Murine gammaherpesvirus-68 productively infects ... - Semantic Scholar

Journal of General Virology (2007), 88, 1896–1905

DOI 10.1099/vir.0.82931-0

Murine gammaherpesvirus-68 productively infects immature dendritic cells and blocks maturation Romana Hochreiter,1 Catherine Ptaschinski,1 Steven L. Kunkel2 and Rosemary Rochford1 Correspondence Rosemary Rochford [email protected]

Received 19 February 2007 Accepted 1 March 2007

1

Department of Microbiology and Immunology, SUNY Upstate Medical University, 750 East Adams Sweet, Syracuse, NY, USA

2

Department of Pathology, University of Michigan, Ann Arbor, MI, USA

Many viruses have evolved mechanisms to evade host immunity by subverting the function of dendritic cells (DCs). This study determined whether murine gammaherpesvirus-68 (cHV-68) could infect immature or mature bone-marrow-derived DCs and what effect infection had on DC maturation. It was found that cHV-68 productively infected immature DCs, as evidenced by increased viral titres over time. If DCs were induced to mature by exposure to LPS and then infected with cHV-68, only a small percentage of cells was productively infected. However, limiting-dilution assays to measure viral reactivation demonstrated that the mature DCs were latently infected with cHV-68. Electron microscopy revealed the presence of capsids in the nucleus of immature DCs but not in mature DCs. Interestingly, infection of immature DCs by cHV-68 did not result in upregulation of the co-stimulatory molecules CD80 and CD86 or MHC class I and II, or induce cell migration, suggesting that the virus infection did not induce DC maturation. Furthermore, cHV-68 infection of immature DCs did not result in elevated interleukin-12, an important cytokine in the induction of T-cell responses. Finally, lipopolysaccharide and poly(I : C) stimulation of cHV-68-infected immature DCs did not induce increases in the expression of co-stimulatory molecules and MHC class I or II compared with mock-treated cells, suggesting that cHV-68 infection blocked maturation. Taken together, these data demonstrate that cHV-68 infection of DCs differs depending on the maturation state of the DC. Moreover, the block in DC maturation suggests a possible immunoevasion strategy by cHV-68.

INTRODUCTION Dendritic cells (DCs) play a key role in the initiation of a primary T-cell response (Banchereau et al., 2000; Hart, 1997) and also function in the maintenance of adaptive immune responses. Several features of myeloid DCs contribute to their function. For example, DCs are very efficient at antigen uptake and processing and are able to present antigen for both MHC class I and class II pathways (Banchereau et al., 2000; Banchereau & Steinman, 1998). Of equal importance is their capacity to migrate through tissue as part of the peripheral immune surveillance (Caux et al., 2000). Induction of T-cell immunity against microbial pathogens is thought to occur following the recruitment of immature DCs from the periphery to lymphoid tissues and their subsequent differentiation into mature DCs (Kelsall et al., 2002). Numerous phenotypic and functional features distinguish immature from mature DCs. Immature DCs phagocytose pathogens; this ability is lost following maturation. Maturation of DCs is associated with increases in expression of the co-stimulatory molecules CD80 (B7.1) and CD86 (B7.2), upregulation of MHC 1896

class I and II (Ardavin, 2003) and expression of cytokines such as interleukin (IL)-12 (Trinchieri et al., 2003). Maturation also results in the upregulation of the chemokine receptor CCR7 directing the DCs to the lymph nodes where they prime a T-cell response (Forster et al., 1999; Martin-Fontecha et al., 2003). Because of the primary role of DCs in the initiation of adaptive immunity, viruses have evolved numerous strategies to subvert DC function (reviewed by Moll, 2003). One strategy observed in several viral infections is the inhibition of DC maturation (Engelmayer et al., 1999; Moutaftsi et al., 2002; Pollara et al., 2003; Salio et al., 1999; Smith et al., 2005). Two important human pathogens, the gammaherpesviruses Epstein–Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) infect DCs (Li et al., 2002; Rappocciolo et al., 2006). EBV can infect precursor myeloid cells and interferes with DC development, thus preventing the differentiation of sufficient numbers of DCs (Li et al., 2002). Patients with Kaposi’s sarcoma have been reported to have functionally impaired DCs (Stebbing et al., 2003), but whether this is due to infection with KSHV is 0008-2931 G 2007 SGM Printed in Great Britain

