Complement Suppresses the Initial Type 1 Interferon Response to Ocular Herpes Simplex Virus Type 1 Infection in Mice.

The complement system (CS) contributes to the initial containment of viral and bacterial pathogens and clearance of dying cells in circulation. We previously reported mice deficient in complement component 3 (C3KO mice) were more sensitive than wild-type (WT) mice to ocular HSV-1 infection, as measured by a reduction in cumulative survival and elevated viral titers in the nervous system but not the cornea between days three and seven post infection (pi). The present study was undertaken to determine if complement deficiency impacted virus replication and associated changes in inflammation at earlier time points in the cornea. C3KO mice were found to possess significantly (p < 0.05) less infectious virus in the cornea at 24 h pi that corresponded with a decrease in HSV-1 lytic gene expression at 12 and 24 h pi compared to WT animals. Flow cytometry acquisition found no differences in the myeloid cell populations residing in the cornea including total macrophage and neutrophil populations at 24 h pi with minimal infiltrating cell populations detected at the 12 h pi time point. Analysis of cytokine and chemokine content in the cornea measured at 12 and 24 h pi revealed that only CCL3 (MIP-1α) was found to be different between WT and C3KO mice with >2-fold increased levels (p < 0.05, ANOVA and Tukey's post hoc t-test) in the cornea of WT mice at 12 h pi. C3KO mouse resistance to HSV-1 infection at the early time points correlated with a significant increase in type I interferon (IFN) gene expression including IFN-α1 and IFN-ß and downstream effector genes including tetherin and RNase L (p < 0.05, Mann-Whitney rank order test). These results suggest early activation of the CS interferes with the induction of the type I IFN response and leads to a transient increase in virus replication following corneal HSV-1 infection.

HSV-1 0∆NLS vaccine elicits a robust B lymphocyte response and preserves vision without HSV-1 glycoprotein M or thymidine kinase recognition.

Effective experimental prophylactic vaccines against viral pathogens such as herpes simplex virus type 1 (HSV-1) have been shown to protect the host through T and/or B lymphocyte-driven responses. Previously, we found a live-attenuated HSV-1 mutant, 0ΔNLS used as a prophylactic vaccine, provided significant protection against subsequent ocular HSV-1 challenge aligned with a robust neutralizing antibody response. Yet, how the virus mutant elicited the humoral immune response relative to parental virus was unknown. Herein, we present the characterization of B cell subsets in vaccinated mice at times after primary vaccination and following boost compared to the parental virus, termed GFP105. We found that 0∆NLS-vaccinated mice possessed more CD4+ follicular helper T (TFH) cells, germinal B cells and class-switched B cells within the first 7 days post-vaccination. Moreover, 0∆NLS vaccination resulted in an increase in plasmablasts and plasma cells expressing amino-acid transporter CD98 along with an elevated titer of HSV-1-specific antibody compared to GFP105-vaccinated animals. Furthermore, O∆NLS-vaccine-induced CD4+ (TFH) cells produced significantly more IL-21 compared to mice immunized with the parental HSV-1 strain. In contrast, there were no differences in the number of regulatory B cells comparing the two groups of immunized mice. In comparing sera recognition of HSV-1-encoded proteins, it was noted antiserum from GFP105-vaccinated mice immunoprecipitated HSV-1 thymidine kinase (TK) and glycoprotein M (gM) whereas sera from 0∆NLS-immunized mice did not even though both groups of vaccinated mice displayed similar neutralizing antibody titers to HSV-1 and were highly resistant to ocular HSV-1 challenge. Collectively, the results suggest (1) the live-attenuated HSV-1 mutant 0∆NLS elicits a robust B cell response that drives select B cell responses greater than the parental HSV-1 and (2) HSV-1 TK and gM are likely expendable components in efficacy of a humoral response to ocular HSV-1 infection.

