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Home医源资料库在线期刊传染病学杂志2003年第187卷第2期

Herpes Simplex Virus Infection of Dendritic Cells: Balance among Activation Inhibition and Immunity

来源:传染病学杂志
摘要:Herpessimplexvirus(HSV)belongstotheHerpesviridiaefamily,whichhavethecharacteristicabilitytoestablishlatencyafterprimaryinfection,andthustopersistinthehumanhostforlife。Viruspreparation。InfectionofDCs。Virusinfectivityassay。...

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1Department of Immunology and Molecular Pathology and 2Department of Oncology, University College London Hospitals, London, United Kingdom

Received 1 April 2002; revised 19 September 2002; electronically published 6 January 2003.

Several lines of evidence suggest that dendritic cells (DCs), the most potent antigen-presenting cells known, play a role in the immunological control of herpes simplex virus (HSV) infections. HSV infection of DCs induced submaximal maturation, but DCs failed to mature further in response to lipopolysaccharide (LPS). LPS induced interleukin (IL)12 secretion, and the induction of primary and secondary T cell responses were impaired by infection. Ultimately, DC infection resulted in delayed, asynchronous apoptotic cell death. However, infected DCs induced HSV recall responses in some individuals. Furthermore, soluble factors secreted by DCs after infection induced DC maturation and primed for IL-12 secretion after LPS stimulation. These data support a pathogenetic model of HSV infection, in which initial delay in the generation of immune responses to HSV at peripheral sites is mediated by disruption of DC function but is overcome by bystander DC maturation and cross-presentation of HSV antigens.

 


     Financial support: International Journal of Experimental Pathology M.B. Ph.D. fellowship program (to G.P.); Cancer Research UK (to K.S.); Wolfson Foundation (G.P.).
     Reprints or correspondence: Prof. Benjamin Chain, Dept. of Immunology and Molecular Pathology, University College, 46 Cleveland St., W1T 4JF London, United Kingdom .


     Herpes simplex virus (HSV) belongs to the Herpesviridiae family, which have the characteristic ability to establish latency after primary infection, and thus to persist in the human host for life. For a virus to do so, it must be able to avoid immune eradication both after primary infection and after subsequent reactivation.

     Although the importance of innate immunity in HSV infection has been well characterized [1], it is the degree of adaptive immunity that best correlates with the severity of consequent clinical disease. Cellular immune defects are more closely associated with severe HSV disease than are humoral immune defects [2], which points to a role for T cells in the control of HSV infection [3]. Precursors of HSV-specific CD4+ and CD8+ T cells are found in peripheral blood, the exact frequency depending on the detection technique used [46], and both of these infiltrate herpetic lesions [7, 8].

     HSV has evolved multiple immune evasion strategies to subvert the immune response and thus enable virus survival and replication in the human host. These include resistance to complement-mediated attack by glycoprotein C [9], prevention of antibody binding [10], and down-regulation of the antigen-presenting machinery through ICP47, which prevents peptide loading onto major histocompatibility complex (MHC) class I and thus interferes with presentation of HSV antigens to T cells [11].

     One possible target site for HSV is at the level of the antigen-presenting cell (APC). The most potent form of APC known is the dendritic cell (DC) [12]. Several viral infections of DCs have been analyzed, and 2 main end points have been observed. In one scenario, DC maturation (increased expression of MHC and costimulatory molecules) occurs, with subsequent enhanced APC activity (e.g., dengue fever virus [13] and influenza [14]). Alternatively, infection results in impaired function and death (e.g., vaccinia virus [15] and measles [16]).

     Networks of DCs in epithelial surfaces, including epidermal Langerhans cells (LCs), are the first professional APCs to encounter HSV on mucosal/cutaneous infection, and the importance of DCs in the generation of immune responses to HSV has been demonstrated directly in murine models. The severity of HSV infections in mice is inversely proportional to the number of CD1a+ LCs in the epidermis [17, 18], and vaccination with UV-inactivated HSV-pulsed DCs has resulted in significant survival and reduced severity of illness after genital HSV-2 infection [19]. Impairment of the expression of costimulatory molecules also reduces HSV-specific CD4+ T cell immunity and survival in mice challenged with HSV-1 [20]. Therefore, the investigation of the consequences of HSV infection of human DCs is of considerable interest.

