Targeting NECTIN-1 Based on CRISPR/Cas9 System Attenuated the Herpes Simplex Virus Infection in Human Corneal Epithelial Cells In Vitro.

PURPOSE: Viral keratitis caused by herpes simplex virus 1 (HSV-1) is a lifelong recurring disease and an unignored cause of blindness worldwide. Current antiviral therapy cannot eliminate the transcriptionally silent HSV-1 in latently infected patients. With the explosive applications of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9 gene-editing system in recent years, we aim to develop a CRISPR/Cas9 system targeting down the major HSV receptor, NECTIN-1 on human corneal epithelial cells (HCECs), to provide a novel strategy for herpes simplex keratitis (HSK) treatment. METHODS: The selected single guide RNAs (sgRNAs) targeting human nectin cell adhesion molecule 1 (NECTIN-1), together with Cas-9, were assembled into lentivirus. HCECs were infected with Lenti-Cas9-gRNAs to establish NECTIN-1 knockdown cells. Following HSV-green fluorescent protein (GFP) infection, cell survival and virus infection were determined by fluorescence microscopy and flow cytometry. Relative HSV DNA amount was also compared through quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Lentivirus packaged with the CRISPR/Cas9 system and the two selected sgRNAs both successfully edited down the protein levels of NECTIN-1 of HCECs. After HSV-GFP infection, the infection rate of HCECs in knockdown groups dramatically decreased, especially in the NECTIN-1 knockdown group 1. In addition, the relative HSV DNA amount of both knockdown groups was only 30% when compared with the control group. CONCLUSIONS: We successfully knocked down the NECTIN-1 expression in vitro by the CRISPR/Cas9 system, which alleviated the HSV infection in HCECs. TRANSLATIONAL RELEVANCE: This study offered a promising target for the cure of HSK.

Modulating glutamine metabolism to control viral immuno-inflammatory lesions.

Infection of the cornea with HSV results in an immune-inflammatory reaction orchestrated by proinflammatory T cells that is a major cause of human vision impairment. The severity of lesions can be reduced if the representation of inflammatory T cells is changed to increase the presence of T cells with regulatory function. This report shows that inhibiting glutamine metabolism using 6-Diazo-5-oxo-l-norleucine (DON) administered via intraperitoneal (IP) starting 6 days after ocular infection and continued until day 15 significantly reduced the severity of herpetic stromal keratitis lesions. The therapy resulted in reduced neutrophils, macrophages as well proinflammatory CD4 Th1 and Th17 T cells in the cornea, but had no effect on levels of regulatory T cells. A similar change in the representation of inflammatory and regulatory T cells occurred in the trigeminal ganglion (TG) the site where HSV infection establishes latency. Glutamine metabolism was shown to be required for the in-vitro optimal induction of both Th1 and Th17 T cells but not for the induction of Treg that were increased when glutamine metabolism was inhibited. Inhibiting glutamine metabolism also changed the ability of latently infected TG cells from animals previously infected with HSV to reactivate and produce infectious virus.

Role of interferon regulatory factor 7 in corneal endothelial cells after HSV-1 infection.

Viral infections of the cornea including herpes simplex virus 1 (HSV-1) cause visual morbidity, and the corneal endothelial cell damage leads to significant visual impairment. Interferon regulatory factor 7 (IRF7) has been identified as a significant regulator in corneal endothelial cells after an HSV-1 infection. To examine the role played by IRF7, the DNA binding domain (DBD) of IRF7 of human corneal endothelial cells (HCEn) was disrupted. An RNAi inhibition of IRF7 and IRF7 DBD disruption (IRF7 ∆DBD) led to an impairment of IFN-ß production. Impaired IFN-ß production by IRF7 ∆DBD was regained by IRF7 DNA transfection. Transcriptional network analysis indicated that IRF7 plays a role in antigen presentation function of corneal endothelial cells. When the antigen presentation activity of HCEn cells were examined for priming of memory CD8 T cells, IRF7 disruption abolished the anti-viral cytotoxic T lymphocyte (CTL) response which was dependent on the major histocompatibility complex (MHC) class I. To further examine the roles played by IRF7 in CTL induction as acquired immunity, the contribution of IRF7 to MHC class I-mediated antigen presentation was assessed. Analysis of IRF7 ∆DBD cells indicated that IRF7 played an unrecognized role in MHC class I induction, and the viral infection induced-MHC class I induction was abolished by IRF7 disruption. Collectively, the IRF7 in corneal endothelial cells not only contributed to type I IFN response, but also to the mediation of viral infection-induced MHC class I upregulation and priming of CD8 arm of acquired immunity.