Tripartite-Motif 21 (TRIM21) Deficiency Results in a Modest Loss of Herpes Simplex Virus (HSV)-1 Surveillance in the Trigeminal Ganglia Following Cornea Infection.

Tripartite-motif 21 (TRIM21) is thought to regulate the type I interferon (IFN) response to virus pathogens and serve as a cytosolic Fc receptor for immunoglobulin. Since herpes simplex virus (HSV)-1 is sensitive to type I IFN and neutralizing antibody, we investigated the role of TRIM21 in response to ocular HSV-1 infection in mice. In comparison to wild type (WT) mice, TRIM21 deficient (TRIM21 KO) mice were found to be no more susceptible to ocular HSV-1 infection than WT animals, in terms of infectious virus recovered in the cornea. Similar pathology, in terms of neovascularization, opacity, and loss of peripheral vision function, was observed in both WT and TRIM21 KO mice. However, TRIM21 KO mice did possess a significant increase in infectious virus recovered in the trigeminal ganglia, in comparison to the WT animals. The increased susceptibility was not due to changes in HSV-1-specific CD4+ or CD8+ T cell numbers or functional capabilities, or in changes in type I IFN or IFN-inducible gene expression. In summary, the absence of TRIM21 results in a modest, but significant, increase in HSV-1 titers recovered from the TG of TRIM21 KO mice during acute infection, by a mechanism yet to be determined.

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 Durability of Vaccine Efficacy against Ocular HSV-1 Infection Using ICP0 Mutants 0∆NLS and 0∆RING Is Lost over Time.

Vaccines to viral pathogens in experimental animal models are often deemed successful if immunization enhances resistance of the host to virus challenge as measured by cumulative survival, reduction in virus replication and spread and/or lessen or eliminate overt tissue pathology. Furthermore, the duration of the protective response against challenge is another important consideration that drives a vaccination regimen. In the current study, we assessed the durability of two related vaccines, 0∆NLS and 0∆RING, against ocular herpes simplex virus type 1 (HSV-1) challenge in mice thirty days (short-term) and one year (long-term) following the vaccine boost. The short-term vaccine efficacy study found the 0∆RING vaccine to be nearly equivalent to the 0∆NLS vaccine in comparison to vehicle-vaccinated mice in terms of controlling virus replication and preserving the visual axis. By comparison, the long-term assessment of the two vaccines found notable differences and less efficacy overall as noted below. Specifically, the results show that in comparison to vehicle-vaccinated mice, the 0∆NLS and 0∆RING vaccinated groups were more resistant in terms of survival and virus shedding following ocular challenge. Moreover, 0∆NLS vaccinated mice also possessed significantly less infectious virus in the peripheral and central nervous systems but not the cornea compared to mice vaccinated with vehicle or 0∆RING which had similar levels. However, all vaccinated groups showed similar levels of blood and lymphatic vessel genesis into the central cornea 30 days post infection. Likewise, corneal opacity was also similar among all groups of vaccinated mice following infection. Functionally, the blink response and visual acuity were 25-50% lower in vaccinated mice 30 days post infection compared to measurements taken prior to infection. The results demonstrate a dichotomy between resistance to infection and functional performance of the visual axis that collectively show an overall loss in vaccine efficacy long-term in comparison to short-term studies in a conventional prime-boost protocol.

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.

Lack of neonatal Fc receptor does not diminish the efficacy of the HSV-1 0ΔNLS vaccine against ocular HSV-1 challenge.

