Noncognate Signals Drive Enhanced Effector CD8+ T Cell Responses through an IFNAR1-Dependent Pathway after Infection with the Prototypic Vaccine, 0ΔNLS, against Herpes Simplex Virus 1.

Previous studies by our group identified a highly efficacious vaccine 0ΔNLS (deficient in the nuclear localization signal of infected cell protein 0) against herpes simplex virus 1 (HSV-1) in an experimental ocular mouse model. However, details regarding fundamental differences in the initial innate and adaptive host immune response were not explored. Here, we present a side-by-side analysis of the primary infection characterizing differences of the host immune response in mice infected with 0ΔNLS versus the parental, GFP105. The results show that local viral infection and replication are controlled more efficiently in mice exposed to 0ΔNLS versus GFP105 but that the clearance of infectious virus is equivalent when the two groups are compared. Moreover, the 0ΔNLS-infected mice displayed enhanced effector CD8+ but not CD4+ T cell responses from the draining lymph nodes at day 7 postinfection measured by gamma interferon (IFN-γ) and tumor necrosis factor alpha production along with changes in cell metabolism. The increased effector function of CD8+ T cells from 0ΔNLS-infected mice was not driven by changes in antigen presentation but lost in the absence of a functional type I IFN pathway. These results are further supported by enhanced local expression of type I IFN and IFN-inducible genes along with increased IL-12 production by CD8α+ dendritic cells in the draining lymph nodes of 0ΔNLS-infected mice compared to the GFP105-infected animals. It was also noted the recall to HSV-1 antigen by CD8+ T cells was elevated in mice infected with HSV-1 0ΔNLS compared to GFP105. Collectively, the results underscore the favorable qualities of HSV-1 0ΔNLS as a candidate vaccine against HSV-1 infection. IMPORTANCE Cytotoxic T lymphocytes (CTLs) play a critical role in the clearance for many viral pathogens including herpes simplex virus 1 (HSV-1). Here, we compared the cellular innate and adaptive immune response in mice infected with an attenuated HSV-1 (0ΔNLS) found to be a highly successful experimental prophylactic vaccine to parental HSV-1 virus. We found that CD8+ T cell effector function is elevated in 0ΔNLS-infected mice through noncognate signals, including interleukin-12 and type I interferon pathways along with changes in CD8+ T cell metabolism, whereas other factors, including cell proliferation, costimulatory molecule expression, and antigen presentation, were dispensable. Thus, an increase in CTL activity established by exposure to HSV-1 0ΔNLS in comparison to parental HSV-1 likely contributes to the efficacy of the vaccine and underscores the nature of the attenuated virus as a vaccine candidate for HSV-1 infection.

Osteopontin contributes to virus resistance associated with type I IFN expression, activation of downstream ifn-inducible effector genes, and CCR2+CD115+CD206+ macrophage infiltration following ocular HSV-1 infection of mice.

Ocular pathology is often associated with acute herpes simplex virus (HSV)-1 infection of the cornea in mice. The present study was undertaken to determine the role of early T lymphocyte activation 1 protein or osteopontin (OPN) in corneal inflammation and host resistance to ocular HSV-1 infection. C57BL/6 wild type (WT) and osteopontin deficient (OPN KO) mice infected in the cornea with HSV-1 were evaluated for susceptibility to infection and cornea pathology. OPN KO mice were found to possess significantly more infectious virus in the cornea at day 3 and day 7 post infection compared to infected WT mice. Coupled with these findings, HSV-1-infected OPN KO mouse corneas were found to express less interferon (IFN)-α1, double-stranded RNA-dependent protein kinase, and RNase L compared to infected WT animals early post infection that likely contributed to decreased resistance. Notably, OPN KO mice displayed significantly less corneal opacity and neovascularization compared to WT mice that paralleled a decrease in expression of vascular endothelial growth factor (VEGF) A within 12 hr post infection. The change in corneal pathology of the OPN KO mice aligned with a decrease in total leukocyte infiltration into the cornea and specifically, in neutrophils at day 3 post infection and in macrophage subpopulations including CCR2+CD115+CD206+ and CD115+CD183+CD206+ -expressing cells. The infiltration of CD4+ and CD8+ T cells into the cornea was unaltered comparing infected WT to OPN KO mice. Likewise, there was no difference in the total number of HSV-1-specific CD4+ or CD8+ T cells found in the draining lymph node with both sets functionally competent in response to virus antigen comparing WT to OPN KO mice. Collectively, these results demonstrate OPN deficiency directly influences the host innate immune response to ocular HSV-1 infection reducing some aspects of inflammation but at a cost with an increase in local HSV-1 replication.

