type i interferon inhibition and dendritic cell activation...

JOURNAL OF VIROLOGY, Sept. 2007, p. 9778–9789 Vol. 81, No. 180022-538X/07/$08.00�0 doi:10.1128/JVI.00360-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Type I Interferon Inhibition and Dendritic Cell Activation duringGammaherpesvirus Respiratory Infection�

Janet L. Weslow-Schmidt,1 Nancy A. Jewell,1 Sara E. Mertz,1 J. Pedro Simas,3Joan E. Durbin,1,2 and Emilio Flano1,2*

Center for Vaccines and Immunity, Columbus Children’s Research Institute, Columbus Children’s Hospital, Columbus,Ohio 432051; The Ohio State University College of Medicine, Columbus, Ohio2; and Instituto de Medicina Molecular,

Universidade de Lisboa, Lisboa, and Instituto Gulbenkian de Ciencia, Oeiras, Portugal3

Received 19 February 2007/Accepted 22 June 2007

The respiratory tract is a major mucosal site for microorganism entry into the body, and type I interferon(IFN) and dendritic cells constitute a first line of defense against viral infections. We have analyzed theinteraction between a model DNA virus, plasmacytoid dendritic cells, and type I IFN during lung infection ofmice. Our data show that murine gammaherpesvirus 68 (�HV68) inhibits type I IFN secretion by dendriticcells and that plasmacytoid dendritic cells are necessary for conventional dendritic cell maturation in responseto �HV68. Following �HV68 intranasal inoculation, the local and systemic IFN-�/� response is below detect-able levels, and plasmacytoid dendritic cells are activated and recruited into the lung with a tissue distributionthat differs from that of conventional dendritic cells. Our results suggest that plasmacytoid dendritic cells andtype I IFN have important but independent roles during the early response to a respiratory �HV68 infection.�HV68 infection inhibits type I IFN production by dendritic cells and is a poor inducer of IFN-�/� in vivo,which may serve as an immune evasion strategy.

Respiratory viral infections are the leading cause of acuteillnesses worldwide, and several members of the herpesvirusfamily are responsible for severe pneumonia in neonates andimmunocompromised patients (52). Herpes simplex virus, cy-tomegalovirus, varicella-zoster virus, Epstein-Barr virus, hu-man herpesvirus 6, and Kaposi’s sarcoma-associated herpesvi-rus (KSHV) have all been associated with respiratory diseases(7, 10, 30, 33, 42, 46). Much of our understanding of immuneresponses to viral infection of the respiratory tract comes fromexperimental animal models. Murine gammaherpesvirus 68(�HV68) is structurally and biologically similar to the humangammaherpesviruses Epstein-Barr virus and KSHV (16, 49,50), and it has become a useful in vivo model of herpesvirusinfection. Intranasal infection of mice with �HV68 causes anacute respiratory infection that is rapidly resolved and followedby the establishment of splenic latency mainly in the B-cellcompartment (16, 34). Analogous to KSHV (35, 38, 41),�HV68 also infects dendritic cells, a process that may act as amechanism of immune evasion (18, 20).

Plasmacytoid dendritic cells are professional type I inter-feron (IFN)-producing cells that quickly respond to most vi-ruses by secreting large amounts of type I IFNs (28). Type IIFN signaling is important for the control of acute �HV68infection (17, 51). In addition, plasmacytoid dendritic cellssecrete cytokines and interact with conventional dendritic cellsand T cells (28) and are critical for the defense against paren-teral and mucosal infections (1, 13, 25, 29). Although plasma-cytoid dendritic cells have been detected in the lungs (12, 14)and have been shown to prevent the development of allergic

asthma, we have limited information regarding their role in theantiviral response of the respiratory tract. This is of specialimportance because the lung is the largest epithelial surface inthe body and constitutes a major portal of entry for microor-ganisms (32). The regulation of immune responses in the res-piratory tract must be tightly controlled to elicit an adequatedefense against invading agents while maintaining tolerance toinnocuous antigens. Dendritic cells have a central role in main-taining homeostasis by discriminating pathogens from harm-less antigens and eliciting the right response to induce immu-nity or tolerance, respectively (44). Not surprisingly, manyviruses have developed strategies for disrupting dendritic cellfunction (36, 37), and the immune system has developed sys-tems such as plasmacytoid dendritic cells to quickly detect andrespond to viruses (28).

In this study, we have looked at the interplay between amodel double-stranded DNA (dsDNA) virus, plasmacytoiddendritic cells, and type I IFNs during lung infection. The datashow that plasmacytoid dendritic cells are necessary for thematuration of conventional dendritic cells and that �HV68inhibits type I IFN secretion by dendritic cells. Following�HV68 infection, plasmacytoid dendritic cells are activatedand recruited into the lung with a tissue distribution that differsfrom that of conventional dendritic cells. No local or systemicIFN-�/� activity was detected following intranasal �HV68 in-stillation, and the production of IFN-� mRNA was limited toscattered epithelial cells within the respiratory tract. Our re-sults indicate that plasmacytoid dendritic cells and type I IFNhave important but independent roles during the early re-sponse to a respiratory DNA virus infection.