     Our previous studies were the first to demonstrate that immature human DCs could be infected efficiently in vitro by HSV at low multiplicities of infection (MOI) [21]. HSV infection of both immature [22, 23] and mature [24] DCs has also been investigated in other studies. In the present study, we have analyzed further the consequences of HSV infection on DC phenotype and function. The results highlight the complexity of the interaction between virus and cell. HSV induces partial maturation of DCs, but this process is counterbalanced by a progressive impairment of function that terminates in delayed cell death. Infected populations of DCs have an impaired ability to stimulate both recall and allogeneic responses but retain the ability to stimulate an HSV recall response in some individuals. The data presented here, taken in the context of previous studies, suggest that DCs may have a dual role, which contributes to both the establishment and the resolution of the herpetic peripheral lesion.

MATERIALS AND METHODS

     Antibodies.     The following monoclonal antibodies (MAbs) were used: CD3 (supernatant mouse MAb UCHT1, IgG1; gift from P. C. L. Beverley [Edward Jenner Institute for Vaccine Research, Compton, UK]), CD2 (mouse MAb MAS 593, IgG2b; Harlan), CD19 (supernatant mouse MAb BU12, IgG1; gift from D. Hardie [Birmingham University, Birmingham, UK]), HLA-DR (supernatant mouse MAb L243, IgG2a; gift from P. C. L. Beverley), HLA-DQ (supernatant mouse MAb Ia3, IgG2a; gift from R. Winchester [New York University School of Medicine]), CD14 (supernatant mouse MAb HB246, IgG2b; gift from P. C. L. Beverley), CD1a (supernatant mouse MAb NA1/34, IgG2a; gift from A. McMichael [John Radcliffe Hospital, Oxford, UK]), CD86 (supernatant mouse MAb BU63, IgG1; gift from D. Hardie), and HLA-ABC (W6/32; Serotec).

     Cell preparation.     Monocyte-derived DCs (MDDCs) were prepared from 120 mL of fresh whole blood samples obtained from healthy volunteers. Mononuclear cells separated on Lymphoprep (Nycomed Pharma) (400 g for 30 min) were incubated into 6-well tissue culture plates for 2 h at 37°C in 5% CO2 in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS; PAA Laboratories), 100 U/mL penicillin, 100 g/mL streptomycin, and 2 mM L -glutamine (all from Clare Hall Laboratories, Imperial Cancer Research Fund; referred to hereafter as "medium"). Nonadherent cells were removed, and the adherent cells were cultured in fresh medium with 100 ng/mL human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng/mL interleukin (IL)4 (both gifts from Schering-Plough Research Institute). After 4 days of incubation, loosely adherent cells were collected, and any remaining lymphocytes were removed by incubation with CD3, CD2, and CD19 MAbs, followed by antimouse IgGcoated immunomagnetic Dynabeads (Dynal). DCs were cultured for another 3 days at a starting concentration of 5 × 105 DCs/mL and then were used as immature DCs for infection. In some experiments, control and infected DCs were stimulated to mature by culture in lipopolysaccharide (LPS; 100 ng/mL ) for another 1640 h. In other experiments, control and infected DCs were plated on to fibronectin (FN)coated glass cover slips that had been precoated overnight in 20 g/mL FN (Sigma) in Hanks' balanced salt solution (HBSS).

     T cells were obtained from the 2-h nonadherent population removed on day 1 and were stored at -70°C until future use. B cells and monocytes were depleted by incubation with CD19, HLA-DR, and CD14 MAbs, followed by antimouse immunoglobulincoated immunomagnetic Dynabeads. This procedure also removed 10% of T cells that express HLA-DR and therefore are considered to be activated.

     Virus preparation.     The HSV-1 construct used in the present study was derived from HSV-1 strain 17+ and contains a cassette that consists of a cytomegalovirus (CMV) promoter that drives expression of green fluorescent protein (GFP; Clontech) and a respiratory syncitial virus promoter that drives expression of -galactosidase. This cassette is inserted into the UL43 gene, as described elsewhere [25]. The virus was propagated in baby hamster kidney (BHK)21 (C13) cells, which were grown in Dulbecco's MEM (DMEM) supplemented with 10% FCS and 100 U/mL penicillin/streptomycin, as described elsewhere [26]. This virus showed the same growth characteristics both in vivo and in vitro as the wild-type virus [27], although, because the function of UL43 remains unclear, the possibility that its deletion has on unknown effects on infected cells cannot be excluded.

     Infection of DCs.     Purified immature DCs were centrifuged and infected with appropriate plaque-forming units, depending on the required MOI and the number of cells in the pellet. The virus-DC mixture was incubated at 37°C in 5% CO2 for 1 h. The cells were washed once and plated at 5 × 105 cells/mL in fresh medium with GM-CSF and IL-4. After 16 and 40 h at 37°C in 5% CO2, GFP expression was monitored by fluorescence microscopy or flow cytometry. Infected DCs were used for T cell proliferation assays after 16 h of infection.