The Chx10-Traf3 Knockout Mouse as a Viable Model to Study Neuronal Immune Regulation.

Uncontrolled inflammation is associated with neurodegenerative conditions in central nervous system tissues, including the retina and brain. We previously found that the neural retina (NR) plays an important role in retinal immunity. Tumor necrosis factor Receptor-Associated Factor 3 (TRAF3) is a known immune regulator expressed in the retina; however, whether TRAF3 regulates retinal immunity is unknown. We have generated the first conditional NR-Traf3 knockout mouse model (Chx10-Cre/Traf3f/f) to enable studies of neuronal TRAF3 function. Here, we evaluated NR-Traf3 depletion effects on whole retinal TRAF3 protein expression, visual acuity, and retinal structure and function. Additionally, to determine if NR-Traf3 plays a role in retinal immune regulation, we used flow cytometry to assess immune cell infiltration following acute local lipopolysaccharide (LPS) administration. Our results show that TRAF3 protein is highly expressed in the NR and establish that NR-Traf3 depletion does not affect basal retinal structure or function. Importantly, NR-Traf3 promoted LPS-stimulated retinal immune infiltration. Thus, our findings propose NR-Traf3 as a positive regulator of retinal immunity. Further, the NR-Traf3 mouse provides a tool for investigations of neuronal TRAF3 as a novel potential target for therapeutic interventions aimed at suppressing retinal inflammatory disease and may also inform treatment approaches for inflammatory neurodegenerative brain conditions.

An intact complement system dampens cornea inflammation during acute primary HSV-1 infection.

Corneal transparency is an essential characteristic necessary for normal vision. In response to microbial infection, the integrity of the cornea can become compromised as a result of the inflammatory response and the ensuing tissue pathology including neovascularization (NV) and collagen lamellae destruction. We have previously found complement activation contributes to cornea pathology-specifically, denervation in response to HSV-1 infection. Therefore, we investigated whether the complement system also played a role in HSV-1-mediated neovascularization. Using wild type (WT) and complement component 3 deficient (C3 KO) mice infected with HSV-1, we found corneal NV was accelerated associated with an increase in inflammatory monocytes (CD11b+CCR2+CD115+/-Ly6G-Ly6Chigh), macrophages (CD11b+CCR2+CD115+Ly6G-Ly6Chigh) and a subpopulation of granulocytes/neutrophils (CD11b+CCR2-CD115+Ly6G+Ly6Clow). There were also increases in select pro-inflammatory and pro-angiogenic factors including IL-1α, matrix metalloproteinases (MMP)-2, MMP-3, MMP-8, CXCL1, CCL2, and VEGF-A that coincided with increased inflammation, neovascularization, and corneal opacity in the C3 KO mice. The difference in inflammation between WT and C3 KO mice was not driven by changes in virus titer. However, viral antigen clearance was hindered in C3 KO mouse corneas suggesting the complement system has a dynamic regulatory role within the cornea once an inflammatory cascade is initiated by HSV-1.

Herpes Simplex Virus 1 (HSV-1) 0ΔNLS Live-Attenuated Vaccine Protects against Ocular HSV-1 Infection in the Absence of Neutralizing Antibody in HSV-1 gB T Cell Receptor-Specific Transgenic Mice.