Gammaherpesvirus infection of DCs

unknown. More recently, KSHV was shown to bind to DCSIGN on myeloid DCs ex vivo, and infection of DCs inhibits endocytosis and antigen presentation (Rappocciolo et al., 2006). Although these studies suggest that infection of DCs could be an integral part of the gammaherpesvirus pathogenesis, there are limitations in the study of EBV and KSHV pathogenesis. An amenable model system to study gammaherpesvirus pathogenesis utilizes murine gammaherpesvirus-68 (cHV68). cHV-68-infected DCs can be isolated from lymphoid compartments (e.g. spleen and mediastinal lymph node) and from lung (Flano et al., 2000, 2003, 2005; Marques et al., 2003). More recently, Flano et al. (2005) demonstrated that bone-marrow-derived DCs could be infected with cHV-68. In their study, both productive and latent infection of DCs was detected following ex vivo infection, but they did not distinguish immature from mature DCs in their analysis (Flano et al., 2005). An important question that remains is whether cHV-68 preferentially infects immature DCs or mature DCs, or whether cHV-68 is capable of infecting both. In this study, we tested whether cHV-68 infection of bonemarrow-derived DCs was dependent on the maturation state of the DCs and whether infection resulted in phenotypic changes. We found that cHV-68 productively infected immature DCs and blocked maturation. In contrast, bonemarrow-derived DCs driven to maturation following lipopolysaccharide (LPS) stimulation were latently infected with cHV-68. In addition, we found that cHV-68 infection of bone-marrow precursor cells drastically reduced the development of DCs. These data suggest that, like the other members of the herpesvirus family, gammaherpesviruses target DCs as one potential strategy to subvert host immunity.

METHODS Cell lines and viruses. Owl monkey kidney (OMK) cells (ATCC

CRL-1556) and NIH 3T3 cells (ATCC CRL-1658) were maintained in culture as described previously (Rochford et al., 2001). MEF-1 cells (ATCC CRL-2214) were maintained in DMEM containing 4.5 g glucose l21, 1 mM sodium pyruvate, 10 % fetal bovine serum (FBS), 4 mM glutamine, 100 IU penicillin ml21 and 100 mg streptomycin ml21. cHV-68 expressing enhanced green fluorescent protein (cHV-eGFP) was a generous gift of Dr Ren Sun (UCLA, Los Angeles, CA, USA) and the generation of this recombinant virus has been described previously (Wu et al., 2001). Generation of virus stocks and determination of viral titre by plaque assay were carried out as described previously (Cardin et al., 1996), with the exception that some plaque assays were done on MEF-1 cells instead of NIH 3T3 cells. To UV-inactivate virus, 1 ml of virus stock was placed in a 24well plate and exposed to UV light for three 5 min exposures in a Stratalinker (Strategene). Plaque assays on virus stocks following UV exposure indicated that the virus had been inactivated (data not shown). For mock infection, uninfected OMK cells were processed exactly as if they had been virus infected (Rochford et al., 2001). The resulting supernatant was used for all mock infections.

cells were flushed out of the tibia and femur of C57Bl/6 mice. After ammonium chloride lysis of red blood cells, 26106 bone-marrow cells were plated on non-cell-culture-treated 100 mm Petri dishes in RPMI 1640 supplemented with 10 % heat-inactivated FBS, 2 mM glutamine, 50 mM 2-mercaptoethanol, 100 IU penicillin ml21, 100 mg streptomycin ml21 and 10 ng granulocyte–macrophage colony-stimulating factor (GM-CSF) ml21. GM-CSF was prepared from the culture supernatant of X63.AG-rGMCSF cells (a gift of S. Dewhurst, University of Rochester, NY, USA) (Zal et al., 1994). Cells were fed on days 3 and 6 by adding 50 % fresh medium containing GM-CSF. By day 9, cells had a typical purity of 80–90 % CD11c+ CD11b+ myeloid DCs (data not shown). For infections, DCs were infected with cHVeGFP at an m.o.i. of 5 for 1 h. Unbound virus was removed by washing and cells were replated for 24–48 h. After transfer to new plates, the immature phenotype of the adherent population was verified by their low expression of CD80, CD86 and MHC II, as well as by high uptake of FITC–dextran (data not shown). In maturation experiments, non-adherent cells (mature) were removed prior to the addition of the toll-like receptor (TLR) ligands LPS 026 : B6 (0.1–1 mg ml21) or poly(I : C) (20 mg ml21) (Sigma) onto the adherent, immature DCs. In some experiments, immature DCs harvested at day 8 of culture were matured with 0.1–1 mg LPS 026 : B6 ml21 or poly(I : C), 24 h prior to infection with cHV-eGFP at an m.o.i. of 5 for 1 h. Ex vivo limiting-dilution reactivation assays and plaque assays of infected DCs. Measurement of the frequency of viable cells that

