CD137 costimulation is associated with reduced herpetic stromal keratitis and with developing normal CD8+ T cells in trigeminal ganglia.

Costimulatory interactions can be critical in developing immune responses to infectious agents. We recently reported that herpes simplex type 1 (HSV-1) infections of the cornea require a functional CD28-CD80/86 interaction to not only reduce the likelihood of encephalitis, but also to mediate herpetic stromal keratitis (HSK) following viral reactivation. In this same spirit we decided to determine the role that CD137 costimulation plays during HSK. Using both B6-CD137L-/- mice, as well as antagonistic and agonistic antibodies to CD137 we characterize the immune response and to what extent CD137 plays an important role during this disease. Immune responses were measured in both the cornea and in the trigeminal ganglia where the virus forms a latent infection. We demonstrate that CD137 costimulation leads to reduced corneal disease. Interestingly, we observed that lack of CD137 costimulation resulted in significantly reduced CD8+ T expansion and function in the trigeminal ganglia. Finally, we showed that viruses that have been genetically altered to express CD137 display significantly reduced corneal disease, though they did present similar levels of trigeminal infection and peripheral virus production following reactivation of a latent infection. CD137 interactions lead to reduced HSK and are necessary to develop robust trigeminal CD8+ T cell responses.

CD28 Costimulation Is Required for Development of Herpetic Stromal Keratitis but Does Not Prevent Establishment of Latency.

Corneal infection with herpes simplex virus 1 (HSV-1) leads to infection of trigeminal ganglia (TG), typically followed by the establishment of latency in the infected neurons. When latency is disrupted, the virus reactivates and migrates back to the cornea, where it restimulates the immune response, leading to lesions in a disease called herpetic stromal keratitis (HSK). HSK requires T cell activation, as in the absence of T cells there is no disease. We decided to determine if CD28 costimulation of T cells was required in HSK. The results indicated that C57BL/6 CD28-/- and BALB/c CD28-/- mice failed to develop recurrent HSK, while their wild-type counterparts did. In order to better understand the dynamics of TG infection in these mice, we evaluated the amount of virus in infected TG and the number of individual neurons harboring latent virus. The results indicated that CD28-/- mice possessed significantly increased genome levels in their TG but many fewer LAT-positive cells than wild-type mice from day 7 to day 30 but that after day 30 these differences became nonsignificant. We next evaluated total and antigen-specific CD8+ T cells in TG. The results indicated that there were significantly fewer CD8 T cells in TG from day 10 to day 25 but that after that the differences were not significant. Taken together, these data suggest that CD28 costimulation is required for HSK but that while initial infection of TG is greater in CD28-/- mice, this begins to normalize with time and this normalization is concurrent with the delayed development of antigen-specific CD8+ T cells.IMPORTANCE We study the pathogenesis of herpes simplex virus-mediated corneal disease. T cells play a critical role both in disease and in the maintenance of latency in neurons. Consequently, the focus of this study was to evaluate the role that T cell costimulation plays both in corneal disease and in controlling the ability of the virus to maintain a stable infection of the ganglia that innervate the cornea. We demonstrate that in the absence of costimulation with CD28, corneal disease does not take place. However, this costimulation does not prevent the ability of CD8+ T cells to develop and, thus, control latent infection of neurons. We conclude from these studies that CD28 costimulation is required for corneal destructive immune responses but that CD8+ T cells develop over time and help to maintain latency.

Anti-IL-17 therapy restricts and reverses late-term corneal allorejection.

