ABSTRACT To assess the role of Fas in lesion development during genital HSV-2 infection, we used a well-established HSV-2 murine model applied to MRL-_Fas__lpr_/J (Fas−/−) and

C3-_Fasl__gld_/J (FasL−/−) C57BL6 mice. _In vitro_ infection of murine keratinocytes and epithelial cells was used to clarify molecular details of HSV-2 infection. Despite upregulation of

Fas and FasL, HSV-2-infected keratinocytes and epithelial cells showed a moderate level of apoptosis due to upregulated expression of the anti-apoptotic factors Bcl-2, Akt kinase and

NF-_κ_B. Inflammatory lesions within the HSV-2-infected epithelium of C57BL6 mice consisted of infected cells upregulating Fas, FasL and Bcl-2, uninfected cells upregulating Fas and

inflammatory response in the vaginal epithelium at the initial stage of HSV-2 infection. SIMILAR CONTENT BEING VIEWED BY OTHERS HYALURONIC ACID IS A NEGATIVE REGULATOR OF MUCOSAL

FIBROBLAST-MEDIATED ENHANCEMENT OF HIV INFECTION Article Open access 11 May 2021 IMMUNE ACTIVATION OF VAGINAL HUMAN LANGERHANS CELLS INCREASES SUSCEPTIBILITY TO HIV-1 INFECTION Article Open

access 25 February 2023 TRPV4 CHANNEL IS INVOLVED IN HSV-2 INFECTION IN HUMAN VAGINAL EPITHELIAL CELLS THROUGH TRIGGERING CA2+ OSCILLATION Article 23 September 2022 MAIN Herpes simplex

viruses (HSV) type 1 and 2 are among the DNA viruses interfering at multiple levels with cellular apoptotic processes important for cellular response to virus infection.1 Moderate level of

apoptosis were however observed in HSV-2-infected epithelial cells, in contrast to HSV-1-infected cells, which displayed consistent levels of apoptosis.2 HSV-2-mediated apoptosis occurs

during HSV-2 infection both _in vivo_ – in dorsal root ganglia and spinal cord pituitary gland 3– and _in vitro_ – in peritoneal macrophages,4 monocytes,5 dendritic cells6 and human

peripheral blood mononuclear cells (PBMCs).7 Other studies, however, have concluded that HSV-2 can prevent apoptosis in a manner similar to HSV-1 and that the viral US3 protein kinase8 and

the ribonucleotide reductase9 participate in blocking of the apoptotic process. Furthermore, it was shown that HSV-2 ICP10PK protein, which has a role in establishment of virus latency and

reactivation, inhibits caspase-3 activation through its ability to activate Ras and the downstream survival pathways Raf-1/MEK/ERK and PI3-K/Akt.9, 10 Clinical and laboratory HSV-2 strains

both induced, and subsequently blocked, apoptosis in infected HEp-2 epithelial cells and these biological features were correlated with NF-kB translocation to the nuclei of HSV-2-infected

cells.11 Infection of human keratinocyte cell line HaCaT with HSV-1 and HSV-2 led to a significant increase of apoptosis,12 an event depending on the p63 protein, involved in the maintenance

of stratified epithelial tissues. Together, these observations suggest that whether HSV-2 infection leads to apoptotic cell death or not, depends on the cell type. The Fas/FasL system has

an important role in skin and epithelial homeostasis, carcinogenesis and inflammatory skin diseases.13, 14 Epidermal keratinocytes express Fas but not FasL.15 Abnormal expression of

lytically active FasL was however found in inflammatory skin diseases as toxic epidermal necrolysis, atopic dermatitis and allergic contact dermatitis.13, 14, 16 FasL elicited a

proinflammatory reaction in human keratinocyte HaCaT cell line and reconstructed human epidermis by triggering the expression of stress-responsive transcription factors and inflammatory

cytokines in absence of an apoptotic response.17 HSV-2, but not HSV-1, was shown to inhibit cell surface expression of FasL in PBMCs and K562 leukemic cell line.18 However, _in vivo_

clearance of HSV-2 infection in a murine model requires Fas and perforin-dependent cytotoxic mechanisms, as mice lacking both perforin- and Fas-mediated cytolytic mechanisms were unable to

completely clear the infection from the vaginal tissue.19 The role of Fas/FasL system in the vaginal epithelium may thus not only be limited to the elimination of HSV-2 infected cells via

apoptosis but also related to the development of inflammatory lesions preceding the induction of the local immune response to the infection. We assessed the role of Fas during HSV-2 vaginal

infection using keratinocytes and epithelial cells _in vitro_ and a murine model of genital herpes. _In vitro_, HSV-2-infected cells upregulated Fas and FasL and anti-apoptotic proteins

(Bcl-2, NF-_κ_B, Akt kinase) and displayed moderate levels of apoptosis only during the later stage of infection. Upregulation of Fas, FasL and Bcl-2 was noticed in the HSV-2-infected

epithelium of C57BL/6 mice. In the mice, the cells undergoing apoptosis were infected cells present at the site of the lesions and uninfected, Fas-expressing, epithelial cells surrounding

the HSV-2-infected sites. Experiments performed in HSV-2-infected Fas (−/−) and FasL (−/−) C57BL mice showed a reduced level of apoptosis among uninfected cells and an increased infiltration

of neutrophils at the vaginal site. Our results suggest a role for Fas/FasL in the regulation of inflammatory responses at the early stages of vaginal HSV-2 infection. RESULTS HSV-2

INFECTION INDUCES MODERATE LEVELS OF APOPTOSIS IN EPITHELIAL CELLS AND KERATINOCYTES A significantly higher number of cells, with caspase-3-specific cytokeratin 18 cleavage (M30-positive

cells), was found in HSV-2-infected mouse keratinocytes (291.03C) and epithelial cells (Hepa 1–6) at 24 and 48 h postinfection (p.i.) in comparison with uninfected cultures (_P_≤0.05)

(Figure 1a). Interestingly, in spite of the fact that both infected epithelial and keratinocyte cultures underwent apoptosis, the total cell number in these cultures was comparable to the

control cell cultures (Figure 1b). At 24 h p.i. (HSV-2 MOI 1–10), the number of HSV-2-infected cells reached approximately 80% of cells in culture (Figure 1c). Of these, 48.23±2.34% of

