Herpes Simplex Virus Type 1-induced FasL Expression in Human Monocytic Cells and Its Implications for Cell Death, Viral Replication, and Immune Evasion

Abstract

Herpes simplex virus type 1 (HSV-1) is a ubiquitously occurring pathogen that infects humans early in childhood. The virus persists as a latent infection in dorsal root ganglia, especially of the trigeminal nerve, and frequently becomes reactivated in humans under conditions of stress. Monocytic cells constitute an important component of the innate and adaptive immune responses. We show here for the first time that HSV-1 stimulates human FasL promoter and induces de novo expression of FasL on the surface of human monocytic cells, including monocytes and macrophages. This virus-induced FasL expression causes death of monocytic cells growing in suspension, but not in monolayers (e.g., macrophages). The addition of a broad-spectrum caspase inhibitor, as well as anti-FasL antibodies, reduced cell death but increased viral replication in the virus-infected cell cultures.

We also show here for the first time that the virus-induced de novo expression of FasL on the cell surface acts as an immune evasion mechanism by causing the death of interacting human CD4+ T cells, CD8+ T cells, and natural killer (NK) cells. Our study provides novel insights on FasL expression and cell death in HSV-infected human monocytic cells and their impact on interacting immune cells.

Introduction

Herpes simplex virus type 1 (HSV-1; hereafter referred to as HSV) is a ubiquitously occurring human herpes virus that infects humans early in life (reviewed in 1–3). It is a member of the α-Herpesviridae subfamily. Primary infections with the virus usually occur in early childhood and are mild or symptomless. However, infected humans can never eliminate the virus and become lifelong carriers. The virus travels from the oral and facial skin nerve endings to dorsal root ganglia, especially of the trigeminal nerve, where it becomes latent. The latent infections frequently become reactivated under conditions of stress, immunosuppression, physical trauma, or exposure to UV radiation (4). These reactivations are often manifested as painful blisters or “cold sores” at the mucocutaneous junctions of the lips. The condition is called herpes labialis. The virus may also infect the cornea and cause keratitis. These conditions cause considerable discomfort and represent a serious health problem. Primary and reactivated latent infections may rarely cause encephalitis, especially in neonates and immunocompetent persons with unknown defects of the immune system (3). HSV infection is the most common cause of sporadic infectious encephalitis in apparently healthy individuals. Effective anti-HSV drugs have been developed; however, the emergence of drug-resistant viruses has also been documented, particularly in immunocompromised individuals (reviewed in 5). Unfortunately, effective vaccines against the virus are not yet available.

 

Monocytes and macrophages represent important cellular elements of the immune system. In response to a viral infection, they release a variety of proinflammatory cytokines and chemokines, and recruit inflammatory cells to the site of infection. Activated macrophages phagocytose pathogens and immune complexes, and present viral antigens to other immune cells. Unlike epithelial cells, in which HSV prevents apoptosis and causes cell death with predominant features of necrosis, HSV infects monocytic cells with different degrees of permissiveness, and appears to induce their cell death via apoptosis (6–8). However, little is known about the mechanism of this virus-induced apoptosis, or its consequences for antiviral immunity as well as for viral replication. We addressed these questions and show here that HSV infection causes apoptosis in human monocytic cells by inducing expression of FasL on their surface. Our data provide experimental evidence showing for the first time that the virus induces FasL at the transcriptional level by stimulating FasL promoter. Interference with this apoptotic pathway prevents cell death, but enhances viral replication. Furthermore, HSV-infected human monocytic cells were able to kill Fas-positive human CD4+ T cells, CD8+ T cells, and natural killer (NK) cells in in vitro co-culture assays. These observations provide valuable insights about the relevance of apoptosis to viral replication and immune evasion in this viral infection.