The contribution of T cell and antibody responses following vaccination in resistance to herpes simplex virus 1 (HSV-1) infection continues to be rigorously investigated. In the present article, we explore the contribution of CD8+ T cells specific for the major antigenic epitope for HSV-1 glycoprotein B (gB498-505, gB) in C57BL/6 mice using a transgenic mouse (gBT-I.1) model vaccinated with HSV-1 0ΔNLS. gBT-I.1-vaccinated mice did not generate a robust neutralization antibody titer in comparison to the HSV-1 0ΔNLS-vaccinated wild-type C57BL/6 counterpart. Nevertheless, the vaccinated gBT-I.1 mice were resistant to ocular challenge with HSV-1 compared to vehicle-vaccinated animals based on survival and reduced corneal neovascularization but displayed similar levels of corneal opacity. Whereas there was no difference in the virus titer recovered from the cornea comparing vaccinated mice, HSV-1 0ΔNLS-vaccinated animals possessed significantly less infectious virus during acute infection in the trigeminal ganglia (TG) and brain stem compared to the control-vaccinated group. These results correlated with a significant increase in gB-elicited interferon-γ (IFN-γ), granzyme B, and CD107a and a reduction in lymphocyte activation gene 3 (LAG-3), programmed cell death 1 (PD-1), and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) expressed by TG infiltrating gB-specific CD8+ T cells from the HSV-1 0ΔNLS-vaccinated group. Antibody depletion of CD8+ T cells in HSV-1 0ΔNLS-vaccinated mice rendered animals highly susceptible to virus-mediated mortality similar to control-vaccinated mice. Collectively, the HSV-1 0ΔNLS vaccine is effective against ocular HSV-1 challenge, reducing ocular neovascularization and suppressing peripheral nerve virus replication in the near absence of neutralizing antibody in this unique mouse model.IMPORTANCE The role of CD8+ T cells in antiviral efficacy using a live-attenuated virus as the vaccine is complicated by the humoral immune response. In the case of the herpes simplex virus 1 (HSV-1) 0ΔNLS vaccine, the correlate of protection has been defined to be primarily antibody driven. The current study shows that in the near absence of anti-HSV-1 antibody, vaccinated mice are protected from subsequent challenge with wild-type HSV-1 as measured by survival. The efficacy is lost following depletion of CD8+ T cells. Whereas increased survival and reduction in virus replication were observed in vaccinated mice challenged with HSV-1, cornea pathology was mixed with a reduction in neovascularization but no change in opacity. Collectively, the study suggests CD8+ T cells significantly contribute to the host adaptive immune response to HSV-1 challenge following vaccination with an attenuated virus, but multiple factors are involved in cornea pathology in response to ocular virus challenge.

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.

"Amyloid-beta accumulation cycle" as a prevention and/or therapy target for Alzheimer's disease.

The cell cycle and its regulators are validated targets for cancer drugs. Reagents that target cells in a specific cell cycle phase (e.g., antimitotics or DNA synthesis inhibitors/replication stress inducers) have demonstrated success as broad-spectrum anticancer drugs. Cyclin-dependent kinases (CDKs) are drivers of cell cycle transitions. A CDK inhibitor, flavopiridol/alvocidib, is an FDA-approved drug for acute myeloid leukemia. Alzheimer's disease (AD) is another serious issue in contemporary medicine. The cause of AD remains elusive, although a critical role of latent amyloid-beta accumulation has emerged. Existing AD drug research and development targets include amyloid, amyloid metabolism/catabolism, tau, inflammation, cholesterol, the cholinergic system, and other neurotransmitters. However, none have been validated as therapeutically effective targets. Recent reports from AD-omics and preclinical animal models provided data supporting the long-standing notion that cell cycle progression and/or mitosis may be a valid target for AD prevention and/or therapy. This review will summarize the recent developments in AD research: (a) Mitotic re-entry, leading to the "amyloid-beta accumulation cycle," may be a prerequisite for amyloid-beta accumulation and AD pathology development; (b) AD-associated pathogens can cause cell cycle errors; (c) thirteen among 37 human AD genetic risk genes may be functionally involved in the cell cycle and/or mitosis; and (d) preclinical AD mouse models treated with CDK inhibitor showed improvements in cognitive/behavioral symptoms. If the "amyloid-beta accumulation cycle is an AD drug target" concept is proven, repurposing of cancer drugs may emerge as a new, fast-track approach for AD management in the clinic setting.

