Clinical Reactivations of Herpes Simplex Virus Type 2 Infection and Human Immunodeficiency Virus Disease Progression Markers

Background

The natural history of HSV-2 infection and role of HSV-2 reactivations in HIV disease progression are unclear.

Methods

Clinical symptoms of active HSV-2 infection were used to classify 1,938 HIV/HSV-2 co-infected participants of the Women''s Interagency HIV Study (WIHS) into groups of varying degree of HSV-2 clinical activity. Differences in plasma HIV RNA and CD4+ T cell counts between groups were explored longitudinally across three study visits and cross-sectionally at the last study visit.

Results

A dose dependent association between markers of HIV disease progression and degree of HSV-2 clinical activity was observed. In multivariate analyses after adjusting for baseline CD4+ T cell levels, active HSV-2 infection with frequent symptomatic reactivations was associated with 21% to 32% increase in the probability of detectable plasma HIV RNA (trend p = 0.004), an average of 0.27 to 0.29 log10 copies/ml higher plasma HIV RNA on a continuous scale (trend p<0.001) and 51 to 101 reduced CD4+ T cells/mm³ over time compared to asymptomatic HSV-2 infection (trend p<0.001).

Conclusions

HIV induced CD4+ T cell loss was associated with frequent symptomatic HSV-2 reactivations. However, effect of HSV-2 reactivations on HIV disease progression markers in this population was modest and appears to be dependent on the frequency and severity of reactivations. Further studies will be necessary to determine whether HSV-2 reactivations contribute to acceleration of HIV disease progression.     

Oxidative Stress Markers Induced by Hyperosmolarity in Primary Human Corneal Epithelial Cells

Oxidative stress has been known to be involved in pathogenesis of dry eye disease. However, few studies have comprehensively investigated the relationship between hyperosmolarity and oxidative damage in human ocular surface. This study was to explore whether and how hyperosmolarity induces oxidative stress markers in primary human corneal epithelial cells (HCECs). Primary HCECs were established from donor limbal explants. The hyperosmolarity model was made in HCECs cultured in isosmolar (312 mOsM) or hyperosmotic (350, 400, 450 mOsM) media. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and anti-oxidative enzymes were analyzed by DCFDA kit, RT-qPCR, immunofluorescent and immunohistochemical staining and Western blotting. Compared to isosmolar medium, ROS production significantly increased at time- and osmolarity-dependent manner in HCECs exposed to media with increasing osmolarities (350–450 mOsM). Hyperosmolarity significantly induced oxidative damage markers in cell membrane with increased toxic products of lipid peroxidation, 4–hydroxynonenal (4-HNE) and malondialdehyde (MDA), and in nuclear and mitochondria DNA with increased aconitase-2 and 8-OHdG. Hyperosmotic stress also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but reduced the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), and glutathione peroxidase-1 (GPX1). In conclusion, our comprehensive findings demonstrate that hyperosmolarity induces oxidative stress in HCECs by stimulating ROS production and disrupting the balance of oxygenases and antioxidant enzymes, which in turn cause cell damage with increased oxidative markers in membrane lipid peroxidation and mitochondrial DNA damage.     

Mitochondrial Dysfunction Enhances Susceptibility to Oxidative Stress by Down-Regulation of Thioredoxin in Human Neuroblastoma Cells

Increasing evidence suggests that Alzheimer’s disease is associated with mitochondrial dysfunction and oxidative damage. To develop a cellular model of Alzheimer’s disease, we investigated the effects of thioredoxin (Trx) expression in the response to mitochondrial dysfunction-enhanced oxidative stress in the SH-SY5Y human neuroblastoma cells. Treatment of SH-SY5Y cells with 15 mM of NaN₃, an inhibitor of cytochrome c oxidase (complex IV), led to alteration of mitochondrial membrane potential but no significant changes in cell viability. Therefore, cells were first treated with 15 mM NaN₃ to induce mitochondrial dysfunction, then, exposed to different concentrations of H₂O₂. Cell susceptibility was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and morphological observation. Expressions of Trx mRNA and protein were determined by RT-PCR; and Western-blot analysis, respectively. It was found that the SH-SY5Y cells with mitochondrial impairment had lower levels of Trx mRNA and protein, and were significantly more vulnerable than the normal cells after exposure to H₂O₂ while no significant changes of Trx mRNA and protein in SH-SY5Y cells exposed to H₂O₂ but without mitochondrial complex IV inhibition. These results, together with our previous study in primary cultured neurons, demonstrated that the increased susceptibility to oxidative stress is induced at least in part by the down-regulation of Trx in SH-SY5Y human neuroblastoma cells with mitochondrial impairment and also suggest the mitochondrial dysfunction-enhanced oxidative stress could be used as a cellular model to study the mechanisms of Alzheimer’s disease and agents for prevention and treatment.     

