The atypical chemokine receptor-2 fine-tunes the immune response in herpes stromal keratitis.

Herpes stromal keratitis (HSK) is a blinding corneal disease caused by herpes simplex virus-1 (HSV-1), a common pathogen infecting most of the world's population. Inflammation in HSK is chemokine-dependent, particularly CXCL10 and less so the CC chemokines. The atypical chemokine receptor-2 (ACKR2) is a decoy receptor predominantly for pro-inflammatory CC chemokines, which regulates the inflammatory response by scavenging inflammatory chemokines thereby modulating leukocyte infiltration. Deletion of ACKR2 exacerbates and delays the resolution of the inflammatory response in most models. ACKR2 also regulates lymphangiogenesis and mammary duct development through the recruitment of tissue-remodeling macrophages. Here, we demonstrate a dose-dependent upregulation of ACKR2 during corneal HSV-1 infection. At an HSV inoculum dose of 5.4 x 105 pfu, but not at higher dose, ACKR2 deficient mice showed prolonged clinical signs of HSK, increased infiltration of leukocytes and persistent corneal neovascularization. Viral clearance and T cell activation were similar in ACKR2-/- and wild type mice, despite a transient diminished expression of CD40 and CD86 in dendritic cells. The data suggest that ACKR2 fine-tunes the inflammatory response and the level of neovascularization in the HSK.

Loss of Osteopontin Expression Reduces HSV-1-Induced Corneal Opacity.

Purpose: Corneal opacity and neovascularization (NV) are often described as outcomes of severe herpes simplex virus type 1 (HSV-1) infection. The current study investigated the role of colony-stimulating factor 1 receptor (CSF1R)+ cells and soluble factors in the progression of HSV-1-induced corneal NV and opacity. Methods: MaFIA mice were infected with 500 plaque-forming units of HSV-1 in the cornea following scarification. From day 10 to day 13 post-infection (pi), mice were treated with 40 µg/day of AP20187 (macrophage ablation) or vehicle intraperitoneally. For osteopontin (OPN) neutralization experiments, C57BL/6 mice were infected as above and treated with 2 µg of goat anti-mouse OPN or isotypic control IgG subconjunctivally every 2 days from day 4 to day 12 pi. Mice were euthanized on day 14 pi, and tissue was processed for immunohistochemistry to quantify NV and opacity by confocal microscopy and absorbance or detection of pro- and anti-angiogenic and inflammatory factors and cells by suspension array analysis and flow cytometry, respectively. Results: In the absence of CSF1R+ cells, HSV-1-induced blood and lymphatic vessel growth was muted. These results correlated with a loss in fibroblast growth factor type 2 (FGF-2) and an increase in OPN expression in the infected cornea. However, a reduction in OPN expression in mice did not alter corneal NV but significantly reduced opacity. Conclusions: Our data suggest that CSF1R+ cell depletion results in a significant reduction in HSV-1-induced corneal NV that correlates with the loss of FGF-2 expression. A reduction in OPN expression was aligned with a significant drop in opacity associated with reduced corneal collagen disruption.

Role of TH17 Responses in Increasing Herpetic Keratitis in the Eyes of Mice Infected with HSV-1.