HSV-2-infected epithelial cells and 45.89±4.01% of HSV-2-infected keratinocytes showed apoptotic features at 24 h p.i. (Figure 1d). At 48 h p.i., the population of HSV-2-infected epithelial

cells, corresponding to 90% of cells in culture (Figure 1c), included 56.55±6.78% of apoptotic cells (Figure 1d), whereas HSV-2-infected keratinocytes included 34.93±1.29% of apoptotic cells

(_P_≤0.05) (Figure 1d). These results show that at 48 h of infection, the rate of apoptosis in keratinocyte cultures decreased and in the culture remained a population of HSV-2-infected,

non-apoptotic cells. HSV-2-infected epithelial cells and keratinocytes (Figure 1e) were not susceptible to staurosporine (ST)-induced apoptosis if the treatment with ST was initiated later

than 4 h p.i., thus indicating that HSV-2 induces an apoptosis-resistant status in the infected epithelial cells and keratinocytes within hours from infection through the induction of

anti-apoptotic proteins. Taking into account that HSV-2-infected cells of epithelial and neuronal origin activate anti-apoptotic mechanisms dependent on Bcl-2-family and NF-_κ_B and

PI3-K/Akt pathways,11, 20 we assessed the expression of Bcl-2, NF-_κ_B and Akt in HSV-2-infected epithelial cells and keratinocytes. We found that Bcl-2 was significantly upregulated in both

types of cells during the entire tested period in comparison with uninfected cells (_P_≤0.05) (Figure 2a). In our experimental conditions, both HSV-2-infected cell lines showed a

significant increase in NF-_κ_B activation, especially at 6 h of infection, in comparison with uninfected cells (_P_≤0.05) (Figure 2b). Between 6 and 18 h p.i., NF-_κ_B activation decreased

to levels close to baseline (_P_≤0.05) (Figure 2b). Similarly, a significantly increased activity of the anti-apoptotic Akt kinase (_P_≤0.05) occurred in HSV-2-infected epithelial and

keratinocyte cultures (Figure 2c). HSV-2-INFECTED EPITHELIAL CELLS AND KERATINOCYTES UPREGULATE FAS AND FASL BUT DO NOT DIE THROUGH THIS RECEPTOR PATHWAY Upon infection with HSV-2, mouse

keratinocyte and epithelial cell cultures significantly upregulated both Fas and FasL (_P_≤0.05) (Figures 3a and b). To assess whether HSV-2-infected cells may influence Fas expression on

uninfected cells, we used HSV-2 at MOI 0.1, which resulted in 50% of HSV-2-infected cells and 50% of non-infected cells in culture. The intensity of Fas expression was not only higher for

HSV-2-infected cells (_P_=0.001) but also increased for non-infected cells at 24 h p.i. (_P_=0.013) (Figure 3c). A similar picture was also observed for keratinocytes, with a significant

increase in Fas expression on both non-infected (_P_=0.01) and infected cells (_P_=0.004) (Figure 3c). To assess the sensitivity of HSV-2-infected epithelial cells and keratinocytes to

Fas-induced apoptosis we used an anti-mouse Fas cytotoxic antibody (Jo-1 clone). In both the Hepa 1–6 epithelial cell cultures and in keratinocytes, addition of the anti-Fas antibody to

uninfected cultures resulted in an increased percentage of annexin V-positive cells after 24 h (_P_<0.001) (Figures 4a and b). Addition of anti-Fas antibody to HSV-2-infected cultures

also significantly increased Fas-mediated apoptosis (_P_<0.05) (Figures 4a and b). FasL blocking antibody (clone MFL-4) did not decrease the numbers of apoptotic cells in HSV-2-infected

epithelial cells and keratinocytes (Figures 4a and b). Fas/FasL system has an important role in apoptosis of keratinocytes in the skin,14 and keratinocytes are the main target cells for both

HSV-1 and HSV-2.21 Therefore, we used the anti-mouse Fas cytotoxic antibody Jo-1 to study the involvement of Fas in apoptosis of HSV-2-infected and uninfected keratinocytes. Addition of the

anti-Fas antibody led to a significant decrease in HSV-2-infected cells at 24 h of infection (_P_=0.02) (Figure 4c), whereas addition of anti-FasL blocking antibody (MFL-4) caused a

significant increase of HSV-2-positive cells (_P_=0.032) (Figure 4c). To verify whether the decrease in HSV-2-infected cells occurring upon incubation with the cytotoxic anti-Fas antibody

could result from bystander apoptosis of uninfected cells, we measured apoptosis in cultures infected with HSV-2 at MOI 0.1, when only 50% of cells were infected at 24 h p.i. (Figure 4d).

Although more HSV-2-infected cells underwent apoptosis in the cultures incubated with anti-Fas and anti-FasL antibodies, this difference was not statistically significant (Figure 4d). On the

contrary, in presence of cytotoxic anti-Fas antibody, more uninfected cells were apoptotic in comparison with untreated control cells and cells treated with FasL blocking antibody

(_P_≤0.05) (Figure 4d). FAS IS INVOLVED IN THE DEVELOPMENT OF INFLAMMATORY MUCOSAL LESIONS _IN VIVO_ During HSV-2 infection of C57BL/6 mice, the vaginal mucosa is subjected to development of

lesions, compromising its integrity and ability to protect from the virus attack.22 In our experiments, HSV-2-infected sites were present within the vaginal epithelium of C57BL/6 mice at 3

days of infection. Staining of the vaginal tissues showed that HSV-2-infected cells in the epithelium upregulated both Fas and FasL (Figures 5a and b), whereas non-infected cells surrounding

the HSV-2-infected cells upregulated only Fas (Figures 5a and b). Fas and FasL expressions were also upregulated on Gr-1-positive cells (neutrophils), with only Fas showing a significant

increase (_P_=0.01) (Figure 5c). In HSV-2-infected sites, apoptotic cells, defined as TUNEL-positive, were found not only among HSV-2-positive cells but also among non-infected cells (Figure

6a). To estimate the role of Fas and FasL in the development of vaginal epithelium lesions induced by HSV-2, we infected mice with either a knockout in Fas gene (B6.MRL-_Fas__lpr_/J) or a

knockout in FasL gene (B6Smn.C3-_Fasl__gld_/J) together with the control background strain (C57BL/6). The control, uninfected Fas (−/−) mice presented with a thin vaginal epithelium,

consisting of densely packed cells, whereas uninfected FasL (−/−) mice showed no progesterone-dependent thinning of the epithelium (Figure 6a). In both Fas and FasL knockout mice, the