MATERIALS AND METHODS

Virus stocks. �HV68 clone WUMS was propagated and titers were deter-mined on monolayers of NIH 3T3 fibroblasts. Respiratory syncytial virus (RSV)

* Corresponding author. Mailing address: Columbus Children’s Re-search Institute, 700 Children’s Drive WA4015, Columbus, OH 43205.Phone: (614) 722-2735. Fax: (614) 722-3680. E-mail: [email protected].

� Published ahead of print on 11 July 2007.

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strain A2 was grown in HEp-2 cells. Newcastle disease virus (NDV) was grownin 10-day-old embryonated chicken eggs, and titers were determined by immu-nofluorescence. Influenza virus A/WSN/33 (H1N1) was grown in Madin-Darbybovine kidney cells, and titers were determined by immunofluorescence.

Animal procedures and virus infection. C57BL/6J mice were purchased fromTaconic Farms or Harlan Sprague Dawley Inc. and housed under specific-pathogen-free conditions in biosafety level 2 containment. IFN-�/� receptor-deficient (IFN-�/�R�/�) and control (129SvEv strain) mice were bred at theColumbus Children’s Research Institute (CCRI). The Institutional Animal Careand Use Committee at CCRI approved all studies described here. Mice wereanesthetized with 2,2,2-tribromoethanol and inoculated with 103 PFU of �HV68,102 PFU influenza virus A/WSN/33 (H1N1), or 5 � 105 PFU NDV in Hanksbalanced salt solution.

Dendritic cell cultures. Dendritic cells were generated from bone marrowcultures in complete tissue culture medium supplemented with 20 ng/ml murinerecombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)(Peprotech) or 100 ng/ml human recombinant Flt3-L (Peprotech). On days 6 to8, the dendritic cells were infected with 1 � 108 to 3.3 � 108 PFU of �HV68,RSV, or NDV and stimulated with 10 �g/ml lipopolysaccharide (LPS) (Sigma) or2 �g/ml CpG ODN1826 (InvivoGen) if needed.

Fluorescence-activated cell sorter (FACS) analysis. Single-cell suspensionswere obtained from tissues after collagenase D (5 mg/ml; Roche) treatment for45 min. Cells were next incubated with 5 mM phosphate-buffered saline (PBS)–EDTA for 10 min at room temperature to disrupt multicellular complexes. Thecells were Fc blocked and stained with combinations of the following antibodies:CD11c, CD11b, B220, CD8a, CD19, NK1.1, CD3, CD80, CD86, Kb, CD54, I-A,and CD40. Samples were washed and resuspended in 1% paraformaldehydediluted in PBS before analysis. Flow cytometry data were acquired on aFACSCalibur or LSR (Becton-Dickinson) apparatus and analyzed usingFlowJo software (TreeStar, Inc.).

Plaque assay. To determine the titer of infectious virus, lungs were storedfrozen and mechanically homogenized. The lytic virus concentration of the lunghomogenates or of dendritic cell culture supernatants was determined in astandard plaque assay on NIH 3T3 fibroblasts.

IFN-�/� bioassay. An IFN-�/� bioassay was performed as described previ-ously (31). Briefly, supernatants were acid treated to inactivate any input virus aswell as other cytokines. Samples were then neutralized, and twofold dilutions ofeach sample were added to murine fibroblast monolayers. The next day, 1.25 �105 PFU of vesicular stomatitis virus (VSV) were added to each well. Controlsincluded untreated monolayers plus and minus VSV infection and IFN-�/�standards. After 2 days of incubation, wells were fixed and stained. IFN-�/�concentrations were determined by a comparison of protection from VSV-in-duced cell killing with that seen with known amounts of IFN-�/�.

Immunohistochemistry. Frozen tissue sections were fixed in cold acetone for10 min. Endogenous peroxidase was neutralized using PBS–0.3% H2O2–0.1%sodium azide. The sections were stained with anti-CD11c (eBioscience), anti-mPDCA-1 (Miltenyi), or anti-M3 (23) antiserum followed by peroxidase-conju-gated anti-immunoglobulin G antibodies (Jackson ImmunoResearch), andstaining was visualized with 3-amino-9-ethylcarbazole. The sections werecounterstained with hematoxylin and viewed on an Axioscop 2plus apparatus.Images were captured using a Axiocam HRc digital camera with Axiocamsoftware.