Role Played by Receptors for Advanced Glycosylation End Products in Corneal Endothelial Cells after HSV-1 Infection.

Senescence, sterile inflammation, and infection cause dysfunction of corneal endothelial cells, leading to visual morbidity that may require corneal transplantation. With increasing age, the extracellular matrix is modified by non-enzymatic glycation forming advanced glycation end products (AGEs). The modifications are primarily sensed by the receptors for the AGEs (RAGE) and are manifested as a type I interferon response. Interestingly, in our study, human corneal endothelial cells (HCEn) cells did not respond to the typical RAGE ligands, including the AGEs, high mobility group box 1 (HMGB1), and serum amyloid-A (SAA). Instead, HCEn cells responded exclusively to the CpG DNA, which is possessed by typical corneal pathogen, herpes simplex virus-1 (HSV-1). Upon HSV-1 infection, the surface expression of RAGE was increased, and endocytosed HSV-1 was associated with RAGE and CpG DNA receptor, TLR9. RAGE DNA transfection markedly increased interferon-ß secretion by CpG DNA or HSV-1 infection. HSV-1 infection-induced interferon-ß secretion was abolished by TLR9 inhibition and partially by RAGE inhibition. Global transcriptional response analysis confirmed that RAGE and TLR9 were both significantly involved in type I interferon responses. We conclude that RAGE is a sensor of HSV-1 infection and provokes a type I interferon response.

Polluted Air Exposure Compromises Corneal Immunity and Exacerbates Inflammation in Acute Herpes Simplex Keratitis.

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

Extracellular matrix changes in corneal opacification vary depending on etiology.

Purpose: The purpose of this study is to examine the expression of tenascin-C and matrilin-2 in three different disorders, which frequently require corneal transplantation. These pathological conditions include bullous keratopathy (BK), Fuchs' endothelial corneal dystrophy (FECD), and corneal scarring in herpetic keratitis. Methods: Histological sections of corneal buttons removed during keratoplasty were analyzed in BK (n = 20), FECD (n = 9), herpetic keratitis (n = 12), and cadaveric control (n = 10) groups with light microscopy following chromogenic immunohistochemistry. The sections were evaluated by three investigators, and semiquantitative scoring (0 to 3+) was applied according to standardized methods at 400X magnification. Each layer of the cornea was investigated; moreover, the stroma was subdivided into subepithelial, middle, and pre-Descemet's membrane areas for more detailed analysis. Results: Excessive epithelial and stromal expression of tenascin-C was identified in all investigated conditions; the results were most pronounced in the pre-Descemet's membrane. Regarding matrilin-2, when examined in BK, there was increased labeling intensity in the epithelium (p<0.001) and stromal layers (p<0.05), and a decrease in the endothelium (p<0.001). In the other investigated conditions, only a low degree of stromal localization (p<0.05) of matrilin-2 was detected. Conclusions: The expression of tenascin-C and matrilin-2 differs when examined in various corneal pathologies resulting in opacification. Both molecules seem to be involved in regeneration and wound healing of the corneal matrix in these diseases.

IL-1α Modulates IFN-γ-Induced Production of CXCL9/MIG during Herpes Simplex Virus Type-1 Corneal Infection.

PURPOSE: Investigating the modulation of neutrophil production of MIG and IP-10 during the inflammatory response to HSV-1 infection. MATERIALS AND METHODS: An ex vivo model of human corneal infection by HSV-1 was used for this study. This model permits the study of cytokine production by human corneal buttons in the presence, or absence, of gradient purified human neutrophils, under conditions of HSV-1 infection. All experimental samples were stimulated with a baseline concentration of recombinant human IFN-γ at 1 ng/mL. The relative levels of production for 12 pro-inflammatory mediators were screened using a multi-analyte ELISA assay. Neutrophil production of chemokines MIG and IP-10, under conditions of IFN-γ and/or HSV-1 stimulation were measured by quantitative ELISA. Lastly, antibody neutralization (goat IgG anti-human IL-1α, 2 µg/mL) of de novo production of IL-1α by corneal tissue was performed to investigated the effect on MIG and IP-10 production in the ex vivo model for HSV-1 infection. RESULTS: Four of the 12 pro-inflammatory mediators screened (IL-8, IL-6, IL-1α and IL-1ß) demonstrated elevated levels of production during corneal cell infection with HSV-1 and communication with neutrophils. Neutrophils were demonstrated to produce significant levels of both MIG and IP-10 under conditions of IFN-γ stimulation, and production of MIG was further upregulated by co-stimulation with IFN-γ and HSV-1. Neutralization of de novo IL-1α production in the model resulted in increased production of the chemokine production MIG but had no observable effect on IP-10 production. CONCLUSIONS: Our data provide evidence demonstrating the potential for expression patterns of MIG and IP-10 to be modulated by IL-1α, during the inflammatory response to HSV-1 corneal infection. Both corneal cells and neutrophils contribute to the production of T cell recruiting chemokines. However, IL-1α has the potential to upregulate MIG production by corneal cells while down-regulating MIG production by neutrophils.