     Virus infectivity assay.     DCs and HeLa cells were infected at an MOI of 1, and aliquots of the supernatants and the infected cells were harvested at 0, 24, 48, 72, and 96 h after infection and were stored at -80°C until future use. Harvested samples were thawed, disrupting the cells, and virus yield was assessed by use of plaque assay. Serial dilutions (10-310-8 pfu) of virus in DMEM were prepared and added to 80% confluent BHK cells in 6-well plates in triplicate. After 1 h of incubation at 37°C in 5% CO2, 2.5 mL of 1 : 2 (vol/vol) dilution of 1.6% (wt/vol) carboxymethyl celluloseDMEM supplemented with 10% FCS was added to each well. Cells then were incubated for 48 h at 37°C in 5% CO2, and the number of plaques in each well was counted to determine the virus titer in plaque-forming units per milliliter.

     Microscopy.     DCs were analyzed by confocal microscopy (Bio-Rad Confocal microscope). GFP fluorescence was recorded by a 488-nm excitation laser and was detected in the fluorescence channel by a 522 ± 32-nm emission filter. The images were analyzed by use of Confocal Assistant and Adobe Photoshop software.

     Flow cytometric analysis.     DCs were stained for various surface markers by incubation, first with the relevant MAb for 30 min at 4°C, followed by 1 : 25-diluted phycoerythrein-conjugated goat antimouse immunoglobulin (Jackson ImmunoResearch) for 30 min at 4°C. Cells were examined using a FACScan flow cytometer (Becton Dickinson) and were analyzed with WinMDI software (Joseph Trotter, Scripps Research Institute). Results are expressed as median fluorescence intensity. T cells were stained with the CD3-APCconjugated antibody (UCHT1; BD Pharmingen) for 30 min at 4°C and were examined in the same manner as DCs. T cells in DCT cell cocultures were resuspended to disrupt heterotypic clusters, stained with CD3-APC antibody, and examined in the same manner as DCs

     Cytokine measurement.     After infection (as described above), DCs were resuspended in medium supplemented with GM-CSF and IL-4 at a concentration of 106 cells/mL and were aliquoted in flat-bottomed 96-well microtiter plates with or without 100 ng/mL LPS (Sigma). After 40 h, supernatants were taken, and the IL-12 produced was measured by use of a commercially available ELISA kit (R&D Systems Europe) that detects the p40 subunit of IL-12.

     In some experiments, the effect of supernatant of infected cells on normal uninfected DCs was tested. Supernatants from uninfected and infected DCs were taken after 16 h of incubation. The supernatant from 3 wells was pooled and centrifuged in a minifuge at 16,000 g for 30 min and was passed through a 0.2-m pore filter (Sartorius) to remove any virus particles and cell debris, before it was added to 7-day syngeneic DCs in triplicate. Direct assay of the supernatants after this treatment by plaque assay and for GFP expression did not find any live virus remaining. The supernatant-treated cells then were incubated at 37°C in 5% CO2 for 16 h and were examined for surface phenotype changes and IL-12 secretion using the same method as that for primary cultures.

     Proliferation assays.     Titrations of DCs, either uninfected or infected and incubated for 16 h, were incubated at 37°C in 5% CO2 with either allogeneic or autologous T cells (105 cells/well) with or without 500 U/mL purified protein derivative (PPD) of Mycobacterium tuberculosis (Evans Medical; final concentration, 500 U/mL) in flat-bottomed 96-well microtiter plates. The cell cultures were incubated for 6 days and then were pulsed with 1 mCi 3H-thymidine (ICN Biomedical) for 16 h. Cells were harvested, and proliferation was measured by liquid scintillation counting. All assays were performed in triplicate. Results are expressed as counts per minute.

     HSV-1 serology.     To test for the presence of neutralizing antibodies to HSV-1, serum samples were obtained from volunteers, heat inactivated to remove the effect of complement components, and incubated at 37°C with 105 pfu/mL HSV-1. DMEM supplemented with 10% FCS was used as a negative control. The resulting HSV-serum mixture was titrated on BHK in a virus infectivity assay, as described above. A >3-log decrease in plaque formation was interpreted as reflecting the presence of neutralizing antiHSV antibodies in the serum sample. Neutralizing antibodies are often type common, and this assay therefore does not distinguish between previous infection with HSV-1 and HSV-2.

