Infected Trigeminal Ganglia Research Articles

Control of the Calcitonin Gene-related Peptide Enhancer by Upstream Stimulatory Factor in Trigeminal Ganglion Neurons

The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. However, the transcription factors controlling CGRP expression in the migraine-relevant trigeminal ganglion neurons are unknown. Previous in vitro studies demonstrated that upstream stimulatory factor (USF) 1 and USF2 bind to the CGRP neuroendocrine-specific 18-bp enhancer, yet discrepant overexpression results in cell lines, and the ubiquitous nature of the USF cast doubts about its role. To test the functional role of USF, we first demonstrated that small interfering RNAs directed against USF1 and USF2 reduced endogenous CGRP RNA and preferentially targeted the USF binding site at the 18-bp enhancer in the neuronal-like CA77 cell line. In cultured rat trigeminal ganglion neurons, knockdown of either USF1 or USF2 reduced CGRP promoter activity. Conversely, overexpression of USF1 or USF2 increased promoter activity. The activation was even greater upon cotransfection with an upstream activator of mitogen-activated protein kinases and was synergistic in a heterologous cell line. To begin to address the paradox of how ubiquitous USF proteins might direct neuronal-specific activity, we examined USF expression and used a series of adenoviral reporters in the cultured ganglia. Unexpectedly, there was more intense USF immunostaining in neurons than nonneuronal cells. Importantly, the 18-bp USF enhancer driving a minimal promoter was sufficient for neuronal specificity, although it was not the only site that directed neuronal expression. These results demonstrate that USF1 and USF2 are important contributors to neuronal-specific and mitogen-activated protein kinase regulation of the CGRP gene in trigeminal ganglion neurons.

The number of herpes simplex virus-infected neurons and the number of viral genome copies per neuron correlate with the latent viral load in ganglia

The latent viral load is the most important factor that predicts reactivation rates of animals latently infected with herpes simplex virus (HSV). To estimate the latent viral load, individual latently infected mouse trigeminal ganglia were dispersed into single cell suspensions and plated into 96-well real-time PCR plates, and HSV-2 genome copies were measured. By assuming a Poisson distribution for both the number of HSV-2 infected cells per well and the number of HSV-2 genome copies per infected cell, the numbers of infected cells and mean genome copies per infected cell were determined. Both the number of HSV-2 infected cells and the mean HSV-2 genome copy per infected cell significantly correlated with the latent viral load ( p < 10 − 4 ), indicating that both factors are responsible for the increase in the latent viral load.

A Recombinant Herpes Simplex Virus Type 1 Expressing Two Additional Copies of gK Is More Pathogenic than Wild-Type Virus in Two Different Strains of Mice

The effect of glycoprotein K (gK) overexpression on herpes simplex virus type 1 (HSV-1) infection in two different strains of mice was evaluated using a recombinant HSV-1 virus that expresses two additional copies of the gK gene in place of the latency-associated transcript (LAT). This mutant virus (HSV-gK3) expressed higher levels of gK than either the wild-type McKrae virus or the parental dLAT2903 virus both in vitro (in cultured cells) and in vivo (in infected mouse corneas and trigeminal ganglia [TG] of BALB/c and C57BL/6 mice). gK transcripts were detected in the TG of both HSV-gK3-infected mouse strains on day 30 postinfection (p.i.), while gB transcripts were detected only in the TG of the HSV-gK3-infected C57BL/6 mice, a finding that suggests that increased gK levels promote chronic infection. C57BL/6 mice infected with HSV-gK3 also contained free virus in their TG on day 30 p.i. Both HSV-gK3-infected mouse strains had significantly higher corneal scarring (CS) than did McKrae-infected mice. T-cell depletion studies in C57BL/6 mice suggested that this CS enhancement in the HSV-gK3-infected mice was mediated by a CD8+ T-cell response. Taken together, these results strongly suggest that increased gK levels promote eye disease and chronic infection in infected mice.