Purpose: TH17 cells play an important role in host defense and autoimmunity yet very little is known about the role of IL17 in herpes simplex virus (HSV)-1 infectivity. To better understand the relationship between IL17 and HSV-1 infection, we assessed the relative impact of IL17A-deficiency and deficiency of its receptors on HSV-1 responses in vivo. Methods: We generated IL17RA-/- and IL17RA-/-RC-/- mice in-house and infected them along with IL17A-/- and IL17RC-/- mice in the eyes with 2 × 105 PFU/eye of wild type (WT) HSV-1 strain McKrae. WT C57BL/6 mice were used as control. Virus replication in the eye, survival, corneal scarring (CS), angiogenesis, levels of latency-reactivation, and levels of CD8 and exhaustion markers (PD1, TIM3, LAG3, CTLA4, CD244, and CD39) in the trigeminal ganglia (TG) of infected mice were determined on day 28 postinfection. Results: No significant differences in virus replication in the eye, survival, latency, reactivation, and exhaustion markers were detected among IL17A-/-, IL17RA-/-, IL17RC-/-, IL17RA-/-RC-/-, and WT mice. However, mice lacking IL17 had significantly less CS and angiogenesis than WT mice. In addition, angiogenesis levels in the absence of IL17RC and irrespective of the absence of IL17RA were significantly less than in IL17A- or IL17RA-deficient mice. Conclusions: Our results suggest that the absence of IL17 protects against HSV-1-induced eye disease, but has no role in protecting against virus replication, latency, or reactivation. In addition, our data provide rationale for blocking IL17RC function rather than IL17A or IL17RA function as a key driver of HSV-1-induced eye disease.

Frontline Science: Aspirin-triggered resolvin D1 controls herpes simplex virus-induced corneal immunopathology.

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye, caused by HSV-1 infection, and a common cause of vision impairment in humans. The inflammatory lesions in the cornea are primarily caused by neutrophils with the active participation of CD4+ T cells. Therefore, the targeting of these immune cell types and their products represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin D1 (RvD1) and its epimer aspirin-triggered RvD1 (AT-RvD1) are lipid mediators derived from docosahexaenoic acid (DHA) and were shown to promote resolution in several inflammatory disease models. In this report, we examined whether AT-RvD1 administration, begun before infection or at a later stage after ocular infection of mice with HSV-1, could control the severity of SK lesions. Treatment with AT-RvD1 significantly diminished the extent of corneal neovascularization and the severity of SK lesions. AT-RvD1-treated mice had fewer numbers of inflammatory cells that included neutrophils as well as Th1 and Th17 cells in the infected cornea. The mechanisms by which AT-RvD1 acts appear to be multiple. These include inhibitory effects on proinflammatory mediators, such as IL-1ß, IL-6, IL-12, CXCL1, MCP-1, MIP-2, vascular endothelial growth factor (VEGF)-A, matrix metalloproteinase 9 (MMP-9), and proinflammatory miRNA, such as miR-155, miR-132, and miR-223, which are involved in SK pathogenesis and corneal neovascularization. In addition, AT-RvD1 attenuated STAT1, which plays an important role in Th1 cell differentiation and IFN-γ expression. These findings demonstrate that AT-RvD1 treatment could represent a useful strategy for the management of virus-induced immunopathological lesions.

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.

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.

Interleukin-17 enhanced immunoinflammatory lesions in a mouse model of recurrent herpetic keratitis.

Interleukin-17 (IL-17), mainly produced by activated (memory) T cells, has been found in the corneas from herpetic stromal keratitis (HSK) patients. To better understand the role of IL-17 and to optimize fidelity to human recurrent HSK, in this study, we utilized a mouse model of recurrent HSK, examined the expression of IL-17 and Th17 cells, and determine the alterability of virus-induced corneal inflammation after anti-IL-17 antibody treatment during murine recurrent HSK. We found that Th17 cells were obviously up-regulated in both cornea and DLNs of recurrent mice. Peak IL-17 protein present in recurrent cornea in conjunction with peak opacity mediated by CD4(+) T cells. Systemic administration of anti-IL-17 antibody resulted in a diminished severity of corneal opacity, neovascularization, and CD4(+) T cells infiltration compared to control. Anti-IL-17 treatment down-regulated the mRNA and protein levels of TNF-α expression in recurrent corneas, and decreased HSV-specific DTH responses. Our results indicate that elevated IL-17 expression may be involved in the development of recurrent HSK. The likely mechanisms of action for IL-17 are through up-regulating TNF-α expression and promoting HSV-specific DTH responses. Thus, IL-17 might constitute a useful target for therapeutic intervention in recurrent HSK.