Corneal allograft rejection has been described as a Th1-mediated process involving IFN-γ production. However, recent evidence also implicated IL-17 as being involved in acute corneal allograft responses. Our data support that IL-17 is involved in early acute corneal allograft acceptance. However, we decided to extend these studies to include a later phase of rejection in which there is a peak of IL-17 production that is >15-fold higher than that seen during acute rejection and occurs >45 d postengraftment at the onset of late-term rejection. We demonstrate that neutralizing IL-17A at this time significantly reduced corneal graft rejection. Surprisingly, when corneal grafts that are undergoing this later phase of rejection are treated with anti-IL-17A, there is a reversal of both opacity and neovascularization. Compared with the early phase of rejection, the cellular infiltrate is significantly less, with a greatly reduced presence of Gr-1(+) neutrophils and a relative increase in CD4(+) T cells and macrophages. We went on to identify that the cells expressing IL-17 were CD4(+) IL-17(+) T cells and, somewhat surprisingly, IL-17(+) F4/80(+) macrophages within the rejecting corneal allografts. Taken together, these findings describe a distinct late phase of corneal allograft rejection that is likely mediated by Th17 cells; therapeutic neutralization of IL-17A reverses this rejection. This further suggests that IL-17 might serve as an excellent therapeutic target to reduce this form of corneal allograft rejection.

CXCL1 but not IL-6 is required for recurrent herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) is characterized by an inflammatory response that includes neutrophils, macrophages, NK cells, and T cells. The factors that are responsible for this inflammation are proinflammatory cytokines and chemokines. Many of these factors have been defined for primary disease, but relatively few have been investigated during recurrent HSK. The present study was designed to determine the role that two of these factors, IL-6 and CXCL1, play during recurrent HSK. Results clearly indicate that unlike primary disease, IL-6 plays no role in recurrent HSK. However, the presence of CXCL1 is required for recurrent HSK as evidenced by the lack of corneal disease in mice treated with anti-CXCL1 Ab. This was confirmed using mice lacking the primary receptor for CXCL1, CXCR2. Corneal disease in this strain was significantly reduced compared with wild-type C57BL/6 controls. Unexpectedly, lack of disease occurs even though CXCL1 knockout mice display increased viral shedding at the cornea. The primary mechanism that CXCL1 plays during disease is its ability to stimulate neutrophils to infiltrate the cornea following reactivation. This paper provides further evidence that primary HSK and recurrent HSK possess overlapping yet distinct disease mechanisms.

Mice with mutations in Fas and Fas ligand demonstrate increased herpetic stromal keratitis following corneal infection with HSV-1.

HSV-1 infection of the cornea leads to a potentially blinding immunoinflammatory lesion of the cornea, termed herpetic stromal keratitis. It has also been shown that one of the factors limiting inflammation of the cornea is the presence of Fas ligand (FasL) on corneal epithelium and endothelium. In this study, the role played by FasL expression in the cornea following acute infection with HSV-1 was determined. Both BALB/c and C57BL/6 (B6) mice with HSV-1 infection were compared with their lpr and gld counterparts. Results indicated that mice bearing mutations in the Fas Ag (lpr) displayed the most severe disease, whereas the FasL-defective gld mouse displayed an intermediate phenotype. It was further demonstrated that increased disease was due to lack of Fas expression on bone marrow-derived cells. Of interest, although virus persisted slightly longer in the corneas of mice bearing lpr and gld mutations, the persistence of infectious virus in the trigeminal ganglia was the same for all strains infected. Further, B6 mice bearing lpr and gld mutations were also more resistant to virus-induced mortality than were wild-type B6 mice. Thus, neither disease nor mortality correlated with viral replication in these mice. Collectively, the findings indicate that the presence of FasL on the cornea restricts the entry of Fas(+) bone marrow-derived inflammatory cells and thus reduces the severity of HSK.

UVB induced reactivation leads to HSV1 in the corneas of virtually all latently infected mice and requires STING to develop corneal disease.

Reactivation of latent herpes simplex type 1 results in virus returning to the cornea leading to recurrent herpetic stromal keratitis (rHSK). We compare two competing models to reactivate viruses from latency, UV-B irradiation and cyclophosphamide (CP). Results revealed that while both result in corneal recrudescence, only UV-B irradiation results in rHSK. To better understand the dynamics of reactivation, we analyzed corneas for both the presence of infectious viruses and the dynamics of exposure to multiple reactivations using UV-B. We noted that multiple reactivations result in progressively worse corneal disease. We also noted that expression of IFNα and STING, surragate markers for the presence of virus, are induced by the presence of reactivated virus. Studies to determine the importance of STING to the development of HSK revealed that in the absence of STING, mice do not develop significant HSK and the magnitude of the infiltrate of CD45+ cells in these corneas is significantly reduced. The resulting paucity of CD45+CD11b+GR-1+F4/80-neutrophils, and to a lesser extent CD45+CD11b+GR-1-F4/80+ macrophages in B6-STING KO mice following reactivation is likely the underlying cause for lack of rHSK as has been noted by ourselves and others. These results underscore the critical importance of STING's role in developing rHSK.