Relaxed Repression of Herpes Simplex Virus Type 1 Genomes in Murine Trigeminal Neurons

The expression of herpes simplex virus (HSV) genomes in the absence of viral regulatory proteins in sensory neurons is poorly understood. Previously, our group reported an HSV immediate early (IE) mutant (d109) unable to express any of the five IE genes and encoding a model human cytomegalovirus immediate early promoter-green fluorescent protein (GFP) transgene. In cultured cells, GFP expressed from this mutant was observed in only a subset of infected cells. The subset exhibited cell type dependence, as the fractions of GFP-expressing cells varied widely among the cell types examined. Herein, we characterize this mutant in murine embryonic trigeminal ganglion (TG) cultures. We found that d109 was nontoxic to neural cultures and persisted in the cultures throughout their life spans. Unlike with some of the cultured cell lines and strains, expression of the GFP transgene was observed in a surprisingly large subset of neurons. However, very few nonneuronal cells expressed GFP. The abilities of ICP0 and an inhibitor of histone deacetylase, trichostatin A (TSA), to activate GFP expression from nonexpressing cells were also compared. The provision of ICP0 by infection with d105 reactivated quiescent genomes in nearly every cell, whereas reactivation by TSA was much more limited and restricted to the previously nonexpressing neurons. Moreover, we found that d109, which does not express ICP0, consistently reactivated HSV type 1 (KOS) in latently infected adult TG cultures. These results suggest that the state of persisting HSV genomes in some TG neurons may be more dynamic and more easily activated than has been observed with nonneuronal cells.

Spontaneous Reactivation of Herpes Simplex Virus Type 1 in Latently Infected Murine Sensory Ganglia

Careful studies of mouse trigeminal ganglia (TG) latently infected with herpes simplex virus type 1 (HSV-1) indicate the presence of productive cycle viral gene products and persistent immune response, suggesting ongoing spontaneous viral reactivation in these tissues. In the present study we set out to determine whether infectious virus is present in murine TG latently infected with HSV-1 (KOS). At 37 days after ocular inoculation we found a small amount of infectious virus in ca. 6% of latently infected murine TG. Furthermore, the amount of infectious virus that we detected (PFU per viral antigen-positive neuron) was similar to that detected in acutely infected ganglia. We conclude that spontaneous reactivation of infectious HSV-1 occurs in the mouse TG and is likely the principle cause of viral protein expression in these tissues. We next examined the role of latency-associated transcript (LAT) in spontaneous ganglionic reactivation by examining ganglia latently infected with KOS dlLAT1.8, a LAT deletion virus. Through the use of immunocytochemistry we found that KOS dlLAT1.8 had a rate of spontaneous ganglionic reactivation very similar to that of HSV-1 (KOS). Studying spontaneous ganglionic reactivation of HSV in the mouse TG allows a direct study of viral reactivation from latently infected neurons without the potential confounders and complicating downstream events that accompany the study of viral reactivation by explantation or peripheral viral shedding. Since most cases of human viral shedding and reactivation are not associated with a known trigger, spontaneous ganglionic reactivation of HSV-1 may be a better model of human disease than existing models.

Expression of Extremely Low Levels of Thymidine Kinase from an Acyclovir-Resistant Herpes Simplex Virus Mutant Supports Reactivation from Latently Infected Mouse Trigeminal Ganglia

A single-cytosine-deletion in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. A laboratory strain engineered to carry this mutation did not generate sufficient TK activity for detection by plaque autoradiography, which detected 0.25% wild-type activity. However, a drug sensitivity assay suggested that extremely low levels of TK are generated by this virus. The virus was estimated to express 0.09% of wild-type TK activity via a ribosomal frameshift 24 nucleotides upstream of the mutation. Remarkably, this appeared to be sufficient active TK to support a low level of reactivation from latently infected mouse trigeminal ganglia.

Simian Varicella Virus Expresses a Latency-Associated Transcript That Is Antisense to Open Reading Frame 61 (ICP0) mRNA in Neural Ganglia of Latently Infected Monkeys

Simian varicella virus (SVV) and varicella-zoster virus (VZV) are closely related alphaherpesviruses that cause varicella (chickenpox) in nonhuman primates and humans, respectively. After resolution of the primary disease, SVV and VZV establish latent infection of neural ganglia and may later reactivate to cause a secondary disease (herpes zoster). This study investigated SVV gene expression in neural ganglia derived from latently infected vervet monkeys. SVV transcripts were detected in neural ganglia, but not in liver or lung tissues, of latently infected animals. A transcript mapping to open reading frame (ORF) 61 (herpes simplex virus type 1 [HSV-1] ICP0 homolog) was consistently detected in latently infected trigeminal, cervical, and lumbar ganglia by reverse transcriptase PCR. Further analysis confirmed that this SVV latency-associated transcript (LAT) was oriented antisense to the gene 61 mRNA. SVV ORF 21 transcripts were also detected in 42% of neural ganglia during latency. In contrast, SVV ORF 28, 29, 31, 62, and 63 transcripts were not detected in ganglia, liver, or lung tissues of latently infected animals. The results demonstrate that viral gene expression is limited during SVV latency and that a LAT antisense to an ICP0 homolog is expressed. In this regard, SVV gene expression during latency is similar to that of HSV-1 and other neurotropic animal alphaherpesviruses but differs from that reported for VZV.

Lytic granule components are required for the maintenance of HSV-1 neuronal latency (43.35)

Abstract Using a murine model of HSV-1 corneal infection, our data suggest that secretion of CD8 T cell lytic granules (LGs), in addition to IFN-γ, is required for the maintenance of viral latency within trigeminal ganglia (TG). First, HSV-specific CD8 T cells (HSV-CD8), which recognize an immunodominant HSV-1 glycoprotein B (gB498–505) epitope, surround neurons within latently infected TG and contain functional LGs, with GrB polarizing to the contact site with these neurons. HSV-CD8 use GrB and IFN-γ to block HSV-1 reactivation from latency in a non-redundant manner in ex vivo TG cultures, possibly due to a lack of IFN-γ receptors on some latently infected neurons. Like WT mice, Pfn and GrB KO mice establish latency in the TG by 8 days post-infection (dpi) with a similar viral genome copy number, but by 14 dpi, they exhibit a transient rise in viral genome copy number not seen in WT mice suggestive of HSV-1 reactivation from latency. A similar transient rise in copy number is also seen in WT mice depleted of CD8 T cells. Further, neither endogenous CD8 T cells within Pfn KO nor exogenous HSV-CD8 from GrB KO latently infected TG maintain latency as efficiently as CD8 T cells from WT TG in ex vivo TG cultures. Based on the current lack of evidence that CD8 T cell protection leads to neuronal death, we hypothesize a non-lethal LG-mediated block in HSV-1 reactivation from latency and are currently investigating possible mechanisms of protection.

CTCF-Dependent Chromatin Boundary Element between the Latency-Associated Transcript and ICP0 Promoters in the Herpes Simplex Virus Type 1 Genome

Cells latently infected with herpes simplex virus (HSV) contain nucleosomal DNA similar to that of host cell chromatin. Recent studies have demonstrated that histones in the latency-associated transcript (LAT) promoter and intron regions contain histone modifications permissive for transcription. However, those histones associated with the lytic-specific ICP0 gene, which lies only 5 kb away, contain modifications typical of silenced chromatin. How this active chromatin is kept separate from the repressed chromatin in the nearby ICP0 region remains crucial to the understanding of the HSV lytic cycle. In this study, we show that the LAT intron region contains an insulator. Specifically, we show that an 800-bp region from the LAT intron can block enhancers in both tissue culture cells and Drosophila melanogaster embryos. Importantly, the 800-bp HSV insulator protects a LAT transgene from positional effects in Drosophila eye tissue. The 800-bp region contains nine copies of 16-bp repeats. In vitro electrophoretic mobility shift assay revealed that CTCF interacts with the CTCCC sequence within the repeats. In vivo chromatin immunoprecipitation assay demonstrated that CTCF interacts with these repeats in latently infected trigeminal ganglion neurons. The deletion of these repeats impaired insulator activity in human K562 cells and Drosophila embryos. Finally, double-spaced RNA knockdown of CTCF disrupts enhancer-blocking activity of the LAT insulator in transfected Drosophila S3 cells. These results strongly support the hypothesis that the 800-bp DNA in the LAT intron region works as a chromatin boundary during latency to separate active chromatin associated with the LAT promoter region from repressed chromatin in the ICP0 gene.

