSK pathogenesis, but the involvement of its binding partners and its connection to the pathogenic T cell response have not been elucidated. In this study, we investigated the role of HVEM and its binding partners in mediating the T cell response using a murine model of ocular HSV-1 infection. By infecting mice lacking the binding partners, we demonstrated that multiple HVEM binding partners were required for HSK pathogenesis. Surprisingly, while LIGHT -/- , BTLA -/- , and CD160 -/- mice did not show differences in disease compared to wild-type mice, BTLA -/- LIGHT -/- and CD160 -/- LIGHT -/- double knockout mice showed attenuated disease characterized by decreased clinical symptoms, increased retention of corneal sensitivity, and decreased infiltrating leukocytes in the cornea. We determined that the attenuation of disease in HVEM -/- , BTLA -/- LIGHT -/- , and CD160 -/- LIGHT -/- mice correlated with a decrease in gamma interferon (IFN-γ)-producing CD4 + T cells. Together, these results suggest that HVEM cosignaling through multiple binding partners induces a pathogenic Th1 response to promote HSK. This report provides new insight into the mechanism of HVEM in HSK pathogenesis and highlights the complexity of HVEM signaling in modulating the immune response following ocular HSV-1 infection. IMPORTANCE Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, is capable of causing a progressive inflammatory ocular disease called herpes stromal keratitis (HSK). HSV-1 ocular infection leads to persistent inflammation in the cornea resulting in outcomes ranging from significant visual impairment to complete blindness. Our previous work showed that herpesvirus entry mediator (HVEM) promotes the symptoms of HSK independently of viral entry and that HVEM expression on CD45 + cells correlates with increased infiltration of leukocytes into the cornea during the chronic inflammatory phase of the disease. Here, we elucidated the role of HVEM in the pathogenic Th1 response following ocular HSV-1 infection and the contribution of HVEM binding partners in HSK pathogenesis. Investigating the molecular mechanisms of HVEM in promoting corneal inflammation following HSV-1 infection improves our understanding of potential therapeutic targets for HSK., (Copyright © 2020 Park et al.)