UV-B induced HSV-1 reactivation leads to infectious virus in the corneas of virtually all latently infected mice and requires an intact STING to develop herpetic stromal keratitis.

Reactivation of latent herpes simplex type 1 results in virus returning to the cornea leading to recurrent herpetic stromal keratitis (rHSK). We compare two competing models to reactivate viruses from latency, UV-B irradiation and cyclophosphamide. Results revealed that while both result in corneal recrudescence, only UV-B irradiation results in rHSK. To better understand the dynamics of reactivation, we analyzed corneas for both the presence of infectious viruses and the dynamics of exposure to multiple reactivations using UV-B. We noted that multiple reactivations result in progressively worse corneal disease. We also noted that expression of IFNα and STING, surragate markers for the presence of virus, are induced by the presence of reactivated virus. Studies to determine the importance of STING to the development of HSK revealed that in the absence of STING, mice do not develop significant HSK and the magnitude of the infiltrate of CD45 + cells in these corneas is significantly reduced. The resulting paucity of CD45 + CD11b + GR-1 + F4/80-neutrophils, and to a lesser extent CD45 + CD11b + GR-1-F4/80 + macrophages in B6-STING KO mice following reactivation is likely the underlying cause for lack of rHSK as has been noted by ourselves and others. These results underscore the critical importance of STING's role in developing rHSK.

Recurrent herpetic stromal keratitis in mice: a model for studying human HSK.

Herpes simplex virus 1 (HSV-1) infection of the cornea leads to a potentially blinding disease, termed herpetic stromal keratitis (HSK) that is characterized by lesions of an immunoinflammatory nature. In spite of the fact that HSK typically presents as a recurrent disease due to reactivation of virus which latently infects the trigeminal ganglia, most murine studies of HSK have employed a primary and not recurrent model of the disease. This report documents the several recurrent models of HSK that have been developed and how data generated from these models differs in some important aspects from data generated following primary infection of the cornea. Chief among these differences is the fact that recurrent HSK takes place in the context of an animal that has a preexisting anti-HSV immune response, while primary HSK occurs in an animal that is developing such a response. We will document both differences and similarities that derive from this fundamental difference in these models with an eye towards possible vaccines and therapies that demonstrate promise in treating HSK.

Role of NK T cells in transplantation with particular emphasis on corneal transplantation.

Natural killer T cells (NKT cells) are a unique subset of the immune system that possess characteristics of both an innate and adaptive immune response. This study reviews the reported roles of NKT cells in different solid transplantations such as cardiac, skin, liver, and corneal grafts as well as investigates a novel role of NKT cells in steroid-resistant corneal rejections. It is unknown why there is late corneal graft rejection despite being treated with immunosuppression. Our experimental data suggests NKT cells are playing a crucial part in steroid-resistant late graft rejections. While the pathophysiology of acute rejection is better understood, the process of chronic graft rejection is much less clear. Our data suggests NKT cells as a potential therapeutic target to prevent chronic transplant rejection which needs further investigation.

Sensory Nerve Retraction and Sympathetic Nerve Innervation Contribute to Immunopathology of Murine Recurrent Herpes Stromal Keratitis.