could reactivate from latency was done by a limiting-dilution reactivation assay essentially as described previously (van Dyk et al., 2000; Weck et al., 1996), with the exception that serial threefold dilutions of cells were carried out. To detect pre-formed virus, a duplicate aliquot of cells was disrupted by three rounds of freeze-thawing. To determine viral titres in infected DCs, cells were infected for 1 h, washed and plated in 12-well plates. Supernatant and cells were harvested at various times post-infection (p.i.), cells were disrupted by three rounds of freeze-thawing, and plaque assays were carried out as described previously (Cardin et al., 1996). The yield of virus for each time point was calculated as number of p.f.u. per input number of DCs in each well. Electron microscopy. Immature and mature DCs were infected with cHV-68 for 1 h at an m.o.i. of 5. After 48 h, DCs were harvested and

centrifuged. The resulting pellets were fixed overnight in 2.5 % phosphate-buffered glutaraldehyde, post-fixed in 1 % OsO4, dehydrated in a graded ethanol series followed by propylene oxide and then embedded in Araldite 502 epoxy resin. Ultrathin sections were stained with uranyl acetate and Reynold’s lead citrate before examination using a Tecnai BioTWIN 12 transmission electron microscope (FEI Co.). At least 30 cells were scanned in each preparation. Immunofluorescence staining and flow cytofluorimetric analyses. All methods, media formulations, reagents and strategies for

single-colour and multi-colour immunofluorescence staining and cell sorting have been detailed elsewhere (Hobbs et al., 1993), with the exception that FC-Block (BD-Pharmingen) was added. Multi-colour immunofluorescent-stained cells were analysed with an LSRII flow cytometer (Becton Dickinson). Live cells were collected by gating on forward–side scatter characteristics. Data acquisition was performed with FACSDIVA software (BD Biosciences) and data were further analysed with FLOWJO (Tree Star Inc.) and WINMDI 2.8 software (The Scripps Research Institute). Anti-CD11c, anti-CD80, anti-CD86, anti-MHC class I (H-2Kb), anti-MHC class II (I-A/I-E) and antiCD11b monoclonal antibodies (mAbs) were obtained from BD PharMingen. Cytokine ELISA. Supernatants of DC cultures were harvested at

Culture of DCs. Bone-marrow-derived myeloid DCs were generated

by a method adapted from Lutz et al. (1999). Briefly, bone-marrow http://vir.sgmjournals.org

24 h p.i. and stored at 280 uC until analysis. The p40 subunit of IL-12 (IL-12p40) was quantified by sandwich ELISA using the BD 1897

R. Hochreiter and others OptEIA system (BD Pharmingen) according to the manufacturer’s instructions. IL-10 was detected by a sandwich ELISA technique as described previously (Wen et al., 2006). Transwell migration assay. DC migration was determined by measuring cells migrating through a polycarbonate filter (5 mm pore size) in 24-well Transwell chambers (Costar Corning). cHV-eGFP-

infected DCs were harvested at 24 h p.i. and tested for migration towards CCL19 chemokine (Peprotech), a CCR7 ligand. The lower chambers of the Transwell plates were filled with 600 ml DMEM supplemented with 1 % BSA and 10 mM HEPES with CCL19 (100 ng ml21). DCs (56105) were added in 100 ml DMEM/BSA/ HEPES into the upper chamber and cells were incubated at 37 uC for 3 h. Input cells as well as migrated cells were stained with phycoerythrin (PE)-conjugated anti-CD86 and analysed by flow cytometry.

RESULTS The maturation state of DCs affects their susceptibility to productive cHV-68 infection To investigate the susceptibility of DCs of different maturation states to cHV-68 infection, we first isolated and cultured DCs from C57BL/6 bone marrow. After 8 days of culture, the ex vivo-expanded DCs were left untreated (immature) or treated with LPS to induce maturation. After 24 h, cells were infected with a recombinant cHV-68 that expressed eGFP under control of the human cytomegalovirus (HCMV) promoter (cHV-eGFP). Cells were harvested 24 h later and stained with allophycocyanin (APC)-conjugated anti-CD11c mAb (a DC cell-surface marker) and PE-conjugated anti-CD86 mAb (a maturation marker). In a representative set of over ten experiments, greater than 70 % of cells were immature CD11c+ CD86lo DCs. Of these immature cells, 70 % were infected, as indicated by the percentage of CD11c+ cells that were GFP positive (Fig. 1a). In contrast, DCs driven to maturation by treatment with LPS and then infected with cHV-eGFP appeared to be refractory to cHV-eGFP infection, as indicated by the very low number of GFP-expressing cells (Fig. 1a). To determine whether the immature DCs were productively infected with cHV-68, we performed plaque assays on both cell-associated virus and supernatant harvested from immature DCs and mature DCs at various times p.i. Wells were infected in duplicate and the mean titre in p.f.u. per 106 input DCs from a representative experiment is shown (Fig. 1b). We observed that the supernatant virus titre in the infected immature DCs increased between 8 and 24 h p.i., but reached a plateau by 48 h p.i. The cellassociated virus titre reached its peak at 24 h p.i. and then declined. In contrast, following infection of mature DCs, virus titres did not increase significantly from the input levels, suggesting that they were not productively infected. Cell viability of both mature and immature DCs was slightly lower than mock-infected cells at 24 h p.i. and began to decline by 48 h p.i. (Fig. 1c). However, even at 48 h p.i., there was still greater than 70 % cell viability of infected DCs. 1898