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.

Effect of Dry Eye Disease on the Kinetics of Lacrimal Gland Dendritic Cells as Visualized by Intravital Multi-Photon Microscopy.

The lacrimal gland (LG) is the main source of the tear film aqueous layer and its dysfunction results in dry eye disease (DED), a chronic immune-mediated disorder of the ocular surface. The desiccating stress (DS) murine model that mimics human DED, results in LG dysfunction, immune cell infiltration, and consequently insufficient tear production. To date, the immune cell kinetics in DED are poorly understood. The purpose of this study was to develop a murine model of intravital multi-photon microscopy (IV-MPM) for the LG, and to investigate the migratory kinetics and 3D morphological properties of conventional dendritic cells (cDCs), the professional antigen presenting cells of the ocular surface, in DED. Mice were placed in a controlled environmental chamber with low humidity and increased airflow rate for 2 and 4 weeks to induce DED, while control naïve transgenic mice were housed under standard conditions. DED mice had significantly decreased tear secretion and increased fluorescein staining (p < 0.01) compared to naïve controls. Histological analysis of the LG exhibited infiltrating mononuclear and polymorphonuclear cells (p < 0.05), as well as increased LG swelling (p < 0.001) in DED mice compared to controls. Immunofluorescence staining revealed increased density of cDCs in DED mice (p < 0.001). IV-MPM of the LG demonstrated increased density of cDCs in the LGs of DED mice, compared with controls (p < 0.001). cDCs were more spherical in DED at both time points compared to controls (p < 0.001); however, differences in surface area were found at 2 weeks in DED compared with naïve controls (p < 0.001). Similarly, 3D cell volume was significantly lower at 2 weeks in DED vs. the naïve controls (p < 0.001). 3D instantaneous velocity and mean track speed were significantly higher in DED compared to naïve mice (p < 0.001). Finally, the meandering index, an index for directionality, was significant increased at 4 weeks after DED compared with controls and 2 weeks of DED (p < 0.001). Our IV-MPM study sheds light into the 3D morphological alterations and cDC kinetics in the LG during DED. While in naïve LGs, cDCs exhibit a more dendritic morphology and are less motile, they became more spherical with enhanced motility during DED. This study shows that IV-MPM represents a robust tool to study immune cell trafficking and kinetics in the LG, which might elucidate cellular alterations in immunological diseases, such as DED.

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.

HSV-1 Hijacks the Host DNA Damage Response in Corneal Epithelial Cells through ICP4-Mediated Activation of ATM.

Purpose: Herpes simplex virus type I (HSV-1) infection of corneal epithelial cells activates ataxia telangiectasia mutated (ATM), an apical kinase in the host DNA damage response pathway, whose activity is necessary for the progression of lytic HSV-1 infection. The purpose of this study is to investigate the mechanism of ATM activation by HSV-1 in the corneal epithelium, as well as its functional significance. Methods: Mechanistic studies were performed in cultured human corneal epithelial cell lines (hTCEpi, HCE), as well as in esophageal (EPC2) and oral (OKF6) cell lines. Transfection-based experiments were performed in HEK293 cells. HSV-1 infection was carried out using the wild-type KOS strain, various mutant strains (tsB7, d120, 7134, i13, n208), and bacterial artificial chromosomes (fHSVΔpac, pM24). Inhibitors of ATM (KU-55933), protein synthesis (cycloheximide), and viral DNA replication (phosphonoacetic acid) were used. Outcomes of infection were assayed using Western blotting, qRT-PCR, immunofluorescence, and comet assay. Results: This study demonstrates that HSV-1-mediated ATM activation in corneal epithelial cells relies on the viral immediate early gene product ICP4 and requires the presence of the viral genome in the host nucleus. We show that ATM activation is independent of viral genome replication, the ICP0 protein, and the presence of DNA lesions. Interestingly, ATM activity appears to be necessary at the onset of infection, but dispensable at the later stages. Conclusions: This study expands our understanding of HSV-1 virus-host interactions in the corneal epithelium and identifies potential areas of future investigation and therapeutic intervention in herpes keratitis.