Purpose: Herpes stromal keratitis (HSK) represents a spectrum of pathologies which is caused by herpes simplex virus type 1 (HSV-1) infection and is considered a leading cause of infectious blindness. HSV-1 infects corneal sensory nerves and establishes latency in the trigeminal ganglion (TG). Recently, retraction of sensory nerves and replacement with "unsensing" sympathetic nerves was identified as a critical contributor of HSK in a mouse model where corneal pathology is caused by primary infection. This resulted in the loss of blink reflex, corneal desiccation, and exacerbation of inflammation leading to corneal opacity. Despite this, it was unclear whether inflammation associated with viral reactivation was sufficient to initiate this cascade of events. Methods: We examined viral reactivation and corneal pathology in a mouse model with recurrent HSK by infecting the cornea with HSV-1 (McKrae) and transferring (intravenous [IV]) human sera to establish primary infection without discernible disease and then exposed the cornea to UV-B light to induce viral reactivation. Results: UV-B light induced viral reactivation from latency in 100% of mice as measured by HSV-1 antigen deposition in the cornea. Further, unlike conventional HSK models, viral reactivation resulted in focal retraction of sensory nerves and corneal opacity. Dependent on CD4+ T cells, inflammation foci were innervated by sympathetic nerves. Conclusions: Collectively, our data reveal that sectoral corneal sensory nerve retraction and replacement of sympathetic nerves were involved in the progressive pathology that is dependent on CD4+ T cells after viral reactivation from HSV-1 latency in the UV-B induced recurrent HSK mouse model.

Developments in Vaccination for Herpes Simplex Virus.

Herpes simplex virus (HSV) is an alpha herpes virus, with two subtypes: HSV-1 and HSV-2. HSV is one of the most prevalent sexually transmitted infections. It is the cause of severe neonatal infections and a leading cause of infectious blindness in the Western world. As of 2016, 13.2% of the global population ages 15-49 were existing with HSV-2 infection and 66.6% with HSV-1. This high prevalence of disease and the fact that resistance to current therapies is on the rise makes it imperative to develop and discover new methods of HSV prevention and management. Among the arsenal of therapies/treatments for this virus has been the development of a prophylactic or therapeutic vaccine to prevent the complications of HSV reactivation. Our current understanding of the immune responses involved in latency and reactivation provides a unique challenge to the development of vaccines. There are no approved vaccines currently available for either prophylaxis or therapy. However, there are various promising candidates in the pre-clinical and clinical phases of study. Vaccines are being developed with two broad focuses: preventative and therapeutic, some with a dual use as both immunotherapeutic and prophylactic. Within this article, we will review the current guidelines for the treatment of herpes simplex infections, our understanding of the immunological pathways involved, and novel vaccine candidates in development.

Acanthamoeba Keratitis, Pathology, Diagnosis and Treatment.

Acanthamoeba keratitis is an unusual corneal infection that is recently increasing in frequency and is often contracted by contact lens wearers, someone who experienced recent eye trauma, or someone exposed to contaminated waters. Acanthamoeba survive in air, soil, dust, and water. Therefore, eye trauma and poor contact lens hygiene practices lead to the entrapment of debris and thus infection. Acanthamoeba keratitis results in severe eye pain, inflammation, and defects of the epithelium and stroma that can potentially result in vision loss if not diagnosed early and treated promptly. The disease can be diagnosed using corneal scrape/biopsy, polymerase chain reactions, impression cytology, or in vivo confocal microscopy. Once diagnosed, it is usually treated with an antimicrobial combination therapy of biguanide and aromatic diadine eye drops for several months. Advanced stages of the disease result in vision loss and the need for corneal transplants. Avoiding the risk factors and diagnosing the disease early are the most effective ways to combat Acanthamoeba keratitis.

Delivery of Interferon-gamma by an adenovirus vector blocks herpes simplex virus Type 1 reactivation in vitro and in vivo independent of RNase L and double-stranded RNA-dependent protein kinase pathways.

HSV-1 is a significant human pathogen that can result in the loss of sight as a result of episodic reactivation of latent virus from sensory ganglion neurons. In this study the potential efficacy of anti-viral cytokine expression in preventing latent virus reactivation was investigated. Both type I (IFN-beta) and type II (IFN-gamma) IFN transgene expression following transduction of trigeminal ganglion explant cultures significantly reduced the incident of HSV-1 reactivation that in the case of IFN-beta was dependent on the presence of double stranded RNA-dependent protein kinase and RNase L. In vivo, expression of the IFN-gamma but not IFN-beta transgene significantly delayed and reduced the frequency of reactivation of latent mice exposed to UV light without discernable inflammation. This result is the first report that demonstrates the ability to block reactivation using an ectopic cytokine expression system and warrants further exploration as a means to prevent HSV-1 reactivation.