HSV-2-infected epithelium was shedding off (Figure 6a) and apoptotic bodies could be found at all inflammatory sites (Figure 6a). A significantly higher number of TUNEL-positive cells were,

however, found at the HSV-2-infected sites in Fas- and FasL-deficient mice (Figure 6b). The comparison of Fas- and FasL-deficient mice revealed that Fas (−/−) and FasL (−/−) mice showed more

infected TUNEL-positive cells at the HSV-2-infected sites (Figure 6c). In Fas and FasL knockout mice, a significant increase in infiltrating Gr-1-positive cells (neutrophils) was found

within the HSV-2-infected epithelium, in comparison with infected wild-type (WT) mice (_P_≤0.05) (Figure 6d). A PCR method was used to measure DNA titers in vaginal lavages collected at day

2 of infection in mice; the results showed no differences in the titers of HSV-2 shed into the vaginal lumen in WT and Fas- and FasL-deficient mice (Figure 6e). To verify whether Bcl-2

expression is affected by the depletion of Fas and FasL, we stained for Bcl-2 during HSV-2 infection of the knockout mice. The stainings detected a common pattern of Bcl-2 expression in the

middle part of the infection sites in all infected mice strains (Figure 6f). FAS IS INVOLVED IN THE REGULATION OF KERATINOCYTE INFLAMMATORY RESPONSE _IN VITRO_ To assess whether stimulation

through the Fas/FasL pathway influences the induction of inflammatory responses by HSV-2-infected keratinocytes and epithelial cells, we evaluated the expression of CXCL1/2 chemokines,

TNF-_α_ and IL-1_β_ cytokines. HSV-2 infection of keratinocytes _in vitro_ led to a significant upregulation of CXCL1/2, TNF-_α_ and IL-1_β_ expression at 18 h of infection (_P_≤0.001)

(Figures 7a–c), whereas HSV-2-infected epithelial cells upregulated CXCL1/2 (Figure 7d), TNF-_α_ and IL-1_β_ (data not shown). Addition of anti-Fas cytotoxic antibody to control uninfected

keratinocytes led to a significant upregulation of CXCL1/2 and TNF-_α_ expression at 18 h of incubation (_P_≤0.001) (Figures 7a and b). Interestingly, addition of the cytotoxic anti-Fas

antibody to HSV-2-infected keratinocyte and epithelial cultures significantly abrogated CXCL1/2 expression, whereas, upon these conditions, TNF-_α_ and IL-1_β_ expression was abrogated only

in keratinocytes (_P_≤0.05, Figures 7a–d). To analyze how the blocking of Fas/FasL interaction influences CXCL1/2, TNF-_α_ and IL-1_β_ expression in HSV-2-infected keratinocyte, we used an

anti-FasL blocking antibody. The anti-FasL blocking antibody showed no influence on CXCL1/2, TNF-_α_ and IL-1_β_ expression in uninfected keratinocytes and epithelial cells (Figures 7a–d).

The biological effect of the anti-FasL blocking antibody was significantly different in HSV-2-infected cells. In fact, upon blocking of FasL, the HSV-2-infected keratinocytes significantly

upregulated CXCL1/2 and IL-1_β_ expression (_P_≤0.05), but not TNF-_α_ (Figures 7a–c). HSV-2-infected epithelial cells incubated with anti-FasL blocking antibody significantly upregulated

only CXCL1/2 (_P_≤0.05) (Figure 7d). To verify whether the inflammatory cytokines and chemoattractants produced from keratinocytes infected and/or stimulated with the cytotoxic anti-Fas

antibody exerted a chemotactic role for migration of neutrophils into HSV-2-infected vaginal tissue, we allowed neutrophils to migrate toward keratinocytes subjected to different stimuli

(Figure 7e). The transwell experiment showed that Fas stimulation of uninfected and HSV-2-infected keratinocytes induced a significant migration of Gr-1-positive cells, in comparison with

migration toward control, uninfected cells (_P_≤0.001) (Figure 7e). FasL blocking antibody significantly upregulated migration toward HSV-2-infected cells (_P_≤0.05), whereas Fas-stimulating

antibody significantly decreased migration toward HSV-2-infected keratinocytes (_P_≤0.001) (Figure 7e). Furthermore, staining for CXCL1/2 and IL-1_β_ of the vaginal tissues from

HSV-2-infected WT, Fas- and FasL-deficient mice showed a significantly increased numbers of both infected and uninfected cells expressing CXCL1/2 (Figures 7f and g) and IL-1_β_ (Figures 7f

and g) in Fas- and FasL-deficient mice (_P_≤0.05). DISCUSSION The goal of this study was to investigate the role of Fas pathway during _in vitro_ HSV-2 infection of mouse keratinocyte and

epithelial cells and during _in vivo_ HSV-2 vaginal infection of mice. In our study, both epithelial cells and keratinocytes underwent moderate apoptosis upon HSV-2 infection but not earlier

than 12 h p.i. because of the induction of anti-apoptotic pathways early during infection. Indeed, we could observe NF-_κ_B induction already at 6 h p.i. in both cell lines together with

upregulated expression of the anti-apoptotic factor Bcl-2. Decreased NF-_κ_B levels could be observed before apoptosis induction, remaining at low levels throughout the follow-up time of

infection in both types of infected cell cultures. Activation of the NF-_κ_B transcription factor is one of the anti-apoptotic mechanisms operating in epidermis and epithelial cells.11, 14,

23 Observations similar to the findings presented in this paper were made during infection with HSV-1, whose genome is collinear with HSV-2.24, 25 PI3K/Akt signaling is important in

apoptosis suppression of epithelial cells and keratinocytes.14, 26 In HSV-2-infected cultures of epithelial cells and keratinocytes, we detected an increase in cells positive for the active

form of Akt kinase, thus implying a role for this kinase in apoptosis suppression during the later stages of HSV-2 infection _in vitro_. The involvement of Akt in protection from apoptosis

was previously shown for HSV-2-infected neurons27 and during HSV-1 infection of human epithelial HEp-2 cells.28 Bcl-2 overexpression increased the capacity of HSV-2-infected monocytoid cells

to sustain a fully productive infection, while protecting against HSV-2-induced apoptosis.29 In our experiments, Bcl-2 expression was upregulated during the initial 12 h of infection, to

remain at a stable, low level in the analyzed period. The induction of anti-apoptotic genes, during early events of HSV-2 infection of epithelial cells and keratinocytes, was verified by

experiments conducted in presence of the proapoptotic substance ST. It was previously shown30 that human epithelial HEp-2 cells infected with HSV-1 were resistant to Fas-induced apoptosis.