In situ hybridization. Tissues were harvested on days 2, 3, and 7 after infectionwith influenza virus or �HV68 or mock infection. To detect type I IFN transcriptsin the lung and mediastinal lymph node (MLN) sections, we synthesized digoxi-genin-labeled riboprobes of murine IFN-�4 and IFN-� genes using a digoxigenin(DIG) RNA labeling kit (Roche) according to the manufacturer’s instructions.After deparaffinization and prehybridization of the tissue sections, DIG-labeledriboprobes at 60 ng/sample were diluted into hybridization buffer and incubatedwith the sections overnight at 42°C. Next, the sections were stained with anti-DIG alkaline phosphatase (Roche), and the signal was detected with nitrobluetetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate). The sections werewashed in water and counterstained with nuclear fast green. Adjacent serialtissue sections were stained with hematoxylin and eosin.

RESULTS

Plasmacytoid dendritic cells are necessary for in vitro acti-vation of conventional dendritic cells in response to �HV68.Previous studies of the interaction between �HV68 and den-dritic cells have shown that �HV68 infection of bone marrow-

derived dendritic cells cultured with GM-CSF does not inducedendritic cell maturation, although �HV68 does not preventthe activation of infected dendritic cells by other stimuli (19).Despite the impact of �HV68 on dendritic cell function, in-fected mice eventually mount an immune response that con-trols infectious �HV68 but never clears latent infection (16). Itis unclear whether plasmacytoid dendritic cells or other den-dritic cells mediate the recognition of �HV68 and how theimmune response to this virus is initiated at the site of infec-tion. It is thus possible that cells other than conventional den-dritic cells first detect the presence of �HV68 infection andinitiate the adaptive immune response. Recognition of dsDNAviruses is mediated by Toll-like receptor 9 (TLR-9) and plas-macytoid dendritic cells (25, 26). However, in the mouse, bothconventional and plasmacytoid dendritic cells can be activatedin vivo by TLR-4, TLR-7, or TLR-9, but they differ in theirrequirements for type I IFNs for activation and migration (2).

To initially characterize the response of plasmacytoid den-dritic cells to �HV68, we generated bone marrow-derived den-dritic cells in the presence of GM-CSF or Flt3-L, infected thecultures with �HV68, and monitored cell activation by cellsurface analysis of the expression of several costimulatory andmajor histocompatibility complex molecules. As previously re-ported (19), �HV68 infection of dendritic cells grown in GM-CSF did not up-regulate the surface expression of CD80,CD86, CD40, or I-A molecules compared with mock-infectedcontrols (Fig. 1A). However, the data show that �HV68 infec-tion of dendritic cells generated in the presence of Flt3-Linduced robust cell surface up-regulation of all the activationmarkers analyzed. Dendritic cells generated in the presence ofGM-CSF constitute a homogeneous population with 95%CD11c� CD11b� conventional dendritic cells (5; data notshown). Dendritic cells generated with Flt3-L are heteroge-neous and contain a mixture of 20 to 30% CD11c� B220�

plasmacytoid dendritic cells and 70 to 80% CD11c� CD11b�

conventional dendritic cells (5; data not shown). Next, we ques-tioned whether the activation of Flt3-L-derived dendritic cellsby �HV68 was homogeneous or whether conventional andplasmacytoid dendritic cell subpopulations had distinct re-sponses to the virus. We analyzed the activation status of eachsubpopulation of dendritic cells in Flt3-L cultures under dif-ferent stimulatory conditions (Fig. 1B). �HV68 induced theup-regulation of surface molecules on plasmacytoid dendriticcells to the same extent as LPS, a TLR-4 agonist. In addition,stimulation with the TLR-9 agonist CpG induced a more ro-bust response by plasmacytoid dendritic cells, and �HV68 in-fection did not prevent the changes induced by LPS or CpG.Analysis of the conventional dendritic cell subset showedequivalent up-regulation of CD54, CD80, CD86. CD40, Kb,and I-A in response to �HV68, LPS, or CpG. Taken together,our results indicate that plasmacytoid dendritic cells are nec-essary for conventional dendritic cell activation in response to�HV68 infection.

�HV68 inhibits type I IFN production by dendritic cells. Tofurther investigate the consequences of the interaction of�HV68 with dendritic cells for the induction of immunity, wecompared dendritic cell activation and type I IFN productionduring �HV68 infection with that of two model viruses, RSVand NDV. RSV is a poorly immunogenic virus with reinfec-tions occurring throughout life and a model of inhibition of