　

　

Discussion

We have shown here that HSV-1 productively infects human monocytes and macrophages, as well as a monocytic human cell line THP-1. These conclusions were reached using both a wild-type HSV-1, as well as a recombinant virus in which the coding sequences for green fluorescent protein (GFP) were fused in frame with those of the viral gB (12). It is noteworthy that gB is transcribed as a late (γ1) gene. GFP expression could clearly be seen under a confocal microscope in human monocytes, macrophages, and THP-1 cells. Furthermore, these cell types also produced infectious virions, as shown by the ability of their culture supernatants to infect Vero cells, which are known to be very permissive for replication of HSV-1. Previous studies showed that monocytes were resistant to HSV-1 infection, and only become susceptible to it upon differentiation towards macrophages (21–23). Another study showed that human peritoneal macrophages become partially permissive to viral infection upon treatment with thioglycollate, and are fully permissive after prior infection with Corynebacterium parvum (24). We show here that freshly isolated human monocytes and monocyte-derived macrophages, as well as the monocytic cell line THP-1, are susceptible to infection with the virus, albeit with different degrees of permissiveness. The rate of virus replication is much lower in these cells than in Vero cells. Furthermore, we show here that another human monocytic cell line, U937, is not permissive to HSV-1 replication. These results are in agreement with those of Feng et al. (25), who showed that the virus did not replicate in U937 cells, but was able to induce activation of NF-κB in them. The cell line, however, also becomes permissive to viral replication upon differentiation with phorbol myristate acetate, but not with dimethyl sulfoxide or all-trans retinoic acid (23). The cell line expresses the herpesvirus entry mediator (HVEM), a specific receptor for gD of the virion. The viral glycoprotein gD binds HVEM and activates NF-κB (26). Collectively, these data suggest that freshly isolated human CD14+ monocytes, macrophages, and certain monocytic cell lines can be productively infected with HSV-1.

 

We show here for the first time that monocytic cells undergo apoptosis upon infection with HSV-1, and that this occurs due to the virus-induced expression of FasL on the surface of these cells. The addition of anti-FasL antibodies reduced virus-induced cell death in our study. Similar effects of the virus were reported earlier on human monocyte-derived macrophages (27,28). The infected macrophages were reported to induce death of interacting CD8+ T cells. The induction of FasL by these cells served as an immune evasion strategy. We extend these observations and show here for the first time that FasL-expressing HSV-infected macrophages also kill human CD4+ T cells and NK cells. In this regard another human virus, the human immunodeficiency virus type 1 (HIV-1), has also been reported to induce FasL expression on infected macrophages (28a).

 

Given the widespread expression of Fas on the surface of human cells, the HSV-induced expression of FasL on human monocytic cells may induce death of the virus-infected cells via apoptosis, and the infected cells may also kill Fas-positive immune cells (e.g., T cells, neutrophils, and NK cells), and thus evade the antiviral immune response. The first consequence may benefit the host by causing early death of the infected cells, and hence may result in reduced viral production. Indeed our results show that treating HSV-1-infected monocytic cells with a broad-spectrum caspase inhibitor reduces cell death, and increases viral titers in the culture medium. In this respect we have shown that the virus-induced apoptosis is a host beneficial response, as its inhibition with a broad-spectrum caspase inhibitor or with antagonistic anti-FasL antibodies decreases cell death, but increases viral replication. This conclusion is supported by an in-vivo study with HSV-2, a virus that is very closely related to HSV-1, showing that the virus causes more deaths and replicates to higher titers in mice lacking Fas or the FasL gene than in wild-type mice (29). The FasL-positive CD4+ T cells were shown to play a protective role in this study.

 

We also show here for the first time that HSV-1 enhances FasL expression in monocytic cells at the transcriptional level, as the infection stimulates FasL promoter and increases expression of the human FasL promoter-driven reporter gene. Interestingly, replication of the virus seems to be absolutely essential for the virus-mediated induction of FasL expression and cell death in human monocytic cells. Treating the infected cells with acyclovir, which inhibits viral DNA polymerase and viral DNA replication (19), inhibited FasL expression and prevented cell death. Furthermore, we show here that the virus induces de novo FasL expression on the surface of virus-infected, but not on uninfected bystander, cells.