DNase Induced After Infection of KB Cells by Herpes Simplex Virus Type 1 or Type 2 II. Characterization of an Associated Endonuclease Activity

Purified preparations of the "exonuclease" specified by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) possess an endonuclease activity. The exonuclease and endonuclease activities copurify and cosediment in a sucrose density gradient. Endonuclease activity is only observed in the presence of a divalent cation, and Mg(2+) or Mn(2+) is equally effective as a cofactor with an optimal concentration of 2 mM. A slight amount of endonuclease activity is observed in the presence of Ca(2+), whereas no activity occurs in the presence of Zn(2+). In the presence of Mg(2+), Ca(2+) and Zn(2+) are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn(2+) greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg(2+). The endonuclease was affected by putrescine and spermidine to the same extent as the exonuclease activity, but in marked contrast the endonuclease was inhibited by a 10-fold-lower concentration of spermine compared to the exonuclease. The activity specified by HSV-1 and HSV-2 has very similar properties. HSV-1 and HSV-2 endonuclease cleave covalently closed circular DNA to yield, firstly, nicked circles and then linear DNA which is subsequently hydrolyzed to small oligonucleotides. Cleavage does not appear to be base sequence specific. Conversion of nicked circles to linear DNA and subsequent degradation of linear DNA occurs more rapidly in the presence of Mg(2+) than Mn(2+) presumably by virtue of the presence of the exonuclease activity. Nonsuperhelical covalently closed circular duplex DNA is cleaved by the endonucleases at a rate 60 times slower than the rate observed on the supercoiled form. These data indicate that the HSV-1 and HSV-2 endonuclease preferentially recognize single-stranded DNA regions.     

