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Current Eye Research, 34(10), 896–904, 2009Copyright © 2009 Informa Healthcare USA, Inc.ISSN: 0271-3683 print/ 1460-2202 onlineDOI: 10.3109/02713680903184250

ORIGINAL ARTICLE

Expression of Herpes Virus Entry Mediator (HVEM) in the Cornea and Trigeminal Ganglia

of Normal and HSV-1 Infected Mice

S. Krisztian Kovacs1, Vaibhav Tiwari2, Emese Prandovszky1, Sandor Dosa1, Sarolta Bacsa2, Klara Valyi-Nagy1, Deepak Shukla2, and Tibor Valyi-Nagy1

1Department of Pathology, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, USA2Departments of Ophthalmology and Visual Sciences and Microbiology and Immunology, University of Illinois at

Chicago, College of Medicine, Chicago, Illinois, USA

INTRODUCTION

Infection of the human cornea by herpes simplex viruses (HSV) is primarily due to herpes sim-plex virus type 1 (HSV-1) in adults and herpes

Received 13 April 2009; revised 11 July 2009; accepted 14 July 2009

Correspondence: Tibor Valyi-Nagy, M.D., Ph.D., University of Illinois at Chicago, College of Medicine, Department of Pathology, Room 130, 840 South Wood St., M/C 847, Chicago, IL 60612. E-mail: tiborv@uic.edu

ABSTRACT

Purpose: Herpes virus entry mediator (HVEM) plays a critical role in the regulation of inflammation through interaction with its natural ligands LIGHT and lymphotoxin alpha and also serves as one of the entry receptors of herpes simplex virus (HSV). The purpose of this study was to better understand the expression of HVEM in the cornea and trigeminal ganglia (TG), which are important targets of HSV infection. Materials and Methods: Immunohistochemistry was used to define HVEM expression in the cornea and TG of normal and HSV-1 infected mice euthanized 2 to 5 days or 7 months following corneal inoculation of virus. Results: We found that HVEM is widely expressed in the normal corneal epithelium and endothelium, is weakly and focally expressed in the corneal stroma, and is expressed in a portion of neurons and non-neuronal cells in the TG. Acute HSV-1 keratitis and ganglionitis were associated with increased HVEM expression in the corneal epithelium and stroma and in neurons and non-neuronal cells of TG, and many inflammatory cells in these tissues also expressed HVEM. TG derived from mice 7 months after virus inoculation demonstrated latent HSV-1 infection that was associated with increased HVEM expression in neurons and non-neuronal cells relative to uninfected control tissues. Latent TG also contained focal infiltrates of mononuclear inflammatory cells, many of which expressed HVEM. Corneas derived from latently infected mice demonstrated chronic keratitis, with no evidence of virus replication or increased HVEM expression in the corneal epithelium, and inflammatory cells present showed only weak HVEM expression. Conclusions: HVEM is expressed in the cornea and TG and therefore may serve as an HSV entry receptor in these tissues. Furthermore, these findings raise the possibility that changes in HVEM expression following ocular HSV-1 infection can modulate HSV spread and infection-induced inflammation in the cornea and TG.

KEYWORDS: Cornea; Herpes simplex virus; Herpes virus entry mediator; HveA; HVEM; TNFRS14; Trigeminal ganglia

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simplex virus type 2 (HSV-2) in neonates, and is an important cause of ocular disease.1–4 Latent HSV infection of the trigeminal ganglia (TG) plays an important role in the pathogenesis of recurrent HSV keratitis as a source of reactivated virus.3 Following corneal infection, HSV infects nerve endings, moves by retrograde axonal transport to nerve cell bodies in the TG, and establishes latent infection in neurons.3,5–7 During latent HSV-1 infec-tion of neurons, viral genomic DNA is present in neuronal nuclei, abundant viral gene expression is limited to the latency-associated transcripts (LAT), and viral proteins or infectious virus are usually not detectable. The virus periodically reactivates with production of infectious virus in the ganglia and consequently spread to peripheral tissues, including the cornea. In HSV keratitis, visual morbidity usu-ally results from recurrent disease through corneal scarring, thinning, and neovascularisation, with the immune response playing a key role in the develop-ment of these pathologic changes.4,8

Expression of cellular receptors of HSV entry is a critically important determinant of viral pathogen-esis, as these receptors will determine which tissues and cells become infected during primary and recur-rent infections. Although significant progress has been made in studies of the molecular basis of virus-host interactions leading to HSV entry into cells,9–12 the expression of HSV entry receptors in ocular and peripheral nervous system tissues is not completely understood.