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type I IFNs (39, 43). NDV is a potent inducer of both type IIFN production and dendritic cell maturation (6, 22). First, weanalyzed levels of IFN-�/� bioactivity in response to viral stim-ulation in dendritic cell cultures. The data reported in Fig. 2Ashow that NDV induced strong type I IFN production with aresponse 10-fold greater than that of RSV, which correlateswith previous observations (31). �HV68 did not induce anysignificant amounts of type I IFN bioactivity in dendritic cellcultures. A similar pattern of type I IFN bioactivity for all threeviruses was obtained using dendritic cells cultured with GM-CSF or with Flt3-L. Second, we analyzed dendritic cell activa-tion after �HV68, RSV, or NDV infection. As shown in Fig. 2B(left column), both RSV and NDV induced robust activationof GM-CSF dendritic cells, as measured by the up-regulationof surface expression of CD40 and I-A molecules. As expected,�HV68 did not induce the up-regulation of the activationmarkers analyzed. All three viruses induced the activation ofFlt3-L-derived dendritic cells although to a different extent(Fig. 2B, right column). Taken together, these results indicatethat �HV68 is a poor inducer of type I IFN production bydendritic cells. In addition, the data suggest that the lack ofIFN-�/� bioactivity is independent of the maturation state ofthe dendritic cells. To test whether the lack of IFN-�/� bioac-tivity in culture supernatants is due to an active viral processthat requires �HV68 replication or is due to the poor immu-nogenicity of �HV68 particles, we used UV-inactivated �HV68

to stimulate dendritic cell cultures. The data show that UV-inactivated �HV68 induced 1,000-fold more IFN-�/� synthesisby dendritic cells than did live �HV68 (Fig. 2C). These dataindicate that the inhibition of type I IFN production by den-dritic cells is an active process that requires �HV68 replication.

Anatomical distribution of dendritic cell subsets within thelung. In vivo dendritic cell activation by microbial products andviruses has been shown to induce dendritic cell redistributionin the spleen (2, 9). However, we have limited informationregarding the distribution of different subsets of dendritic cellsin the respiratory tract. Until recently, the existence of plas-macytoid dendritic cells in the lung and their immunoregula-tory role in response to inhaled antigens were unknown (12).We analyzed the distribution of conventional dendritic cellsand plasmacytoid dendritic cells within the lung and the spleenat early time points after intranasal infection with �HV68.Because of the low level of expression of CD11c on plasmacy-toid dendritic cells in vivo, immunohistochemical staining dis-tinguishes between plasmacytoid dendritic cells and conven-tional dendritic cells (2). In naıve control mice (Fig. 3A),numerous CD11c� cells were found interdigitating betweenrespiratory epithelial cells and within the submucosa of con-ducting airways. In addition, scattered CD11c� cells werepresent in the alveolar spaces. After �HV68 infection (Fig. 3Band C), CD11c� cells were more numerous, forming a contin-uous layer beneath the bronchiolar epithelium and concentrat-

FIG. 1. Dendritic cell activation in response to �HV68 infection. (A) Bone marrow-derived dendritic cells grown in the presence of Flt3-L (leftcolumn) or GM-CSF (right column) were infected (empty histograms) or not infected (gray histograms) with �HV68 as described in Materials andMethods. Forty hours later, the cells were surface stained with antibodies against CD11c and the indicated activation markers to analyze theirfluorescence intensity on a flow cytometer. (B) Bone marrow-derived dendritic cells grown in the presence of Flt3-L were infected or not infectedwith �HV68 and stimulated with CpG-ODN or LPS as described in Materials and Methods. Forty-eight hours after treatment, the cells wereharvested and stained with antibodies against the indicated cell surface markers to analyze their fluorescence intensity. Dendritic cells werepreviously gated as plasmacytoid dendritic cells (CD11c� B220�) or conventional dendritic cells (CD11c� CD11b�). The data are representativeof three independent experiments. The data shown in A and B are from two independent experiments.

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ing in areas of inflammation. Cells positive for mPDCA-1, amarker specific for plasmacytoid dendritic cells, could not bevisualized in uninfected control mice (Fig. 3D). However, after�HV68 infection, mPDCA-1� cells were found scattered or insmall clusters in areas of inflammation of the lung parenchymaadjacent to blood vessels (Fig. 3D). These results show thatconventional and plasmacytoid dendritic cells are distributeddifferently within the lung.

To determine whether plasmacytoid dendritic cells were re-cruited toward infection sites or to the lung in general, we

analyzed the distribution of the �HV68 antigen M3 in thelung of infected mice. As shown in Fig. 4, M3 expression isdetected on cells of the airway epithelium, mononuclearcells in the airways, and individual cells in areas of inflam-mation of the lung parenchyma. Altogether, these data sug-gest that mPDCA-1� plasmacytoid dendritic cells and�HV68 antigens localize in inflamed areas of the lungparenchyma of infected mice.