Interleukin-18 Protects Mice against Acute Herpes Simplex Virus Type 1 Infection

We examined the effects of interleukin-18 (IL-18) in a mouse model of acute intraperitoneal infection with herpes simplex virus type 1 (HSV-1). Four days of treatment with IL-18 (from 2 days before infection to 1 day after infection) improved the survival rate of BALB/c, BALB/c nude, and BALB/c SCID mice, suggesting innate immunity. One day after infection, HSV-1 titers were higher in the peritoneal washing fluid of control BALB/c mice than in that of IL-18-treated mice. A genetic deficiency of gamma interferon (IFN-γ), however, diminished the survival rate and the inhibition of HSV-1 growth at the injection site in the mice. Anti-asialo GM1 treatment had no influence on the protective effect of IL-18 in infected mice. IL-18 augmented IFN-γ release in vitro by peritoneal cells from uninfected mice, while no appreciable IFN-γ production was found in uninfected mice administered IL-18. Although IFN-γ has the ability to induce nitric oxide (NO) production by various types of cells, administration of the NO synthase inhibitor N^G-monomethyl-l-arginine resulted in superficial loss of the improved survival, but there was no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. Based on these results, we propose that IFN-γ produced before HSV-1 infection plays a key role as one of the IL-18-promoted protection mechanisms and that neither NK cells nor NO plays this role.Interleukin-18 (IL-18) is a newly cloned murine and human cytokine (28, 36) previously called gamma interferon (IFN-γ)-inducing factor. It is synthesized by activated macrophages and has a structural relationship to the IL-1 family (5). Precursor IL-18 is processed by IL-1β-converting enzyme and is cleaved into mature IL-18 (11). IL-18 induces IFN-γ production by murine helper T cells and NK cells and stimulates T-cell proliferation and NK activation (18, 28). Moreover, IL-18 augments the Fas ligand-mediated cytotoxic activity of the Th1 clone and the NK cell clone (8, 35). Thus, IL-18 shares some biological activities with IL-12, although no significant homology between the two cytokines has been detected at the protein level (34). Furthermore, treatment with IL-12 and IL-18 has a synergistic effect on IFN-γ production (2, 14, 38, 40).According to a review by Nash (27), not only nonspecific or innate immunity, such as that from IFN, NK cells, or macrophages, but also specific or adaptive immunity is important in protection against herpesvirus infection. Herpes simplex virus is known to be an IFN inducer (13). IFN is produced at an early stage of virus infection. In addition to the direct inhibition of viral replication, it enhances the efficiency of the adaptive (specific) immune response by stimulating increased expression of major histocompatibility complex class I and II or by activating macrophages and NK cells. In protection from infection by herpesviruses, especially cytomegalovirus, NK cells have been major effector cells because of the correlation of increased susceptibility to cytomegalovirus infection with the absence or reduction of NK cell activity, as seen in Chediak-Higashi syndrome patients and beige mice (27). Upon target cell disruption, NK and cytotoxic T cells share not only the perforin but also the Fas ligand as an effector molecule (4, 20, 37). Recently, nitric oxide (NO) was reported to be involved in host defense against bacteria, fungi, parasites, and viruses (10, 16, 19, 39). NO produced by herpes simplex virus type 1 (HSV-1)-infected macrophages is reported to inhibit viral replication (7). CD4⁺ T cells, macrophages, IFN-γ, and tumor necrosis factor (TNF) are important in adaptive immunity against HSV-1 infection. The Th2 response exacerbates HSV-1-induced disease (25).Recently a protective role of IL-18 was reported in microbial infections (6, 17). Here, we demonstrate that IL-18 treatment protects mice from acute viral infection via both IFN-γ-dependent and -independent pathways. Although IFN-γ has the ability to induce NO production by a variety of cells, including macrophages (9), it is not likely to be important, at least in the inhibition of HSV-1 proliferation at the injection site of IL-18-treated mice. Furthermore, the protective effect of IL-18 on HSV-1 infection also does not seem to require complete NK cell activity in our experimental system, whereas our colleagues have already reported that deletion of NK cells by administration of anti-asialo GM1 antibody resulted in lowering of the improved survival rate of tumor-bearing mice treated with IL-18 (23).     

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.     

Herpes Simplex Virus 1 Infection Alters the mRNA Translation Processing in L-02 Cells

HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry （MALDI-TOF-MS）. The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.     

Recognition of Herpes Simplex Virus Type 2 Tegument Proteins by CD4 T Cells Infiltrating Human Genital Herpes Lesions

The local cellular immune response to herpes simplex virus (HSV) is important in the control of recurrent HSV infection. The antiviral functions of infiltrating CD4-bearing T cells may include cytotoxicity, inhibition of viral growth, lymphokine secretion, and support of humoral and CD8 responses. The antigens recognized by many HSV-specific CD4 T cells localizing to genital HSV-2 lesions are unknown. T cells recognizing antigens encoded within map units 0.67 to 0.73 of HSV DNA are frequently recovered from herpetic lesions. Expression cloning with this region of DNA now shows that tegument protein VP22 and the viral dUTPase, encoded by genes U_L49 and U_L50, respectively, are T-cell antigens. Separate epitopes in VP22 were defined for T-cell clones from each of three patients. Reactivity with the tegument protein encoded by U_L21 was identified for an additional patient. Three new epitopes were identified in VP16, a tegument protein associated with VP22. Some tegument-specific CD4 T-cell clones exhibited cytotoxic activity against HSV-infected cells. These results suggest that herpes simplex tegument proteins are processed for antigen presentation in vivo and are possible candidate compounds for herpes simplex vaccines.     

Role for Plasmacytoid Dendritic Cells in the Immune Control of Recurrent Human Herpes Simplex Virus Infection

Plasmacytoid dendritic cells (pDC) are an important component of the innate immune response, producing large amounts of alpha interferon in response to viral stimulation in vitro. Under noninflammatory conditions, pDC are not found in the skin and are restricted in location to the blood and lymph nodes. Therefore, their role in mucosal and cutaneous herpes simplex virus (HSV) infection has not been well-defined. In this study we show a role for human pDC in the immune response to HSV infection. First, by confocal microscopy we showed that pDC infiltrate the dermis of recurrent genital herpes simplex lesions at early and late phases, often at the dermo-epidermal junction. We then showed that pDC in vitro are resistant to HSV infection despite expressing the entry receptors CD111, CD112, and HVE-A. Within the lesions, pDC were found closely associated with CD3⁺ lymphocytes and NK cells, especially those which were activated (CD69⁺). Furthermore, these HSV-exposed pDC were able to stimulate virus-specific autologous T-lymphocyte proliferation. We conclude from this work that pDC may contribute to the immune control of recurrent herpes virus infection in vivo.     

Herpes Simplex Virus Type 1 Renders Infected Cells Resistant to Cytotoxic T-Lymphocyte-Induced Apoptosis

Many viruses interfere with apoptosis of infected cells, presumably preventing cellular apoptosis as a direct response to viral infection. Since cytotoxic T lymphocytes (CTL) induce apoptosis of infected cells as part of the “lethal hit,” inhibition of apoptosis could represent an effective immune evasion strategy. We report here herpes simplex virus type 1 (HSV-1) interference with CTL-induced apoptosis of infected cells and show that HSV-1 inhibits the nuclear manifestations of apoptosis but not the membrane changes. The HL-60 cell line (human promyelocytic leukemia) undergoes apoptosis in response to many stimuli, including incubation with ethanol. After HSV-1 infection (strains E115 and 17+), ethanol-treated cells did not produce oligonucleosomal DNA fragments characteristic of apoptosis, as assayed by gel electrophoresis and enzyme-linked immunosorbent assay. Inhibition was detected 2 h after infection and increased over time. Importantly, HSV-1-infected cells were resistant to apoptosis induced by antigen-specific CD4⁺ CTL, despite the fact that CTL recognition and degranulation in response to infected targets remained intact. Unlike HSV-1, HSV-2 (strains 333 and HG52) did not inhibit DNA fragmentation. In contrast to the inhibition of DNA fragmentation by HSV-1, none of the HSV-1 or -2 strains interfered with the ethanol-induced exposure of surface phosphatidylserine characteristic of apoptosis, as determined by annexin V binding. These results demonstrate that genes of HSV-1 inhibit the nuclear manifestations of apoptosis but not the membrane manifestations, suggesting that these may be mediated via separate pathways. They also suggest that HSV-1 inhibition of CTL-induced apoptosis may be an important mechanism of immune evasion.     

Alpha and Gamma Interferons Inhibit Herpes Simplex Virus Type 1 Infection and Spread in Epidermal Cells after Axonal Transmission

The ability of alpha interferon (IFN-alpha) and IFN-gamma to inhibit transmission of herpes simplex virus type 1 (HSV-1) from neuronal axon to epidermal cells (ECs), and subsequent spread in these cells was investigated in an in vitro dual-chamber model consisting of human fetal dorsal root ganglia (DRG) innervating autologous skin explants and compared with direct HSV-1 infection of epidermal explants. After axonal transmission from HSV-1-infected DRG neurons, both the number and size of viral cytopathic plaques in ECs was significantly reduced by addition of recombinant IFN-gamma and IFN-alpha to ECs in the outer chamber in a concentration-dependent fashion. Inhibition was maximal when IFNs were added at the same time as the DRG were infected with HSV-1. The mean numbers of plaques were reduced by 52% by IFN-alpha, 36% by IFN-gamma, and by 62% when IFN-alpha and IFN-gamma were combined, and the mean plaque size was reduced by 64, 43, and 72%, respectively. Similar but less-inhibitory effects of both IFNs were observed after direct infection of EC explants, being maximal when IFNs were added simultaneously or 6 h before HSV-1 infection. These results show that both IFN-alpha and IFN-gamma can interfere with HSV-1 infection after axonal transmission and subsequent spread of HSV-1 in ECs by a direct antiviral effect. Therefore, both IFN-alpha and -gamma could contribute to the control of HSV-1 spread and shedding in a similar fashion in recurrent herpetic lesions.