Although less than 8 % of cells were GFP positive following infection of mature DCs, it remained possible that GFP was not expressed if the cells were latently infected. To test this possibility, we performed a limiting-dilution reactivation assay to determine whether there were latently infected mature DCs. In this assay, both viable cells and cells that had been mechanically disrupted to release pre-formed virus were plated onto susceptible MEFs and the percentage of cells showing cytopathic effect (CPE) was calculated to determine the frequency of infected cells. As shown in Fig. 1(d), for immature DCs, there was no difference in the percentage of wells showing CPE when live cells were plated compared with cell lysates, indicating that the infected immature DCs were productively infected and there were no latently infected cells. In contrast, we observed a difference in the frequency of wells showing CPE between wells that received live cells (1 : 3.5) versus disrupted cells (1 : 9). This meant that the majority of mature DCs were latently infected with cHV-68. To compare cHV-68 infection of immature and mature DCs further, electron microscopy studies were performed. Immature and mature DCs were infected for 48 h, and cells were washed and then fixed before being processed for electron microscopy. As shown in Fig. 2(a, b), nucleated capsids were clearly seen in the nucleus of infected immature DCs. Interestingly, we also observed infected DCs being phagocytosed by other DCs in the culture (Fig. 2c). Higher magnification of the phagocytosed infected DCs revealed that the capsids appeared to be empty in contrast to the capsids from infected but non-phagocytosed DCs (compare Fig. 2b and d). No capsids were observed in the nucleus of mature DCs (data not shown). Taken together, the data presented demonstrate that the maturation state of the DCs affects their ability to sustain a productive infection (immature DCs) or a latent infection (mature DCs).

cHV-68 infection of immature DCs blocks maturation Immature DCs are characterized by their ability to phagocytose and have relatively low-level expression of costimulatory molecules (CD80 and CD86), as well as MHC class I and II. In contrast, mature DCs lack the ability to phagocytose but have high-level expression of MHC class I and II as well as co-stimulatory molecules. To determine whether cHV-68 infection of immature DCs induced their maturation, DCs infected with cHV-eGFP or mock infected for 24 h were harvested and stained with APCconjugated anti-CD11c and PE-conjugated antibodies specific for MHC class I and II, co-stimulatory molecules (CD80 and CD86) and CD11b (highly expressed on myeloid DCs). Cells were gated on CD11c+ cells, and GFP expression and histograms for individual surface markers are shown in Fig. 3(a). As a control, immature DCs were treated with LPS to verify that all of the cells were susceptible to a known DC maturation signal. After 24 h of Journal of General Virology 88


Fig. 1. cHV-68 infection of immature and mature DCs. (a) Immature DCs or DCs matured for 24 h with 100 ng LPS ml”1 were infected with cHV-eGFP and harvested 24 h later. Cells were stained with APC-conjugated CD11c and PE-conjugated CD86 mAbs. Cells were gated on CD11c+ (top panel) and analysed for expression of CD86 (maturation marker) and GFP (virus infection) as shown in the bottom panel. (b) Immature DCs or DCs matured for 24 h with 100 ng LPS ml”1 were infected with cHV-eGFP. At 1.5, 4, 8, 24, 48 and 72 h p.i., supernatants (filled bars) and cell lysates [i.e. cell-associated virus; empty bars] were harvested and virus titres were determined by plaque assays. The number of p.f.u. is reported based on the input number of DCs per well. (c) Immature DCs or DCs matured for 24 h with 100 ng LPS ml”1 were infected or not with cHV-eGFP (filled bars, mock infection; shaded bars, cHV-68; empty bars, UV-inactivated cHV-68). After 24, 48 or 72 h p.i., cell viability was determined by a trypan blue exclusion assay. (d) Limiting-dilution reactivation assay. Immature (upper panel) and LPS-matured (lower panel) DCs were infected for 24 h at an m.o.i. of 5. Cells were harvested and plated on MEFs in limiting dilution. In parallel, a separate aliquot of cells was disrupted by freeze-thawing and plated on MEFs. The results are shown as the percentage of wells positive for CPE relative to the value scored positive for viral CPE 3 weeks after plating. Twenty-four wells were plated per cell dilution in each experiment. The results shown are representative experiments from a minimum of three different experiments. X, Total; &, lysate.