Latent Virus Influences the Generation and Maintenance of CD8+ T Cell Memory

The influence of latent virus on CD8+ T cell memory is poorly understood. HSV type 1 specifically establishes latency in trigeminal ganglia (TG) after corneal infection of mice. In latently infected TG, IL-15 deprivation reduced the following: 1) accumulation of HSV-specific CD8+ effector T cells (HSV-CD8(eff)), 2) accumulation of CD127(+) putative HSV-CD8 memory precursors, and 3) the size and functionality of the memory (HSV-CD8(mem)) population. Although compromised in IL-15(-/-) mice, the HSV-CD8(mem) pool persisted in latently infected tissue, but not in noninfected tissue of the same mice. Anti-IL-2 treatment also dramatically reduced the size of the HSV-CD8(eff) population in the TG, but did not influence the concomitant generation of the CD127+ putative HSV-CD8(mem) precursor population or the size or functionality of the HSV-CD8(mem) pool. Thus, the size of the memory pool appears to be determined by the size of the CD127+ CD8(mem) precursor population and not by the size of the overall CD8(eff) pool. HSV-CD8(mem) showed a higher basal rate of proliferation in latently infected than noninfected tissue, which was associated with a reduced population of CD4+FoxP3+ regulatory T cells. Thus, the generation, maintenance, and function of memory CD8+ T cells is markedly influenced by latent virus.

Herpes simplex virus type 1 latently infected neurons differentially express latency-associated and ICP0 transcripts.

During the latent phase of herpes simplex virus type 1 (HSV-1) infection, the latency-associated transcripts (LATs) are the most abundant viral transcripts present in neurons, but some immediate-early viral transcripts, such as those encoding ICP0, have also been reported to be transcribed in latently infected mouse trigeminal ganglia (TG). A murine oro-ocular model of herpetic infection was used to study ICP0 gene expression in the major anatomical sites of HSV-1 latency, including the TG, superior cervical ganglion, spinal cord, and hypothalamus. An HSV-1 recombinant strain, SC16 110LacZ, revealed ICP0 promoter activity in several neurons in latently infected ganglia, and following infection with wild-type HSV-1 strain SC16, in situ hybridization analyses identified ICP0 transcripts in the nuclei of neurons at times consistent with the establishment of latency. Reverse transcription (RT)-PCR assays performed on RNA extracted from latently infected tissues indicated that ICP0 transcripts were detected in all anatomical sites of viral latency. Furthermore, quantitative real-time RT-PCR showed that neurons differentially expressed the LATs and ICP0 transcripts, with splicing of ICP0 transcripts being dependent on the anatomical location of latency. Finally, TG neurons were characterized by high-level expression of LATs and detection of abundant unspliced ICP0 transcripts, a pattern markedly different from those of other anatomical sites of HSV-1 latency. These results suggest that LATs might be involved in the maintenance of HSV-1 latency through the posttranscriptional regulation of ICP0 in order to inhibit expression of this potent activator of gene expression during latency.