Corneal Epithelial Cells Exhibit Myeloid Characteristics and Present Antigen via MHC Class II.

Purpose: To explore the impact of ocular surface insults on the immunomodulatory capacity and phenotype of corneal epithelial cells (CECs) with a focus on epithelial-mesenchymal transition (EMT). Methods: Corneas were harvested from mice 6 days following scratch injury, ragweed pollen-induced allergy, or herpes simplex virus type 1 (HSV-1) infection and compared to healthy tissue controls. Corneas were enzymatically digested and CECs phenotypically characterized using flow cytometry. CECs were defined as epithelial cell adhesion molecule (EpCAM)-positive CD45-negative cells. CECs were assessed by PCR to evaluate EMT-associated transcripts. Recombinant HSV-1 and transgenic mice were utilized to investigate the role of vascular endothelial growth factor A (VEGFA) on the phenotype observed. The immunomodulatory potential of CECs was assessed in coculture assays with ovalbumin-specific CD4 T cells. Results: Ectopic expression of classic "myeloid" antigens Ly6G, CCR2, and CX3CR1 was identified in CEC subsets from all groups with evidence supporting an underlying partial EMT event resulting from loss of cell-cell contacts. Corneal HSV-1 infection induced Ly6C expression and major histocompatibility complex (MHC)-II upregulation in CECs through a VEGFA-linked mechanism. These Ly6C+ MHC-II+ CECs were found to function as amateur antigen-presenting cells and induced CD4 T cell proliferation in vitro. Conclusions: This study characterizes a novel immunomodulatory CEC phenotype with possible implications for immune privilege, chronic inflammation, and tissue fibrosis. Moreover, the identification of CECs masquerading with multiple "myeloid" antigens warrants careful evaluation of flow cytometry data involving corneal digests.

Colony Stimulating Factor-1 Receptor Expressing Cells Infiltrating the Cornea Control Corneal Nerve Degeneration in Response to HSV-1 Infection.

Purpose: Herpes simplex virus type-1 (HSV-1) is a leading cause of neurotrophic keratitis, characterized by decreased or absent corneal sensation due to damage to the sensory corneal innervation. We previously reported the elicited immune response to infection contributes to the mechanism of corneal nerve regression/damage during acute HSV-1 infection. Our aim is to further establish the involvement of infiltrated macrophages in the mechanism of nerve loss upon infection. Methods: Macrophage Fas-Induced Apoptosis (MAFIA) transgenic C57BL/6 mice were systemically treated with AP20187 dimerizer or vehicle (VEH), and their corneas, lymph nodes, and blood were assessed for CD45+CD11b+GFP+ cell depletion by flow cytometry (FC). Mice were ocularly infected with HSV-1 or left uninfected. At 2, 4, and/or 6 days post infection (PI), corneas were assessed for sensitivity and harvested for FC, nerve structure by immunohistochemistry, viral content by plaque assay, soluble factor content by suspension array, and activation of signaling pathways by Western blot analysis. C57BL6 mice were used to compare to the MAFIA mouse model. Results: MAFIA mice treated with AP20187 had efficient depletion of CD45+CD11b+GFP+ cells in the tissues analyzed. The reduction of CD45+CD11b+GFP+ cells recruited to the infected corneas of AP20187-treated mice correlated with preservation of corneal nerve structure and function, decreased protein concentration of inflammatory cytokines, and decreased STAT3 activation despite no changes in viral content in the cornea compared to VEH-treated animals. Conclusions: Our results suggest infiltrated macrophages are early effectors in the nerve regression following HSV-1 infection. We propose the neurodegeneration mechanism involves macrophages, local up-regulation of IL-6, and activation of STAT3.

Long-term consequences of topical dexamethasone treatment during acute corneal HSV-1 infection on the immune system.

Herpes simplex virus type 1 (HSV-1) is a leading cause of neurotrophic keratitis (NTK). NTK is characterized by decreased corneal sensation from damage to the corneal sensory fibers. We have reported on the regression of corneal nerves and their function during acute HSV-1 infection. That nerve loss is followed by an aberrant process of nerve regeneration during the latent phase of infection that lacks functional recovery. We recently showed the elicited immune response in the infected cornea, and not viral replication itself, is part of the mechanism responsible for the nerve degeneration process after infection. Specifically, we showed infected corneas topically treated with dexamethasone (DEX) significantly retained both structure and sensitivity of the corneal nerve network in comparison to mice treated with control eye drops, consistent with decreased levels of proinflammatory cytokines and reduced influx of macrophages and CD8+ T cells into the cornea. This study was undertaken to analyze the long-term effect of such a localized, immunosuppressive paradigm (DEX drops on the cornea surface during the first 8 d of HSV-1 infection) on the immune system and on corneal pathology. We found the profound immunosuppressive effect of DEX on lymphoid tissue was sustained in surviving mice for up to 30 d postinfection (p.i.). DEX treatment had prolonged effects, preserving corneal innervation and its function and blunting neovascularization, as analyzed at 30 d p.i. Our data support previously reported observations of an association between the persistent presence of inflammatory components in the latently infected cornea and structural and functional nerve defects in NTK.

Cornea lymphatics drive the CD8+ T-cell response to herpes simplex virus-1.

Herpes simplex virus-1 (HSV-1) infection of the cornea induces vascular endothelial growth factor A (VEGF-A)-dependent lymphangiogenesis. However, the extent to which HSV-1-induced corneal lymphangiogenesis impacts the adaptive immune response has not been characterized. Here, we used floxed VEGF-A mice to study the importance of newly created corneal lymphatic vessels in the host adaptive immune response to infection. Whereas the mice infected with the parental virus (strain SC16) exhibited robust corneal lymphangiogenesis, mice that received the recombinant virus (SC16 ICP0-Cre) that expresses Cre recombinase under the control of infected cell protein 0 (ICP0), an HSV-1 immediate-early gene, showed a significant reduction in lymphangiogenesis. There was no difference in virus recovered from the cornea of mice infected with SC16 vs SC16 ICP0-Cre. However, viral loads were significantly elevated in the trigeminal ganglia (TG) of mice with reduced corneal lymphangiogenesis. The increase in viral titer correlated with a significant loss of HSV-1-specific CD8+ T cells that traffic to the TG of mice infected with the recombinant virus. Intrastromal delivery of size-exclusion dye (fluorescein isothiocyanate-dextran) revealed a time-dependent defect in the ability of the lymphatic vessels in SC16 ICP0-Cre-infected mice to transport soluble antigen from the cornea to the draining lymph nodes. We interpret these results to suggest that the newly created lymphatic vessels in the cornea driven by HSV-1 infection are critical in the delivery of soluble viral antigen to the draining lymph node and subsequent development of the CD8+ T-cell response to HSV-1.

IL-6 Contributes to Corneal Nerve Degeneration after Herpes Simplex Virus Type I Infection.