CCL3 protects mice from corneal pathology during recurrent HSV-1 infection.

CCL2 and CCL3 are proinflammatory chemokines that are produced during the early stages of inflammation and are known to stimulate the migration of mononuclear cells to the site of inflammation,. Previous studies addressing the role of these chemokines during primary herpetic stromal keratitis (HSK), have suggested that CCL2 is involved in reducing corneal disease and that CCL3 is involved in promoting this disease. We addressed the role that these chemokines play in a recurrent model of HSK. Results from these studies did not demonstrate a significant role for CCL2 except for very early time points following reactivation of virus. Surprisingly, mice deficient in CCL3 did not have significantly reduced recurrent disease, , but in fact showed significantly enhanced disease. This argues that CCL3 might play an ameliorative role during recurrent HSK. In addition, we observed that these same CCL3 deficient mice showed increased resistance to viral-induced mortality following infection with HSV-1. Taken together, these results suggest that CCL3 plays a significant protective role during recurrent HSK and is involved in enhancing lethality.

Understanding the Role of Chemokines and Cytokines in Experimental Models of Herpes Simplex Keratitis.

Herpes simplex keratitis is a disease of the cornea caused by HSV-1. It is a leading cause of corneal blindness in the world. Underlying molecular mechanism is still unknown, but experimental models have helped give a better understanding of the underlying molecular pathology. Cytokines and chemokines are small proteins released by cells that play an important proinflammatory or anti-inflammatory role in modulating the disease process. Cytokines such as IL-17, IL-6, IL-1α, and IFN-γ and chemokines such as MIP-2, MCP-1, MIP-1α, and MIP-1ß have proinflammatory role in the destruction caused by HSV including neutrophil infiltration and corneal inflammation, and other chemokines and cytokines such as IL-10 and CCL3 can have a protective role. Most of the damage results from neutrophil infiltration and neovascularization. While many more studies are needed to better understand the role of these molecules in both experimental models and human corneas, current studies indicate that these molecules hold potential to be targets of future therapy.

Herpes simplex keratitis: challenges in diagnosis and clinical management.

Herpes simplex virus is responsible for numerous ocular diseases, the most common of which is herpetic stromal keratitis. This is a recurrent infection of the cornea that typically begins with a subclinical infection of the cornea that establishes a latent infection of sensory ganglia, most often the trigeminal ganglia. Recurring infections occur when the virus is reactivated from latency and travels back to the cornea, where it restimulates an inflammatory response. This inflammatory response can lead to decreased corneal sensation, scarring, and blindness. The diagnosis of these lesions as the result of a recurrent herpes simplex virus infection can at times be problematic. Currently, herpetic stromal keratitis is diagnosed by its clinical presentation on the slit-lamp examination, but the literature does not always support the accuracy of these clinical findings. Other diagnostic tests such as polymerase chain reaction assay, enzyme-linked immunosorbent assay, immunofluorescent antibody, and viral cultures have provided more definitive diagnosis, but also have some limitations. That said, accurate diagnosis is necessary for proper treatment, in order to prevent serious consequences. Current treatment reduces the severity of lesions and controls further viral spread, but does not provide a cure.

CXCL9 compensates for the absence of CXCL10 during recurrent Herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) is a disease that is typically associated with reactivation of a latent HSV-1 infection. This disease is driven, in part, by chemokines that recruit leukocytes to the cornea. Surprisingly, neutralization of CXCL10 significantly reduced disease, while B6-CXCL10-/- mice exhibited worse disease compared with similarly infected wild-type controls. We hypothesized that compensatory up-regulation of CXCL9 occurs in the absence of CXCL10. Analysis of CXCL9 expression in HSV-1-infected B6 mice and B6-CXCL10-/- mice revealed significantly more CXCL9 in B6-XCL10-/- mice. Treatment of B6 and B6-CXCL10-/- mice with neutralizing antibodies to CXCL9 reduced HSK scores in B6-CXCL10-/-, but not B6 mice. We conclude that CXCL10 production worsens HSK and that CXCL9 may compensate in CXCL10-deficient animals. These studies identify the critical role that CXCL10 plays in the pathogenesis of recurrent HSK, and that CXCL9 displays its importance when CXCL10 is absent.