Low-Level Expression and Reversion both Contribute to Reactivation of Herpes Simplex Virus Drug-Resistant Mutants with Mutations on Homopolymeric Sequences in Thymidine Kinase

Many acyclovir-resistant herpes simplex virus isolates from patients contain insertions or deletions in homopolymeric sequences in the thymidine kinase (TK) gene (tk). Viruses that have one (G8) or two (G9) base insertions in a run of seven G's (G string) synthesize low levels of active TK (TK-low phenotype), evidently via ribosomal frameshifting. These levels of TK can suffice to permit reactivation from latently infected mouse ganglia, but in a majority of ganglia, especially with the G9 virus, reactivation of virus that has reverted to the TK-positive phenotype predominates. To help address the relative contributions of translational mechanisms and reversion in reactivation, we generated viruses with a base either inserted or deleted just downstream of the G string. Both of these viruses had a TK-low phenotype similar to that of the G8 and G9 viruses but with less reversion. Both of these viruses reactivated from latently infected trigeminal ganglia, albeit inefficiently, and most viruses that reactivated had a uniformly TK-low phenotype. We also generated viruses that have one insertion in a run of six C's or one deletion in a run of five C's. These viruses lack measurable TK activity. However, they reactivated from latently infected ganglia, albeit inefficiently, with the reactivating viruses having reverted to the wild-type TK phenotype. Therefore, for G-string mutants, levels of active TK as low as 0.25% generated by translational mechanisms can suffice for reactivation, but reversion can also contribute. For viruses that lack TK activity due to mutations on other homopolymeric sequences, reactivation can occur via reversion.

A mutant deleted for most of the herpes simplex virus type 1 (HSV-1) UOL gene does not affect the spontaneous reactivation phenotype in rabbits

The mechanisms involved in the herpes simplex virus type 1 (HSV-1) latency-reactivation cycle are not fully understood. The latency-associated transcript (LAT) is the only HSV-1 RNA abundantly detected during neuronal latency. LAT plays a significant role in latency because LAT(-) mutants have a reduced reactivation phenotype. Several novel viral transcripts have been identified within the LAT locus, including UOL, which is located just upstream of LAT. The authors report here on a mutant, DeltaUOL, which has a 437-nucleotide deletion that deletes most of UOL. DeltaUOL replicated similarly to its wild-type parental McKrae HSV-1 strain in infected cells, the eyes, trigeminal ganglia, and brains of mice and rabbits. It was indistinguishable from wild-type virus as regards explant-induced reactivation in mice, and spontaneous reactivation in rabbits. In contrast, DeltaUOL was significantly less virulent in mice. Thus, UOL appears to be dispensable for the wild-type reactivation phenotype while appearing to play a role in neurovirulence in ocularly infected animals.

A homologous in vitro model to study interactions between alphaherpesviruses and trigeminal ganglion neurons

A key aspect in the life cycle of alphaherpesviruses is their neurotropic behaviour. Sensory neurons of the trigeminal ganglion (TG) are important target cells for many alphaherpesviruses (including herpes simplex virus 1, pseudorabies virus (PRV), bovine herpesvirus 1) and constitute major sites for latent infections. The aim of this study was to develop an in vitro model that simulates the in vivo infection pattern of TG neurons by alphaherpesviruses. To this end, we developed a homologous in vitro two-chamber model using PRV and porcine TG neurons. TG of 4- to 6-week-old piglets were dissociated and cultured in the inner chamber of the in vitro model, which is separated from the outer chamber by a medium- and virus-impermeable silicon barrier. Outgrowth of axons from neuronal cell bodies in the inner chamber through the silicon barrier into the outer chamber could be observed after 2-3 weeks of cultivation. Subsequent addition of PRV to the outer chamber resulted in exclusive infection of the TG neurons by transport of virus through the axons, subsequently giving rise to productively infected TG neurons that transmitted virus to contacting neurons and non-neuronal cells in the inner chamber. Thus, we established a homologous in vitro model that mimics the natural route of alphaherpesvirus infection of TG neurons that can be used to study interactions between these viruses and this pathogenetically very important cell type.