In our study, HSV-2-infected murine epithelial cells and keratinocytes upregulated Fas and FasL. This event should render the HSV-2-infected cells sensitive to apoptosis delivered either by

FasL present on neighboring cells or through an autocrine pathway (Figure 8). However, a closer analysis showed that induction of apoptosis in the infected epithelial and keratinocytes

cultures by a cytotoxic Fas antibody involved a large portion of uninfected cells, which also upregulated Fas, therefore leading to a decrease in the number of potential target cells for

HSV-2 infection; this process could be diverted by the addition of FasL blocking antibody. These results show that HSV-2-infected cells are relatively resistant to Fas-induced apoptosis and

that cell death of these cells likely occurs through other pathways. Fleck _et al._4 also showed that, although murine macrophages upregulated Fas and TNFR1 receptors upon HSV-2 infection,

inhibition of Fas by addition of soluble Fas and blocking of TNF-_α_ did not prevent HSV-2-induced apoptosis. In the human T-cell line Jurkat, HSV-2 induced apoptosis through extrinsic

(death receptors) and intrinsic (mitochondrial) pathways.31 Therefore, it is likely that mitochondrial pathway may also be involved in apoptosis during HSV-2 infection of epithelial cells

and keratinocytes. The role of Fas/FasL in induction of apoptosis is generally recognized.32 However, evidence accumulates on Fas as a mediator of apoptosis-independent processes, including

proliferation, angiogenesis, fibrosis and inflammation.33 In cells resistant to Fas-induced apoptosis, activation of Fas was shown to induce activation of PI3K/AKT pathway, ultimately

leading to cell migration.34 Therefore, it is possible that keratinocytes may possess the ability to activate non-apoptotic pathways through Fas receptor. In our experimental model,

consisting of murine keratinocyte 03C cells, we demonstrated an increased expression of CXCL1/2, TNF-_α_ and IL-1_β_ in HSV-2-infected keratinocytes. Interestingly, the expression of CXCL1/2

and TNF-_α_ also increased significantly when uninfected keratinocytes were stimulated with the cytotoxic Fas antibody showing their ability to mount a proinflammatory response mediated

through the Fas-dependent pathway (Figure 8). However, the combination of HSV-2 infection and paracrine stimulation of the Fas pathway led to downregulation of the proinflammatory response.

On the contrary, blocking of FasL during HSV-2 infection of keratinocytes led to upregulation of CXCL1/2 and IL-1_β_ responses. The production of the inflammatory cytokines and

chemoattractants was reflected in the migration of Gr1-positive cells toward infected keratinocytes, in that both HSV-2 infection and anti FasL blocking antibody were important mediators for

this phenomenon. Furthermore, the HSV-2-infected sites in Fas- and FasL-deficient mice showed a stronger expression of CXCL1/2 and IL-1_β_ in comparison with HSV-2-infected WT mice,

indicating the role of Fas/FasL in regulation of the inflammatory responses within the vaginal mucosa (Figure 8). CXCL1 and CXCL2 expression is upregulated in the vagina and in the CNS of

mice during acute HSV-2 infection.35 These chemokines specifically targets neutrophils through the CXCR2 receptor, promoting their chemotaxis and activation.35 IL-1_β_ and TNF-_α_ facilitate

the recruitment of neutrophils to inflammatory sites and TNF-_α_ stimulates differentiation of natural killer (NK) cells, a cell population critical in the control of genital HSV-2

infection.36, 37 Vaginal lesions developed during HSV-2 infection destroy the integrity of the local mucosa and lead to increased infiltration of different types of innate immune cells,

including NK cells, macrophages and neutrophils. In our study we considered the possibility that Fas/FasL system may be directly involved in the development of vaginal lesions, as part of

apoptosis induction following HSV-2 infection of mice. The HSV-2-infected epithelial cells upregulated Fas, FasL and also Bcl-2 _in vivo_. These findings, together with the increased

infiltration of neutrophils in Fas- and FasL-deficient mice, suggest that HSV-2-infected cells within the epithelium may be resistant to apoptosis and that Fas/FasL interactions may serve as

a mechanism to reduce inflammation (Figure 8). Depletion of Fas and FasL did not lead to significant changes of HSV-2 titers in the vaginal fluid. It is, however, possible that

Fas-dependent pathway may still influence the amounts of virus retained within the tissue and affect its latency in the ganglia. In this respect, Ishikawa _et al._38 examined HSV-2 lethal

infection in Fas- or FasL-deficient mice. Both the latter types of mice exhibited higher mortality than WT C57BL/6 mice after HSV-2 infection and showed significantly increased viral titers

in the spinal cord compared with WT mice. However, the study38 did not assess vaginal histology. Lack of Fas and FasL in knockout mice increased the numbers of apoptotic-infected cells found

at the infection sites, but decreased the numbers of uninfected cells undergoing apoptosis. However, in the deficient mice, the sites without any visible infiltration of neutrophils

corresponded to sites with scarce or absent apoptotic cells; on the contrary, the sites with infiltration showed significantly higher amounts of apoptotic cells in comparison with similar

sites found in HSV-2-infected WT mice. Neutrophils are the early and predominant innate immune cells, which may contribute to virus clearance through the release of the antiviral cytokines