Role of miR-132 in angiogenesis after ocular infection with herpes simplex virus.

MicroRNAs (miRNAs) are small regulatory molecules that control diverse biological processes that include angiogenesis. Herpes simplex virus (HSV) causes a chronic immuno-inflammatory response in the eye that may result in corneal neovascularization during blinding immunopathological lesion stromal keratitis (SK). miR-132 is a highly conserved miRNA that is induced in endothelial cells in response to growth factors, such as vascular endothelial growth factor (VEGF). In this study, we show that miR-132 expression was up-regulated (10- to 20-fold) after ocular infection with HSV, an event that involved the production of both VEGF-A and IL-17. Consequently, blockade of VEGF-A activity using soluble VEGF receptor 1 resulted in significantly lower levels of corneal miR-132 after HSV infection. In addition, low levels of corneal miR-132 were detected in IL-17 receptor knockout mice after HSV infection. In vivo silencing of miR-132 by the provision of anti-miR-132 (antagomir-132) nanoparticles to HSV-infected mice led to reduced corneal neovascularization and diminished SK lesions. The anti-angiogenic effect of antagomir-132 was reflected by a reduction in angiogenic Ras activity in corneal CD31-enriched cells (presumably blood vessel endothelial cells) during SK. To our knowledge, this is one of the first reports of miRNA involvement in an infectious ocular disease. Manipulating miRNA expression holds promise as a therapeutic approach to control an ocular lesion that is an important cause of human blindness.

Galectin-1 reduces the severity of herpes simplex virus-induced ocular immunopathological lesions.

Stromal keratitis is a chronic immunopathological lesion of the eye caused by HSV-1 infection that can result in blindness. Because the inflammatory lesions are primarily orchestrated by Th1 cells, and to a lesser extent by Th17 cells, inhibiting their activity represents a useful form of therapy. In this study we evaluated the therapeutic potential of galectin-1 (gal-1), an endogenous lectin that in some autoimmune diseases was shown to suppress the functions of Th1 and Th17 cells. Treatment was begun at different times after ocular infection with HSV and the outcome was assessed clinically as well as for effects on various immune parameters. Treatment with recombinant gal-1 significantly diminished stromal keratitis lesion severity and the extent of corneal neovascularization. Treated mice had reduced numbers of IFN-γ- and IL-17-producing CD4(+) T cells, as well as neutrophil infiltration in the cornea. Furthermore, disease severity was greater in gal-1 knockout mice compared with their wild-type counterparts. The many effects of gal-1 treatment include reduction in the production of proinflammatory cytokines and chemokines, increased production of IL-10, and inhibitory effects on molecules involved in neovascularization. To our knowledge, our findings are the first to show that gal-1 treatment represents a useful approach to control lesion severity in a virally induced immunopathological disease.

Ocular neovascularization caused by herpes simplex virus type 1 infection results from breakdown of binding between vascular endothelial growth factor A and its soluble receptor.

The normal cornea is transparent, which is essential for normal vision, and although the angiogenic factor vascular endothelial growth factor A (VEGF-A) is present in the cornea, its angiogenic activity is impeded by being bound to a soluble form of the VEGF receptor-1 (sVR-1). This report investigates the effect on the balance between VEGF-A and sVR-1 that occurs after ocular infection with HSV, which causes prominent neovascularization, an essential step in the pathogenesis of the vision-impairing lesion, stromal keratitis. We demonstrate that HSV-1 infection causes increased production of VEGF-A but reduces sVR-1 levels, resulting in an imbalance of VEGF-A and sVR-1 levels in ocular tissues. Moreover, the sVR-1 protein made was degraded by the metalloproteinase (MMP) enzymes MMP-2, -7, and -9 produced by infiltrating inflammatory cells that were principally neutrophils. Inhibition of neutrophils, inhibition of sVR-1 breakdown with the MMP inhibitor marimastat, and the provision of exogenous recombinant sVR-1 protein all resulted in reduced angiogenesis. Our results make the novel observation that ocular neovascularization resulting from HSV infection involves a change in the balance between VEGF-A and its soluble inhibitory receptor. Future therapies aimed to increase the production and activity of sVR-1 protein could benefit the management of stromal keratitis, an important cause of human blindness.

