Role of IL-27 in HSV-1-Induced Herpetic Stromal Keratitis.

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-ß 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.

Corneal dendritic cells and the subbasal nerve plexus following neurotoxic treatment with oxaliplatin or paclitaxel.

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/mm2) compared to healthy controls (Md = 10.1 cells/mm2, p = 0.001), but not in the paclitaxel group (Md = 10.6 cells/mm2). 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.

Time-Dependent Serial Changes of Antigen-Presenting Cell Subsets in the Ocular Surface Are Distinct between Corneal Sterile Inflammation and Allosensitization in a Murine Model.

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, CD11bintCD11chiMHCIIhiCD86hi cells increased until 4 weeks with an increase in IFNγhi T cells. In Syn, both CD11bintCD11chiMHCIIhiCD86hi and CD11bhiCD11chiMHCIIhiCD86hi APC subsets increased until 4 weeks with a brief increase in CD69hi T cells at 2 weeks. In Allo, CD11bintCD11chiMHCIIhiCD86hi and CD11bhiCD11chiMHCIIhiCD86hi APC subsets increased until 4 weeks, and an early increase in CD69hi T cells was observed at 2 weeks followed by a late increase in IFNγhi T cells at 4 weeks. The frequency of the IFNγhi T cell subset was positively correlated with the frequency of the CD11bintCD11chiMHCIIhiCD86hi 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.

Healing of Ocular Herpetic Disease Following Treatment With an Engineered FGF-1 Is Associated With Increased Corneal Anti-Inflammatory M2 Macrophages.

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.

TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread.

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.

Resident Innate Immune Cells in the Cornea.

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.

The cornea IV immunology, infection, neovascularization, and surgery chapter 1: Corneal immunology.

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.

Supplementing the Diet with Sodium Propionate Suppresses the Severity of Viral Immuno-inflammatory Lesions.

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.

Effect of IRT5 probiotics on dry eye in the experimental dry eye mouse model.

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_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.

Production of the Cytokine VEGF-A by CD4+ T and Myeloid Cells Disrupts the Corneal Nerve Landscape and Promotes Herpes Stromal Keratitis.

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.

Distinguishing Features of High- and Low-Dose Vaccine against Ocular HSV-1 Infection Correlates with Recognition of Specific HSV-1-Encoded Proteins.

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 × 103 to 1 × 105 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 × 104-1 × 105 PFU) of the HSV-1 0∆NLS-vaccinated mice, displaying reduced viral latency, ocular pathology, or inflammation in comparison with the lowest dose (1 × 103 PFU) or vehicle vaccine employed. Fifteen HSV-1-encoded proteins were uniquely recognized by antisera from high-dose (1 × 105 PFU)-vaccinated mice in comparison with low-dose (1 × 103 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.

Lentivirus-mediated IL-10-expressing Bone Marrow Mesenchymal Stem Cells promote corneal allograft survival via upregulating lncRNA 003946 in a rat model of corneal allograft rejection.

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.

Immune Checkpoints Contribute Corneal Immune Privilege: Implications for Dry Eye Associated with Checkpoint Inhibitors.

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.

Herpesvirus Entry Mediator Binding Partners Mediate Immunopathogenesis of Ocular Herpes Simplex Virus 1 Infection.

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.

Nerolidol inhibits the LOX-1 / IL-1ß signaling to protect against the Aspergillus fumigatus keratitis inflammation damage to the cornea.

PURPOSE: Nerolidol, a naturally occurring sesquiterpene has both anti-microbial and anti-inflammatory properties. The current study aims to investigate the antifungal and the anti-inflammatory effects of nerolidol against mouse Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The minimum inhibitory concentration (MIC) and cytotoxicity tests were used to study the antifungal ability. For in vivo and in vitro studies, the mouse corneas and the human corneal epithelial cells (HCECs) infected with A. fumigatus spores were intervented with nerolidol or phosphate buffer saline (PBS). Thereafter, the effect of the nerolidol on the response against inflammation was analyzed using the following parameters: recruitment of the neutrophils or macrophages and the expression of the lectin-type oxidized low density lipoprotein receptor-1 (LOX-1) and interleukin 1ß (IL-1ß). Techniques used were the slit lamp, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: Nerolidol directly inhibits the growth of A. fumigatus. The administration of nerolidol reduced the severity of fungal keratitis with infiltration of fewer inflammatory cells and reduced levels of the LOX-1, as well the anti-inflammatory cytokines such as IL-1ß were reduced compared with the PBS group. Additionally, in vitro studies showed that treatment with nerolidol inhibited the production of the LOX-1 / IL-1ß levels in A. fumigatus stimulated HCECs. CONCLUSION: Nerolidol attenuated the A. fumigatus keratitis inflammatory response by inhibiting the growth of A. fumigatus, reducing the recruitment of the neutrophils and the macrophages, and inhibiting the LOX-1/ IL-1ß signaling.

Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin-10.

The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft-attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating the host's alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using 2 well-established models of corneal transplantation (low-risk and high-risk models), we show that Tregs derived from low-risk graft recipients have a superior capacity in protecting CEnCs against effector T cell-mediated and interferon-γ and tumor necrosis factor-α-induced cell death compared to Tregs derived from high-risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low-risk hosts occurs independently of direct cell-cell contact and is mediated by the immunoregulatory cytokine IL-10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL-10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.

Genomic foundations of evolution and ocular pathogenesis in human adenovirus species D.

Human adenovirus commonly causes infections of respiratory, gastrointestinal, genitourinary, and ocular surface mucosae. Although most adenovirus eye infections are mild and self-limited, specific viruses within human adenovirus species D are associated with epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular surface infection, which can lead to chronic and/or recurrent, visually disabling keratitis. In this review, we discuss the links between adenovirus ontogeny, genomics, immune responses, and corneal pathogenesis, for those viruses that cause EKC.

Role of Herpes Simplex Envelope Glycoprotein B and Toll-Like Receptor 2 in Ocular Inflammation: An ex vivo Organotypic Rabbit Corneal Model.

It has been recently reported, using in vitro studies, that the herpes simplex virus 1 (HSV-1) encoded envelope glycoprotein B (gB1) interacts with cell surface toll-like receptor 2 (TLR2) and induces the secretion of interleukin-8 (IL8), a representative marker of inflammatory cytokine activation. The purpose of this study is to investigate the role of gB1 in activating host inflammatory responses by using a secreted form of gB1 (gB1s) and an ex vivo organotypic rabbit corneal model. Abraded corneas exposed to gB1s alone or to the recombinant protein mixed with anti gB polyclonal antibody were cultured in an air-liquid interface. The corneas exposed to gB1s show the appearance of mydriasis and high levels of TLR2 and IL-8 mRNAs transcripts were detected in the superficial layer of corneal epithelial cells. Histological stain and immunohistochemical analyses revealed morphological changes in the epithelium of the treated corneas and variations in expression and localization of TLR2. Collectively these findings provide new insight into the pathogenesis of HSV-1 ocular infection by demonstrating the leading role of gB in activating an inflammatory response and in the appearance of mydriasis, a sign of HSV-1 anterior uveitis.

Cytopathic Change and Inflammatory Response of Human Corneal Epithelial Cells Induced by Acanthamoeba castellanii Trophozoites and Cysts.

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.

CCR2+ migratory macrophages with M1 status are the early-responders in the cornea of HSV-1 infected mice.

Complex interactions between HSV-1 and infiltrating immune cells play important roles in establishing localized, acute virus replication as well as chronic latent infection. The extent and duration of initial virus replication are the key determinants of subsequent pathologic inflammatory responses and therefore, the accumulation of immune cell populations at this time point is a key target for prevention. Therefore, we evaluated the role of various immune cell infiltrates between 1 h and 28 days post-infection (PI) using mice infected with virulent HSV-1 strain McKrae without corneal scarification. The effect of corneal scarification on immune cell infiltrates was also determined. We first determined the activation status and origin of macrophage infiltrates as early as 1 h PI. We found a sharp increase in the total macrophage population after 12 h PI, that was primarily due to infiltration of CCR2+ migratory macrophages, mostly in M1 status (MHC II+). The number of CCR2- resident macrophages, mostly unpolarized (M0), increased gradually over time and peaked at 48 h PI. Interestingly, some of the resident macrophages gained an M2-like phenotype (CD206Low), which peaked at 12 h PI, concurrent with M1 macrophage infiltration. From 1-7 days PI, infiltration of various immune cells correlated strongly with HSV-1 replication, with neutrophils showing the biggest increase, and NKT cells the biggest decrease, after infection. The presence of geographical ulcer did not correlate with increased infiltration, while mice with corneal scarring had significantly more immune cell infiltration than those without corneal scarring. Overall, we showed time-dependent infiltration of various immune cells in the eye of HSV-1 infected mice. Initial infiltration of macrophages followed by infiltration of T cells at later times PI demonstrates the importance of targeting macrophages rather than other immune cells type, for therapeutic treatment of HSV-1.