TNF-_α_, IFN-_α_, IFN-_γ_, GM-CSF or oxygen and nitrogen metabolites.37 However, neutrophil-induced tissue damage by release of oxidants and proteases seen during inflammation can be

controlled in part by neutrophil apoptosis,39 in which Fas/FasL pathway have an important role.39 Our study shows that lack of Fas and FasL in HSV-2-infected mice resulted in lack of control

of the local inflammation induced by neutrophils, facilitating for apoptosis induction. Fas/FasL pathway in the vaginal mucosa appears to participate in the regulation of the local

inflammation. Upon stimulation with extrinsic source of FasL (like NK cells, cytotoxic T cells, etc.), Fas-expressing cells produce proinflammatory cytokines, promoting the recruitment of

neutrophils, NK cells and macrophages. However, the upregulation of FasL by epithelial cells appears to block the effects of extrinsic Fas stimulation, probably functioning as a

self-regulatory mechanism. Fas and FasL upregulation by HSV-2 infected cells appears to have the role of protecting the virus from the effects of immune competent cells infiltrating the

HSV-2 lesion, thus ensuring virus survival and further spreading (Figure 8). MATERIALS AND METHODS VIRUS The HSV-2 strain 333 was grown and titrated in African green monkey kidney cells

(GMK-AH1) and prepared by one cycle of freeze–thaw and subsequent removal of cellular debris by centrifugation.40 CELL LINES AND _IN VITRO_ HSV-2 INFECTION The mouse epithelial Hepa 1–6

cells and mouse keratinocyte cell line 291.03C were kindly provided by M Kulesz-Martin (Department of Dermatology, Oregon Health and Science University, Portland, OR, USA). Hepa 1–6 cells

were maintained in Dulbecco's modified Eagle medium (Thermo Fisher Scientific, Lafayette, CO, USA) with 5% glucose, 10% fetal bovine serum (FBS) and 1% antibiotics (Thermo Fisher

Scientific). The 291.03C line is a 7,12-dimethylbenz[_a_]anthracene-initiated clone, derived from non-transformed 291 cells41 and was cultured in DMEM supplemented with 5% FBS (Thermo Fisher

Scientific), 10 ng/ml epidermal growth factor (Sigma, St. Louis, MO, USA) and 1% antibiotic (Sigma). The cell lines were infected with HSV-2 333 strain at MOI=10 or 0.1, incubated for up to

48 h and then collected by trypsinization or scratching. The cells were further stained with antibodies for external and internal antigens. APOPTOSIS DETECTION Early apoptosis was detected

using Annexin V-Apoptosis detection kit I (BD Biosciences, Franklin Lakes, NJ, USA), according to the producer's protocol. The annexin V-positive, propidium iodide-negative cells were

scored as apoptotic cells, whereas all propidium iodide-positive cells were considered to be necrotic when analyzed in FACS Scan (BD Biosciences). Staurosporine at 5 _μ_M was used as an

inductor of apoptosis (positive control). To detect executive phase of apoptosis, M30 CytoDEATH Fluorescein kit was used (Roche, Indianapolis, IN, USA); the M30 antibody cytoDEATH recognizes

a specific cleavage site within cytokeratin 18 that is not detectable in native cytokeratin 18 of normal cells. Before staining, the samples were permeabilized with Cytofix/Cytoperm

fixation/permeabilization kit (BD Biosciences), according to the manufacturer's protocol and once stained, analyzed in FACS Scan (BD Biosciences) or with Leica fluorescence microscope

equipped with Hamatsu C4880 cold CCD camera (Leica Microsystems GmbH, Wetzlar, Germany), after mounting in medium containing Hoechst 33342 (1 _μ_g/ml) (Sigma). ANTIBODIES AND IMMUNOSTAINING

For detection of cell surface antigens Fas and FasL, infected cells were collected by trypsinization and washed in 1% FBS/PBS. FITC-conjugated monoclonal hamster anti-mouse Fas antibody and

PE-conjugated monoclonal hamster anti-mouse FasL antibody were used to detect surface expression of Fas and FasL (BD Biosciences). Intra-cellular antigens were detected using

Cytofix/Cytoperm fixation/permeabilization kit (BD Biosciences), according to the manufacturer's protocol and by using the following antibodies: FITC-conjugated monoclonal hamster

anti-Bcl-2 antibody (BD Biosciences), PE-conjugated monoclonal mouse anti-p65-NF-_κ_B antibody (BD Biosciences) and polyclonal rabbit anti-p-Akt (Ser 473) (Santa Cruz Biotechnology, Santa

Cruz, CA, USA). HSV-2 antigens were detected using rabbit polyclonal HSV antibody (Dako, Glostrup, Denmark). TNF-_α_, CXCL1/2 and IL-1_β_ were detected using Cytofix/Cytoperm

fixation/permeabilization kit with Golgi plug (BD Biosciences) as described above, using polyclonal goat anti-GRO _α_/_β_ (CXCL1/2) antibody (Santa Cruz Biotechnology), monoclonal hamster

anti-IL-1_β_ antibody (BD Biosciences) and Alexa Fluor 488-conjugated monoclonal rat anti-TNF-_α_ antibody (BD Biosciences). Following incubation with primary antibodies, appropriate

anti-rabbit PE or FITC-conjugated anti-goat or anti-hamster antibodies were used, where necessary (BD Biosciences). Cell suspensions from the vaginal tissue were prepared as follows: vaginal

tissues were cut into small pieces with scissors and then treated with collagenase/dispase (1 mg/ml) (Roche) in Iscove's medium at 37°C for 40 min. Treated tissues were pressed through

a 70 _μ_m cell strainer and washed in PBS. Gr-1-positive cells were detected using anti-CD11c-APC (BD Biosciences) and anti-Ly-6G-PE (BD Biosciences), Fas, FasL and HSV-2 were detected as

described above, using FITC-conjugated antibodies. For all stainings, appropriate isotype control antibodies were used. The stained cell suspensions were analyzed in FACS Scan for the

percentage of positively stained cells and/or mean intensity of fluorescence. The data were further analyzed using FlowJo software (Celeza GmbH, Olten, Switzerland). HSV-2 INFECTION OF MICE

Female mice, 6- to 8-week old, were used for all experiments. B6. MRL-_Fas__lpr_/J (Fas−/−) and B6Smn.C3-_Fasl__gld_/J (FasL−/−) mice were purchased from Charles River (Dortmund, Germany).

C57BL/6 mice were used as controls. Mice were injected s.c. with 2.0 mg/kg of medroxyprogesterone (Depo-Provera; Upjohn, Puurs, Belgium) in 100 _μ_l of PBS. At 6 days later, the mice were

infected by intravaginal inoculation of 104PFU per mice (100 LD50) of HSV-2 strain 333 in 20 _μ_l of HBSS. The animals were kept in ventilated cages under specific pathogen-free conditions

at the Department of Experimental Biomedicine at the Gothenburg University. The studies were approved by the Ethics Committee for Animal Experimentation, Gothenburg, Sweden. At 3 days

following intra-vaginal HSV-2 infection, the animals were killed and the vaginal tissue was used for preparation of cryostat sections. IMMUNOFLUORESCENCE MICROSCOPY OF ANIMAL TISSUE Vaginal

tissue was removed, fixed in 2% paraformaldehyde in PBS for 4 h, then washed twice in 10% sucrose in PBS over 16 h before freezing and cryosectioning. Slides were washed in PBS and used for

apoptosis detection via TUNEL (Millipore, Billerica, MA, USA) or M30 CytoDEATH Fluorescein kit (Roche) according to appropriate manufacturer's protocols. TUNEL and M30 CytoDEATH

Fluorescein detection of apoptosis in tissues was followed by incubation with anti-HSV-2 antibody (1 : 50) (Dako) and secondary anti-rabbit-IgG-PE antibody (Vector Laboratories, Burlingame,

CA, USA). For Fas, FasL, Bcl-2, IL-1_β_ and CXCL1/2 and HSV-2 double stainings, the slides were blocked with 3% BSA in PBS/0.1% Tween for 30 min and incubated overnight at 4°C with

anti-Bcl-2, anti-Fas, anti-FasL, anti-IL-1_β_ (1 : 100) (BD Biosciences) and anti-CXCL1/2 (1 : 100) (Santa Cruz Biotechnology) and anti-HSV-2 (1 : 250) antibodies in 1% BSA in PBS/0.1%

Tween. This procedure was followed by 30 min incubation with goat anti-rabbit IgG-PE antibody (Dako) and goat anti-hamster-IgG-FITC or donkey anti-goat IgG-FITC antibody (Santa Cruz

Biotechnology) in 1% goat serum on PBS/0.1% Tween. After mounting in medium containing Hoechst 33342 (1 _μ_g/ml), fluorescence was captured with Leica fluorescence microscope equipped in

Hamatsu C4880 cold CCD camera. For all stainings, appropriate isotype control antibodies were used. An HSV-2-infected site was defined as the area of HSV-2 positive staining within the

epithelium and the surrounding infiltration of neutrophils, without the sub-mucosal stromal layer. VIRUS TITRATION The virus titers were determined as described by Namvar _et al._42 Briefly,

a 118-nucleotide segment of the gB region from HSV-2 region was amplified using a HSV-2 probe labeled with JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein) in a real-time PCR

instrument ABI Prism 7000 (Applied Biosystems, Carlsbad, CA, USA) and titrated as described.42 NEUTROPHILS ISOLATION AND TRANSWELL MIGRATION ASSAY Neutrophils were isolated as described by

Siemsen _et al._43 and stained with PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma) as indicated by the manufacturer. Purity of isolation was determined by

Gr-1-positive staining and measured by FACS. Neutrophils migration was assayed using 24 well Transwell inserts (BD Biosciences) with 3 _μ_m pores. One million of isolated neutrophils were

loaded into the top well and allowed to migrate for 6 h toward keratinocytes treated with anti-Fas antibody, anti-FasL blocking antibody, HSV-2-infected keratinocytes and HSV-2-infected

keratinocytes treated with anti-Fas antibody or anti-FasL blocking antibody. Migrated cells were then collected and measured using FACS. Extent of migration was expressed as a migration

index, which was defined as the number of cells that migrated in a particular assay by the number of cells that migrated toward cells kept in standard tissue culture of uninfected

keratinocytes. STATISTICAL METHODS Quantitative data were presented as means±S.E.M. In the case of normal distribution of values, confirmed by Shapiro's test, statistical comparisons

were performed using the Student's _t_-test. With non-Gaussian distributions, non-parametric Kruskal–Wallis and Wilcoxon tests were applied. In every analysis values of _P_<0.05 were

considered significant. ABBREVIATIONS * Bcl-2: B-cell lymphoma 2 * CXCL1/2: chemokine ligand 1/2 * GM-CSF: granulocyte macrophage colony stimulating factor * HSV-1 or 2: herpes simplex

virus type 1 or 2 * IFN: interferon * IL: interleukin * MOI: multiplicity of infection * Nf-_κ_B: nuclear factor kappa-light-chain-enhancer of activated B cells * NK cells: natural killer

cells * p.i.: post infection * PBMC: human peripheral blood mononuclear cell * PFU: plaque forming unit * PI3K: phosphatidylinositol 3-kinase * MEK: mitogen-activated protein kinase kinase *

ERK: extracellular signal-regulated kinase * Raf-1: rapidly accelerated fibrosarcoma * TNF: tumour necrosis factor * TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labelling *

JOE: 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein * FITC: fluorescein isothiocyanate * PE: phycoerythrin REFERENCES * Koyama AH, Adachi A . Induction of apoptosis by herpes simplex

virus type 1. _J Gen Virol_ 1997; 78: 2909–2912. Article  CAS  PubMed  Google Scholar  * Koyama AH, Akari H, Adachi A, Goshima F, Nishiyama Y . Induction of apoptosis in HEp-2 cells by

infection with herpes simplex virus type 2. _Arch Virol_ 1998; 143: 2435–2441. Article  CAS  PubMed  Google Scholar  * Ozaki N, Sugiura Y, Yamamoto M, Yokoya S, Wanaka A, Nishiyama Y .

Apoptosis induced in the spinal cord and dorsal root ganglion by infection of herpes simplex virus type 2 in the mouse. _Neurosci Lett_ 1997; 228: 99–102. Article  CAS  PubMed  Google

Scholar  * Fleck M, Mountz JD, Hsu HC, Wu J, Edwards III CK, Kern ER . Herpes simplex virus type 2 infection induced apoptosis in peritoneal macrophages independent of Fas and tumor necrosis

factor-receptor signaling. _Viral Immunol_ 1999; 12: 263–275. Article  CAS  PubMed  Google Scholar  * Mastino A, Sciortino MT, Medici MA, Perri D, Ammendolia MG, Grelli S _et al_. Herpes

simplex virus 2 causes apoptotic infection in monocytoid cells. _Cell Death Differ_ 1997; 4: 629–638. Article  CAS  PubMed  Google Scholar  * Jones CA, Fernandez M, Herc K, Bosnjak L,

Miranda-Saksena M, Boadle RA _et al_. Herpes simplex virus type 2 induces rapid cell death and functional impairment of murine dendritic cells _in vitro_. _J Virol_ 2003; 77: 11139–11149.

Article  CAS  PubMed  PubMed Central  Google Scholar  * Han JY, Sloan DD, Aubert M, Miller SA, Dang CH, Jerome KR . Apoptosis and antigen receptor function in T and B cells following

exposure to herpes simplex virus. _Virology_ 2007; 359: 253–263. Article  CAS  PubMed  Google Scholar  * Hata S, Koyama AH, Shiota H, Adachi A, Goshima F, Nishiyama Y . Antiapoptotic

activity of herpes simplex virus type 2: the role of US3 protein kinase gene. _Microbes Infect_ 1999; 1: 601–607. Article  CAS  PubMed  Google Scholar  * Perkins D, Pereira EF, Aurelian L .

The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) functions as a dominant regulator of apoptosis in hippocampal neurons involving activation of the ERK survival pathway and

upregulation of the antiapoptotic protein Bag-1. _J Virol_ 2003; 77: 1292–1305. Article  CAS  PubMed  PubMed Central  Google Scholar  * Smith CC, Nelson J, Aurelian L, Gober M, Goswami BB .

Ras-GAP binding and phosphorylation by herpes simplex virus type 2 RR1 PK (ICP10) and activation of the Ras/MEK/MAPK mitogenic pathway are required for timely onset of virus growth. _J

Virol_ 2000; 74: 10417–10429. Article  CAS  PubMed  PubMed Central  Google Scholar  * Yedowitz JC, Blaho JA . Herpes simplex virus 2 modulates apoptosis and stimulates NF-kappaB nuclear

translocation during infection in human epithelial HEp-2 cells. _Virology_ 2005; 342: 297–310. Article  CAS  PubMed  Google Scholar  * Megyeri K, Orosz L, Kormos B, Pasztor K, Seprenyi G,

Ocsovszki I _et al_. The herpes simplex virus-induced demise of keratinocytes is associated with a dysregulated pattern of p63 expression. _Microbes Infect_ 2009; 11: 785–794. Article  CAS 

PubMed  Google Scholar  * Trautmann A, Akdis M, Kleemann D, Altznauer F, Simon HU, Graeve T _et al_. T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in

eczematous dermatitis. _J Clin Invest_ 2000; 106: 25–35. Article  CAS  PubMed  PubMed Central  Google Scholar  * Lippens S, Hoste E, Vandenabeele P, Agostinis P, Declercq W . Cell death in

the skin. _Apoptosis_ 2009; 14: 549–569. Article  PubMed  Google Scholar  * Viard-Leveugle I, Bullani RR, Meda P, Micheau O, Limat A, Saurat JH _et al_. Intracellular localization of

keratinocyte Fas ligand explains lack of cytolytic activity under physiological conditions. _J Biol Chem_ 2003; 278: 16183–16188. Article  CAS  PubMed  Google Scholar  * Viard I, Wehrli P,

Bullani R, Schneider P, Holler N, Salomon D _et al_. Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. _Science_ 1998; 282: 490–493. Article

  CAS  PubMed  Google Scholar  * Farley SM, Dotson AD, Purdy DE, Sundholm AJ, Schneider P, Magun BE _et al_. Fas ligand elicits a caspase-independent proinflammatory response in human

keratinocytes: implications for dermatitis. _J Invest Dermatol_ 2006; 126: 2438–2451. Article  CAS  PubMed  Google Scholar  * Sieg S, Yildirim Z, Smith D, Kayagaki N, Yagita H, Huang Y _et

al_. Herpes simplex virus type 2 inhibition of Fas ligand expression. _J Virol_ 1996; 70: 8747–8751. CAS  PubMed  PubMed Central  Google Scholar  * Dobbs ME, Strasser JE, Chu CF, Chalk C,

Milligan GN . Clearance of herpes simplex virus type 2 by CD8+ T cells requires gamma interferon and either perforin- or Fas-mediated cytolytic mechanisms. _J Virol_ 2005; 79: 14546–14554.

Article  CAS  PubMed  PubMed Central  Google Scholar  * Gober MD, Laing JM, Thompson SM, Aurelian L . The growth compromised HSV-2 mutant DeltaRR prevents kainic acid-induced apoptosis and

loss of function in organotypic hippocampal cultures. _Brain Res_ 2006; 1119: 26–39. Article  CAS  PubMed  PubMed Central  Google Scholar  * Taylor TJ, Brockman MA, McNamee EE, Knipe DM .

Herpes simplex virus. _Front Biosci_ 2002; 7: 752–764. Article  Google Scholar  * Parr MB, Kepple L, McDermott MR, Drew MD, Bozzola JJ, Parr EL . A mouse model for studies of mucosal

immunity to vaginal infection by herpes simplex virus type 2. _Lab Invest_ 1994; 70: 369–380. CAS  PubMed  Google Scholar  * Seitz CS, Freiberg RA, Hinata K, Khavari PA . NF-kappaB

determines localization and features of cell death in epidermis. _J Clin Invest_ 2000; 105: 253–260. Article  CAS  PubMed  PubMed Central  Google Scholar  * Goodkin ML, Ting AT, Blaho JA .