CD8 T cells mediate transient herpes stromal keratitis in CD4-deficient mice.

PURPOSE: To evaluate the role of CD4(+) T cells in the development of murine herpes stromal keratitis (HSK). METHODS: The corneas of wild-type (WT) BALB/c mice and three types of CD4-deficient BALB/c mice (CD4(-/-), CD4-depleted, CD4 and CD8 double-depleted) were infected with different doses of HSV-1 RE, and HSK incidence and severity were monitored. Corneal infiltrates were quantitatively and functionally assayed by flow cytometric analysis of individually digested diseased corneas and documented histologically. RESULTS: At a relatively high infectious dose (1 x 10(5) pfu/cornea): (1) CD4-deficient and WT BALB/c mice had severe HSK with a similar incidence (80%-100%), whereas HSK did not develop in mice deficient in both CD4(+) and CD8(+) T cells; (2) neutrophils were the predominate leukocyte in the corneas of CD4-deficient and WT mice; (3) the corneas of WT mice had activated, HSV-1-specific CD4(+) T cells, but few if any CD8(+) T cells; (4) the corneas of CD4-deficient mice had activated, HSV-1-specific CD8(+) T cells; and (5) HSK in CD4-deficient mice was transient, showing loss of CD8(+) T cells at 2 to 3 weeks after infection (pi) followed by a loss of neutrophils. At a relatively low infectious dose of HSV-1 (10(3) pfu/cornea) severe HSK developed in 80% to 90% of WT mice, but in only 30% to 40% of CD4-deficient mice. CONCLUSIONS: CD4(+) T cells preferentially mediate HSK, but, in their absence, a high infectious dose of HSV-1 can induce histologically similar but transient HSK that is mediated by CD8(+) T cells.

Dendritic Cell Autophagy Contributes to Herpes Simplex Virus-Driven Stromal Keratitis and Immunopathology.

UNLABELLED: Herpetic stromal keratitis (HSK) is a blinding ocular disease that is initiated by HSV-1 and characterized by chronic inflammation in the cornea. Although HSK immunopathology of the cornea is well documented in animal models, events preceding this abnormal inflammatory cascade are poorly understood. In this study, we have examined the activation of pathological CD4(+) T cells in the development of HSK. Dendritic cell autophagy (DC-autophagy) is an important pathway regulating major histocompatibility complex class II (MHCII)-dependent antigen presentation and proper CD4(+) T cell activation during infectious diseases. Using DC-autophagy-deficient mice, we found that DC-autophagy significantly and specifically contributes to HSK disease without impacting early innate immune infiltration, viral clearance, or host survival. Instead, the observed phenotype was attributable to the abrogated activation of CD4(+) T cells and reduced inflammation in HSK lesions. We conclude that DC-autophagy is an important contributor to primary HSK immunopathology upstream of CD4(+) T cell activation. IMPORTANCE: Herpetic stromal keratitis (HSK) is the leading cause of infectious blindness in the United States and a rising cause worldwide. HSK is induced by herpes simplex virus 1 but is considered a disease of inappropriately sustained inflammation driven by CD4(+) T cells. In this study, we investigated whether pathways preceding CD4(+) T cell activation affect disease outcome. We found that autophagy in dendritic cells significantly contributed to the incidence of HSK. Dendritic cell autophagy did not alter immune control of the virus or neurological disease but specifically augmented CD4(+) T cell activation and pathological corneal inflammation. This study broadens our understanding of the immunopathology that drives HSK and implicates the autophagy pathway as a new target for therapeutic intervention against this incurable form of infectious blindness.