Role of Insulin-Like Growth Factor Binding Protein-3 in the Pathogenesis of Herpes Stromal Keratitis.

Purpose: The goal of this study was to determine the role of insulin-like growth factor-binding protein-3 (IGFBP-3) in the pathogenesis of herpes stromal keratitis (HSK). Methods: In an unbiased approach, a membrane-based protein array was carried out to determine the level of expression of pro- and anti-angiogenic molecules in uninfected and HSV-1 infected corneas. Quantitative RT-PCR and ELISA assays were performed to measure the amounts of IGFBP-3 at mRNA and protein levels. Confocal microscopy documented the localization of IGFBP-3 in uninfected and infected corneal tissue. Flow cytometry assay showed the frequency of immune cell types in infected corneas from C57BL/6J (B6) and IGFBP-3 knockout (IGFBP-3-/-) mice. Slit-lamp microscopy was used to quantitate the development of opacity and neovascularization in infected corneas from both groups of mice. Results: Quantitation of protein array dot blot showed an increased level of IGFBP-3 protein in HSV-1 infected than uninfected corneas and was confirmed with ELISA and quantitative RT-PCR assays. Cytosolic and nuclear localization of IGFBP-3 were detected in the cells of corneal epithelium, whereas scattered IGFBP-3 staining was evident in the stroma of HSK developing corneas. Increased opacity and hemangiogenesis were noted in the corneas of IGFBP-3-/- than B6 mice during the clinical period of HSK. Furthermore, an increased number of leukocytes comprising of neutrophils and CD4 T cells were found in HSK developing corneas of IGFBP-3-/- than B6 mice. Conclusions: Our data showed that lack of IGFBP-3 exacerbates HSK, suggesting the protective effect of IGFBP-3 protein in regulating the severity of HSK.

Herpes Simplex DNA in Tears of Atypical Dendritic Keratitis and Multiple Punctate Subepithelial Stromal Opacity: A Case Report.

PURPOSE: To report an atypical presentation of herpes simplex virus (HSV) keratitis followed up using expression levels of HSV DNA in tears. METHODS: A 22-year-old Japanese woman with hyperemia and foreign body sensation in her left eye was diagnosed with atypical dendritic keratitis. A slit-lamp examination at presentation indicated the presence of a rush of dendritic lesions with a sparse branching pattern and poor development of terminal bulbs; follicular conjunctivitis was also observed. Positivity for house-dust-mite- and cedar pollen-specific IgE antibodies in her serum indicated atopic diathesis. The HSV DNA levels in her tears were measured by a real-time polymerase chain reaction. RESULTS: At the initial visit, the HSV DNA levels in tears were 6.4 × 10 copies/sample in the right eye and 1.6 × 10 copies/sample in the left eye. The keratitis improved after treatment with topical acyclovir ointment, 5 times a day for 7 days, and systemic valacyclovir 1000 mg/d for 5 days. Multiple punctate subepithelial opacities developed in her left eye on day 7, with undetectable HSV DNA in tears, bilaterally. CONCLUSIONS: We have successfully monitored the HSV DNA levels in tears using quantitative real-time polymerase chain reaction in HSV keratitis where the corneal findings progressed from atypical dendritic keratitis to multiple punctate corneal subepithelial opacities during the treatment period.

Blockade of IL-27 signaling ameliorates herpes stromal keratitis with upregulated CD4+ Foxp3+ regulatory T cells influx in mice.

