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Herpes simplex virus type 1 (HSV-1) has the ability to delay its clearance from the eye during ocular infection. Here, we show that ocular infection of mice with HSV-1 suppressed expression of the costimulatory molecule CD80 but not CD86 in the cornea. The presence of neutralizing anti-HSV-1 antibodies did not alleviate this suppression. At the cellular level, HSV-1 consistently downregulated the expression of CD80 by dendritic cells (DCs) but not by other antigen-presenting cells. Furthermore, flow cytometric analysis of HSV-1-infected corneal cells during a 7-day period reduced CD80 expression in DCs but not in B cells, macrophages, or monocytes. This suppression was associated with the presence of virus. Similar results were obtained using infected or transfected spleen cells or bone marrow-derived DCs. A combination of roscovitine treatment, transfection with immediate early genes (IE), and infection with a recombinant HSV-1 lacking the ICP22 gene shows the importance of ICP22 in downregulation of the CD80 promoter but not the CD86 promoter in vitro and in vivo At the mechanistic level, we show that the HSV-1 immediate early gene ICP22 binds the CD80 promoter and that this interaction is required for HSV-1-mediated suppression of CD80 expression. Conversely, forced expression of CD80 by ocular infection of mice with a recombinant HSV-1 exacerbated corneal scarring in infected mice. Taken together, these studies identify ICP22-mediated suppression of CD80 expression in dendritic cells as central to delayed clearance of the virus and limitation of the cytopathological response to primary infection in the eye. IMPORTANCE HSV-1-induced eye disease is a major public health problem. Eye disease is associated closely with immune responses to the virus and is exacerbated by delayed clearance of the primary infection. The immune system relies on antigen-presenting cells of the innate immune system to activate the T cell response. We found that HSV-1 utilizes a robust and finely targeted mechanism of local immune evasion. It downregulates the expression of the costimulatory molecule CD80 but not CD86 on resident dendritic cells irrespective of the presence of anti-HSV-1 antibodies. The effect is mediated by direct binding of HSV-1 ICP22, the product of an immediate early gene of HSV-1, to the promoter of CD80. This immune evasion mechanism dampens the host immune response and, thus, reduces eye disease in ocularly infected mice. Therefore, ICP22 may be a novel inhibitor of CD80 that could be used to modulate the immune response., (Copyright © 2019 American Society for Microbiology.)

HSV-1-induced corneal scarring (CS), also broadly referred to as Herpes Stromal Keratitis (HSK), is the leading cause of infectious blindness in developed countries. It is well-established that HSK is in fact an immunopathological disease. The contribution of the potentially harmful T cell effectors that lead to CS remains an area of intense study. Although the HSV-1 gene(s) involved in eye disease is not yet known, we have demonstrated that gK, which is one of the 12 known HSV-1 glycoproteins, has a crucial role in CS. Immunization of HSV-1 infected mice with gK, but not with any other known HSV-1 glycoprotein, significantly exacerbates CS, and dermatitis. The gK-induced eye disease occurs independently of the strain of the virus or mouse. HSV-1 mutants that lack gK are unable to efficiently infect and establish latency in neurons. HSV-1 recombinant viruses expressing two additional copies of the gK (total of three gK genes) exacerbated CS as compared with wild type HSV-1 strain McKrae that contains one copy of gK. Furthermore, we have shown that an 8mer (ITAYGLVL) within the signal sequence of gK enhanced CS in ocularly infected BALB/c mice, C57BL/6 mice, and NZW rabbits. In HSV-infected "humanized" HLA-A * 0201 transgenic mice, this gK 8mer induced strong IFN-γ-producing cytotoxic CD8 + T cell responses. gK induced CS is dependent on gK binding to signal peptide peptidase (SPP). gK also binds to HSV-1 UL20, while UL20 binds GODZ (DHHC3) and these quadruple interactions are required for gK induced pathology. Thus, potential therapies might include blocking of gK-SPP, gK-UL20, UL20-GODZ interactions, or a combination of these strategies.

Recently, we have reported that CD8α(+) DCs, rather than CD8(+) T cells, are involved in the establishment and maintenance of HSV-1 latency in the trigeminal ganglia (TG) of ocularly infected mice. In the current study, we investigated whether similar results can be obtained using Batf3(-/-) mice that previously were reported to lack CD8α(+) DCs. However, our results demonstrate that Batf3(-/-) mice, without any known infection, express CD8α(+) DCs. Consequently, due to the presence of CD8α(+) DCs, no differences were detected in the level of HSV-1 latency between Batf3(-/-) mice compared with wild type control mice., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The development of immunization strategies to protect against ocular infection with herpes simplex virus 1 (HSV-1) must address the issue of the effects of the strategy on the establishment of latency in the trigeminal ganglia (TG). It is the reactivation of this latent virus that can cause recurrent disease and corneal scarring. CD8(+) T cells and dendritic cells (DCs) have been implicated in the establishment and maintenance of latency through several lines of inquiry. The objective of the current study was to use CD8α(-/-) and CD8β(-/-) mice to further evaluate the contributions of CD8(+) T cells and the CD8α(+) and CD8α(-) subpopulations of DCs to the protection afforded against ocular infection by immunization against HSV-1 and their potential to increase latency. Neutralizing antibody titers were similar in immunized CD8α(-/-), CD8β(-/-), and wild-type (WT) mice, as was virus replication in the eye. However, on day 3 postinfection (p.i.), the copy number of HSV-1 glycoprotein B (gB) was higher in the corneas and TG of CD8α(-/-) mice than those of WT mice, whereas on day 5 p.i. it was lower. As would be anticipated, the lack of CD8α(+) or CD8β(+) cells affected the levels of type I and type II interferon transcripts, but the effects were markedly time dependent and tissue specific. The levels of latent virus in the TG, as estimated by measurement of LAT transcripts and in vitro explant reactivation assays, were lower in the immunized, ocularly challenged CD8α(-/-) and WT mice than in their CD8β(-/-) counterparts. Immunization reduced the expression of PD-1, a marker of T-cell exhaustion, in the TG of ocularly challenged mice, and mock-immunized CD8α(-/-) mice had lower levels of PD-1 expression and latency than mock-immunized WT or CD8β(-/-) mice. The expansion of the CD8α(-) subpopulation of DCs through injection of WT mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA reduced the amount of latency and PD-1 expression in the TG of infected mice. In contrast, injection of FMS-like tyrosine kinase 3 ligand (Flt3L) DNA, which expanded both subpopulations, was less effective. Our results suggest that the absence of both CD8α(+) T cells and CD8α(+) DCs does not reduce vaccine efficacy, either directly or indirectly, in challenged mice and that administration of GM-CSF appears to play a beneficial role in reducing latency and T-cell exhaustion. Importance: In the past 2 decades, two large clinical HSV vaccine trials were performed, but both vaccine studies failed to reach their goals. Thus, as an alternative to conventional vaccine studies, we have used a different strategy to manipulate the host immune responses in an effort to induce greater protection against HSV infection. In lieu of the pleiotropic effect of CD8α(+) DCs in HSV-1 latency, in this report, we show that the absence of CD8α(+) T cells and CD8α(+) DCs has no adverse effect on vaccine efficacy. In line with our hypothesis, we found that pushing DC subpopulations from CD8α(+) DCs toward CD8α(-) DCs by injection of GM-CSF reduced the amount of latent virus and T-cell exhaustion in TG. While these studies point to the lack of a role for CD8α(+) T cells in vaccine efficacy, they in turn point to a role for GM-CSF in reducing HSV-1 latency., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid (CSF) of both human neonates with preterm hydrocephalus, and P8 hydrocephalic mice. We hypothesized CSF CSPGs are synthesized by neural precursors, separated from ventricular CSF by ependyma, which is often disrupted in hydrocephalus. Western blotting demonstrates that neural precursors cultured as neurospheres secrete CSPGs (> 30 microg/ml) into their media which appear to be very similar to these CSF CSPGs. Some CSPGs bear the stage-specific embryonic antigen-1 (ssea-1), associated with embryonic/neural stem cells. Neurospheres transcribe many CSPG genes, including the entire aggrecan/lectican family, phosphacan, and tenascin. Phosphacan can be detected in media by Western blotting. Aggrecan can be detected in media after purification using hyaluronic acid affinity chromatography. During differentiation, neurospheres downregulate CSPGs. This is the first report to show that proliferating neural precursors synthesize lecticans, including aggrecan, which are downregulated with differentiation. These observations suggest novel links between CSPGs and CNS precursor biology.