Plasmacytoid dendritic cell recruitment to the lung follow-ing �HV68 infection. We have used enzymatic tissue digestionand flow cytometry to analyze the migration of dendritic cellsin the respiratory tract in response to �HV68 infection. Wehave compared this information with that for the spleen, wheredendritic cell subsets are well described, and bone marrow,where dendritic cell precursors are generated. During thestudy, lineage-positive cells (CD19�, CD3�, and NK1.1�) andmacrophages (CD11b� CD11cdim and/or large forward-scat-ter/side-scatter autofluorescent cells) were excluded from theanalysis. Dendritic cells were gated using forward scatter/sidescatter and low autofluorescence as plasmacytoid dendriticcells (CD11cdim B220�) and conventional dendritic cells(CD11chigh CD8�� or CD11chigh CD11b�). Figure 5 shows arepresentative plot of the CD11c� population in lung, bonemarrow, and spleen 5 days after infection and the B220 ex-pression profile of three different subsets of dendritic cells. Thedifferent subsets of respiratory tract dendritic cells presentedthe same B220 staining profile as their splenic counterparts.

We next did a temporal kinetic analysis of the numbers ofdendritic cells in various tissues following �HV68 infection.The data in Fig. 6A show an increase in the absolute numbersof dendritic cells after �HV68 infection in all the tissues ana-lyzed. This increase starts at day 3 after infection in lung andspleen, although at that time, viral replication is restricted tothe respiratory tract (8, 45). By the time that lytic virus iscleared from the respiratory tract and viral latency peaks in thespleen at day 14 (8), the dendritic cell numbers have increasedtwo- to threefold in lung, spleen, and bone marrow. To analyzethe composition of the different subsets of dendritic cells andthe frequency of plasmacytoid dendritic cells present in thelung at different times after �HV68 infection, we digestedwhole lungs and compared them with spleen and bone marrow.As observed in the representative plots shown in Fig. 6B,plasmacytoid dendritic cells migrate into the lung after intra-nasal instillation with �HV68, and by days 3 to 7, a distinctpopulation can be observed in the FACS contour plots. Simul-taneously, the frequency of plasmacytoid dendritic cells in thespleen also increased, with a distinct population becomingevident by day 7. An analysis of the frequency of plasmacytoiddendritic cells in lung showed a twofold increase in the per-centage of plasmacytoid dendritic cells from days 3 to 14 afterinfection and up to a sixfold increase in the spleen at day 7after infection (Fig. 6C). Altogether, these data indicate that(i) plasmacytoid dendritic cells are differentially recruited intothe lung after gammaherpesvirus infection and (ii) plasmacy-toid dendritic cells are also being recruited into the spleen,although viral replication is restricted to the lung.

Kinetics of dendritic cell activation and type I IFN secretionin response to �HV68 infection. Dendritic cells play a centralrole in the induction of adaptive and innate immune responsesto respiratory tract infections. Although “myeloid” dendritic

FIG. 2. �HV68 inhibits type I IFN production by dendritic cells.(A) Type I IFN induction by �HV68, RSV, or NDV was measured incell culture supernatants 40 h after infection using an IFN-�/� bioas-say. The dendritic cells were grown in the presence of GM-CSF (leftcolumn) or Flt3-L (right column). (B) The relative abilities of �HV68,RSV, or NDV to induce maturation of dendritic cells were tested usingbone marrow-derived dendritic cells grown in the presence of GM-CSF (left column) or Flt3-L (right column). The cell cultures wereinfected as described in Materials and Methods, and 40 h later, thecells were surface stained with antibodies against CD11c, CD40, andI-A. The histograms shown have been previously gated as CD11c�

cells. (C) Type I IFN induction by UV-inactivated �HV68 in dendriticcell cultures supplemented with GM-CSF. The data presented are themeans and standard deviations of triplicate dendritic cell cultures.


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cells constitute the predominant population of pulmonary den-dritic cells in humans (47) and mice (11), other subsets alsoplay essential roles in the response to inhaled antigens (12, 48).Our previous in vitro results suggest that plasmacytoid den-dritic cells are essential for host detection of �HV68 infection.Thus, we questioned which subset(s) of respiratory dendriticcells was activated in response to infection in vivo. To investi-gate the kinetics of plasmacytoid and conventional dendriticcell activation in the lung, we monitored the surface expressionof several costimulatory (CD80, CD86, and CD40) and majorhistocompatibility complex (I-A) molecules on B220� plasma-cytoid dendritic cells, conventional CD11b� dendritic cells,and conventional CD8�� dendritic cells. Plasmacytoid den-dritic cells up-regulated all activation markers analyzed by 24 h

after infection, and this state of activation was maintained untilday 5 after infection (Fig. 7A). Conventional dendritic cells didnot show any phenotypic changes until days 2 to 3 and thenonly partially up-regulated some of the markers analyzed:CD80 (CD11b� dendritic cells) and class II (CD11b� andCD8�� dendritic cells). Thus, plasmacytoid dendritic cells arethe first dendritic cell subpopulation at the site of infection thatup-regulates activation markers in response to �HV68. In ad-dition, plasmacytoid dendritic cells display a more robust stateof activation than their conventional dendritic cell counter-parts in the lung. These data suggest that plasmacytoid den-dritic cells are the first lung dendritic cell population to detect�HV68 infection after intranasal inoculation.