Purpose: The purpose of this study was to investigate the production of IL-27 p28 and EBI3 in the ocular inflammatory sites, and the role of IL-27 signaling in a model of HSV-1 induced herpetic stromal keratitis (HSK). Methods: The BALB/c mice were injected intraperitoneally (24 h before infection) with anti-IL-27 antibody or IgG antibody as control, infected with HSV-1 via corneal scarification, and then injected intraperitoneally with anti-IL-27 antibody or IgG antibody at 1, 3, and 5 days postinfection. Slit lamp and histopathology were used to assess disease outcome. The levels of IL-27 p28 and EBI3 in corneas were determined by western blotting and immunofluorescence. Furthermore, viral titers were determined, and immune cell infiltrates were collected and analyzed by flow cytometry. Results: We found that the levels of IL-27 p28 and EBI3 in corneas were elevated significantly at the peak of HSK, and both of them were expressed simultaneously in the epithelium, stroma, and endothelium of corneas. In the group of anti-IL-27 treatment, the severity of the corneal lesion and CD4+ T cells infiltration were significantly decreased, and the percentage of CD4+ Foxp3+ Tregs was upregulated markedly in the spleen, DLNs and cornea of HSK mice compared to IgG treatment. Conclusion: These results provided evidence that IL-27 as a pathogenic pro-inflammatory cytokine controlled CD4+ Foxp3+ Tregs production in HSK, which ultimately resulted in promoting the progression of HSK and poor prognosis.

NLRP3, NLRP12, and IFI16 Inflammasomes Induction and Caspase-1 Activation Triggered by Virulent HSV-1 Strains Are Associated With Severe Corneal Inflammatory Herpetic Disease.

The crosstalk between the host's inflammasome system and the invading virulent/less-virulent viruses determines the outcome of the ensuing inflammatory response. An appropriate activation of inflammasomes triggers antiviral inflammatory responses that clear the virus and heal the inflamed tissue. However, an aberrant activation of inflammasomes can result in a harmful and overwhelming inflammation that could damage the infected tissue. The underlying host's immune mechanisms and the viral virulent factors that impact severe clinical inflammatory disease remain to be fully elucidated. In this study, we used herpes simplex virus type 1 (HSV-1), the causative agent of corneal inflammatory herpetic disease, as a model pathogen to determine: (i) Whether and how the virulence of a virus affects the type and the activation level of the inflammasomes; and (ii) How triggering specific inflammasomes translates into protective or damaging inflammatory response. We showed that, in contrast to the less-virulent HSV-1 strains (RE, F, KOS, and KOS63), corneal infection of B6 mice with the virulent HSV-1 strains (McKrae, 17 or KOS79): (i) Induced simultaneous expression of the NLRP3, NLRP12, and IFI16 inflammasomes; (ii) Increased production of the biologically active Caspase-1 and pro-inflammatory cytokines IL-1ß and IL-18; (iii) Heightened recruitment into the inflamed cornea of CD45highLy6C+Ly6G-F4/80+CD11b+CD11c- inflammatory monocytes and CD45highCD11b+F4/80-Ly6GhiLy6Cmed neutrophils; and (iv) This intensified inflammatory response was associated with a severe corneal herpetic disease, irrespective of the level of virus replication in the cornea. Similarly, in vitro infection of human corneal epithelial cells and human monocytic THP-1 cells with the virulent HSV-1 strains triggered a synchronized early expression of NLRP3, NLRP12 and IFI16, 2 h post-infection, associated with formation of single and dense specks of the adapter molecule ASC in HSV(+) cells, but not in the neighboring bystander HSV(-) cells. This was associated with increased cleavages of Caspase-1, IL-1ß, and IL-18. These findings suggest a previously unappreciated role of viral virulence in a synchronized early induction of the NLRP3, NLRP12, and IFI16 inflammasomes that lead to a damaging inflammatory response. A potential role of common virus virulent factors that stimulate this harmful inflammatory corneal disease is currently under investigation.

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.

The US11 Gene of Herpes Simplex Virus 1 Promotes Neuroinvasion and Periocular Replication following Corneal Infection.

Herpes simplex virus 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation, US11 through preventing PKR activation and ICP34.5 through mediating dephosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). While profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11-null virus and examined its properties in vitro and in vivo In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2α phosphorylation late in infection. However, the effect was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2α unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2α correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for the US11-null virus than for the wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia were unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of antiviral response of infected cells and promotes the efficient emergence of virus following reactivation.IMPORTANCE Alphaherpesviruses are ubiquitous DNA viruses and include the human pathogens herpes simplex virus 1 (HSV-1) and HSV-2 and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent coexistence with host immune defenses are dependent on the ability of these viruses to counter the antiviral response. HSV-1 and HSV-2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses.