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Herpes simplex virus type 2 (HSV-2) induces acute local infection followed by latent infection in the nervous system and often leads to the development of lethal encephalitis in immunocompromised hosts. The mechanisms of immune protection against lethal HSV-2 infection, however, have not been clarified. In this study, we examined the roles of Fas-Fas ligand (FasL) signaling in lethal infection with HSV-2 by using mice with mutated Fas (lpr) or FasL (gld) in C57BL/6 background. Both lpr and gld mice exhibited higher mortality than wild-type (WT) C57BL/6 mice after infection with virulent HSV-2 strain 186 and showed significantly increased viral titers in the spinal cord compared with WT mice 9 days after infection, just before the mice started to die. There were no differences in the numbers of CD4+ and CD8+ T cells infiltrated in the spinal cord or in the levels of HSV-2-specific gamma interferon produced by those cells in a comparison of lpr and WT mice 9 days after infection. Adoptive transfer studies demonstrated that CD4+ T cells from WT mice protected gld mice from lethal infection by HSV-2. Furthermore, CD4+ T cells infiltrated in the spinal cord of HSV-2-infected WT mice expressed functional FasL that induced apoptosis of Fas-expressing target cells in vitro. These results suggest that FasL-mediated cytotoxic activity of CD4+ T cells plays an important role in host defense against lethal infection with HSV-2.
Fas-Fas ligand (FasL) signaling-induced apoptotic cell death has pleiotropic roles in T-cell-mediated host defense mechanisms. First, Fas and FasL are expressed on activated T cells and thereby limit their number by inducing suicide or fratricide. It is generally accepted that Fas-mediated activation-induced cell death plays a predominant role during chronic infection, whereas starvation-induced cell death mediated by the proapoptotic BH3-only subgroup of the Bcl-2 protein family is the main mechanism for T-cell death during termination of immune responses in acute infection (30). Fas-FasL signaling might also play a role in T-cell development, as suggested by an accumulation of T-cell receptor αβ-positive (TCR αβ+) CD4− CD8− T cells expressing B220 in lymphoid organs of mice with mutated Fas (lpr) or FasL (gld) although the origin and functions of such double-negative T cells are still a matter of debate (21). Lastly, Fas-FasL interaction can be directly involved in host defense by inducing apoptosis of infected cells to facilitate pathogen clearance (23). Therefore, the roles of Fas-FasL signaling in immune responses for host defense might vary depending on the pathogen.
Herpes simplex virus type 2 (HSV-2) is an alphaherpesvirus that causes genital herpes, the most common viral sexually transmitted disease (29). After initial infection in the vaginal epithelium, HSV-2 invades local nerve termini, travels via retrograde axonal transport to neuronal cell bodies in sensory ganglia, and establishes latent infection (13). However, especially in neonates and immunocompromised hosts, HSV-2 can cause lethal central nervous system (CNS) infection, which indicates the importance of immune systems in limiting the pathogenicity of HSV-2. Immune responses against HSV-2 have been studied in various murine models using different strains of virus and routes of inoculation, with or without vaccination with an attenuated strain of HSV-2. In such vaccination models, CD4+ T cells producing gamma interferon (IFN-γ) predominantly conferred protection against challenge with a virulent strain of HSV-2 (11, 19), whereas various subsets of lymphocytes, including NK cells, NK T cells, and TCR γδ T cells as well as CD4+ T cells were reported to be involved in host defense against primary infection with virulent HSV-2 (3, 15, 24), in which IFN-γ also played an important role (9). Fas-FasL signaling was shown to be dispensable for the clearance of an attenuated strain of HSV-2, which lacks thymidine kinase and causes only transient mild vaginal pathologies but not neurologic diseases (6, 16). Similarly Fas-mediated apoptosis was not involved in the vaccination effect of the attenuated HSV-2 (11). However, the roles of Fas-FasL signaling in host defense against a virulent strain of HSV-2 have not been clarified.
In this study, we examined the roles of Fas-FasL signaling in a murine model of HSV-2 infection by using a highly virulent HSV-2 strain 186 with lpr and gld mice. We found that FasL-Fas signaling plays an important role in host defense against lethal HSV-2 infection.
C57BL/6 mice with mutated Fas (lpr) and mutated FasL (gld) as well as wild-type (WT) C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). C57BL/6 Ly5.1-congenic mice were purchased from Charles River Japan (Hino, Japan). Jα281 knockout (KO) mice with a C57BL/6 background were provided by Masaru Taniguchi (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan) (5). Female mice at 6 to 8 weeks of age were used for the experiments. The Ethics Committee on Animal Experiments of the Faculty of Medicine, Kyushu University, Fukuoka, Japan, approved this study. Experiments were carried out according to the Guidelines for Animal Experiments.
HSV-2 strain 186 was provided by Fred Rapp (Pennsylvania State University College of Medicine, Hershey, PA). The viral stock was grown in monolayer cultures of Vero cells overlaid with minimal essential medium supplemented with 5% calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. The stock was stored frozen at 2 × 107 to 5 × 107 PFU/ml. The virus was diluted in phosphate-buffered saline (PBS) just before infection. Mice were infected intravaginally or subcutaneously with various doses of strain 186 in 10 μl of PBS. For intravaginal infection, mice were injected subcutaneously with 2 ml/mouse β-estradiol 17-cypionate (Sigma Chemical Co., St. Louis, MO) 5 days before infection to synchronize their estrous cycles at the progesterone-dominant stage.
Fluorescein isothiocyanate-conjugated anti-CD8 monoclonal antibody (MAb; 53-6.7), anti-CD44 MAb (IM7) and streptavidin; biotin-conjugated anti-H-2Kb MAb (AF6-88.5) and anti-CD45.1 MAb (A20); phycoerythrin-conjugated anti-CD4 (RM4-5), anti-NK1.1 (PK136), and anti-IFN-γ (XMG1.2) MAbs; and allophycocyanin-conjugated anti-CD3 MAb (145-2C11), anti-CD8 MAb (53-6.7), and streptavidin were purchased from e-Bioscience (San Diego, CA). The cells were incubated with saturating amounts of the MAbs for 30 min at 4°C. Stained cells were run on a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using CellQuest software (BD Biosciences). To calculate cell numbers in each organ by using a flow cytometer, we first applied a standard sample of a known cell concentration and then ran the test samples at the same flow rate for a constant acquisition time.
Lymphocytes infiltrated in the spinal cord were isolated as follows: samples were cut into small pieces with scissors and then treated with collagenase (0.5 mg/ml) (Sigma-Aldrich, St. Louis, MO) at 37°C for 90 min. Treated samples were pressed through a 200-gauge stainless steel mesh and suspended in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μl/ml streptomycin, and 10 mM HEPES. Cell suspension was filtered through a double 74-μm mesh and fractionated by using 33% Percoll gradient centrifugation.
Lymphocytes from the spinal cord were cultured in 96-well flat-bottomed plates (Falcon; Becton Dickinson Ltd., Oxford, United Kingdom) at a density of 2 × 105 cells/well with the same number of mitomycin C-treated spleen cells from naïve C57BL/6 as antigen-presenting cells with or without 2 × 106 PFU UV-inactivated HSV-2 (1.5 J/cm2) for 48 h. IFN-γ in the culture supernatant was measured by using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN).
Lymphocytes from the spinal cord were stimulated with 5 μg/ml of an immunodominant Kb-restricted HSV-2-specific glycoprotein B (gB) peptide consisting of residues 498 to 505 ([gB498-505] SSIEFARL) for 7 or 20 h in a CO2 incubator at 37°C. Brefeldin A (10 μg/ml) was added for the last 5 h of incubation. Spleen cells from naive C57BL/6 Ly5.1-congenic mice were used as antigen-presenting cells and were gated out from analysis by using anti-CD45.1 MAb. Intracellular staining was performed according to the manufacturer's instructions (BD Biosciences). In brief, 100 μl of BD Cytofix/Cytoperm solution (BD Biosciences) was added to the cell suspension with mild mixing and placed for 20 min at 4°C. Fixed cells were washed with 250 μl of BD Perm/Wash solution (BD Biosciences) twice and were stained intracellularly with anti-IFN-γ MAb for 30 min at 4°C.
Spleen cells of WT mice were passed through nylon wool columns, and CD4+ or CD8+ T cells were removed by using an Auto-MACS separator (Miltenyi Biotech, Bergisch Gladbach, Germany). Whole and CD4+ or CD8+ T-cell-depleted spleen cells were adjusted to keep the original number of each cell subset (whole WT or gld T cells, 1 × 107/mouse; CD4-depleted cells, 5 × 106/mouse; CD8-depleted cells, 5.6 × 106/mouse) in PBS and transferred intravenously into naïve gld recipient mice 1 day before HSV-2 infection.
WT WR19L cells (ATCC TIB52; T lymphoma derived from H-2Kd mice) and WR19L cells transfected with a full-length Fas cDNA (W4.5 cells) were used as the targets, which were kindly provided by Takashi Suda (Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University) (26). A total of 1 × 103 target cells were incubated with effector CD4+ T cells for 12 h at 37°C in U-bottomed, 96-well microplates. The number of surviving target cells detected as negative for H-2Kb and propidium iodide (Sigma Chemical Co.) staining was analyzed by a flow cytometer. Percent cytotoxicity was calculated as follows: [(number of living targets without effectors − number of living targets with effectors)/number of living targets without effectors] × 100.
Kaplan-Meier survival curves were analyzed by a log rank test. Differences in viral burdens in mice were analyzed by a Mann-Whitney test. For the other experiments, the statistical significance of the data was determined by a Student's t test. P values of less than 0.05 were considered statistically significant.
In preliminary experiments, we inoculated HSV-2 strain 186 intravaginally to recapture the physiological infection process. To synchronize the estrous cycle, we injected 2 mg of β-estradiol 17-cypionate to each mouse 5 days before infection. However, it was difficult to reproducibly establish latent infection with this highly virulent strain of HSV-2. Viral DNA was not always detected in the spinal cord of surviving mice, whereas all mice died at higher doses of the virus (data not shown). We next injected the virus subcutaneously into the footpads, a model that is also known to induce CNS infection (14), and found much superior reproducibility of latent infection. Therefore, to determine precise roles of Fas-FasL signaling in CNS infection, we used the subcutaneous route of infection with HSV-2 throughout the experiments.
After a subcutaneous injection of 2 × 105 PFU of HSV-2 strain 186, which was about a 50% lethal dose for WT C57BL/6 mice, all lpr mice died (P < 0.01) (Fig. (Fig.1A).1A). About half of the lpr mice died after infection with 2 × 104 PFU of HSV-2 although all WT mice survived (P < 0.01) (Fig. (Fig.1B).1B). gld and lpr mice showed similar susceptibility to lethal HSV infection. Most of the mice that died after HSV-2 infection exhibited paralysis of limbs with the appearance of infectious virions in the brain (data not shown). HSV-2 viral titers in the spinal cord of lpr and gld mice were significantly higher than titers of WT mice on day 9 after infection, which was just before lpr or gld mice started to die (P < 0.05) (Fig. (Fig.1C).1C). Therefore, it was revealed that Fas-FasL signaling plays a significant role in protection against lethal HSV-2 infection.
The results described above suggest the involvement of Fas-FasL signaling in the clearance of HSV-2. However, it is also possible that an impaired activation-induced cell death due to the lack of Fas-FasL signaling caused excess infiltration in the CNS, leading to increased mortality in lpr and gld mice. Therefore, we performed flow cytometric analysis to quantitatively evaluate cellular infiltration in the spinal cord. There was no significant difference in the number of CD4+ T cells or CD8+ T cells infiltrated in the spinal cord on day 9 after HSV-2 infection (Fig. 2A and B). There was also no difference in the number of macrophages, neutrophils, NK cells, and NK T cells (data not shown). Lymphocytes including T cells were rarely found even in lpr mice before infection (data not shown). The numbers of CD4+ T cells and CD8+ T cells did not differ between WT and lpr mice, and these numbers were not changed significantly by HSV-2 infection (Fig. 2C and D). In contrast, there was a significant increase in CD4− CD8− T cells in the spleen of lpr mice after HSV-2 infection. These CD4− CD8− cells expressed B220 (data not shown). However, although CD4− CD8− T cells reached nearly half the number of T cells in the spleen, they were much less abundant in the CNS (Fig. (Fig.2A).2A). In addition, there was no statistically significant difference in the number of CD4− CD8− T cells in the CNS between WT and lpr mice (Fig. (Fig.2B2B).
Although there was no significant difference in the total number of CD4+ or CD8+ T cells, the lack of Fas-FasL signaling might affect HSV-2-specific T-cell responses. We measured IFN-γ production of CD4+ T cells infiltrated in the spinal cord in response to UV-inactivated HSV-2 by an ELISA. It was revealed that HSV-2-specific IFN-γ production by CD4+ T cells did not differ between WT and lpr mice on day 9 after infection (Fig. (Fig.3A).3A). Flow cytometric analysis for HSV-2 peptide-specific IFN-γ production revealed that a large part of CD8+ T cells in the spinal cord produced IFN-γ in response to HSV-2 peptide (Fig. (Fig.3B).3B). There was no difference in the number of IFN-γ-positive CD8+ T cells between WT and lpr mice. There was also no difference in the number of HSV-2-specific CD8+ T cells in the spleen of WT and lpr mice (data not shown).
FasL-mediated killing of infected cells is an alternative host defense mechanism against viral infection (23). As FasL can be expressed on various kinds of cells, whose importance varies depending on the pathogen, we next examined FasL expression to determine which cell population was important for protection against HSV-2. As significant staining of FasL was not detected by flow cytometry even after infection with HSV-2, adoptive transfer experiments were undertaken by using gld mice as recipients (Fig. (Fig.4A).4A). CD4+ or CD8+ cell-depleted nylon wool nonadherent splenocytes from WT mice were used as donors. All the gld mice to which CD8+ cell-depleted donor cells were transferred survived after HSV-2 infection, which is similar to results in unmanipulated WT mice. However, transfer of CD4+ cell-depleted WT cells did not improve the survival of gld mice infected with HSV-2. These results indicate that FasL expressed on CD4+ T cells but not on either CD8+ T cells or on CD4− CD8− T cells was critical to the protection against lethal HSV-2 infection. Because a subset of NK T cells also express CD4 and exhibit FasL-mediated cytotoxicity (31), we examined the role of NK T cells in HSV-2 infection by using Jα281 KO mice, which selectively lack NK T cells (5). However, no significant difference in survival rates was detected between WT and Jα281 KO mice after HSV-2 infection, even when WT mice received a 50% lethal dose (Fig. (Fig.4B).4B). Therefore, FasL expression on conventional CD4+ T cells but not on CD4+ NK T cells is likely to be responsible for the protection against lethal infection with HSV-2.
We lastly examined whether the CD4+ T cells infiltrated in the spinal cord of HSV-2-infected WT mice expressed functional FasL that induces apoptosis of Fas-expressing cells by an in vitro cytotoxic assay using Fas-transfectant lymphoma cells (W4.5) as the targets (Fig. (Fig.5A).5A). It was previously shown that W4.5 cells underwent apoptosis by Fas-mediated signaling (26). The parental nontransfectants (WR19L) were used as the negative control. CD4+ T cells isolated from the spinal cord of HSV-2-infected WT or gld mice were incubated ex vivo with the target cells. CD4+ T cells in WT mice showed much higher cytotoxic activity against Fas-expressing targets than CD4+ T cells in gld mice while there was no difference in the cytotoxic activity against Fas-negative targets between WT and gld CD4+ T cells (Fig. (Fig.5B5B).
Fas-FasL signaling is involved in apoptosis of cells of immune systems as well as infected target cells. Thus, Fas-mediated apoptosis has pleiotropic functions in host defense, which may vary depending on the pathogen. In the present study, we found that Fas-FasL signaling plays a protective role against lethal infection with a virulent strain of HSV-2.
There are several possible explanations for the increased susceptibility to HSV-2 infection by the lack of Fas-FasL signaling. First, an impaired activation-induced cell death and the resulting deleterious excess immune responses might cause the increased mortality in lpr or gld mice. However, it seems unlikely to be the case as we did not detect any difference between WT and lpr mice in the numbers of CD4+ and CD8+ T cells in the spinal cord and the spleen. There was also no difference in the levels of HSV-2-specific IFN-γ production by CD4+ T and CD8+ T cells. Nevertheless, the viral titer in the spinal cord was higher in lpr and gld mice, suggesting an impaired virus clearance from CNS responsible for the increased mortality. As we found an increase in CD4− CD8− B220+ T cells in the spleen of HSV-2-infected lpr mice, this unusual population of T cells might be involved in the increased susceptibility to HSV-2 infection. In fact, immunosuppressive functions of the CD4− CD8− B220+ T cells in lpr and gld mice were reported (8). However, in the spinal cord, the number of CD4− CD8− B220+ T cells did not differ between WT and lpr mice. Furthermore, adoptive transfer of relatively small numbers of WT spleen cells restored resistance against HSV-2 infection of gld mice even in the presence of CD4− CD8− B220+ T cells, strongly arguing against this possibility. Therefore, it is most likely that a lack of Fas-mediated apoptosis of infected cells is responsible for the increased virus titer in the spinal cord of lpr and gld mice. Although Fas is not usually expressed on neural cells, it has been shown that inflammation as well as viral infection induces expression of Fas (7, 17, 22).
T-cell-mediated cytotoxicity against infected cells is widely accepted as one of the major host defense mechanisms against viral infection. Generally, CD8+ T cells with cytotoxic granules, which contain perforin and granzymes, play predominant roles while the presence of cytotoxic CD4+ T cells and the involvement of Fas-mediated cytotoxicity have also been shown in some cases (1, 28). West Nile virus (WNV) is a neurotropic flavivirus that, similar to HSV, causes meningitis and encephalitis especially in immunocompromised hosts (32). It was shown that gld mice were highly susceptible to lethal WNV infection (27) although, interestingly, Fas-FasL signaling was not important in protection against a less virulent strain of WNV (33). It should be noted that neural cells are not able to regenerate. Therefore, Fas-mediated apoptosis of infected cells is beneficial only when limiting virus replication outweighs neuronal injury. Although CD8+ T cells play a predominant role in the case of WNV infection, the results of cell transfer experiments indicated that FasL expression on CD4+ T cells was essential for host defense against HSV-2 infection. This is consistent with an earlier report showing the involvement of cytotoxic CD4+ T cells in HSV-2 infection although their dependency on Fas was not verified (18). The presence of Fas-dependent cytotoxic CD4+ T cells was also shown in β2-microglobulin-deficient mice infected with lymphocytic choriomeningitis virus, in which cytotoxic CD4+ cells contributed to lethal pathology (34). Interestingly, an involvement of Fas-mediated cytotoxicity by CD4+ T cells in neurodegenerative diseases was recently demonstrated (2, 4). Thus, FasL-expressing CD4+ T cells might play multifaceted roles in the homeostasis of the CNS.
There have been several studies examining the role of Fas-FasL signaling in murine models of HSV-2 infection. Milligan reported that gld mice exhibited normal levels of protection against intravaginal infection with an attenuated strain of HSV-2, which causes transient local infection but does not spread to the CNS (20). In another study, using attenuated HSV-2 expressing ovalbumin with ovalbumin-specific TCR transgenic T cells, it was shown that either Fas or perforin, but not both, was required to reduce the local viral titer (6). This resembles the case of WNV infection and further supports the hypothesis that the importance of Fas-mediated apoptosis emerges only during lethal infection. Recently, Iijima et al. showed that vaccination with an avirulent strain of HSV-2 induced protection against the virulent 186 strain of HSV-2, in which IFN-γ produced by Th1 cells, but not Fas-mediated apoptosis, was involved (11). It seems to contradict our results at first sight, but it is possible that the importance of FasL-mediated apoptosis diminishes in a situation where a large amount of IFN-γ is rapidly induced by memory Th1 CD4+ T cells. In fact, these investigators showed that local administration of IFN-γ at 2 h, but not 48 h, after infection provided similar enhancement of protection against HSV-2 (11). Therefore, different host defense mechanisms might predominate in immunized and nonimmunized mice. Alternatively, vaccination with an avirulent strain of HSV-2 might not have been able to induce FasL-expressing effector CD4+ T cells. There have also been studies on the role of Fas-FasL signaling in HSV-1 infection. While gld mice showed impaired viral clearance after infection with HSV-1 strain SC16, lpr mice showed normal clearance of the HSV-1 KOS strain (12, 25), further indicating differential requirements for Fas-FasL signaling depending on the strain of the virus.
The present study focuses on roles of Fas-FasL signaling in the acute phase of lethal infection with HSV-2, in which we did not detect a difference in the numbers of T cells. However, it is possible that Fas-mediated death of activated T cells is induced in the surviving mice toward the latent phase of HSV-2 infection because the importance of Fas-mediated activation-induced cell death is pronounced during chronic immune responses (10). In fact, a small portion of lpr and gld mice died at a relatively late phase after HSV-2 infection (Fig. (Fig.1B).1B). Further studies are required to clarify the roles of Fas-FasL signaling in the regulation of T-cell responses during the late to latent phases of HSV-2 infection.
In summary, we found in this study a previously unappreciated role of Fas-FasL signaling in protection against lethal infection with HSV-2. This may further an understanding of the pathogenesis of HSV-2-induced encephalitis for the development of a novel treatment/prevention strategy.
We thank Kazue Hirowatari, Yoko Tagawa, and Hideki Oimomi for their excellent technical assistance. We also thank Takashi Suda (Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University) and Masaru Taniguchi (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan) for providing W4.5 cells and Jα281 KO mice, respectively.
This work was supported in part by the Program of Founding Research Centers for Emerging and Reemerging Infectious Disease and was launched as a project commissioned by the Ministry of Education, Culture, Sports, Science and Technology, Japan; by a Grant-in-Aid for Japan Society for Promotion of Science; and by grants from the Japanese Ministry of Education, Science and Culture (Y.Y.).
We have no financial/commercial conflicts of interests.
Published ahead of print on 9 September 2009.