The recognition of dsDNA viruses by plasmacytoid dendritic

FIG. 3. Lung dendritic cell location after �HV68 infection. Lungs were sampled from naıve mice (A and D) or from �HV68-infected mice at7 days postinfection (B and E). Serial sections were stained with anti-CD11c (left panels) or anti-mPDCA-1 (right panels) as described in Materialsand Methods. Objective magnification, �20. C and F show a detail of the tissue area adjacent to the bronchi (b) or blood vessel (v) from B andE, respectively. One representative staining out of three mice per group is shown from three independent experiments. a, arteriole.


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cells triggers IFN-� secretion (28), and the activation and mi-gration of plasmacytoid dendritic cells is thought to be depen-dent on type I IFN (2). To determine the role of IFN-�/�during �HV68 respiratory infection, we measured the amountof type I IFN present in bronchoalveolar lavage (BAL) sam-ples and sera of �HV68-infected mice. Mice intranasally in-fected with NDV were used as positive controls. The data inFig. 7B show that no IFN-�/� activity could be detected inBAL samples or sera of �HV68-infected mice. In addition,although intranasal infection with NDV induces potent IFN-

�/� bioactivity in BAL samples, the amount of systemic type IIFNs in serum was below the level of detection of our bioassay.

Type I IFN production and dendritic cell activation during�HV68 infection of the lung. Type I IFNs are critical in thedefense against viral infections by direct inhibition of viralreplication in infected cells as well as through immunoregula-tory effects (4). IFN-�/� has been shown to be important forplasmacytoid dendritic cell activation and for conventionaldendritic cell activation and migration (2, 22). Our previousresults suggested that plasmacytoid dendritic cell activationmay be critical for the induction of a conventional dendriticcell response to �HV68 in mice and demonstrated that �HV68does not induce a detectable type I IFN response on dendriticcell cultures or in the lung. Next, we analyzed lung virus titersand dendritic cell activation and recruitment into the lung of�HV68-infected mice using IFN-�/�R�/� and wild-type mice(Fig. 8). As expected, IFN-�/�R�/� mice showed a 100-foldincrease in infectious virus compared with normal mice. Thesedata corroborate the important role of type I IFN signaling inthe control of �HV68 lytic infection (17, 51). In addition, anddespite the increased virus production, IFN-�/�R�/� miceshowed a two- to threefold decrease in the recruitment ofdendritic cells into the lung after �HV68 infection (Fig. 8B).The frequency of activated dendritic cells as measured by classII expression was also lower in IFN-�/�R�/� mice (40%) thanin wild-type mice (80%) after �HV68 infection (Fig. 8C andD). These data suggest that type I IFN signaling enhances, butis not an absolute requirement, for dendritic cell recruitmentinto and activation in the lung after �HV68 infection.

There is a discrepancy between the important role of IFN-�/� in controlling �HV68 respiratory infection and our inabil-ity to detect IFN-�/� bioactivity in BAL and serum samples ofinfected mice. To resolve this apparent contradiction, we usedin situ hybridization to analyze the production of type I IFN inlung and draining lymph nodes and to determine which cellsare responsible for its production in vivo. We analyzed infectedtissues on days 2, 3, and 7 after intranasal viral inoculationusing probes for IFN-�4 and for IFN-�. Mice infected with 102

PFU of influenza virus were used as positive controls. The datashow that in influenza virus-infected mice, a strong IFN-�/�signal is detected in the epithelium of the bronchi and in a fewscattered cells in the airways (Fig. 9A to C). On the contrary,�HV68-infected mice showed a very weak IFN-�/�-positive

FIG. 4. Location of viral antigens in the lung of �HV68-infected mice. Lungs were sampled from �HV68-infected mice at 7 days postinfection,and sections were stained with anti-�HV68 M3 antiserum. (A) Area of inflammation in the lung parenchyma. Magnification, �20. (B) Detail ofthe bronchi (b) from A. Magnification, �40.

FIG. 5. Identification of dendritic cell subsets in respiratory tractmPDCA-1� cells. (A) Expression of CD8� and CD11b in previouslygated CD11c� cells defines different dendritic cell subsets in the lung,bone marrow, and spleen of mice 5 days after �HV68 infection: 1,plasmacytoid dendritic cells; 2, CD8 conventional dendritic cells; 3,conventional CD11b dendritic cells. (B) B220 cell surface expressionon mouse dendritic cell subsets as previously gated in A. Data arerepresentative of three independent experiments.


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signal in lung sections exclusively in scattered bronchiolar epi-thelial cells (Fig. 9D to F). The analysis of tissue sections fromMLN of influenza virus-infected mice revealed IFN-�/�-posi-tive cells distributed throughout the lymph node (Fig. 9G andH). Mice infected with �HV68 showed no IFN-�/� signal inthe draining lymph node (Fig. 9I and J). Similar results wereobtained using the � and � probes and at all different timepoints analyzed. Altogether, the data indicate that intranasal�HV68 infection does not induce a potent type I IFN responsein either the lung or its draining lymph nodes.