Successful HSV infection depends upon inter-action between the viral particle and cell surface receptors required for viral entry. Viral glycopro-teins B and C (gB and gC) provide the initial binding interaction between the virus and cell-surface gly-cosaminoglycans, preferentially heparan sulphate.9–11 Following this attachment, HSV glycoprotein D (gD) interacts with one of three known cell-surface receptors that are necessary for viral entry.10–11 A spe-cific modification in heparan sulfate generated by certain 3-O-sulfotransferases generates a family of gD receptors that are specific for HSV-1.13 Two cell-surface proteins are known to mediate the entry of HSV-1 and HSV-2 into cultured cells by interacting with gD: nectin-1 and herpes virus entry mediator (HVEM).

Among HSV entry receptors, the expression of nectin-1 in the cornea and TG is relatively well characterized, while the expression of HVEM and 3-O-sulfated heparan sulfate receptors is less well understood. Nectin-1 is a member of the immun-globulin (Ig) superfamily closely related to the poliovirus receptor.14–17 Nectin-1 and the related proteins nectin-2 and nectin-3 are cell adhesion

molecules found at Ca2+-dependent cadherin-based cell adhesion junctions in epithelial cells, synapses, and other types of cells. Nectin-1 is widely expressed in the corneal epithelium and endothelium and in TG neurons.18–20 This widespread expression and the observation that nectin-1 is the primary entry receptor of HSV-1 into sensory neurons21–22 suggest that nectin-1 plays an important role in the patho-genesis of HSV keratitis. However, there is grow-ing evidence that HSV entry receptors other than nectin-1 also have a role in the pathogenesis of HSV ocular infections. For instance, 3-O-sulfated heparan sulfate has been shown to mediate HSV-1 entry into human corneal fibroblasts.23 HVEM in turn mediates HSV-2 entry into corneal fibroblasts24 and HSV-1 entry into the trabecular meshwork25 and conjuncti-val epithelial cells.26

HVEM, also called herpes virus entry protein A (HveA) and tumor necrosis factor receptor 14 (TNFRS14), is a member of the tumor necrosis factor receptor superfamily (TNFR) of proteins.27 HVEM plays a critical role in the regulation of inflammation through interaction with its natural ligands which are LIGHT and lymphotoxin alpha (Lt).28–30 Both ligands are structurally related to tumor necrosis fac-tor (TNF), exist as trimers,31–32 and presumably signal by inducing or altering receptor aggregation on the cell surface.28,33–35 The expression of HVEM in various tissues and cell types is not well understood. Studies using human tissue RNA blots found relatively high HVEM mRNA levels in the lung, spleen, and thymus, but no significant HVEM expression was detected in the brain, liver, or skeletal muscle.36 HVEM is expressed in T-cells, B-cells, natural killer cells (NK), dendritic cells (DCs), neutrophils, and myeloid cells.36 HVEM is expressed in ocular trabecular meshwork cells, cultured corneal fibroblasts, and conjuncti-val epithelial cells,23–26 and HVEM expression was detected in primary cultures of rat and mouse sen-sory neurons.21 HVEM expression was also detected in epithelial cells, fibroblasts, and endothelial cells in gingival tissue.37

To better understand the expression of HVEM in ocular and peripheral nervous system tissues, in the current study, we have used a well-established murine model of HSV-1 pathogenesis and immuno-histochemistry to determine HVEM expression in the cornea and TG. In addition to normal tissues, mouse eye and TG tissues derived from mice during the acute and latent stages of HSV-1 infection were also studied to determine if HSV-1 infection had an effect of HVEM expression. We show here that HVEM is widely expressed in murine cornea and TG, and that HSV-1 infection leads to changes in HVEM expression in these tissues.

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MATERIAL AND METHODS

Virus Stocks

HSV-1 strain 17+ was propagated and titered by plaque assay using baby hamster kidney (BHK) 21 clone 13 cells.

Infection of Mice, Recovery of Tissues, and Tissue Processing

To establish a collection of tissues for studies of HVEM expression in normal and HSV-1 infected eyes and trigeminal ganglia, 4- to 6-week-old female BALB/c mice were inoculated on their corneas with 105 PFU of HSV-1 strain 17+ after corneal scarification, as previ-ous described.38 Control mice were either not treated or were inoculated with an equivalent volume (5 µl) of sterile tissue-culture medium using the same inocu-lation technique (mock infection). Groups of at least three mice were euthanized 2–5 days (expected times of acute, productive HSV-1 infection) or at 7 months (expected time of latent HSV-1 infection) after HSV-1 inoculation. The eye bulbs and TG were aseptically removed, embedded in paraffin, and sectioned. Tissue sections were either stained with hematoxylin and eosin or were examined by immunohistochemistry for HSV-1 protein and HVEM expression. TG tissues derived from mice 7 months post infection (p.i.) were also examined by in situ hybridization for the expres-sion of HSV-1 LAT. All animal procedures followed an IAUCUC-approved protocol and the NIH guidelines for laboratory animal care as outlined in NIH publica-tion No. 86-23 (revised 1985).

Immunohistochemical Detection of HSV-1 Proteins

Tissue sections for immunohistochemistry were deparaffinized with xylene and rehydrated through a series of graded ethanols. Endogenous peroxidase activity was quenched using a 0.3% H2O2-methanol bath followed by several washes with phosphate-buffered saline. HSV-1 antigens were detected using a 1:1000 dilution of an HSV-1-specific antiserum raised in a rabbit (DAKO, Carpinteria, CA, USA) as previ-ously described.18,39 Tissue sections were incubated with primary antibody at 43°C for 32 min prior to the addition of biotinylated anti-rabbit immunoglobu-lin secondary antibody, avidin-horseradish peroxi-dase, and 3,3′-diaminobenzidine tetrahydrochloride (0.04%) in 0.05 M Tris-HCL (pH 7.4) and 0.025% H2O2 as a chromogen (Ventana Medical Systems, Tucson,

AZ, USA). Before staining, binding of secondary antibodies and conjugates was blocked by appropri-ate reagents provided by the manufacturer.

Immunohistochemical Detection of HVEM

Tissue sections for HVEM immunohistochemistry were deparaffinized with xylene and rehydrated through a series of graded ethanols. Endogenous peroxidase activity was quenched using a 0.3% H2O2-methanol bath followed by several washes with phosphate-buffered saline. Tissue sections were hydrated with distilled water, and antigen retrieval was performed using the DAKO Target Retrieval Solution 10X Concentrate. Nonspecific staining was blocked using an H2O2 solution for 10 min followed by a protein block for 10 min. Sections were incubated with a rabbit polyclonal antibody against HVEM (courtesy of Professor Patricia Spear, Northwestern University, Chicago, IL, USA) at 1:200 at room tem-perature for 1 hr, as previously described.26 HVEM staining was detected using the DAKO Envision+ kit. To characterize HVEM expression in tissues, a semi-quantitative scoring system was designed and used that placed HVEM expression in a given cell type into one of three categories: 1+ = focal weak HVEM expres-sion, 2+ = widespread moderate HVEM expression, 3+ = widespread strong HVEM expression.

In situ Hybridization for HSV-1-LAT Gene Expression

In situ hybridization was performed using a nick-translated 35S-labeled DNA probe specific for HSV-1 LAT (Bst2 ± Bst2 fragment) according to previously described techniques.40 The probe used in these experiments had a specific activity of .108 cpm/mg.

RESULTS

Establishment of a Collection of Tissues for Studies of HVEM Expression in Normal and HSV-1 Infected Murine Cornea and TG

To establish a collection of tissues for studies of HVEM expression in normal and HSV-1 infected eyes and TG, 4- to 6-week-old female BALB/c mice were inoculated on their corneas with 105 PFU of HSV-1. Control mice were either not treated or were inoculated with sterile tissue-culture medium (mock infection). HSV-1 inoculated mice euthanized at 2 or 5 days after HSV-1 inoculation demonstrated keratitis

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(focal ulceration of the corneal epithelium associ-ated with moderate to severe inflammatory infiltrates composed of neutrophils and mononuclear inflamma-tory cells primarily involving the corneal stroma) and HSV-1 protein expression in the corneal epithelium and stroma (Figure 1A). Uninfected mice showed no evidence of significant corneal inflammation, and no HSV-1 protein expression was detected in the cornea by immunohistochemistry (data not shown). Similar to uninfected mice, no HSV-1 protein expression was detected in the corneas of mock infected mice, and, probably due to the gentle and superficial nature of the corneal scarification procedure, no significant inflammation was detected in the the corneas of mock infected mice (Figure 1B).

The TG of mice euthanized 5 days after virus inoculation demonstrated focal inflammation and ex-pression of HSV-1 proteins in some neurons and non-neuronal cells (Figure 1C), consistent with HSV-1 repli-cation. No evidence of significant inflammation and no HSV-1 protein expression were detected in the TG of un-infected and mock infected mice (Figure 1D) or in mice that were euthanized 2 days after virus inoculation.

The corneas of mice euthanized at 7 months fol-lowing virus inoculation demonstrated moderate to severe chronic inflammation associated with

significant vascularization of the corneal stroma ( Figure 2A). However, no HSV-1 protein expression was detected. No evidence of significant inflamma-tion and no HSV-1 protein expression were detected in the corneas of mock infected mice 7 months after mock infection (Figure 2B). The trigeminal ganglia of mice euthanized 7 months following virus inoculation showed focal mononuclear inflammatory cell infil-trates and no evidence of HSV-1 protein expression (Figure 2C). However, many neurons demonstrated LAT expression by in situ hybridization (Figure 2D). These findings indicate that corneas collected at 2 and 5 days after virus inoculation and trigeminal ganglia collected 5 days after virus inoculation were involved by acute productive HSV-1 infection. These findings also indicate that extensive productive HSV-1 infec-tion subsided in the corneas and trigeminal ganglia of mice by 7 months after virus inoculation without the clearance of latent HSV-1 infection from the trigemi-nal ganglia.

A B

C D

FIGURE 1 HSV-1 protein expression in murine cornea and TG tissues during the acute phase of HSV-1 infection as detected by immunohistochemistry. Brown staining indicates HSV-1 proteins. (A) Cornea tissue derived from a mouse 5 days after corneal HSV-1 inoculation. Black arrow points to an area of the corneal epithelium expressing HSV-1 proteins. White arrow points to an area of the corneal stroma expressing HSV-1 proteins. (B) Cornea tissue derived from a mouse 2 days after corneal mock infection. There is no evidence of HSV-1 protein expression. (C) TG tissue derived from a mouse 5 days after corneal HSV-1 inoculation. White arrow points to a neuron expressing HSV-1 proteins. (D) TG tissue derived from a mouse 2 days after corneal mock infection. There is no evidence of HSV-1 protein expression. Magnification ×400.

A B

C D

FIGURE 2 No evidence of HSV-1 protein expression in murine cornea and TG tissues 7 months after corneal HSV-1 inoculation by immunohistochemistry. (A) Cornea tissue derived from a mouse 7 months after corneal HSV-1 inoculation. There is no evidence of HSV-1 protein expression (brown staining). However, there is evidence of chronic inflammation. White arrow points to an area of inflammation in the corneal stroma. Magnification ×400. (B) Cornea tissue derived from a mouse 7 months after corneal mock infection. There is no evidence of HSV-1 protein expression (brown staining) or inflammation. Magnification ×400. (C) TG tissue derived from a mouse 7 months after corneal HSV-1 inoculation. There is no evidence of HSV-1 protein expression (brown staining); however, there is evidence of chronic inflammation (white arrow). Magnification ×400. (D) Detection of HSV-1 LAT by in situ hybridization in TG tissue derived from a mouse 7 months after corneal HSV-1 inoculation. Black signal indicates LAT expression. White arrow points to a LAT expressing cell. Magnification ×100.

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HVEM Expression in Normal Murine Cornea and TG

To define the expression of HVEM in the cornea and TG of mice by immunohistochemistry and to deter-mine whether the immunohistochemical technique used was capable of HVEM detection in a pattern con-sistent with that previously reported,21,23–26,37 corneal and TG sections derived from healthy adult BALB/c mice were incubated with an anti-HVEM antibody as previously described.26 In the normal mouse cornea, moderate HVEM expression was detected in the cor-neal epithelium and endothelium (Figure 3A. Table 1).

Only a few stromal fibroblasts showed weak HVEM expression (Figure 3A, Table 1). Immunostaining per-formed without primary antibody showed no stain-ing of corneal epithelium, fibroblasts, or endothelium (data not shown).

In sections of TG derived from healthy adult mice, weak HVEM expression was detected in some neu-rons and non-neuronal cells (perineuronal satellite cells, Schwann cells); however, the majority of these cells were HVEM negative (Figure 3B, Table 1). These findings demonstrate that HVEM is widely expressed in the normal corneal epithelium and endothelium and that HVEM expression is weak and focal in the corneal stroma and in the TG.

Increased HVEM Expression in the Murine Eye and TG During Acute HSV-1 Infection

To determine whether acute HSV-1 infection has an effect on HVEM expression in the cornea and trigemi-nal ganglia, immunohistochemistry was used to detect HVEM expression in the cornea and TG of mice euthanized 2 or 5 days after corneal HSV-1 inocula-tion or mock infection. The corneas of mock infected mice demonstrated HVEM expression similar to that of uninfected mice (data not shown). However, in corneal tissues derived from HSV-1 infected animals, increased HVEM expression was detected among cells of the corneal epithelium and among stromal fibroblasts at apparent sites of infection and inflam-mation (Figure 3C, Table 1). In addition, the stroma of infected corneas contained many cells with morpho-logic features of mononuclear inflammatory cells that expressed HVEM (Figure 3C, Table 1).

TG derived from mock infected mice and those derived from mice 2 days after HSV-1 corneal inocula-tion demonstrated HVEM expression similar to that of uninfected mice (data not shown). In contrast, in TG derived from mice 5 days after HSV-1 inoculation, HVEM expression was increased relative to unin-fected and mock infected tissues: moderately strong HVEM expression was observed in many neurons and non-neuronal cells (Figure 3D, Table 1). An increased strength of HVEM expression was associated with

A B

C D

FIGURE 3 HVEM expression in the normal, uninfected murine cornea and TG and in murine cornea, and TG tissues during the acute phase of HSV-1 infection, as detected by immunohistochemistry. Brown staining indicates HVEM expression. (A) Cornea tissue derived from a normal, uninfected mouse. White arrow points to corneal epithelium demonstrating moderate HVEM expression, and the black arrow points to the corneal stroma that shows no significant HVEM expression. (B) TG tissue derived from a normal, uninfected mouse. White arrow points to a neuron that is weakly positive for HVEM. (C) Cornea tissue derived from a mouse 2 days after corneal HSV-1 inoculation. Black arrow points to the corneal epithelium, and the white arrow points to the corneal stroma. Note the increased expression of HVEM relative to panel (A). (D) TG tissue derived from a mouse 5 days after corneal HSV-1 inoculation. White arrow points to a neuron demonstrating moderate HVEM expression. Magnification ×400.

TABLE 1 Detection of HVEM expression by immunohistochemistry in corneal and trigeminal ganglion tissues of normal mice and mice euthanized 5 days or 7 months after corneal inoculation of HSV-1 or sterile medium (mock infection)

TreatmentCornea Trigeminal ganglia

Epithelium Stroma Endothelium Inflam. cells Neurons Satellite cells Inflam. cellsUntreated/mock* 2+ 1+ 1+ N/A 1+ 1+ N/A5 days after HSV inoc 3+ 3+ 2+ 3+ 2+ 2+ 2+7 months after HSV inoc 2+ 1+ 2+ 1+ 3+ 2+ 2+*Untreated and mock inoculated mice showed identical HVEM expression. 1+ = focal weak HVEM expression, 2+ = widespread moderate HVEM expression, 3+ = widespread strong HVEM expression, N/A = nonapplicable, no inflammatory cells present.

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an increased number of HVEM-positive neurons in infected tissues as the majority of TG neurons were HVEM positive. In inflamed foci in the trigeminal gan-glia, many mononuclear inflammatory cells showed HVEM expression (Figure 3D, Table 1). These findings indicate that acute HSV-1 infection leads to enhanced HVEM expression in the cornea and TG.

HVEM Expression in the Murine Cornea and TG 7 Months after Corneal HSV-1 Inoculation

To determine whether latent HSV-1 infection in the TG modifies the expression of HVEM in the cornea and trigeminal ganglia, tissues derived from mice 7 months following HSV-1 inoculation were examined by immunohistochemistry using an HVEM-specific antiserum. As demonstrated above, corneal and TG tissues derived from these mice showed no evidence of HSV-1 protein expression but HSV-1 LAT expres-sion was readily detectable by in situ hybridization in the TG, consistent with latent HSV-1 infection in the TG. Consistent with previous reports,40–42 both latently infected TG and corneal tissues derived from these mice frequently demonstrated focal chronic inflam-mation (Figures 4A and 4B). Corneal tissues of latently infected mice demonstrated weaker HVEM immuno-reactivity in the corneal epithelium and stroma than those derived from acutely infected mice, and HVEM signal strength and distribution were similar to that detected in uninfected and mock infected control corneas. HVEM-positive cells could be observed pri-marily in the upper layer of corneal epithelium and in the endothelium and in occasional cells in the stroma (Figure 4A, Table 1). In inflamed foci in the cornea, moderate to severe chronic inflammation and signifi-cant vascularization was associated with only weak and focal HVEM expression in mononuclear inflam-matory cells (Figure 4A, Table 1).

In latently infected TG, widespread and strong HVEM expression was detected among neurons (Figure 4B, Table 1). Relative to uninfected and mock infected tissues, increased HVEM expression was also detected among non-neuronal cells. In inflamed foci in the trigeminal ganglia, many mononuclear inflam-matory cells showed HVEM expression, and neuronal HVEM immunoreactivity was stronger and denser in areas of chronic inflammation. The vast majority of neurons stained more intensively positive for HVEM than those in uninfected trigeminal ganglia.

These findings indicate that, similar to acute HSV-1 ganglionitis, latent HSV-1 infection is associated with in-creased neuronal and non-neuronal HVEM expression in the TG. However, in the cornea of latently infected mice, HVEM expression was reduced relative to acutely HSV-1 infected tissue and was similar to that detected in mock infected and uninfected cornea. Interestingly, HVEM expression was weak and infrequent in inflam-matory cells in the corneas of latently infected mice.

DISCUSSION

We show here that HVEM is expressed widely in the normal corneal epithelium and endothelium and is weakly and focally expressed in the corneal stroma. HVEM is also expressed in some neurons and non-neuronal cells in the normal murine TG. Acute HSV-1 keratitis and ganglionitis are associated with increased HVEM expression in the corneal epithelium and stroma and in TG neurons and non-neuronal cells, and many inflammatory cells in these tissues also express HVEM. Furthermore, we also show here that HSV-1 latency in TG is associated with increased HVEM expression in neurons and non-neuronal cells relative to uninfected control tissues and many mononuclear inflammatory cells in latently infected TG express HVEM. Corneas derived from latently infected mice demonstrated chronic keratitis with no evidence of virus replication or increased HVEM expression in the corneal epithe-lium and endothelium, and inflammatory cells present showed only weak HVEM expression.

These findings indicate that several different cell types in the murine cornea and trigeminal ganglia express HVEM and thus suggest that HVEM may serve as an entry receptor for HSV within these tis-sues. The finding that HVEM expression is modified by the HSV-1 infection in the cornea and trigeminal ganglia is novel and may be of clinical significance. Increased expression of HVEM in the corneal epithe-lium and stroma and in TG neurons and non-neuronal cells during acute infection may increase the number of cells susceptible to HSV entry. This may be par-ticularly important in the case of corneal stromal

A B

FIGURE 4 HVEM expression in murine cornea and TG tissues 7 months after corneal HSV-1 inoculation. Brown staining indicates HVEM expression. (A) Cornea tissue derived from a mouse 7 months after corneal mock infection. Black arrow points to HVEM expressing corneal epithelium. (B) TG tissue derived from a mouse 7 months after corneal HSV-1 inoculation. White arrow points to a neuron strongly positive for HVEM. Magnification ×400.

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fibroblasts that showed little or no HVEM expres-sion in the absence of HSV-1 infection, but demon-strated increased HVEM expression following HSV-1 inoculation. Similarly, an increased number of HVEM-expressing neurons and non-neuronal cells in the TG during HSV-1 infection may increase the number of cells susceptible for virus entry.

The mechanism by which HSV-1 infection modi-fies HVEM infection in the cornea and TG is unclear from our studies. Corneas and TG of mock infected (scarified, but not virus exposed) mice showed no evidence of increased HVEM expression, thus the increased HVEM expression detected in the HSV-1 inoculated mice could not be due to the scarification process. HVEM expression was increased in a much larger number of cells in the cornea and TG than the number of cells that expressed HSV-1 proteins during acute HSV-1 infection. Furthermore, HVEM expression was increased in a large number of neu-rons and non-neuronal cells in latently infected TG in the absence of HSV-1 protein expression. These find-ings suggest that HSV-1 replication was not directly responsible for the induction of HVEM expression in the cornea and TG. It is possible that HSV-1 infection-induced host inflammatory responses and associated changes in cellular activation may have contributed to the observed increased HVEM expression. It is well known that HVEM expression can be dependent on cell differentiation and activation. For instance, naive T-cells express high levels of HVEM, but expression decreases during activation, with high levels being re-expressed at the end of the activation phase.43–44 Interestingly, our studies indicated that HVEM expres-sion was not increased in the cornea of mice 7 months after HSV-1 inoculation, although these tissues har-bored prominent chronic inflammation. These find-ings suggest that the inflammation associated with chronic herpetic keratitis differs from acute keratitis in its capacity to induce HVEM expression in cells of the corneal epithelium and stroma.

HVEM plays a critical role in the regulation of inflammation through interaction with its natural ligands LIGHT and lymphotoxin alpha (Lt).28 Con-sistent with previous reports, we found evidence of HVEM expression among inflammatory cells in HSV-1 infected corneal and trigeminal ganglion tissues. It was very interesting to note that HVEM expression was widespread among inflammatory cells in the acutely infected cornea and ganglia and in the latently infected ganglia, but was quite weak in inflammatory cells present in the cornea 7 months after virus inocu-lation. The significance of these findings is not clear at this point; however, it is possible that differential expression of HVEM in nervous system tissues and in

the cornea has a role in a site-specific regulation of the immune response in these tissues.

In summary, observations reported here indicate that HVEM is widely expressed in the cornea and TG, and therefore may serve as an HSV entry receptor in these tissues. Furthermore, our findings raise the pos-sibility that special and temporal changes in HVEM expression following ocular HSV-1 infection play a role in the regulation of HSV spread in the cornea and TG and in the regulation of HSV-1 infection-induced inflammation in the cornea and TG.

ACKNOWLEDGEMENTS

This work was supported by fellowship grants to S.K.K., E.M., S.D. and S.B. by the Rosztoczy Foundation.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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