     Cell viability.     DCs were infected and cultured for 16 and 40 h at 37°C in 5% CO2; 105 DCs/group were cultured in triplicate in a 96-well plate with 20 L of 5-mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) at 37°C in 5% CO2 for 4 h. Then 100 L of 10% SDS (Sigma) was added, and the plate was incubated at 37°C in 5% CO2 overnight. Subsequently, the plate was read in a microplate reader at a wavelength of 570 nm, with wavelength correction at 630 nm. Results are expressed as mean optical density.

     Apoptosis.     To identify whether the cells had undergone apoptosis, cells were washed once and resuspended in HBSS to give 106 cells/mL. Aliquots were fixed by the dropwise addition of an equal volume of 70% ethanol (stored at -20°C), to give a final concentration of 35% ethanol. The cells then were left on ice for 30 min, washed twice, and treated with DNase-free RNase A (Sigma; final concentration, 500 g/mL) at 37°C for 10 min. Propidium iodide (PI; Sigma) was added (final concentration, 50 g/mL), and the cells were analyzed immediately by flow cytometry.

     Statistical analysis.     The means of paired groups were analyzed by a 2-tailed Student's t test.

RESULTS

     HSV infects DCs efficiently and expresses the GFP transgene.     The aim of the initial experiment was to confirm the efficiency of infection by HSV strain 17+ with a CMV-driven GFP-expressing cassette inserted into the nonessential UL43 gene [25]. DCs infected at an MOI of 1 were analyzed by flow cytometry for GFP expression. GFP was detected as early as 2 h after infection. The amount of GFP per cell increased steadily, until a plateau was reached 18 h after infection. This was maintained for at least another 6 h. During this period, the percentage of DCs expressing GFP continued to increase (). The percentage of infected cells reached a plateau after 24 h and later decreased as viability fell (data not shown).  shows that the number of DCs expressing GFP was dependent on the number of virus particles to which the DCs were exposed (). Although HSV infection of DCs is highly efficient, HSV replication within DCs is negligible, relative to a permissive epithelial cell line (), which is consistent with a very low level of immediate early gene ICP0 and ICP22 expression in DCs, compared with that of the permissive cell line (Western blot data not shown).

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Figure 1.        Time- and dose-dependent green fluorescent protein (GFP) expression and virus production in dendritic cells (DCs) after herpes simplex virus type 1 (HSV-1) infection. DCs were infected with HSV-1, and GFP expression was monitored by flow cytometry every 6 h for 24 h after infection (A) or at 16 h after infection (B). A, Mean of median GFP fluorescence (n = 3). B, Representative of 3 experiments. C, DCs and HeLa cells were infected at an MOI of 1, and supernatant and cell lysates were collected for 4 days after infection. Infectious virus was quantified by plaque assay. Representative experiment (n = 3).

     HSV-1 infection of DCs results in morphological changes.     The GFP expression allowed for analysis of the morphology of the infected DCs by fluorescence microscopy. DCs form long processes after adherence to FN () [28]. The ability of DCs to develop these dendritic processes was impaired after infection. In contrast, uninfected DCs in the same culture adhered to FN and formed processes as usual ( and ). Strikingly, at higher cell concentrations, many DC clusters that were composed of a mixture of both infected and uninfected DCs were observed ( and )


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Figure 2.        Effect of herpes simplex virus type 1 (HSV-1) infection on dendritic cell (DC) morphology. DCs were infected with HSV-1 at an MOI of 0.3 and were plated on fibronectin-coated glass coverslips for 16 h. Scale bars represent 50 m. AD, Phase contrast channel. E and F, GFP fluorescence. Representative of 3 experiments.

     HSV infection of DCs induces phenotypic maturation.     The observations made by microscopy suggested that HSV was causing changes in both infected and uninfected DCs. Therefore, the cell-surface expression of a panel of characteristic DC surface markers was analyzed by flow cytometry. Immature DCs were infected, and the surface phenotype of these DCs was analyzed after 16 h of culture in the absence or presence of 100 ng/mL LPS to provide a well-characterized maturation stimulus.