DISCUSSION

In this paper, we show that �HV68 infection inhibits type IIFN production in dendritic cells and is a poor inducer ofIFN-�/� in vivo. In addition, our data show that dendritic cell

activation and recruitment to the lung after �HV68 respiratoryinfection occur in spite of an IFN-�/� response that is belowthe limit of detection or in mice that lack IFN-�/� signaling.Our data also indicate that plasmacytoid dendritic cells play animportant role in the response to gammaherpesviruses by de-tecting �HV68 and promoting the activation of conventionaldendritic cells regardless of the weak IFN-�/� response toinfection.

Type I IFNs are essential for the defense against viral infec-tions (4), and �HV68 is not an exception. IFN-�/� is importantfor the control of acute �HV68 infection (17, 51) and also forthe control of latency (3). Thus, it is not surprising that �HV68has evolved strategies to subvert type I IFN responses. Ourdata showing a lack of type I IFN production in response to�HV68 infection by cultured dendritic cells generated in thepresence of GM-CSF or Flt3-L suggest the existence of specific

FIG. 6. Dendritic cell migration into the lung and spleen in response to �HV68 infection. (A) Time course analysis of the absolute dendriticcell numbers in bone marrow, lung, and spleen after �HV68 infection. The data presented are the means and standard deviations of threeindependent experiments, each containing three mice. (B) Plasmacytoid dendritic cells migrate into the lung and spleen after �HV68 infection.Numbers indicate the percentages of cells inside the gate. One representative of three experiments is shown. (C) Time course analysis of thefrequency of plasmacytoid dendritic cells (DC) in bone marrow, lung, and spleen after �HV68 infection. The data presented are the means andstandard deviations of three independent experiments, each containing three mice. In all the panels shown, dendritic cells were analyzed asCD11c� and lineage-negative (CD3, CD19, and NK1.1) nonautofluorescent cells.


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IFN-inhibitory mechanisms. The activation of Flt3-L-deriveddendritic cells by infectious �HV68 in the absence of type IIFN production also supports this conclusion. In addition, theability of UV-inactivated, but not live, �HV68 to induce IFN-�/� production in dendritic cell cultures gives strong support tothe hypothesis that �HV68 inhibition of type I IFN productionis an active process. Several mechanisms common to gamma-herpesviruses may account for the following findings: (i) M2gene expression inhibits IFN-mediated transcriptional activa-tion by down-regulating STAT1 and STAT2 (27), and (ii)ORF45, a gene conserved among the gammaherpesviruses thatis essential for �HV68 replication (24), blocks interferon reg-ulatory factor 7 (IRF-7) phosphorylation and nuclear accumu-lation (54). Regardless of the mechanism inhibiting IFN-�/�production, the data show that �HV68 inhibits type I IFNsynthesis by cultured dendritic cells, a process that may helpthe virus to evade immune control in vivo. This idea is alsosupported by (i) in vivo data showing the lack of detectableIFN-�/� bioactivity in BAL and serum samples of �HV68-

infected mice, (ii) in situ hybridization data showing weakIFN-� and IFN-� signals only from respiratory epithelial cells,and (ii) previous studies showing that �HV68 infects dendriticcells (18–20).

Our results indicate that plasmacytoid dendritic cells are thefirst dendritic cell population in the lung to show signs ofactivation in response to �HV68 intranasal inoculation andthat �HV68 induces the activation of conventional dendriticcells in vitro only when external “help” in the form of plasma-cytoid dendritic cells is present. These data correlate with arequirement for plasmacytoid dendritic cell “help” in conven-tional dendritic cell function during cutaneous herpes simplexvirus infection (53). However, in a model of genital herpessimplex virus type 2 infection, plasmacytoid dendritic cellswere not required to mediate Th1 immunity (29). Thus, itseems that the type of herpesvirus and/or the route of infectionis likely to contribute to fundamental differences in the re-sponse. In addition, our data demonstrate that in response toa gammaherpesvirus, (i) plasmacytoid dendritic cell activation

FIG. 7. Dendritic cell maturation and IFN-�/� production in �HV68-infected mice. (A) Dendritic cell subsets undergo differential maturationin the lung in response to �HV68 infection. Lung dendritic cells were analyzed at different time points after �HV68 infection (days 0 to 7) for thelevel of cell surface expression of several activation markers (CD80, CD86, CD40, and I-A). Histograms are previously gated as CD11c� B220�

(plasmacytoid dendritic cells) (first row), CD11c� CD11b� (conventional dendritic cells) (second row), or CD11c� CD8a� (conventional dendriticcells) (third row). (B) Type I IFN bioactivity in BAL fluid of �HV68- and NDV-infected mice at different time points after infection. (C) TypeI IFN bioactivity in serum of �HV68- and NDV-infected mice at different time points after infection. The data presented are the means andstandard deviations for three to four mice.


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in culture is independent of IFN-�/�, (ii) plasmacytoid den-dritic cell recruitment and activation in the lung occur in thepresence of a local and systemic IFN-�/� response that isbelow the limits of detection by bioassay, and (iii) dendritic cellactivation and recruitment into the lung, albeit reduced, stilloccur in IFN-�/�R�/� mice. Altogether, our findings suggestthat type I IFN signaling is important but not an absoluterequirement for dendritic cells to respond to �HV68 infection.These findings contrast with the previously observed require-ment for IFN-�/� signaling during the activation of conven-tional dendritic cells in culture (22) as well as the need for typeI IFN signaling for plasmacytoid dendritic cell migration andactivation in response to TLR-7 and TLR-9 ligands (2). It ispossible that low levels of IFN-�/� below the limit of detectionof our assays may contribute to the recruitment and activationof dendritic cells and that dendritic cell activation can also beinduced by alternative mechanisms such as a CD40/CD40Linteraction or membrane-bound interleukin-15. In addition, itis likely that the response to a DNA virus infection in vivo ismore complex and may account for the differences observedbetween experimental systems.

The ability of plasmacytoid dendritic cells to recognizeand respond to viruses is critical for providing a first line ofdefense at mucosal surfaces. The importance of plasmacy-toid dendritic cells during the immune response to �HV68 issupported by our analysis of infected mice. The immunohis-tochemistry and flow cytometry data show that plasmacytoiddendritic cells are rapidly recruited to the lung after intra-

nasal instillation of �HV68. This increase in the number ofrespiratory plasmacytoid dendritic cells is accompanied bythe early induction of an activation phenotype. The tissuedistribution and migration of plasmacytoid dendritic cellsinto the respiratory tract are less well defined in comparisonto those of conventional dendritic cells. Our data are con-sistent with human and mouse data indicating that conven-tional dendritic cells in the lung are mainly CD11b� or“myeloid” dendritic cells and that they form a contiguoussubepithelial network (40, 47). Lung plasmacytoid dendriticcells have recently been described as BDCA2�/CD123�

cells in humans (14) and as Gr-1�/B220� cells scatteredthroughout the lung interstitium in mice (12). Our analysisindicates that plasmacytoid dendritic cells constitute a smallfraction (2%) of pulmonary dendritic cells and that theywere not visualized during steady-state conditions by immu-nohistochemistry using mPDCA-1. However, after respira-tory �HV68 inoculation, plasmacytoid dendritic cells(CD11c� B220�) are recruited into the lung, constitute 10%of lung dendritic cells, and can be visualized as mPDCA-1�

cells in areas of inflammation. Thus, the distribution ofplasmacytoid dendritic cells in the lung is different from thatof conventional CD11c� dendritic cells. While conventionaldendritic cells mostly form a dense network underneath theepithelium and in areas of inflammation, plasmacytoid den-dritic cells appear to migrate into the lung in response to aninflammatory stimulus.

It is becoming increasingly evident that plasmacytoid den-

FIG. 8. Role of type I IFN signaling in virus control and dendritic cell recruitment and activation in the lung. (A) Infectious virus titersin the lung of 129SvEv or IFN-�/�R�/� mice were determined by plaque assay on day 5 after virus inoculation. (B) Frequency of CD11c�

dendritic cells in the lung of �HV68-infected mice. (C) Representative histograms of the level of expression of class II molecules on CD11c�

cells in the lung. Numbers indicate the percentage of cells inside the gate. (D) Frequency of class II expression on CD11c� cells in the lung.FACS analysis was performed on naıve and �HV68-infected mice on day 7. The data presented are the means and standard deviations forthree individual mice.


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dritic cells play an essential dual role in the initiation of anti-viral responses: as professional type I IFN producer cells andin regulating the function of conventional dendritic cells byIFN-independent pathways (9, 28). The data presented heresupport this hypothesis using the �HV68 model of infection.Due to the key role of type I IFNs in antiviral defense, it is notsurprising that many viruses have developed immune evasionmechanisms to block their production (21). Plasmacytoid den-dritic cells are resistant to many of these strategies eitherbecause they cannot be infected by the virus or because thevirus targets IRF-3-dependent pathways, which are not essen-tial for type I IFN induction by TLR-7 and TLR-9 (15). Theexceptions to this rule include some highly successful patho-

gens including measles virus, RSV, KSHV (21, 54), and�HV68.

ACKNOWLEDGMENTS

We thank the Morphology Core at CCRI for technical help.This work was supported by NIH grant AI-59603 and by CCRI.

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