LPS treatment, we observed a significant increase in DC maturation markers. In contrast, MHC class II and CD80 and CD86 levels remained significantly lower in the infected GFP+ cells, suggesting that viral infection did not induce differentiation. No changes in MHC class I expression were observed following infection. To test that binding of virus particles induced DC maturation, DCs were infected with cHV-68 that had been inactivated by UV irradiation. Increases in CD86 and MHC II were observed following infection with UV-inactivated virus, suggesting that inhibition of maturation by cHV-68 requires virus replication. In Fig. 3(b), the mean fluorescence intensity http://vir.sgmjournals.org

from six to eight experiments is shown. These data showed that infection of immature DCs did not upregulate CD80, CD86 or MHC class II compared with LPS-matured DCs or DCs infected with UV-inactivated virus.

cHV-68 infection of DCs inhibits chemotaxis In addition to upregulation of co-stimulatory molecules, maturation of DCs is also characterized by changes in the expression of the chemokine receptors (Sallusto et al., 1998; Vecchi et al., 1999). Maturing DCs downregulate CCR1, CCR5 and CXCR1 and upregulate CXCR4, CCR4 1899

R. Hochreiter and others

To assess the effect of cHV-68 on the expression of IL-12, we infected DCs with cHV-eGFP or treated the cells with UV-inactivated virus or mock lysate. After 24 h, IL-12p40 and IL-10 were measured in the cell-culture supernatants by ELISA (Fig. 3d). Significantly lower levels of IL-12 were observed in the cHV-eGFP-infected wells compared with cells treated with UV-inactivated virus or mock infected. In contrast, both mock- and cHV-eGFP-infected DCs produced IL-10, although the level of IL-10 in the infected cells was not significantly higher than in the mock-infected cells. No IL-10 was observed in the DCs infected with UVinactivated virus.

cHV-68 infection of DCs blocks LPS and poly(I : C)-induced maturation

Fig. 2. Visualization of cHV-68-infected DCs. Immature DCs were infected with cHV-68 at an m.o.i. of 5, and at 48 h p.i. cells were harvested and visualized under an electron microscope as described in Methods. The images in (b) and (d) represent the boxed insets in (a) and (c), respectively. Bars, 2 mm (a, c); 0.5 mm (b, d).

and CCR7. In particular, CCR7, which responds to the chemokines CCL19 and CCL21, is important for directing DCs to the lymphoid organs (Martin-Fontecha et al., 2003; Sallusto et al., 1998). To test whether infected DCs were responsive to CCL19, we performed a Transwell migration assay to measure the capacity of infected DCs to migrate. DCs were infected with cHV-eGFP for 24 h and the adherent population was harvested and placed in the upper chamber of a Transwell. Cells were routinely greater than 80 % viable at the time of transfer. The lower chamber contained the CCR7 ligand CCL19 (100 ng ml21). Only a small percentage of infected DCs was able to migrate towards CCL19 (Fig. 3c). Even stimulation with LPS for 24 h after infection did not increase the number of migrating cHV-68-infected cells (data not shown). This suggested that cHV-68 infection of DCs not only inhibited the upregulation of co-stimulatory molecules, but also prevented the induction of DC migration in infected cells.

cHV-68 infection of DCs inhibits IL-12 expression but not IL-10 expression Activation and maturation of DCs causes secretion of IL-12, a potent stimulator of gamma interferon (IFN-c) and inducer of Th1 responses (Lamont & Adorini, 1996). 1900

DCs can be induced to mature via stimulation with signalling through TLRs. To determine whether cHV-68 infection impaired DC maturation induced by LPS (a TLR4 ligand) or poly(I : C) (a TLR3 ligand), immature DCs were infected with cHV-eGFP or mock infected. After 24 h, non-adherent cells were removed and discarded and the ability of the immature, adherent cells to mature was tested by adding 1 mg LPS ml21, 20 mg poly(I : C) ml21 or medium alone for an additional 24 h (Fig. 4). After a total of 48 h of infection, cells were stained with appropriate antibodies and flow cytometry was carried out to assess changes in DC phenotype. As expected, we observed increased levels of CD80, CD86 and MHC class II in response to LPS and poly(I : C) in mock-infected cells, demonstrating the susceptibility of ex vivo-generated, bone-marrow-derived DCs to maturation signals. However, the majority of infected DCs (GFP+ DCs) showed limited responsiveness to LPS or to poly(I : C) stimulation as measured by the low-level expression of CD80, CD86 and MHC class II. Together, these data suggested that cHV-68 infection not only failed to induce DC maturation, but also blocked maturation induced by the TLR ligands LPS and poly(I : C). Supernatant from mature DCs blocks productive infection of MEFs To test whether mature DCs express a factor that blocks lytic infection, we treated immature DCs with LPS to induce maturation or left them untreated. After 24 h, the supernatant was harvested from untreated and LPSmatured DCs. MEFs, which are very sensitive to productive cHV-68 infection, were then incubated with the supernatant for 1 or 24 h, infected for 1 h, and incubated for 6 days to determine the viral titre. We found that there was a significantly lower viral titre following infection of MEFs that had been pre-treated with the supernatant from mature DCs compared with MEFs that were treated with supernatant from immature DCs or were incubated with medium alone (Fig. 5). This suggested that the mature DCs secreted a soluble protein that actively inhibited lytic replication. Journal of General Virology 88


Fig. 3. Phenotype and function of cHV-68-infected immature DCs. (a) Immature DCs were infected with cHV-eGFP or UV-inactivated virus, or were mock infected or treated with LPS. Twenty-four hours later, adherent cells were harvested and stained with APC-conjugated anti-CD11c mAb and PE-conjugated anti-CD80, -CD86, -MHC class I or -MHC class II. Shown is a representative FACS analysis, gated on live cells positive for CD11c and GFP for cHV-eGFP-infected cells. (b) Mean fluorescence intensity (MFI) of UV-inactivated virus-treated and GFP+ (infected) DCs from multiple experiments were determined at 24 h p.i. and normalized to the MFI of mock-infected cells. The graph shows the mean±SEM of six to eight independent experiments. GFP+ DCs expressed significantly lower levels of CD80, CD86 and MHC class II compared with UV-inactivated virus-treated DCs. *P,0.05 (paired Student’s t-test). (c) DCs that had been infected for 24 h with cHV-eGFP were seeded in the upper chamber of a Transwell and allowed to migrate towards the lower chamber, which contained the CCR7 ligand CCL19 (100 ng ml”1). After 3 h, input cells and migrated cells were stained with PE-conjugated anti-CD86 mAb and analysed by flow cytometry. Circled cells represent immature DCs. (d) Immature DCs were infected with cHV-eGFP or UV-inactivated virus, or were mock infected. Supernatants were harvested at 24 h p.i., and IL-12 and IL-10 levels were determined by ELISA. Results are shown as the mean±SEM of at least four experiments per treatment. cHV-68-infected cells produced significantly lower amounts of IL-12p40 compared with mock-infected and UV-inactivated virus-treated DCs. *P,0.05 (paired Student’s t-test).

cHV-68 infection of precursor DCs blocks development The immature DCs used in the previous studies were derived following isolation of bone-marrow cells and 9 days growth ex vivo. To determine whether cHV-68 infection blocked DC development, freshly isolated bonemarrow cells were infected with cHV-68 or UV-inactivated cHV-68 or mock-infected for 1 h, and then cultured in standard culture medium supplemented with GM-CSF to drive DC differentiation. At 9 days p.i., we analysed the cells for expression of CD11c and CD11b by flow cytometry (Fig. 6a). The majority of cells that were mock http://vir.sgmjournals.org

infected or infected with UV-inactivated cHV-68 expressed high levels of CD11c and CD11b (84.6 and 93.4 %, respectively), indicative of immature DCs. In contrast, following cHV-68 infection, only 36.8 % of cells were CD11chi CD11bhi. Interestingly, the majority of the cells were CD11clo CD11bhi, indicating that they were more typical of granulocytes, a population not seen in the mock-infected or UV-inactivated virus-treated cultures. Analysis of the cell yield also revealed that cHV-68 infection resulted in a significant reduction in the total cell yield (Fig. 6b). Thus, infection of bone-marrow cells with cHV-68 depleted immature DCs and promoted outgrowth of granulocytes. 1901


Fig. 4. Effect of cHV-68 infection on the expression of maturation markers induced by LPS or poly(I : C) stimulation. Immature DCs were infected with cHV-eGFP at an m.o.i. of 5 or were mockinfected. Twenty-four hours later, the medium was replaced and cells were left untreated or stimulated with 1 mg LPS ml”1 or 20 mg poly(I : C) ml”1 for an additional 24 h. Cells were harvested and stained with APC-conjugated anti-CD11c mAb and PE-conjugated anti-CD80, -CD86 and -MHC class I, -MHC class II mAbs or isotype controls (not shown). For analysis, cells were gated on CD11c and GFP. Histograms show CD11c+ GFP+ cells (shaded histogram) in comparison with CD11c+ mock-infected cells (black line). Similar results were obtained in two additional experiments.

DISCUSSION Herpesviruses have evolved a variety of mechanisms to escape the immune response in order to establish life-long latency in the host. Because of their central role in initiating antiviral immune responses, DCs have been targeted by viruses from several different viral families (reviewed by Tortorella et al., 2000). In this study, we have reported that cHV-68 can infect immature DCs and block their

Fig. 5. Effects of supernatant (SN) from mature DCs on virus replication. MEFs were incubated with supernatant harvested from immature DCs, DCs induced to mature for 24 h with LPS or medium for 1 or 24 h prior to infection with cHV-68 at an m.o.i. of 5. Cells were then incubated for 6 days and virus titre was determined by plaque assay. Results are shown as the mean±SEM of three experiments. A significantly lower titre was found in mature supernatant-treated MEFs compared with mock or immature supernatant-treated MEFs at 24 h. *P,0.01 (paired Student’s t-test). 1902

Fig. 6. Infection of DC precursors. Bone-marrow cells were infected with cHV-eGFP at an m.o.i. of 5, or were treated with mock lysate or UV-inactivated virus. (a) After 9 days of culture in GM-CSF, non-adherent cells were harvested and stained with APC-conjugated anti-CD11c mAb and PE-conjugated antiCD11b mAb and analysed by flow cytometry. (b) Cell yield was determined by counting cells using a haemocytometer. Cell yield in mock-treated cultures was set at 100 % and the cell numbers in the cHV-68-infected cultures were normalized to that value. The graph shows the mean±SEM of three independent experiments.

maturation, suggesting that this is another strategy used to subvert host immunity. Previous studies have found that bone-marrow-derived DCs can be infected with cHV-68 and that both latently and lytically infected cells are detected in the cultures (Flano et al., 2005). Our studies confirmed these results and extended them by demonstrating that lytic infection was predominantly restricted to the immature DCs, whilst LPS-matured DCs were latently infected. The different patterns of viral infection in immature DCs versus mature DCs is concordant with observations from in vivo infection of mice, where lung DCs, which are predominantly immature, harbour lytic infections, whereas DCs isolated from lymph nodes, which are predominantly mature, harbour latent infections (Flano et al., 2005). We initially utilized a recombinant virus, cHV-eGFP, where GFP was under the control of the HCMV promoter, to distinguish infected from uninfected DCs in our culture. However, limitingdilution reactivation assays revealed that the mature DCs, whilst being GFP negative, were indeed latently infected with the virus. Electron microscopy also confirmed the absence of capsids in mature DCs. Thus, the HCMV promoter was suppressed in the latently infected cells. Whether this was due to the cellular environment or to the insertion site in the viral genome remains to be determined. None the less, the demonstration that a latent infection is established following infection of LPS-matured DCs opens up a new tool to examine cHV-68 latency in an ex vivo model. Currently, the only model system for studying cHV-68 latency ex vivo is the S11 B-cell line derived from tumour cells isolated from a cHV-68-infected mouse (Usherwood et al., 1996). We observed that if immature DCs were infected with cHV-68 for 24 h and then treated with LPS, maturation

was blocked. This block was not seen following infection of Journal of General Virology 88


immature DCs with replication-deficient, UV-inactivated virus. This observation differs from previous studies by Flano et al. (2005) who did not observe this block in maturation following LPS stimulation of infected cells. However, in their study, they treated DCs with LPS concurrent with cHV-68 infection. Together, these results support the idea that a viral protein(s) is necessary to block maturation, possibly through blocking maturation signals. One possibility is that cHV-68 infection results in loss of TLR-4, which is essential for LPS responsiveness (Poltorak et al., 1998). We have observed that TLR4 expression is maintained in DCs following cHV-68 infection (C. Ptaschinski, R. Hochreiter & R. Rochford, unpublished observations), suggesting that the block must occur further downstream in the LPS signalling pathway. Furthermore, a similar block in DC maturation was observed after stimulation with poly(I : C), a TLR3 ligand. In contrast to TLR4 signalling, which can be MyD88-dependent and -independent, TLR3 signalling occurs only via an MyD88independent pathway (Takeda & Akira, 2004). This again argues for a cHV-68-mediated inhibitory effect of DC maturation on a broader range, rather than interference with single TLR ligand expression or inhibition early in the signalling cascade. A general inhibitory effect on protein synthesis after cHV-68 infection was not observed up to 48 h p.i., as MHC class I, CD11b and CD11c expression were not affected. Infection of immature DCs and subsequent inhibition of maturation has also been reported for herpes simplex virus type 1 (HSV-1) (Pollara et al., 2003; Salio et al., 1999), HCMV (Moutaftsi et al., 2002), murine cytomegalovirus (Andrews et al., 2001), human herpes virus 6 (HHV-6) (Smith et al., 2005) and vaccinia virus (Engelmayer et al., 1999), suggesting that this is a common viral strategy for immune evasion. HSV-1 and HCMV have been shown to interfere with the IFN signalling JAK/STAT pathway (Miller et al., 1998; Yokota et al., 2004). As DCs are exquisitely sensitive to maturation signals, ex vivo culture of bone-marrow-derived DCs routinely results in a mixed population of cells that contain both phenotypically immature and mature DCs. By using the cHV-68 recombinant virus expressing eGFP, we were able to determine that cHV-68 preferentially productively infects immature DCs, characterized by low-level expression of co-stimulatory molecules, low-level expression of both MHC class I and II, and an inability to migrate towards CCL19. In the infected immature DCs, we did observe a small fraction of GFP-positive cells expressing higher levels of CD86 (albeit not as high as in the mature DCs). We hypothesize that these were cells that had already received the signal for maturation before infection and thus productive infection was not blocked. This would fit in with the data of Flano et al. (2005), who found that LPS treatment simultaneous with infection did not block maturation. Our data also suggest that maturation of DCs decreases the susceptibility to productive cHV-68 infection. One possibility for decreased susceptibility is the production of IFNs that may induce an antiviral state in http://vir.sgmjournals.org