     Gating on GFP-negative and GFP-positive DCs after infection was used to distinguish between uninfected and infected DC populations, respectively. We cannot exclude the possibility that a proportion of GFP-negative DCs were infected but failed to express the transgene. Therefore, we infected DCs at an MOI of 0.3, to ensure the presence of an uninfected population.  shows that HSV infection results in significant phenotypic maturation, as demonstrated most obviously by the increased expression of CD86 in both GFP-negative and GFP-positive DCs. HLA-DQ expression on the surface of DCs also was consistently higher in infected cultures. MHC class I levels were elevated only on GFP-negative DCs. High surface expression of CD1a and low expression of CD14 were not affected by infection, which indicates that neither GFP-negative nor GFP-positive cells reverted to monocytes.

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Figure 3.        Phenotypic changes after herpes simplex virus type 1 (HSV-1) infection of dendritic cells (DCs). Immature DCs were infected with HSV-1 at an MOI of 0.3 and then were cultured for 16 h in presence or absence of lipopolysaccharide (LPS; 100 ng/mL). A, Representative of 3 experiments. B, Mean of median fluorescence intensity (MFI; 3 experiments). MHC I, major histocompatibility complex class I.

     A critical function of DCs is the ability to mature under appropriate stimuli and, as a result, present antigen to T cells more efficiently and in the right context [12]. This is mimicked in vitro by the addition of exogenous LPS, which results in increased HLA-DQ, CD86, and MHC class I expression. Strikingly, in response to LPS stimulation, only GFP-negative DCs were able to up-regulate CD86 to a level higher than that induced by HSV alone, whereas no increase occurred in GFP-positive DCs. This difference was even more apparent for MHC class I expression, which is consistent with the expression of ICP47 in DC, which is known to prevent the expression of MHC class I on the surface of infected cells [11].

     Cytokine secretion after HSV infection.     The phenotypic maturation of DCs by LPS is paralleled by secretion of IL-12. Therefore, we analyzed whether the maturation induced by infection also resulted in IL-12 production (). Although HSV infection did not result in IL-12 production, stimulation of DCs with LPS resulted in significant secretion of the cytokine. Furthermore, IL-12 production in response to LPS stimulation was impaired in a significant dose-dependent manner, with the maximum inhibition observed at the highest MOI (P < .03 for uninfected DCs vs. infection at an MOI of 0.3).

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Figure 4.        Interleukin (IL)12 secretion by dendritic cells (DCs) infected with herpes simplex virus type 1 (HSV-1) in the absence or presence of lipopolysaccharide (LPS; 100 ng/mL). DCs were infected with HSV-1 and were cultured for 40 h in presence or absence of LPS. IL-12 (p40) levels in the supernatant were measured by ELISA. *P < .03, uninfected DCs vs. all other groups. Data are mean ± SEM for 4 experiments. Statistical analysis was done by Student?s t test.

     Uninfected DCs were present in the GFP-negative population. One hypothesis that would account for the phenotypic maturation observed in these cells is that a soluble mediator was secreted in the supernatant after infection (). Thus, DCs were cultured in syngeneic supernatant from uninfected and infected DCs in the absence or presence of 100 ng/mL LPS.  demonstrates that soluble factors in the medium after infection were able to induce DC maturation analogous to that seen with LPS stimulation, as determined by increased expression of CD86 and HLA-DR. However,  demonstrates that the supernatant alone was not a significant stimulus for IL-12 production. Nevertheless, the addition of LPS to these cultures revealed that the supernatant was able to prime the DCs for IL-12 secretion over and above the levels induced by LPS stimulation of uninfected DC (P < .02 for uninfected DC and LPS; P < .04 for supernatant from uninfected DC cultures).

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Figure 5.        Effects of soluble factors released after infection. Dendritic cells (DCs) were infected with herpes simplex virus type 1 (HSV-1) at an MOI of 1, and supernatants were collected 16 h after infection. Supernatants from both groups then were treated, as described in , and then were added to fresh syngeneic DCs. A, Changes in cell surface expression of HLA-DR and CD86 were analyzed by flow cytometry. Representative of 3 experiments. B, Secretion of interleukin-12 (IL-12) (p40) was measured by ELISA. *P < .02, DCs in infected supernatant and lipopolysaccharide (LPS) vs. uninfected DCs plus LPS; #P < .04, DCs in infected supernatant and LPS vs. DCs in uninfected supernatant plus LPS. Data are mean ± SEM of 3 experiments. Statistical analysis was done by Student?s t test.

     T cell responses.     Although the infection of DCs resulted in partial phenotypic maturation, IL-12 secretion was not induced, and the response to inflammatory stimuli was impaired. Therefore, the outcome of HSV infection was explored to see how these effects translate in the DC induction of T cell proliferation. The results are shown in  and .

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Figure 6.        Autologous T cell responses in the presence of herpes simplex virus type 1 (HSV-1)infected dendritic cells (DCs). DCs were infected and cultured for 16 h before the addition of autologous T cells (105), and proliferation was assessed by incorporation of 3H-thymidine. Data are mean ± SEM for triplicate cultures from individual experiments. HSV serostatus of individuals is indicated in box inserts.

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Figure 7.        Memory and primary T cell proliferation elicited by herpes simplex virus type 1 (HSV-1)infected dendritic cells (DCs). A, DCs were infected and cultured for 16 h before the addition of autologous T cells (105) in the presence of purified protein derivative (PPD; 500 U/mL). Data are mean ± SEM of 7 experiments. B, DCs were infected and cultured for 16 h before the addition of allogeneic T cells (105). Data are mean ± SEM of 6 experiments. Proliferation was assessed by incorporation of 3H-thymidine. DC-only proliferation was 56 ± 10 cpm. T cellonly proliferation was 57 ± 11. *P < .05, proliferation in presence of uninfected DCs relative vs. other conditions. Statistical analysis was done by Student?s t test.

     Three healthy individuals were identified whose T cells showed proliferative responses in the presence of HSV-infected DCs (left column). A noteworthy feature was the reverse dose-dependent nature of the responses observed, with the greatest response observed at the lowest MOI. In contrast, 3 other individuals in whom responses were not present were identified ( right column). In total, 10 individuals were tested both for the presence of an HSV-specific T cell proliferative response, as shown in , and for the presence of neutralizing antibodies to HSV in serum. All 5 responders were seropositive, whereas all 5 nonresponders were seronegative. There were no discrepancies between responder status and seropositivity. This close relationship between HSV serostatus and T cell proliferation supports the interpretation that proliferation represents a memory response.

     In those individuals who showed no HSV recall responses, proliferation to another recall antigen, PPD, was analyzed (). All individuals tested had been vaccinated with Bacille Calmette-Guerin. The ability to elicit PPD responses was impaired in infected DC populations in a dose-dependent manner, with the greatest inhibition observed at the highest MOI.

     The capacity of HSV-infected DCs to elicit primary T cell proliferation also was tested in an allogeneic mixed lymphocyte reaction (MLR).  shows that HSV infection resulted in an impaired ability to induce proliferation of allogeneic T cells, with the greatest inhibition again seen at the highest infection rates. The data shown are a mean of 6 individuals, 3 with an HSV recall response and 3 without. The ability to elicit an autologous HSV response did not correlate with the degree of inhibition observed in the allogeneic response.

     It was possible that the functional inhibition observed was being mediated by infection of T cells with HSV from DCs [29]. However, direct flow cytometric evaluation of T cells within the MLR cultures did not detect any significant numbers of CD3 and GFP-positive cells at days 1, 3, or 5 of the allogeneic cultures with infected DCs, although some infected DCs continued to express GFP throughout this period (data not shown).

     Effect of HSV infection on DC viability.     The apparent paradox between DC maturation but impaired cytokine secretion and T cell stimulation required explanation. Hence, DC viability after infection was investigated. Flow cytometric examination of infected DC cultures showed that the forward light scatter of DCs (a measure of cell size) decreased rapidly 1664 h after infection . Three different approaches were used to assess DC viability after infection.

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Figure 8.        Modification of dendritic cell (DC) size after infection with herpes simplex virus type 1 (HSV-1) infection. DCs were infected at an MOI of 1 and were cultured for 16, 40, and 64 h. Forward scatter, a measure of cell size, was assessed by flow cytometry. Representative of 3 experiments.

     Preliminary studies by trypan blue exclusion analysis revealed only a very slight decrease in DC viability in infected cultures 2 days after infection (data not shown).  shows that the reduction of MTT, a measure of overall cellular metabolic activity [30], was slightly but consistently decreased in DCs 16 h after infection. This was not statistically significant. However, there was a significant reduction in viability seen 24 h later (P < .02).

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Figure 9.        Changes in dendritic cell (DC) viability after infection with herpes simplex virus type 1 (HSV-1). DCs were infected at an MOI of 1 and were cultured for 16 or 40 h. MTT reduction was assessed, as described in . *P < .02, mean optical density of uninfected DCs vs. infected cultures. Data are mean optical density ± SEM of 3 experiments. Statistical analysis was done by Student' t test.

     Finally,  shows the result of a representative experiment in which DNA degradation was assessed by PI staining of ethanol-fixed DCs, which is an indication of apoptosis. In uninfected populations of DCs, >90% of the cells were in G0 phase of the cell cycle, as shown by the presence of a single peak of DNA staining. After infection, there was a gradual increase in the proportion of DCs in the sub-G0 zone, which is indicative of DNA degradation, and apoptosis. The asynchronous death of DCs prevented the formation of a distinct sub-G0 peak. The number of DCs in the sub-G0 zone 40 h after infection at different MOIs also was measured and was related directly to the percentage of infected DCs . However, at an MOI of 1, with infection efficiencies typically 40%50%, there were still only 15%20% more apoptotic cells than in uninfected cultures.

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Figure 10.        Induction of apoptosis after herpes simplex virus type 1 (HSV-1) infection of dendritic cells (DCs). DCs were infected at an MOI of 1. Proportion of cells undergoing apoptosis was defined as no. of cells in the sub-G0 phase, as determined by flow cytometry. A, Representative of 3 experiments. B, Mean percentage of cell in sub-G0 phase after 40 h of infection. Data are mean percentage of 3 experiments.

DISCUSSION

     In this study, we have shown that the consequences of HSV infection of DCs are morphological, phenotypic, and functional changes, which reflect both the activation and inhibition of DCs. However, several of the observations documented in this study, including the maturation of neighboring uninfected DCs, the HSV recall responses elicited, and the apoptotic result of infection, all contribute to a model that may be relevant to the in vivo immunopathology and resolution observed at peripheral sites of infection.

     As previously reported, MDDCs are infected efficiently by HSV-1 [21], although immature DCs are not permissive for virus replication, which is analogous to the observation by Kruse et al. [24]. Although the supernatants of infected cultures did contain low levels of HSV, as also reported by Mikloska et al. [23], these levels were very low compared with those in a permissive cell line (e.g., HeLa cells). Infection of DCs leads to a complex series of changes. First, infected cells lost their characteristic DC morphology and formed clusters. This feature of the clustering of DCs themselves, as opposed to the clustering of DCs and T cells, has not been reported elsewhere. In contrast, uninfected cells within the same cultures showed morphological changes characteristic of DC maturation (e.g., increased adherence and formation of long dendritic processes). Second, partial maturation of the DCs was seen, characterized by an up-regulation of MHC class I and class II and CD86. This partial maturation was seen both in infected cells (as evidenced by expression of GFP transgene) and in uninfected cells within the cultures. However, the infected but not the uninfected cells became refractory to full maturation, showing an inability to up-regulate CD86 or MHC class I in response to LPS. Salio et al. [22] reported that immature DCs phenotypically mature after HSV infection in a manner similar to that described here. However, Mikloska et al. [23] did not observe such an acquisition of mature DC phenotype after infection. Both the MOI and the strains of HSV used in these studies were different, which made direct comparison difficult.

     The phenotypic changes described above were accompanied by a reduced ability to produce the Th1-driving cytokine IL-12 after LPS stimulation and by the functional inhibition observed in the stimulation of both primary immune responses (allogeneic MLR) and recall responses to memory antigens (PPD). Nonetheless, despite the decreased function of DCs after infection, we have demonstrated for the first time that HSV recall responses were still elicited in some individuals.

     Finally, infected DCs began to enter apoptosis. Although the reduced viability of DCs after HSV infection by trypan blue exclusion has been reported elsewhere [23], this is the first report in which an apoptotic program has been induced. Entry into apoptosis was extremely slow, with only very slight changes seen 1 day after infection, and only 30% of cells were affected after 40 h, although transgene expression can be detected within 2 h of infection. HSV can prevent apoptosis after infection in some cell types [3133] but not in other cell types [34]. Apoptosis of hematopoietic cells (peripheral blood mononuclear cells [35] and activated T cells [36]) has been noted after HSV infection; therefore, it is not surprising that apoptosis is not prevented in such crucial immune cells as DCs. The relatively low levels of HSV gene expression in DCs may result in production of antiapoptotic proteins that is insufficient to offset the apoptotic stimulus of infection [34].

     Cell death could, in principle, explain some of the functional defects observed. However, HSV alters DC shape and function long before death. First, the changes in DC morphology, which imply significant cytoskeletal rearrangement, can be seen immediately after infection. The ability of HSV to alter cytoskeletal proteins has been noted elsewhere [37], and this ability may be particularly important in DCs, where cytoskeletal structure is critical to function [28, 3840]. Because the vast majority of IL-12 secretion occurs within the first 12 h of LPS stimulation [41], changes in cytokine secretion and cell-surface phenotype also precede any evidence of DC death. Finally, short functional assays of DC and T cell activation (e.g., concanavalin Adependent T cell activation) also showed impaired proliferative responses, despite the reduced relative contribution of DC death over this time period (data not shown). Furthermore, a pronounced inhibition in T cell proliferation has been observed after HSV infection of mature DCs, despite little change in surface phenotype and viability [22, 24], which supports a specific inhibition of DC function.

     The signal induced by HSV that leads to DC maturation has not been elucidated, but HSV binding to its receptor on the DC surface, herpes virus entry mediator (HVEM), may play a role [22]. HVEM is a tumor necrosis factor (TNF) receptor family member that after ligation transmits nuclear factor (NF)Bactivating signals through TNF receptorassociated factors [42]. Recent studies have confirmed that HSV infection does induce NF-B activation [43], the same pathway that can mediate DC maturation [4447].

     The effects of HSV on DCs are rapid, which points to an effect of an early gene product or a protein component of the infectious virus particle viral capsid protein. One of the tegument proteins that the virus carries into infected cells is the virion host shutoff (vhs) protein, which destabilizes host and viral mRNA, enabling accelerated viral mRNA turnover [48]. The impaired ability to secrete IL-12 and the inability to up-regulate CD86 and MHC class I could be explained by the actions of this protein. In contrast, MHC class II molecules are "stored" in endosomes and after maturation associate with the relevant peptides and are shuttled to the surface [49]. Because this process occurs simultaneously with the transcriptional down-regulation of MHC class II [50], vhs would not be expected to have any effect.

     The reduced ability to stimulate T cells may seem paradoxical, in view of the more "mature" phenotype of infected DCs. However, the failure to stimulate T cells effectively might result from cytoskeletal changes in the DCs, as recently suggested in the context of HSV-infected B lymphoblastoid cells [51]. Alternatively, functional inhibition could be mediated during the interaction between DCs and T cells. One possibility would be that the virus blocks an essential "back-signal" by the T cells into the DCs (e.g., CD40/CD40L). Another is that viral glycoprotein insertion into the plasma membrane of DC after infection hinders T cell activation or proliferation, as has been proposed for other viral infections of DCs [16, 52]. The infection of T cells themselves might occur when in coculture with infected DCs [53], but we were unable to document this in the cocultures.

     It is important to relate the in vitro observations to in vivo events. From a virus's viewpoint, subverting adaptive immune responses is often advantageous to its life cycle. In HSV infection, the importance of HSV-specific adaptive immunity in limiting viral replication has been well documented [2, 3, 8]. Because DCs are crucial in initiating adaptive immunity, subversion of this cell type may contribute to the virus's survival. Nevertheless, HSV stimulates cytokine and chemokine production at the site of infection [54]. These mediators may stimulate neighboring uninfected DCs to take up HSV antigen-containing apoptotic/necrotic debris, migrate, and then cross-present to T cells in the draining lymph nodes, as has been suggested for other viruses [55]. This model is consistent with our observation that soluble factors secreted from DCs after HSV infection phenotypically matured and primed uninfected DCs for cytokine secretion.

     The autologous HSV-specific responses that we obtained in vitro also are consistent with this mechanism of cross-priming, because the proliferation was greatest at the lowest MOI, in the presence of the greatest number of uninfected DCs left in coculture. Because T cells were not infected by HSV in coculture, the possibility that the reverse dose-dependent observation resulted from T cell deletion in the presence of a greater number of infected DCs is excluded.

     This model of cutaneous/mucosal infection by HSV-1 would be applicable both for primary environmental infection and for reactivation from neuronal latency. HSV has the potential to infect, among others, epithelial cells (ECs) (e.g., keratinocytes) and DCs. In the first instance, the virus makes many copies of itself in ECs, and these cells die because of cytopathic effects. Some DCs also are infected, in which migratory [22] and HSV-presenting ability is impaired, and die ultimately by apoptosis. DCs neighboring the initial site of infection take up material containing HSV antigens. On receiving maturation stimuli in the inflammatory milieu of the lesion, these cells migrate to the draining lymph nodes and stimulate an HSV-specific immune response that eventually clears any remaining virus particles at the original site of infection. However, the inhibition of DC function may delay the generation of immunity, allowing sufficient time for viral particles to reach and enter cutaneous dermal peripheral nerve endings. Retrograde travel to the dorsal root/trigeminal ganglion permits the virus to establish itself in a latent form [56].

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作者: Gabriele Pollara Katharina Speidel Laila Samady 2007-5-15
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