VEGF-A expression by HSV-1-infected cells drives corneal lymphangiogenesis.

Inflammatory lymphangiogenesis plays a crucial role in the development of inflammation and transplant rejection. The mechanisms of inflammatory lymphangiogenesis during bacterial infection, toll-like receptor ligand administration, and wound healing are well characterized and depend on ligands for the vascular endothelial grow factor receptor (VEGFR) 3 that are produced by infiltrating macrophages. But inflammatory lymphangiogenesis in nonlymphoid tissues during chronic viral infection is unstudied. Herpes simplex virus 1 (HSV-1) infection of the cornea is a leading cause of blindness and depends on aberrant host immune responses to antigen within the normally immunologically privileged cornea. We report that corneal HSV-1 infection drives lymphangiogenesis and that corneal lymphatics persist past the resolution of infection. The mechanism of HSV-1-induced lymphangiogenesis was distinct from the described mechanisms of inflammatory lymphangiogenesis. HSV-1-elicited lymphangiogenesis was strictly dependent on VEGF-A/VEGFR-2 signaling but not on VEGFR-3 ligands. Macrophages played no role in the induction of lymphangiogenesis and were not a detectable source of VEGF-A. Rather, using VEGF-A reporter transgenic mice, we have identified infected epithelial cells as the primary source of VEGF-A during HSV-1 infection. Our results indicate that HSV-1 directly induces vascularization of the cornea through up-regulation of VEGF-A expression.

Role of Tim-3/galectin-9 inhibitory interaction in viral-induced immunopathology: shifting the balance toward regulators.

Controlling chronic immunoinflammatory diseases such as lesions in the eye caused by infection with HSV represents a therapeutic challenge. Since CD4(+) T cells are the primary orchestrators of lesions, targeting activated CD4(+) T cell subsets and increasing the representation of cells that express regulatory function would be a logical therapeutic approach. We show that this outcome can be achieved by therapy, systemic or local, with the lectin family member galectin-9. This molecule, which is a natural product of many cell types, acts as a ligand to the inhibitory molecule TIM-3 (T cell Ig and mucin-3) that is expressed by activated but not naive T cells. We show that 50% or more of T cells in ocular lesions caused by HSV in mice express TIM-3 and that blocking signals from its natural ligand with a mAb results in more severe lesions. More importantly, the provision of additional galectin-9, either systemically or more effectively by local subconjuctival administration, diminished the severity of stromal keratitis lesions as well as the extent of corneal neovascularization. Multiple mechanisms were involved in inhibitory effects. These included apoptosis of the orchestrating effector T cells with consequent reduction of proinflammatory cytokines and an increase in the representation of two separate subtypes of regulatory cells as well as inhibitory effects on the production of molecules involved in neovascularization, an essential component of stromal keratitis pathogenesis. Our results indicate that galectin-9 therapy may represent a useful approach to control HSV-induced lesions, the most common cause of infectious blindness in the Western world.

HSV immune complex (HSV-IgG: IC) and HSV-DNA elicit the production of angiogenic factor VEGF and MMP-9.