The neonatal Fc receptor (FcRn) is constitutively expressed in the cornea and is up-regulated in response to herpes simplex virus type 1 (HSV-1). Previously, we found targeting cornea FcRn expression by small interfering RNA-mediated knockdown reduced the local efficacy of HSV-1 0ΔNLS vaccinated C57BL/6 mice against ocular challenge with HSV-1. The current study was undertaken to evaluate the HSV-1 0ΔNLS vaccine efficacy in FcRn deficient (FcRn KO) mice challenged with HSV-1. Whereas there was little neutralizing antibody detected in the serum of HSV-1 0ΔNLS vaccinated FcRn KO mice, these mice exhibited the same degree of protection against ocular challenge with HSV-1 as wild type (WT) C57BL/6 mice as measured by cumulative survival, infectious virus shed or retained in tissue, and corneal pathology including opacity and neovascularization. Mock-vaccinated FcRn KO mice were found to be more sensitive to ocular HSV-1 infection compared to mock-vaccinated (WT) mice in terms of cumulative survival and virus shedding. In addition, the FcRn KO mice generated significantly fewer effector (CD3+CD44+CD62L-) and central (CD3+CD44+CD62L+) memory CD8+ T cells compared to the WT mice 7 days post infection. Collectively, mock-vaccinated FcRn KO mice are susceptible to ocular HSV-1 infection but HSV-1 0ΔNLS vaccinated FcRn KO mice are resistant suggesting that in addition to the FcRn, other pathways are involved in mediating the protective effect of the HSV-1 0ΔNLS vaccine against subsequent HSV-1 challenge.

Assessment of Two Novel Live-Attenuated Vaccine Candidates for Herpes Simplex Virus 2 (HSV-2) in Guinea Pigs.

Treatment to ameliorate the symptoms of infection with herpes simplex virus 2 (HSV-2) and to suppress reactivation has been available for decades. However, a safe and effective preventative or therapeutic vaccine has eluded development. Two novel live-attenuated HSV-2 vaccine candidates (RVx201 and RVx202) have been tested preclinically for safety. Hartley guinea pigs were inoculated vaginally (n = 3) or intradermally (n = 16) with either vaccine candidate (2 × 107 PFU) and observed for disease for 28 days. All animals survived to study end without developing HSV-2-associated disease. Neither vaccine candidate established latency in dorsal root or sacral sympathetic ganglia, as determined by viral DNA quantification, LAT expression, or explant reactivation. Infectious virus was shed in vaginal secretions for three days following vaginal inoculation with RVx202, but not RVx201, although active or latent HSV-2 was not detected at study end. In contrast, guinea pigs inoculated with wild-type HSV-2 MS (2 × 105 PFU) vaginally (n = 5) or intradermally (n = 16) developed acute disease, neurological signs, shed virus in vaginal secretions, experienced periodic recurrences throughout the study period, and had latent HSV-2 in their dorsal root and sacral sympathetic ganglia at study end. Both vaccine candidates generated neutralizing antibody. Taken together, these findings suggest that these novel vaccine candidates are safe in guinea pigs and should be tested for efficacy as preventative and/or therapeutic anti-HSV-2 vaccines.

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.

Neuroretinal-Derived Caveolin-1 Promotes Endotoxin-Induced Inflammation in the Murine Retina.

Purpose: The immune-privileged environment and complex organization of retinal tissue support the retina's essential role in visual function, yet confound inquiries into cell-specific inflammatory effects that lead to dysfunction and degeneration. Caveolin-1 (Cav1) is an integral membrane protein expressed in several retinal cell types and is implicated in immune regulation. However, whether Cav1 promotes or inhibits inflammatory processes in the retina (as well as in other tissues) remains unclear. Previously, we showed that global-Cav1 depletion resulted in reduced retinal inflammatory cytokine production but paradoxically elevated retinal immune cell infiltration. We hypothesized that these disparate responses are the result of differential cell-specific Cav1 functions in the retina. Methods: We used Cre/lox technology to deplete Cav1 specifically in the neural retinal (NR) compartment to clarify the role NR-specific Cav1 (NR-Cav1) in the retinal immune response to intravitreal inflammatory challenge induced by activation of Toll-like receptor-4 (TLR4). We used multiplex protein suspension array and flow cytometry to evaluate innate immune activation. Additionally, we used bioinformatics assessment of differentially expressed membrane-associated proteins to infer relationships between NR-Cav1 and immune response pathways. Results: NR-Cav1 depletion, which primarily affects Müller glia Cav1 expression, significantly altered immune response pathway regulators, decreased retinal inflammatory cytokine production, and reduced retinal immune cell infiltration in response to LPS-stimulated inflammatory induction. Conclusions: Cav1 expression in the NR compartment promotes the innate TLR4-mediated retinal tissue immune response. Additionally, we have identified novel potential immune modulators differentially expressed with NR-Cav1 depletion. This study further clarifies the role of NR-Cav1 in retinal inflammation.

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.

"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.

Ocular Glands Become Infected Secondarily to Infectious Keratitis and Play a Role in Corneal Resistance to Infection.

Ocular glands play a critical role in eye health through the secretion of factors directly onto the ocular surface. The cornea is a normally transparent tissue necessary for visual acuity located in the anterior segment of the eye. Corneal damage can occur during microbial infection of the cornea, resulting in potentially permanent visual deficits. The involvement of ocular glands during corneal infection has been only briefly described. We hypothesized that ocular glands contribute to resistance as an arm of the eye-associated lymphoid tissue and may also be susceptible to infection secondary to microbial keratitis. Utilizing a mouse model of herpes simplex virus 1 (HSV-1) keratitis, we found that infection of corneas resulted in subsequent infection of ocular glands, including harderian glands (HGs) and extraorbital glands. Similarly, infection of corneas with Pseudomonas aeruginosa resulted in secondary infection of ocular glands. A robust immune response, characterized by increased numbers of immune cells and inflammatory mediators, occurred within ocular glands following HSV-1 keratitis. Removal of HGs altered corneal resistance to HSV-1, as measured by increased viral load, decreased corneal edema, and decreased inflammatory cell infiltration. These novel findings suggest that ocular glands are involved in microbial keratitis through their susceptibility to secondary infection and contribution to corneal resistance.IMPORTANCE Microbial keratitis accounts for up to 700,000 clinical visits annually in the United States. The involvement of ocular glands during microbial keratitis is not readily appreciated, and treatment options do not address the consequences of ocular gland dysfunction. The present study shows that ocular glands are susceptible to direct infection by common ocular pathogens, including HSV-1 and Pseudomonas aeruginosa, subsequent to microbial keratitis. Additionally, ocular glands contribute soluble factors that play a role in corneal resistance to HSV-1 and alter viral load, corneal edema, and immune cell infiltration. Further studies are needed to elucidate the mechanisms by which this occurs.

Vaccine-induced antibodies target sequestered viral antigens to prevent ocular HSV-1 pathogenesis, preserve vision, and preempt productive neuronal infection.

The cornea is essential for vision yet highly sensitive to immune-mediated damage following infection. Generating vaccines that provide sterile immunity against ocular surface pathogens without evoking vision loss is therefore clinically challenging. Here, we tested a prophylactic live-attenuated vaccine against herpes simplex virus type 1 (HSV-1), a widespread human pathogen that can cause corneal blindness. Parenteral vaccination of mice resulted in sterile immunity to subsequent HSV-1 challenge in the cornea and suppressed productive infection of the nervous system. This protection was unmatched by a relevant glycoprotein subunit vaccine. Efficacy of the live-attenuated vaccine involved a T-dependent humoral immune response and complement C3 but not Fcγ-receptor 3 or interferon-α/ß signaling. Proteomic analysis of viral proteins recognized by antiserum revealed an unexpected repertoire dominated by sequestered antigens rather than surface-exposed envelope glycoproteins. Ocular HSV-1 challenge in naive and subunit-vaccinated mice triggered vision loss and severe ocular pathologies including corneal opacification, scar formation, neovascularization, and sensation loss. However, corneal pathology was absent in mice receiving the live-attenuated vaccine concomitant with complete preservation of visual acuity. Collectively, this is the first comprehensive report of a prophylactic vaccine candidate that elicits resistance to ocular HSV-1 infection while fully preserving the cornea and visual acuity.

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.