LPS-matured DCs, reducing virus replication. This model is supported by our observation that treatment of MEFs with the supernatant from LPS-matured DCs but not from immature DCs prior to cHV-68 infection significantly reduced the viral yield. In order to activate T cells, DCs need to provide three signals: presentation of antigenic peptide in the context of MHC molecules, high-level expression of co-stimulatory molecules and secretion of cytokines (Lipscomb & Masten, 2002). Together, these signals not only induce T-cell proliferation but also drive CD4 T cells down the Th1 or Th2 differentiation pathways, as well as inducing T regulatory cells (Maldonado-Lopez & Moser, 2001; Maldonado-Lopez et al., 1999). Importantly, secretion of IL-12 by DCs is critical for the induction of Th1 differentiation, as well as for proliferation and enhanced cytotoxic activity of natural killer cells (Trinchieri, 2003). We observed that DCs infected with cHV-68 produced only very low levels of IL-12 compared with LPS-stimulated DCs or DCs infected with UV-inactivated virus. This suggests that cHV-68infected DCs would have limited function in inducing a Th1 response. An inability to secrete IL-12 following CD40 ligation is also observed in measles virus-infected DCs as well as in HHV-6-infected DCs after LPS and IFN-c stimulation (Smith et al., 2005), suggesting that this might be a common immunoevasion strategy (Fugier-Vivier et al., 1997). Flano et al. (2005) observed that, after an additional activation signal (LPS stimulation), cHV-68-infected DCs produced elevated levels of IL-10, which was found to be responsible for the limited proliferation of allogeneic T cells. In contrast, in the absence of LPS stimulation, Flano et al. (2005) failed to detect IL-10 with or without cHV-68 infection in DC cultures after 96 h p.i. In our study, we were unable to detect significant differences in IL-10 production between mock-treated and cHV-68-infected DCs at 24 h p.i. It is possible that differences in IL-10 levels would be more apparent by 96 h p.i. We also observed that no IL-10 was detected in immature DCs infected with UV-inactivated virus. It is possible that the higher IL-12 levels induced could counteract IL-10 production in these cells. In addition to the inhibitory effects on DC maturation and activation, cHV-68 infection also had a dramatic effect on the generation of DCs from bone-marrow precursor cells. Inhibition of DC development from bone-marrow cells has been observed after lymphocytic choriomeningitis virus clone 13 and measles virus infection (Hahm et al., 2005; Sevilla et al., 2004). Also, the more closely related human gammaherpesvirus EBV has been found to inhibit DC development by inducing apoptosis in monocyte precursors (Guerreiro-Cacais et al., 2004; Li et al., 2002). However, Li et al. (2002) demonstrated that the apoptosispromoting effect of EBV is restricted to the early stages of DC differentiation and is independent of viral gene expression (Li et al., 2002). This finding contradicts our results where we observed that UV-inactivated virus had no inhibitory effect on DC differentiation. 1903


In summary, we found that cHV-68 productively infects immature DCs, whilst LPS-matured DCs are susceptible to latent infection. Importantly, cHV-68 failed to induce DC maturation following infection and infected cells could not be induced to mature fully following LPS or poly(I : C) treatment. Interference of DC maturation may be mediated by a viral protein(s) that inhibits signalling pathways involved in DC maturation. Certain viruses such as EBV, human T cell leukemia virus I, herpesvirus saimiri and KSHV can alter STAT activity to increase the virus persistence, replication and oncogenic potential (Chen et al., 2001; Lund et al., 1997; Migone et al., 1995; Weber-Nordt et al., 1996). Studies are under way to determine whether this is the critical point in DC maturation blocked by cHV68 infection.

ACKNOWLEDGEMENTS The authors thank Nancy Fiore and Pamela Lincoln for excellent technical assistance and Maureen Barcza and Joyce Qui from the Department of Pathology at SUNY Upstate Medical University for transmission electron microscopy preparation and imaging. This work was supported by the Hendricks Foundation and Department of Microbiology and Immunology, SUNY Upstate Medical University. The authors thank Drs Ren Sun (UCLA) and Steven Dewhurst (University of Rochester) for generous sharing of reagents.

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