Angiogenesis and inflammatory mediators are critical pathogenic factors in herpetic stromal keratitis (HSK). Since disease progresses without infectious virus, HSV-DNA and HSV-IgG complexes (HSV-IC) may contribute to HSK by triggering these factors. Production of VEGF and MMP-9 was studied in vitro using corneal epithelial cells (HCE), fibroblasts (HCRF) and macrophages (THP-1). VEGF was elevated in HCRF and THP-1 following treatment with HSV-DNA and HSV-IC. MMP-9 was elevated in THP-1 but not in corneal cells. When anti-HSV-IgG(Fab')2 complexes stimulated THP-1, MMP-9 was reduced to control levels. Pretreatment of THP-1 with anti-TLR-2 and -3 inhibited MMP-9 production. Thus, HSV-IC may stimulate THP-1 through the Fc receptor and TLRs. Proinflammatory cytokines (IL-1b, IL-6, and TNF-alpha) increased VEGF and MMP-9 in corneal cells and macrophages. These studies indicate that the continued presence of HSV-DNA and HSV-IC contribute to angiogenesis and inflammation in HSK. Thus, cytokines and TLRs may be potential targets for intervention.

Effect of human apolipoprotein E genotype on the pathogenesis of experimental ocular HSV-1.

The isoform-specific role of human apolipoprotein E (apoE) has been assessed in a mouse model of ocular herpes. Female, age-matched transgenic mice knocked-in for the human allele apoE3 or apoE4 and their parent C57Bl/6 mice were inoculated corneally with HSV-1 strain KOS. Ocular HSV-1 pathogenesis was monitored through viral replication and clinical progression of stromal opacity and neovascularization by slit-lamp examination. Establishment of latency was determined by analysis of HSV-1 DNA (copy number) by specific real-time PCR in the cornea, trigeminal ganglia (TG), and brain. Representative groups of transgenic mice were sacrificed for the analysis of gene expression of vascular endothelial growth factor (VEGF) by reverse-transcription PCR, and apoE expression by Western blot analysis. At 6days post-infection (P.I.), the ocular infectious HSV-1 titer was significantly higher (p<0.05) in apoE4 mice compared with apoE3 and C57Bl/6 mice. Corneal neovascularization in apoE4 mice was significantly higher (p<0.05) than apoE3 and C57Bl/6 mice. The onset of corneal opacity in apoE4 mice was accelerated during days 9-11 P.I.; however, no significant difference in severity was seen on P.I. days 15 and beyond. At 28 days P.I., infected mice of all genotypes had no significant differences in copy numbers (range 0-15) of HSV-1 DNA in their corneas, indicating that HSV-1 DNA copy numbers in cornea are independent of apoE isoform regulation. At 28 days P.I., both apoE4 and C57Bl/6 mice had a significantly higher (p=0.001) number of copies of HSV-1 DNA in TG compared with apoE3. ApoE4 mice also had significantly higher (p=0.001) copies of HSV-1 DNA in their TGs compared with C57Bl/6 mice. In brain, both apoE4 and C57Bl/6 mice had significantly higher numbers (p

The effect of viral inoculum level and host age on disease incidence, disease severity, and mortality in a murine model of ocular HSV-1 infection.

It has been previously shown that the strain of virus, immune competence of the host, and innate resistance of the host have an effect on the severity of ocular disease induced by topical infection with herpes simplex virus type 1 (HSV-1). This study has expanded on earlier work by examining the effect of virus inoculum and host age on mortality, incidence of ocular disease, and severity of ocular disease. BALB/c mice were infected with inocula ranging from 2 x 10(3) to 1 x 10(6) pfu of HSV-1 strain CJ394. The most significant effect of variation in the inoculum was on the percent of mice developing disease. Increasing the inoculum resulted in significantly increased disease incidence, but at 5 x 10(3) pfu/mouse or higher, there was little difference in disease severity in those animals exhibiting symptoms. Decreasing host age also resulted in a significant increase in the incidence of ocular disease, but the dependence of disease severity on host age varied with the symptom being scored. In animals exhibiting disease, the peak severity of stromal keratitis and vascularization of the cornea were unaffected by host age. However, the severity of blepharitis was significantly reduced in older mice. Increasing host age also resulted in increased resistance to encephalitis. Three to four-week old mice were very susceptible to encephalitis (100% mortality), while only 20% of 4-5 week old mice died by day 15 post-infection. Mice older than 5 weeks were completely resistant to lethal encephalitis after corneal infection.