 

It is noteworthy that in addition to monocytic cells, HSV-1 has been shown to induce FasL expression in other cell types. For example, the virus was shown to induce FasL expression and fratricide death in activated human CD8+ T lymphocytes (30). The virus induced FasL expression on various cell types in the eye when mice were infected via its anterior chamber. The infection resulted in enhanced apoptosis of various cell types in the eye and brain of the infected mice (31). A relatively recent study has shown that HSV-1 induces expression of FasL on neonatal, but not on adult, neutrophils (32). The study demonstrated hastened death of the virus-infected neonatal neutrophils that could be inhibited with antagonistic anti-Fas or anti-FasL antibodies. Interestingly, HSV-1 does not induce FasL expression in all cell types. For example, the infection induced cell death in immature dendritic cells not by inducing expression of FasL, but rather by causing enhanced proteasomal degradation of the long form of the cellular FILCE (or pro-caspase 8) inhibitory protein (c-FLIP-L) (33,34).

 

Several early and immediate early proteins of HSV-1 have been shown to induce apoptosis in different human cell types, although their exact mode of action remains unknown. The pro-apoptotic action of these early viral proteins is countered by subsequently-expressed gene products (e.g., α-2, US3, and gJ). The net result is that HSV-1-infected cells become resistant to several exogenous death-inducing stimuli (e.g., osmotic shock, thermal shock, Fas, TNF-α and C2 ceramide; 35–37). Furthermore, a micro RNA encoded by exon 1 of the LAT gene of HSV-1 protects neuroblastoma cells from apoptosis by downregulating TGF-β and SMAD3 expression (38). Interestingly, the resistance of HSV-1-infected cells to the death-inducing stimuli depends upon the cell type. Furthermore, the pro-apoptotic and anti-apoptotic effects of different viral proteins also varied with the cell type. It appears that these viral proteins exert their effects by interacting with different cellular factors. The differential expression of these factors in different human cells may be responsible for the differential effects of the viral proteins with respect to their effect on cell death (reviewed in 39).

 

We have shown here that HSV-1 replicates in human monocyte-derived macrophages (MDM), and induces de novo FasL expression on their surface. The virus-infected cells become rounded and detach from monolayers and die. The cell death in these cells could not be prevented by the addition of anti-FasL antibodies (unpublished data), despite the fact that these cells express FasL. The virus-infected cells retain their sensitivity to death by anti-Fas agonistic antibodies (unpublished data). The lack of Fas-FasL-mediated cell death in these cells may be due to the fact that they grow in monolayers and do not interact with each other via Fas-FasL. The inability of anti-FasL antibodies to reduce their death suggests that their ultimate death may be due to virus-induced changes occurring intrinsically, as happens in epithelial cells (35). We have demonstrated in this study that the virus-infected FasL-expressing macrophages are able to induce apoptosis in Fas-positive cells that interact with them. Thus the virus-induced expression of FasL may be more important for the infected cells in evading natural and virus-specific adaptive immunity of the host.

 

It is noteworthy that because of its strong capacity to kill Fas-positive cells, FasL is stored in secretory lysosomes in hematopoietic cells, and is translocated to the cell surface upon activation of the cell, or upon its interaction with a target cell (reviewed in 39). In line with this paradigm, our Western blot and flow cytometry results showed that THP-1 cells constitutively express FasL intracellularly, but not on the cell surface. The HSV-mediated de novo expression of FasL on the cell surface shows that, in addition to increasing FasL expression at the transcriptional level, the virus is also able to translocate intracellular FasL to the cell surface. Further studies are required to understand the mechanism behind the virus-induced translocation of FasL to the cell surface.

 

Overall, our study provides novel insights into HSV-induced apoptosis in human monocytic cells and its impact on viral replication and antiviral immunity.

　

SOURCE:

Alexandre Iannello,1,,2,,3,,6,* Olfa Debbeche,1,,2,,3,,6,* Raoudha El Arabi,1,,2,,3,,6 Suzanne Samarani,1,,2,,3,,6 David Hamel,2,,5,,6 Flore Rozenberg,7 Nikolaus Heveker,2,,4,,6 and Ali Ahmadcorresponding author1,,2,,3,,6

　

Herpes Simplex Virus Type 1-induced FasL Expression in Human Monocytic Cells and Its Implications for Cell Death, Viral Replication, and Immune Evasion  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3117309/