Herpes simplex virus type 1 (HSV-1) is a leading cause of neurotrophic keratitis characterized by decreased corneal sensation because of damage to the corneal sensory fibers. We and others have reported regression of corneal nerves during acute HSV-1 infection. To determine whether denervation is caused directly by the virus or indirectly by the elicited immune response, mice were infected with HSV-1 and topically treated with dexamethasone (DEX) or control eye drops. Corneal sensitivity was measured using a Cochet-Bonnet esthesiometer and nerve network structure via immunohistochemistry. Corneas were assessed for viral content by plaque assay, leukocyte influx by flow cytometry, and content of chemokines and inflammatory cytokines by suspension array. DEX significantly preserved corneal nerve structure and sensitivity on infection. DEX reduced myeloid and T-cell populations in the cornea and did not affect viral contents at 4 and 8 days post infection. The elevated protein contents of chemokines and inflammatory cytokines on infection were greatly suppressed by DEX. Subconjunctival delivery of neutralizing antibody against IL-6 to infected mice resulted in partial preservation of corneal nerve structure and sensitivity. Our study supports a role for the immune response, but not local virus replication in the development of HSV-1-induced neurotrophic keratitis. IL-6 is one of the factors produced by the elicited inflammatory response to HSV-1 infection contributing to nerve regression.

A Highly Efficacious Herpes Simplex Virus 1 Vaccine Blocks Viral Pathogenesis and Prevents Corneal Immunopathology via Humoral Immunity.

UNLABELLED: Correlates of immunologic protection requisite for an efficacious herpes simplex virus 1 (HSV-1) vaccine remain unclear with respect to viral pathogenesis and clinical disease. In the present study, mice were vaccinated with a novel avirulent, live attenuated virus (0ΔNLS) or an adjuvanted glycoprotein D subunit (gD-2) similar to that used in several human clinical trials. Mice vaccinated with 0ΔNLS showed superior protection against early viral replication, neuroinvasion, latency, and mortality compared to that of gD-2-vaccinated or naive mice following ocular challenge with a neurovirulent clinical isolate of HSV-1. Moreover, 0ΔNLS-vaccinated mice exhibited protection against ocular immunopathology and maintained corneal mechanosensory function. Vaccinated mice also showed suppressed T cell activation in the draining lymph nodes following challenge. Vaccine efficacy correlated with serum neutralizing antibody titers. Humoral immunity was identified as the correlate of protection against corneal neovascularization, HSV-1 shedding, and latency through passive immunization. Overall, 0ΔNLS affords remarkable protection against HSV-1-associated ocular sequelae by impeding viral replication, dissemination, and establishment of latency. IMPORTANCE: HSV-1 manifests in a variety of clinical presentations ranging from a rather benign "cold sore" to more severe forms of infection, including necrotizing stromal keratitis and herpes simplex encephalitis. The present study was undertaken to evaluate a novel vaccine to ocular HSV-1 infection not only for resistance to viral replication and spread but also for maintenance of the visual axis. The results underscore the necessity to reconsider strategies that utilize attenuated live virus as opposed to subunit vaccines against ocular HSV-1 infection.

The use of human cornea organotypic cultures to study herpes simplex virus type 1 (HSV-1)-induced inflammation.

PURPOSE: To determine the utility of human organotypic cornea cultures as a model to study herpes simplex virus type 1 (HSV-1)-induced inflammation and neovascularization. METHODS: Human organotypic cornea cultures were established from corneas with an intact limbus that were retrieved from donated whole globes. One cornea culture was infected with HSV-1 (10(4) plaque-forming units), while the other cornea from the same donor was mock-infected. Supernatants were collected at intervals post-culture with and without infection to determine viral titer (by plaque assay) and pro-angiogenic and proinflammatory cytokine concentration by suspension array analysis. In some experiments, the cultured corneas were collected and evaluated for HSV-1 antigens by immunohistochemical means. Another set of experiments measured susceptibility of human three-dimensional cornea fibroblast constructs, in the presence and absence of TGF-ß1, to HSV-1 infection in terms of viral replication and the inflammatory response to infection as a comparison to the organotypic cornea cultures. RESULTS: Organotypic cornea cultures and three-dimensional fibroblast constructs exhibited varying degrees of susceptibility to HSV-1. Fibroblast constructs were more susceptible to infection in terms of infectious virus recovered in a shorter period of time. There were changes in the levels of select pro-angiogenic or proinflammatory cytokines that were dictated as much by the cultures producing them as by whether they were infected with HSV-1 or treated with TGF-ß1. CONCLUSION: Organotypic cornea and three-dimensional fibroblast cultures are likely useful for the identification and short-term study of novel antiviral compounds and virus replication, but are limited in the study of the local immune response to infection.