Histone deacetylase inhibitors induce reactivation of herpes simplex virus type 1 in a latency-associated transcript–independent manner in neuronal cells

Histone acetylation is implicated in the regulation of herpes simplex virus type 1 (HSV-1) latency. However, the role of histone acetylation in HSV-1 reactivation is less clear. In this study, the well-established model system, quiescently infected, neuronally differentiated PC12 (QIF-PC12) cells, was used to address the participation of histone acetylation in HSV-1 reactivation. In this model, sodium butyrate and trichostatin A (TSA), two histone deacetylase inhibitors, stimulated production of infectious HSV-1 progeny from a quiescent state. To identify viral genes responsive to TSA, the authors analyzed representative alpha, beta, and gamma viral genes using quantitative real-time polymerase chain reaction. Only the latency-associated transcript (LAT) accumulated in response to TSA treatment, under culture conditions that restricted virus replication and spread. This led the authors to evaluate the importance of LAT expression on TSA-induced reactivation. In QIF-PC12 cells, the LAT deletion mutant virus dLAT2903 reactivated equivalently with its wild-type parental strain (McKrae) after TSA treatment, as well as forskolin and heat stress treatment. Both viruses also reactivated equivalently from latently infected trigeminal ganglia explants from rabbits. In contrast, there was a marked reduction in the recovery of dLAT2903, as compared to wild-type virus, from the eyes of latently infected rabbits following epinephrine iontophoresis. These combined in vitro, ex vivo, and in vivo data suggest that LAT is not required for reactivation from latently infected neuronal cells per se, but may enhance processes that allow for the arrival of virus at, or close to, the site of original inoculation (i.e., recrudescence).

The cellular response to herpes simplex virus type 1 (HSV-1) during latency and reactivation

In order to learn more about the cellular response to viral gene activity during latency and reactivation of herpes simplex virus type 1 (HSV-1), the authors have employed microarray analysis. On an array of about 1200 cellular genes, approximately 56 genes were found to be differentially regulated in infected trigeminal ganglia of mice, compared to uninfected mice, during latency and reactivation. Of these genes, 10 were examined more closely using quantitative real-time polymerase chain reaction (PCR) to confirm the microarray results. Genes involved in interferon and other signaling pathways appeared to predominate in response to a latent or reactivating HSV infection. Interestingly, some genes found to be differentially regulated in latently infected ganglia are neuronal-specific genes (pro-opiomelanocortinin; zinc finger proteins of the cerebellum 1 and 2). During reactivation, the involvement of several cell signaling molecules that may be important for the initiation of an HSV infection was observed, including various receptors and molecules involved in cell-cell spread.

Microarray analysis in the HSV-1 latently infected mouse trigeminal ganglion.

To review our previous studies regarding alterations in gene expression in HSV-1 latently infected mouse trigeminal ganglia (TGs) following treatment with immunosuppressants and hyperthermia. Uninfected and HSV-1 latently infected mice were treated with immunosuppressants or heat stressed (43 degrees C for 10 minutes). In the immunosuppressant study, 4 groups of animals were examined: (1) uninfected, not treated; (2) uninfected, drug-treated; (3) latently infected, not treated; and (4) latently infected, drug-treated. In the hyperthermia study, TG from 6 groups of mice were studied: (1) uninfected, not stressed; (2) uninfected, heat-stressed; killed at 6 hours after hyperthermia; (3) uninfected, heat-stressed, killed at 24 hours after hyperthermia; (4) latently infected, not stressed; (5) latently infected, heat-stressed, killed at 6 hours after hyperthermia; and (6) latently infected, heat-stressed, killed at 24 hours after hyperthermia. PolyA mRNA from the TGs of each group was reverse-transcribed, labeled with P, incubated on a gene array membrane, and analyzed by phosphorimaging. As a comparison and to confirm microarray results, semiquantitative RT-PCR for selected genes was also performed. The immunosuppressive drugs significantly increased expression of two genes--calpactin 1 light chain and guanine nucleotide-binding protein alpha stimulating activity polypeptide (GNAS)--in the ganglia of uninfected mice compared with untreated, uninfected mice. Ten genes were shown to be significantly increased in the latent TGs from mice treated with the immunosuppressants compared with latently infected untreated mice. These genes were prostaglandin E2 receptor EP4 subtype (PTGER4), insulin promoter factor 1 (IPF1), glutathione S-transferase mu2, cyclin D2, peripherin, plasma glutathione peroxidase, methyl CpG-binding protein 2, retinal S-antigen, ErbB2 protooncogene, and GNAS. Eight genes were shown to be significantly decreased in the HSV-1 latent TGs treated with the drugs compared with untreated latent mice. These genes were peripheral myelin protein 22, decorin, transcription factor AP-1, dystroglycan 1, myelin protein zero, mitogen-activated protein kinase 3, prothymosin beta4, and brain lipid-binding protein. The results obtained by semiquantitative RT-PCR results were similar to those obtained by microarray analysis. Six hours after heat stress, the genes whose expression was altered included the FK506-binding protein gene (decreased), the T-complex protein 1alpha subunit gene (increased), and the 94-kDa glucose-regulated protein gene (increased in uninfected TG, decreased in infected TG). Heat stress increased expression of the DNA excision repair protein ERCC5 gene 24 hours after the treatment. Genes previously reported to exhibit increased transcription 1 hour after heat stress did not continue to show significant transcriptional activation at 6 or 24 hours. Those genes whose expression is altered by immunosuppressive drug treatment may play an important role in ocular HSV-1 recurrence. Changes in gene expression in the prostaglandin pathway, a transcription factor, and an enzyme in the cell cycle are considered of special importance for HSV-1 reactivation by immunosuppression. Altered gene expression at 6 and 24 hours after heat stress was different from previously reported changes in gene expression 1 hour after hyperthermia in HSV-1 latently infected mice.