NF-kappaB is required for apoptosis prevention during herpes simplex virus type 1 infection. _J Virol_ 2003; 77: 7261–7280. Article  CAS  PubMed  PubMed Central  Google Scholar  * Medici MA,

Sciortino MT, Perri D, Amici C, Avitabile E, Ciotti M _et al_. Protection by herpes simplex virus glycoprotein D against Fas-mediated apoptosis: role of nuclear factor kappaB. _J Biol Chem_

2003; 278: 36059–36067. Article  CAS  PubMed  Google Scholar  * Thrash BR, Menges CW, Pierce RH, McCance DJ . AKT1 provides an essential survival signal required for differentiation and

stratification of primary human keratinocytes. _J Biol Chem_ 2006; 281: 12155–12162. Article  CAS  PubMed  Google Scholar  * Laing JM, Smith CC, Aurelian L . Multi-targeted neuroprotection

by the HSV-2 gene ICP10PK includes robust bystander activity through PI3-K/Akt and/or MEK/ERK-dependent neuronal release of vascular endothelial growth factor and fractalkine. _J Neurochem_

2010; 112: 662–676. Article  CAS  PubMed  Google Scholar  * Benetti L, Roizman B . Protein kinase B/Akt is present in activated form throughout the entire replicative cycle of deltaU(S)3

mutant virus but only at early times after infection with wild-type herpes simplex virus 1. _J Virol_ 2006; 80: 3341–3348. Article  CAS  PubMed  PubMed Central  Google Scholar  * Sciortino

MT, Perri D, Medici MA, Grelli S, Serafino A, Borner C _et al_. Role of Bcl-2 expression for productive herpes simplex virus 2 replication. _Virology_ 2006; 356: 136–146. Article  CAS 

PubMed  Google Scholar  * Morton ER, Blaho JA . Herpes simplex virus blocks Fas-mediated apoptosis independent of viral activation of NF-kappaB in human epithelial HEp-2 cells. _J Interferon

Cytokine Res_ 2007; 27: 365–376. Article  CAS  PubMed  Google Scholar  * Vanden Oever MJ, Han JY . Caspase 9 is essential for herpes simplex virus type 2-induced apoptosis in T cells. _J

Virol_ 2010; 84: 3116–3120. Article  CAS  PubMed  PubMed Central  Google Scholar  * Krammer PH . CD95's deadly mission in the immune system. _Nature_ 2000; 407: 789–795. Article  CAS 

PubMed  Google Scholar  * Hohlbaum AM, Saff RR, Marshak-Rothstein A . Fas-ligand – iron fist or Achilles’ heel? _Clin Immunol_ 2002; 103: 1–6. Article  CAS  PubMed  Google Scholar  * Kleber

S, Sancho-Martinez I, Wiestler B, Beisel A, Gieffers C, Hill O _et al_. Yes and PI3K bind CD95 to signal invasion of glioblastoma. _Cancer Cell_ 2008; 13: 235–248. Article  CAS  PubMed 

Google Scholar  * Thapa M, Carr DJ . Chemokines and chemokine receptors critical to host resistance following genital herpes simplex virus type 2 (HSV-2) infection. _Open Immunol J_ 2008; 1:

33–41. Article  CAS  PubMed  PubMed Central  Google Scholar  * Peng T, Zhu J, Klock A, Phasouk K, Huang ML, Koelle DM _et al_. Evasion of the mucosal innate immune system by herpes simplex

virus type 2. _J Virol_ 2009; 83: 12559–12568. Article  CAS  PubMed  PubMed Central  Google Scholar  * Milligan GN, Bourne N, Dudley KL . Role of polymorphonuclear leukocytes in resolution

of HSV-2 infection of the mouse vagina. _J Reprod Immunol_ 2001; 49: 49–65. Article  CAS  PubMed  Google Scholar  * Ishikawa T, Yamada H, Oyamada A, Goshima F, Nishiyama Y, Yoshikai Y .

Protective role of Fas-FasL signaling in lethal infection with herpes simplex virus type 2 in mice. _J Virol_ 2009; 83: 11777–11783. Article  CAS  PubMed  PubMed Central  Google Scholar  *

Akgul C, Edwards SW . Regulation of neutrophil apoptosis via death receptors. _Cell Mol Life Sci_ 2003; 60: 2402–2408. Article  CAS  PubMed  Google Scholar  * Svensson A, Nordstrom I, Sun

JB, Eriksson K . Protective immunity to genital herpes simplex virus type 2 infection is mediated by T-bet. _J Immunol_ 2005; 174: 6266–6273. Article  CAS  PubMed  Google Scholar  *

Kulesz-Martin M, Yoshida MA, Prestine L, Yuspa SH, Bertram JS . Mouse cell clones for improved quantitation of carcinogen-induced altered differentiation. _Carcinogenesis_ 1985; 6:

1245–1254. Article  CAS  PubMed  Google Scholar  * Namvar L, Olofsson S, Bergstrom T, Lindh M . Detection and typing of herpes simplex virus (HSV) in mucocutaneous samples by TaqMan PCR

targeting a gB segment homologous for HSV types 1 and 2. _J Clin Microbiol_ 2005; 43: 2058–2064. Article  CAS  PubMed  PubMed Central  Google Scholar  * Siemsen DW, Schepetkin IA, Kirpotina

LN, Lei B, Quinn MT . Neutrophil isolation from nonhuman species. _Methods Mol Biol_ 2007; 412: 21–34. Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work was

supported in part by grants from the Swedish Medical Research Council, Swedish state under the ALF agreement, Karolinska Institutet (to FC), EIF Marie Curie fellowship no. 221246 (to MK),

Torsten and Ragnar Söderbergs Research Foundation (to KE) and IngaBritt and Arne Lundbergs Research Foundation (to KE). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of

Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden M Krzyzowska & F Chiodi * Department of Rheumatology and Inflammation Research, University of Gothenburg,

Gothenburg, Sweden A Shestakov & K Eriksson Authors * M Krzyzowska View author publications You can also search for this author inPubMed Google Scholar * A Shestakov View author

publications You can also search for this author inPubMed Google Scholar * K Eriksson View author publications You can also search for this author inPubMed Google Scholar * F Chiodi View

author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to M Krzyzowska. ETHICS DECLARATIONS COMPETING INTERESTS Dr. Chiodi owns

stock in the company Imed AB. The present work was not financed from Imed AB. The other authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by P Salomoni RIGHTS AND

PERMISSIONS This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit

http://creativecommons.org/licenses/by-nc-nd/3.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Krzyzowska, M., Shestakov, A., Eriksson, K. _et al._ Role of Fas/FasL in

regulation of inflammation in vaginal tissue during HSV-2 infection. _Cell Death Dis_ 2, e132 (2011). https://doi.org/10.1038/cddis.2011.14 Download citation * Received: 31 August 2010 *

Revised: 01 February 2011 * Accepted: 10 February 2011 * Published: 17 March 2011 * Issue Date: March 2011 * DOI: https://doi.org/10.1038/cddis.2011.14 SHARE THIS ARTICLE Anyone you share

the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer

Nature SharedIt content-sharing initiative KEYWORDS * HSV-2 * apoptosis * Fas/FasL