Tumor necrosis factor alpha and interleukin-6 facilitate corneal lymphangiogenesis in response to herpes simplex virus 1 infection.

UNLABELLED: Herpes simplex virus 1 (HSV-1) is a common human pathogen of clinical significance due to its association with vision impairment and encephalitis. In a mouse model of ocular neovascularization, we have previously shown that HSV-1 elicits the genesis of lymphatic vessels into the cornea proper through epithelial cell expression of vascular endothelial growth factor A (VEGFA) dependent upon expression of VEGFR2 during acute infection. We hypothesized that other factors may be involved in lymphangiogenesis, with proinflammatory cytokines as the leading candidates. In the absence of infection or inflammation, intrastromal administration of tumor necrosis factor alpha (TNF-α) coupled with VEGFA elicited lymphatic vessel genesis significantly above either factor alone as well as a vehicle control. Consistent with this observation, anti-TNF-α antibody (Ab) blocked HSV-1-mediated corneal lymphangiogenesis within the first 5 days postinfection. However, TNF-α-deficient (TNF-α(-/-)) mice displayed a level of corneal vessel growth similar to that shown by wild-type (WT) controls. To investigate the likely redundant nature of cytokines, PCR array analysis of HSV-1-infected TNF-α(-/-) mice was conducted, and it revealed several factors elevated above those found in HSV-1-infected WT mice, including interleukin-1ß (IL-1ß), platelet-derived growth factor, angiopoietin 2, insulin-like growth factor 2, and IL-6. Subconjunctival administration of neutralizing Ab to IL-6 blocked lymphangiogenesis in TNF-α(-/-) mice. Whereas the cornea levels of IL-6 were significantly reduced, there was no appreciable change in the level of IL-1ß or other proangiogenic factors analyzed. Collectively, the results suggest in addition to VEGFA, TNF-α and IL-6 promote and likely synergize with VEGFA in corneal lymphangiogenesis during acute HSV-1 infection. IMPORTANCE: We have identified at least two proinflammatory cytokines expressed locally that are involved in the genesis of lymphatic vessels in the normally avascular cornea in response to HSV-1 infection. This finding provides the basis to target IL-6 and TNF-α as additional proangiogenic factors in the cornea during the development of herpetic stromal keratitis as a means to alleviate further neovascularization and tissue pathology associated with the host immune response to the pathogen.

Caveolin-1 increases proinflammatory chemoattractants and blood-retinal barrier breakdown but decreases leukocyte recruitment in inflammation.

PURPOSE: Caveolin-1 (Cav-1), the signature protein of caveolae, modulates inflammatory responses, and innate immunity. However, Cav-1's role in retinal inflammation has not been rigorously tested. In this study, we examined the effect of Cav-1 ablation on the sensitivity of the retina to inflammation. METHODS: Cav-1 knockout (KO) mice were challenged by intravitreal injection of lipopolysaccharide (LPS) and inflammatory cell recruitment was assessed by flow cytometry and immunohistochemistry. Leukostasis was assessed in retinal flatmounts after perfusion with FITC-labeled Concanavalin A (FITC-ConA). Chemoattractants were measured by multiplex immunoassays. Blood-retinal barrier (BRB) breakdown was assessed quantitatively by a FITC-dextran permeability assay. The ratio of extravascular to total immune cells was determined by CD45 immunohistochemistry of retinal flatmounts. RESULTS: Inflammatory challenge resulted in significant blunting of proinflammatory cytokine (monocyte chemoattractant protein-1 [MCP-1/CCL2], CXCL1/KC, IL-6, and IL-1ß) responses as well as reduced inflammatory BRB breakdown in Cav-1 KO retinas. Paradoxically, Cav-1 deficiency resulted in significantly increased recruitment of immune cells compared with controls as well as increased leukostasis. A similar ratio of extravascular/total leukocytes were found in Cav-1 KO and wild-type (WT) retinas suggesting that Cav-1 deficient leukocytes were as competent to extravasate as those from WT mice. We found increased levels of circulating immune cells in naïve (not challenged with LPS) Cav-1 KO mice compared with controls. CONCLUSIONS: Caveolin-1 paradoxically modulates inflammatory signaling and leukocyte infiltration through distinct mechanisms. We hypothesize that Cav-1 expression may enhance inflammatory signaling while at the same time supporting the physical properties of the BRB.

Microglia-induced IL-6 protects against neuronal loss following HSV-1 infection of neural progenitor cells.

Herpes virus type 1 (HSV-1) is one of the most widespread human pathogens and accounts for more than 90% of cases of herpes simplex encephalitis (HSE) causing severe and permanent neurologic sequelae among surviving patients. We hypothesize such CNS deficits are due to HSV-1 infection of neural progenitor cells (NPCs). In vivo, HSV-1 infection was found to diminish NPC numbers in the subventricular zone. Upon culture of NPCs in conditions that stimulate their differentiation, we found HSV-1 infection of NPCs resulted in the loss of neuronal precursors with no significant change in the percentage of astrocytes or oligodendrocytes. We propose this is due a direct effect of HSV-1 on neuronal survival without alteration of the differentiation process. The neuronal loss was prevented by the addition of microglia or conditioned media from NPC/microglia co-cultures. Using neutralizing antibodies and recombinant cytokines, we identified interleukin-6 (IL-6) as responsible for the protective effect by microglia, likely through its downstream Signal Transducer and Activator of Transcription 3 (STAT3) cascade.

HSV-1 targets lymphatic vessels in the eye and draining lymph node of mice leading to edema in the absence of a functional type I interferon response.

Herpes simplex virus type-1 (HSV-1) induces new lymphatic vessel growth (lymphangiogenesis) in the cornea via expression of vascular endothelial growth factor by virally infected epithelial cells. Here, we extend this observation to demonstrate the selective targeting of corneal lymphatics by HSV-1 in the absence of functional type I interferon (IFN) pathway. Specifically, we examined the impact of HSV-1 replication on angiogenesis using type I IFN receptor deficient (CD118(-/-)) mice. HSV-1-induced lymphatic and blood vessel growth into the cornea proper was time-dependent in immunocompetent animals. In contrast, there was an initial robust growth of lymphatic vessels into the cornea of HSV-1-infected CD118(-/-)mice, but such vessels disappeared by day 5 postinfection. The loss was selective as blood vessel integrity remained intact. Magnetic resonance imaging and confocal microscopy analysis of the draining lymph nodes of CD118(-/-) mice revealed extensive edema and loss of lymphatics compared with wild-type mice. In addition to a loss of lymphatic vessels in CD118(-/-) mice, HSV-1 infection resulted in epithelial thinning associated with geographic lesions and edema within the cornea, which is consistent with a loss of lymphatic vasculature. These results underscore the key role functional type I IFN pathway plays in the maintenance of structural integrity within the cornea in addition to the anti-viral characteristics often ascribed to the type I IFN cytokine family.

Type I interferon and lymphangiogenesis in the HSV-1 infected cornea - are they beneficial to the host?

Herpes simplex virus type 1 (HSV-1) is a highly successful pathogen that can result in significant human morbidity. Within the cornea, it was thought the initial recognition of the pathogen was through Toll-like receptors expressed on/in resident cells that then elicit pro-inflammatory cytokine production, activation of anti-viral pathways, and recruitment of leukocytes. However, our lab has uncovered a novel, TLR-independent innate sensor that supersedes TLR induction of anti-viral pathways following HSV-1 infection. In addition, we have also found HSV-1 induces the genesis of lymphatic vessels into the cornea proper by a mechanism independent of TLRs and unique in the field of neovascularization. This review will focus on these two innate immune events during acute HSV-1 infection of the cornea.