CD8(+) T cells control corneal disease following ocular infection with herpes simplex virus type 1.

The role that T cell subsets play in herpetic stromal keratitis (HSK) has been the subject of intense research efforts. While most studies implicate CD4(+) T cells as the principal cell type mediating primary corneal disease, recent reports using knockout mice have suggested that both CD4(+) and CD8(+) T cell subsets may play integral roles in modulating the disease. Furthermore, recent studies suggest that CD8(+) T cells are directly involved in maintaining virus latency in infected trigeminal ganglia. This work has addressed these discrepancies by infecting the corneas of mice lacking CD4(+) and CD8(+) T cells with herpes simplex virus type 1 (HSV-1) and monitoring both corneal disease and latent infection of trigeminal ganglia. Results indicated that mice lacking CD8(+) T cells had more severe corneal disease than either BALB/c or B6 parental strains. In contrast, mice lacking CD4(+) T cells had a milder disease than parental strains. When mice were evaluated for persistence of infectious virus, only transient differences were observed in periocular tissue and corneas. No significant differences were found in persistence of virus in trigeminal ganglia or virus reactivation from explanted ganglia. These data support the following conclusions. CD4(+) T cells are not required for resistance to infection with HSV-1 and probably mediate HSK. Mice lacking CD8(+) T cells do not display differences in viral loads or reactivation and thus CD8(+) T cells are not absolutely required to maintain latency. Finally, CD8(+) T cells probably play a protective role by regulating the immunopathological response that mediates HSK.

Absence of tumour necrosis factor facilitates primary and recurrent herpes simplex virus-1 infections.

Tumour necrosis factor (TNF) is an important cytokine in the innate immune response against various infections, including herpes simplex virus (HSV) infection. It has recently become a molecular target of anti-cytokine treatment in certain inflammatory diseases. TNF depletion resulted in a more rapid emergence of infectious HSV-1 in the explant cultures of latently infected trigeminal ganglia (TG), compared with controls. To further evaluate the importance of TNF in the host's defence responses against HSV-1, TNF-knockout mice were challenged via scarified cornea. These mice were more susceptible to primary acute corneal HSV-1 infection than controls, as manifested by an increased mortality rate and higher infectious virus titres in the eyes and TG, indicating that TNF is critical for defence during acute HSV infection. These results imply that the administration of anti-inflammatory TNF antagonists might facilitate the propagation of infectious HSV, resulting in an exacerbation of primary and recurrent acute lesions.

Persistent expression of chemokine and chemokine receptor RNAs at primary and latent sites of herpes simplex virus 1 infection

Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1α, MIP-1β and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis.