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Type I interferon inhibition and dendritic cell activation during respiratory gamma-1

herpesvirus infection 2

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Janet L. Weslow-Schmidt1, Nancy A. Jewell1, Sara E. Mertz1, J. Pedro Simas3 Joan E. 4

Durbin12 and Emilio Flaño12* 5

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1Center for Vaccines and Immunity, Columbus Children’s Research Institute, Columbus 7

Children’s Hospital, Columbus, OH 43205 8

2The Ohio State University College of Medicine, Columbus, OH 9

3Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa and Instituto Gulbenkian 10

de Ciência, Oeiras, Portugal 11

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*Corresponding author. Mailing Address: Columbus Children’s Research Institute, 700 15

Children’s Drive WA4015, Columbus, OH 43205. Phone: 614 722 2735. Fax: 614 722 3680. 16

E-mail: [email protected] 17

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Running title: Type I IFN evasion by iHV68 19

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Abstract word count: 175 21

Text word count: 4, 900 22

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.00360-07 JVI Accepts, published online ahead of print on 11 July 2007

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Abstract 1

The respiratory tract is a major mucosal site for microorganism entry into the body, and type I 2

interferon (IFN) and dendritic cells constitute a first line of defense against viral infections. We 3

have analyzed the interaction between a model DNA virus, plasmacytoid dendritic cells, and 4

type I IFN during lung infection of mice. Our data show that murine i-herpesvirus 68 (iHV68) 5

inhibits type I IFN secretion by dendritic cells, and that plasmacytoid dendritic cells are 6

necessary for conventional dendritic cell maturation in response to iHV68. Following iHV68 7

intra-nasal inoculation, the local and systemic IFNc/d response is below detectable levels 8

and plasmacytoid dendritic cells are activated and recruited into the lung with a tissue 9

distribution that differs from that of conventional dendritic cells. Our results suggest that 10

plasmacytoid dendritic cells and type I IFN have important but independent roles during the 11

early response to a respiratory iHV68 infection. iHV68 infection inhibits type I IFN production 12

by dendritic cells and is a poor inducer of IFNc/d in vivo, which may serve as an immune 13

evasion strategy. 14

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Introduction 1

Respiratory viral infections are the leading cause of acute illnesses worldwide, and several 2

members of the herpesvirus family are responsible for severe pneumonia in neonates and 3

immunocompromised patients (52). Herpes simplex, cytomegalovirus, varicella-zoster virus, 4

Epstein-Barr virus, human herpesvirus 6 and Kaposi’s sarcoma-associated herpesvirs 5

(KSHV) have all been associated with respiratory diseases (7, 10, 30, 33, 42, 46). Much of 6

our understanding of immune responses to viral infection of the respiratory tract comes from 7

experimental animal models. Murine i-herpesvirus 68 (iHV68) is structurally and biologically 8

similar to the human i-herpesviruses EBV and KSHV (16, 49, 50), and it has become an 9

useful in vivo model of herpesvirus infection. Intra-nasal infection of mice with iHV68 causes 10

an acute respiratory infection that is rapidly resolved and followed by the establishment of 11

splenic latency, mainly in the B cell compartment (16, 34). Analogous to KSHV (35, 38, 41), 12

iHV68 also infects dendritic cells, a process that may act as a mechanism of immune evasion 13

(18, 20). 14

Plasmacytoid dendritic cells are professional type I IFN-producing cells that quickly respond 15

to most viruses by secreting large amounts of type I IFNs (28). Type I IFN signaling is 16

important for the control of iHV68 acute infection (17, 51). In addition, plasmacytoid dendritic 17

cells secrete cytokines and interact with conventional dendritic cells and T cells (28) and arey 18

are critical for the defense against parenteral and mucosal infections (1, 13, 25, 29). Although 19

plasmacytoid dendritic cells have been detected in the lungs (12, 14) and have been shown 20

to prevent the development of allergic asthma, we have limited information regarding their 21

role in the antiviral response of the respiratory tract. This is of special importance because 22

the lung is the largest epithelial surface in the body and constitutes a major portal of entry for 23

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microorganisms (32). The regulation of immune responses in the respiratory tract must be 1

tightly controlled to elicit adequate defenses against invading agents while maintaining 2

tolerance to innocuous antigens. Dendritic cells have a central role in maintaining 3

homeostasis by discriminating pathogens from harmless antigens and eliciting the right 4

response to induce immunity or tolerance, respectively (44). Not surprisingly many viruses 5

have developed strategies for disrupting dendritic cell function (36, 37), and the immune 6

system has developed systems such as the plasmacytoid dendritic cells to quickly detect and 7

respond to viruses (28). 8

In this study, we have looked at the interplay between a model dsDNA virus, plasmacytoid 9

dendritic cells, and type I IFNs during lung infection. The data show that plasmacytoid 10

dendritic cells are necessary for the maturation of conventional dendritic cells, and that 11

iHV68 inhibits type I IFN secretion by dendritic cells. Following iHV68 infection, plasmacytoid 12

dendritic cells are activated and recruited into the lung with a tissue distribution that differs 13

from that of conventional dendritic cells. No local or systemic IFNc/d activity was detected 14

following intra-nasal iHV68 instillation, and production of IFNc mRNA was limited to 15

scattered epithelial cells within respiratory tract. Our results indicate that plasmacytoid 16

dendritic cells and type I IFN have important but independent roles during the early response 17

to a respiratory DNA virus infection. 18

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Materials and Methods 1

Virus stocks. iHV68, clone WUMS, was propagated and titered on monolayers of NIH-3T3 2

fibroblasts. Respiratory syncytial virus (RSV) A2 strain was grown in HEp-2 cells. Newcastle 3

disease virus (NDV) was grown in 10-day-old embryonated chicken eggs and titers were 4

determined by immunofluorescence. Influenza A/WSN/33 (H1N1) was grown in Madin-Darby 5

bovine kidney cells and titers determined by immunofluorescence. 6

Animal procedures and virus infection. C57BL/6J mice were purchased from Taconic Farms 7

or Harlan Sprague Dawley Inc. and housed under spf conditions in BL2 containment. IFNc/d 8

receptor-deficient (IFN-c/dR-/-) and control mice (129SvEv strain) were bred at CCRI. The 9

Institutional Animal Care and Use Committee at CCRI approved all studies described here. 10

Mice were anesthetized with 2,2,2 tribromoethanol and inoculated with 103 PFU of iHV68, 11

102 PFU WSN or 5x105 PFU NDV in HBSS. 12

Dendritic cell cultures. Dendritic cells were generated from bone marrow cultures in CTM 13

supplemented with 20 ng/ml murine recombinant GM-CSF (Peprotech) or 100 ng/ml human 14

recombinant Flt3-L (Peprotech). On day 6-8, the dendritic cells were infected with 1-3.3 x 108 15

PFU of iHV68, RSV or NDV and stimulated with 10 og/ml LPS (Sigma) or 2 og/ml CpG 16

ODN1826 (InvivoGen) if needed. 17

FACS analysis. Single cell suspensions were obtained from tissues after collagenase D (5 18

mg/ml, Roche) treatment for 45 min. Cells were next incubated with 5mM PBS/EDTA for 10 19

min. at room temperature to disrupt multicellular complexes. The cells were Fc-blocked and 20

stained with combinations of the following antibodies: CD11c, CD11b, B220, CD8a, CD19, 21

NK1.1, CD3, CD80, CD86, Kb, CD54, I-A, and CD40. Samples were washed and 22

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resuspended in 1% paraformaldehyde diluted in PBS before analysis. Flow cytometry data 1

were acquired on a FACScalibur or LSR (Becton-Dickinson) and analyzed using FlowJo 2

(TreeStar, Inc.). 3

Plaque assay. To determine the titer of infectious virus, lungs were stored frozen and 4

mechanically homogenized. The lytic virus concentration of the lung homogenates or of 5

dendritic cell culture supernatants was determined in a standard plaque assay on NIH-3T3 6

fibroblasts. 7

IFNc/d bioassay. It was performed as described (31). Briefly, supernatants were acid treated 8

to inactivate any input virus as well as other cytokines. Samples were then neutralized and 9

two-fold dilutions of each were added to murine fibroblast monolayers. Next day, 1.25 x 105 10

PFU of vesicular stomatitis virus (VSV) were added to each well. Controls included untreated 11

monolayers plus and minus VSV infection and IFNc/d standards. After 2 days of incubation, 12

wells were fixed and stained. IFNc/d concentrations were determined by comparison of 13

protection from VSV-induced cell killing with that seen with known amounts of IFNc/d. 14

Immunohistochemistry. Frozen tissue sections were fixed in cold acetone for 10 min. 15

Endogenous peroxidase was neutralized using PBS - 0.3% H2O2 - 0.1% sodium azide. The 16

sections were stained with anti-CD11c (eBioscience), anti-mPDCA-1 (Miltenyi) or anti-M3 17

antiserum (23) followed by peroxidase-conjugated anti-IgG antibodies (Jackson 18

ImmunoResearch), and staining was visualized with AEC. The sections were counterstained 19

with hematoxilin and viewed on an Axioscop 2plus. Images were captured using a Axiocam 20

HRc digital camera with Axiocam software. 21

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In situ hybridization. Tissues were harvested on day 2, 3 and 7 after infection with influenza, 1

iHV68 or mock infection. To detect type I IFN transcripts in the lung and mediastinal lymph 2

node (MLN) sections, we synthesized digoxigenin-labeled riboprobes of murine IFNc4 and 3

IFNd genes using the DIG RNA Labeling Kit (Roche) according to the manufacturer’s 4

instructions. After deparaffinizing and prehybridization of the tissue sections, DIG-labeled 5

riboprobes at 60 ng/sample were diluted into hybridization buffer and incubated with the 6

sections overnight at 42˚C. Next the sections were stained with anti-DIG alkaline-7

phosphatase (Roche), and the signal detected with NBT/BCIP. The sections were washed in 8

water and counterstained with nuclear fast green. Adjacent serial tissue sections were 9

stained with hematoxylin-eosin. 10

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Results 1

Plasmacytoid dendritic cells are necessary for in vitro activation of conventional dendritic cells 2

in response to iHV68 3

Previous studies of the interaction between iHV68 and dendritic cells have shown that iHV68 4

infection of bone marrow-derived dendritic cells cultured with GM-CSF does not induce 5

dendritic cell maturation, although iHV68 does not prevent the activation of infected dendritic 6

cells by other stimuli (19). Despite the impact of iHV68 on dendritic cell function, infected 7

mice eventually mount an immune response that controls infectious iHV68 but never clears 8

latent infection (16). It is unclear whether plasmacytoid dendritic cells or other dendritic cells 9

mediate the recognition of iHV68 and how the immune response to this virus is initiated at 10

the site of infection. It is thus possible that cells other than conventional dendritic cells first 11

detect the presence of iHV68 infection and initiate the adaptive immune response. 12

Recognition of dsDNA viruses is mediated by TLR-9 and plasmacytoid dendritic cells (25, 13

26). However, in the mouse, both conventional and plasmacytoid dendritic cells can be 14

activated in vivo by TLR-4, -7 or -9, but they differ in their requirements of type I IFNs for 15

activation and migration (2). 16

To initially characterize the response of plasmacytoid dendritic cells to iHV68 we generated 17

bone marrow-derived dendritic cells in the presence of GM-CSF or Flt3-L, infected the 18

cultures with iHV68 and monitored cell activation by cell surface analysis of the expression of 19

several costimulatory and MHC molecules. As previously reported (19), iHV68 infection of 20

dendritic cells grown in GM-CSF did not up-regulate the surface expression of CD80, CD86, 21

CD40 or I-A molecules compared with mock-infected controls (Figure 1A). However, the data 22

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show that iHV68 infection of dendritic cells generated in the presence of Flt3-L induced 1

robust cell surface up-regulation of all the activation markers analyzed. Dendritic cells 2

generated in the presence of GM-CSF constitute a homogeneous population with 95% 3

CD11c+CD11b+ conventional dendritic cells ((5) and data not shown). Dendritic cells 4

generated with Flt3-L are heterogeneous and contain a mixture of 20-30% CD11c+B220+ 5

plasmacytoid dendritic cells and 70-80% CD11c+CD11b+ conventional dendritic cells ((5) and 6

data not shown). Next, we questioned whether activation of Flt3-L-derived dendritic cells by 7

iHV68 was homogeneous or whether conventional and plasmacytoid dendritic cell 8

subpopulations had distinct responses to the virus. We analyzed the activation status of each 9

subpopulation of dendritic cells in Flt3-L cultures under different stimulatory conditions 10

(Figure 1B). iHV68 induced the up-regulation of surface molecules on plasmacytoid dendritic 11

cells to the same extent as LPS, a TLR-4 agonist. In addition, stimulation with the TLR-9 12

agonist CpG induced a more robust response by plasmacytoid dendritic cells, and iHV68 13

infection did not prevent the changes induced by LPS or CpG. Analysis of the conventional 14

dendritic cell subset showed equivalent up-regulation of CD54, CD80, CD86. CD40, Kb and I-15

A in response to iHV68, LPS or CpG. Taken together our results indicate that plasmacytoid 16

dendritic cells are necessary for conventional dendritic cell activation in response to iHV68 17

infection. 18

iHV68 inhibits type I IFN production by dendritic cells 19

To further investigate the consequences of the interaction of iHV68 with dendritic cells for the 20

induction of immunity, we compared dendritic cell activation and type I IFN production during 21

iHV68 infection with that of two model viruses: RSV and NDV. RSV is a poorly immunogenic 22

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virus with reinfections occurring through life and a model of inhibition of type I IFNs (39, 43). 1

NDV is a potent inducer of both type I IFN production and dendritic cell maturation (6, 22). 2

First, we analyzed levels of IFNc/d bioactivity in response to viral stimulation in dendritic cell 3

cultures. The data in Figure 2A show that NDV induced strong type I IFN production with a 4

response 10-fold greater than that of RSV, which correlates with previous observations (31). 5

iHV68 did not induce any significant amounts of type I IFN bioactivity in dendritic cell 6

cultures. A similar pattern of type I IFN bioactivity for all three viruses was obtained using 7

dendritic cells cultured with GM-CSF or with Flt3-L. Second, we analyzed dendritic cell 8

activation after iHV68, RSV or NDV infection. As shown in Figure 2B (left column) both RSV 9

and NDV induced robust activation of GM-CSF dendritic cells, as measured by the up-10

regulation of surface expression of CD40 and I-A molecules. As expected, iHV68 did not 11

induce up-regulation of the activation markers analyzed. All three viruses induced activation 12

of Flt3-L-derived dendritic cells, although to a different extent (Figure 2B, right column). 13

Taken together, these results indicate that iHV68 is a poor inducer of type I IFN production 14

by dendritic cells. In addition, the data suggest that the lack of IFNc/d bioactivity is 15

independent of the maturation state of the dendritic cells. To test whether the lack of IFNc/d 16

bioactivity in culture supernatants is due to an active viral process that requires iHV68 17

replication or is due to the poor immunogenicity of iHV68 particles we used UV-inactivated 18

iHV68 to stimulate dendritic cell cultures. The data show that UV-iHV68 induced 1000-fold 19

more IFNc/d synthesis by dendritic cells than did live iHV68 (Figure 2C). These data indicate 20

that inhibition of type I IFN production by dendritic cells is an active process that requires 21

iHV68 replication. 22

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Anatomical distribution of dendritic cell subsets within the lung 3

In vivo dendritic cell activation by microbial products and viruses has been shown to induce 4

dendritic cell redistribution in the spleen (2, 9). However, we have limited information 5

regarding the distribution of different subsets of dendritic cells in the respiratory tract. Until 6

recently, the existence of plasmacytoid dendritic cells in the lung and their immunoregulatory 7

role in response to inhaled antigens was unknown (12). We analyzed the distribution of 8

conventional dendritic cells and plasmacytoid dendritic cells within the lung and the spleen at 9

early time points after intra-nasal infection with iHV68. Because of the low expression of 10

CD11c on plasmacytoid dendritic cells in vivo, immunohistochemical staining distinguishes 11

between plasmacytoid dendritic cells and conventional dendritic cells (2). In naïve control 12

mice (Figure 3A), numerous CD11c+ cells were found interdigitating between respiratory 13

epithelial cells and within the submucosa of conducting airways. In addition, scattered 14

CD11c+ cells were present in the alveolar spaces. After iHV68 infection (Figure 3B,C), 15

CD11c+ cells were more numerous, forming a continuous layer beneath the bronchiolar 16

epithelium and concentrating in areas of inflammation. Cells positive for mPDCA-1, a marker 17

specific for plasmacytoid dendritic cells, could not be visualized in uninfected control mice 18

(Figure 3D). However, after iHV68 infection mPDCA-1+ cells were found scattered or in 19

small clusters in areas of inflammation of the lung parenchyma adjacent to blood vessels 20

(Figure 3D). These results show that conventional and plasmacytoid dendritic cell are 21

distributed differently within the lung. 22

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To determine whether plasmacytoid dendritic cells were recruited toward infection sites or to 1

the lung in general , we analyzed the distribution of the iHV68 antigen M3 in the lung of 2

infected mice. As shown in Figure 4, M3 expression is detected on cells of the airways 3

epithelium, mononuclear cells in the airways, and on individual cells in areas of inflammation 4

of the lung parenchyma. Altogether, these data suggest that mPDCA-1+ plasmacytoid 5

dendritic cells and iHV68 antigens localize in inflamed areas of the lung parenchyma of 6

infected mice. 7

Plasmacytoid dendritic cells recruitment to the lung following iHV68 infection 8

We have used enzymatic tissue digestion and flow cytometry to analyze the migration of 9

dendritic cells in the respiratory tract in response to iHV68 infection. We have compared this 10

information with that of the spleen, where dendritic cell subsets are well described, and of 11

bone marrow, where dendritic cell precursors are generated. During the study, lineage-12

positive cells (CD19+, CD3+ and NK1.1+) and macrophages (CD11b+CD11cdim and/or large 13

FSC/SSC autofluorescent cells) were excluded from the analysis. Dendritic cells were gated 14

using SSC/FSC and low auto-fluorescence as plasmacytoid dendritic cells (CD11cdimB220+) 15

and conventional dendritic cells (CD11chighCD8c+ or CD11chighCD11b+). Figure 5 shows a 16

representative plot of the CD11c+ population in lung, bone marrow and spleen 5 days after 17

infection and the B220 expression profile of three different subsets of dendritic cells. The 18

different subsets of respiratory tract dendritic cells presented the same B220 staining profile 19

as their splenic counterparts. 20

We next did a temporal kinetic analysis of the numbers of dendritic cells in various tissues 21

following iHV68 infection. The data in Figure 6A show an increase in the absolute numbers 22

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of dendritic cells after iHV68 infection in all the tissues analyzed. This increase starts at day 3 1

after infection in lung and spleen, although at that time viral replication is restricted to the 2

respiratory tract (8, 45). By the time that lytic virus is cleared from the respiratory tract and 3

viral latency peaks in the spleen at day 14 (8) the dendritic cell numbers have increased 2 to 4

3-fold in lung, spleen and bone marrow. To analyze the composition of the different subsets 5

of dendritic cells and the frequency of plasmacytoid dendritic cells present in the lung at 6

different times after iHV68 infection we digested whole lungs and compared them with spleen 7

and bone marrow. As observed in the representative plots of Figure 6B plasmacytoid 8

dendritic cells migrate into the lung after intra-nasal instillation with iHV68, and by day 3-7 a 9

distinct population can be observed in the FACS contour plots. Simultaneously, the frequency 10

of plasmacytoid dendritic cells in the spleen is also increased with a distinct population 11

becoming evident by day 7. An analysis of the frequency of plasmacytoid dendritic cells in 12

lung shows a 2-fold increase in the percentage of plasmacytoid dendritic cells from day 3 to 13

14 after infection, and up to a 6-fold increase in the spleen at day 7 after infection (Figure 14

6C). Altogether, these data indicate that (i) plasmacytoid dendritic cells are differentially 15

recruited into the lung after i-herpesvirus infection, and (ii) that plasmacytoid dendritic cells 16

are also being recruited into the spleen, although viral replication is restricted to the lung. 17

Kinetics of dendritic cell activation and type I IFN secretion in response to iHV68 infection 18

Dendritic cells play a central role in the induction of adaptive and innate immune responses to 19

respiratory tract infections. Although “myeloid” dendritic cells constitute the predominant 20

population of pulmonary dendritic cell in humans (47) and mice (11), other subsets also play 21

essential roles in the response to inhaled antigens (12, 48). Our previous in vitro results 22

suggest that plasmacytoid dendritic cells are essential for host detection of iHV68 infection. 23

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Thus, we questioned which subset/s of respiratory dendritic cells were activated in response 1

to infection in vivo. To investigate the kinetics of plasmacytoid and conventional dendritic cell 2

activation in the lung we monitored the surface expression of several co-stimulatory (CD80, 3

CD86, CD40) and MHC (I-A) molecules on B220+ plasmacytoid dendritic cells, CD11b+ 4

conventional dendritic cells and CD8c+ conventional dendritic cells. Plasmacytoid dendritic 5

cells up-regulated all activation markers analyzed by 24 h after infection, and this state of 6

activation was maintained until day 5 after infection (Figure 7A). Conventional dendritic cells 7

did not show any phenotypic changes until day 2-3, and then only partially up-regulated some 8

of the markers analyzed: CD80 (CD11b+ dendritic cells) and class II (CD11b+ and CD8c+ 9

dendritic cells). Thus, plasmacytoid dendritic cells are the first dendritic cell subpopulation at 10

the site of infection that up-regulate activation markers in response to iHV68. In addition, 11

plasmacytoid dendritic cells display a more robust state of activation than their conventional 12

dendritic cells counterparts in the lung. These data suggest that plasmacytoid dendritic cells 13

are the first lung dendritic cell population to detect iHV68 infection after intra-nasal 14

inoculation. 15

Recognition of dsDNA viruses by plasmacytoid dendritic cells triggers IFNc secretion (28), 16

and the activation and migration of plasmacytoid dendritic cells is thought to be dependent on 17

type I IFN (2). To determine the role of IFNc/d during iHV68 respiratory infection, we 18

measured the amount of type I IFN present in BAL and serum of iHV68-infected mice. Mice 19

intra-nasally infected with NDV were used as positive controls. The data in Figure 7B show 20

that no IFNc/d activity could be detected in BAL or serum of iHV68-infected mice. In addition, 21

although intranasal infection with NDV induces potent IFNc/d bioactivity in BAL, the amount 22

of systemic type I IFNs in serum was below the level of detection of our bioassay. 23

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Type I IFN production and dendritic cell activation during iHV68 infection of the lung 1

Type I IFNs are critical in the defense against viral infections by direct inhibition of viral 2

replication in infected cells as well as through immunoregulatory effects (4). IFNc/d has been 3

shown to be important for plasmacytoid dendritic cell activation and for conventional dendritic 4

cells activation and migration (2, 22). Our previous results suggest that plasmacytoid 5

dendritic cell activation may be critical for the induction of a conventional dendritic cell 6

response to iHV68 in mice and demonstrate that iHV68 does not induce a detectable type I 7

IFN response on dendritic cell cultures or in the lung. Next, we analyzed lung virus titers and 8

dendritic cell activation and recruitment into the lung of iHV68-infected mice using IFN-c/dR-/- 9

and wild-type mice (Figure 8). As expected, IFN-c/dR-/- mice show a 100-fold increase in 10

infectious virus when compared with normal mice. These data corroborate the important role 11

of type I IFN signaling in the control of iHV68 lytic infection (17, 51). In addition, and despite 12

the increased virus production, IFN-c/dR-/- mice show a 2- to 3-fold decrease in the 13

recruitment of dendritic cells into the lung after iHV68 infection (Figure 8B). The frequency of 14

activated dendritic cells as measured by class II expression is also lower in IFN-c/dR-/- mice 15

(40%) than in wild type mice (80%) after iHV68 infection (Figure 8C,D). These data suggest 16

that type I IFN signaling enhances, but is not an absolute requirement, for dendritic cell 17

recruitment and activation into the lung after iHV68 infection. 18

There is a discrepancy between the important role of IFNc/d in controlling iHV68 respiratory 19

infection and our inability to detect IFNc/d bioactivity in BAL and serum of infected mice. To 20

resolve this apparent contradiction we used in situ hybridization to analyze the production of 21

type I IFN in lung and draining lymph nodes, and to determine which cells are responsible for 22

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its production in vivo. We analyzed infected tissues on day 2, 3 and 7 after intranasal viral 1

inoculation using probes for IFNc4 and for IFNd. Mice infected with 102 PFU influenza virus 2

were used as positive controls. The data show that in influenza virus infected mice a strong 3

IFNc/d signal is detected in the epithelium of the bronchi and in few scattered cells in the 4

airways (Figure 9A-C). On the contrary, iHV68 infected mice show a very weak IFNc/d 5

positive signal in lung sections exclusively in scattered bronchiolar epithelial cells (Figure 9D-6

F). The analysis of tissue sections from MLNs of influenza infected mice reveals IFNc/d 7

positive cells distributed throughout the lymph node (Figure 9G,H). Mice infected with iHV68 8

showed no IFNc/d signal in the draining lymph node (Figure 9I,J). Similar results were 9

obtained using the c and d probes and at all different time points analyzed. Altogether, the 10

data indicate that intranasal iHV68 infection does not induce a potent type I IFN response 11

either in the lung or its draining lymph nodes. 12

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Discussion 1

In this manuscript we show that iHV68 infection inhibits type I IFN production in dendritic 2

cells and is a poor inducer of IFNc/d in vivo. In addition, our data show that dendritic cell 3

activation and recruitment to the lung after iHV68 respiratory infection occurs in spite of an 4

IFNc/d response that is below the limit of detection or in mice that lack IFN-c/d signaling. Our 5

data also indicate that plasmacytoid dendritic cells play an important role in the response to i-6

herpesviruses by detecting iHV68 and promoting the activation of conventional dendritic cells 7

regardless of the weak IFNc/d response to infection. 8

Type I IFNs are essential for the defense against viral infections (4), and iHV68 is not an 9

exception. IFNc/d is important for the control of acute iHV68 infection (17, 51) and also for 10

the control of latency (3). Thus, it is not surprising that iHV68 has evolved strategies to 11

subvert type I IFN responses. Our data showing a lack of type I IFN production in response to 12

iHV68 infection by cultured dendritic cells generated in the presence of GM-CSF or Flt3-L 13

suggests the existence of specific IFN inhibitory mechanisms. The activation of Flt3-L-derived 14

dendritic cells by infectious iHV68 in the absence of type I IFN production also supports this 15

conclusion. In addition, the ability of UV-inactivated, but not live, iHV68 to induce IFNc/d 16

production in dendritic cell cultures gives strong support to the hypothesis that iHV68 17

inhibition of type I IFN production is an active process. Several mechanisms common to i-18

herpesviruses may account for these findings: (i) M2 expression inhibits IFN-mediated 19

transcriptional activation by down-regulating STAT1 and STAT2 (27), and (ii) ORF45, a gene 20

conserved amongst the i-herpesviruses that is essential for iHV68 replication (24) blocks 21

IRF-7 phosphorylation and nuclear accumulation (54). Regardless of the mechanism 22

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inhibiting IFNc/d production, the data show that iHV68 inhibits type I IFN synthesis by 1

cultured dendritic cells, a process that may help the virus to evade immune control in vivo. 2

This idea is also supported by in vivo data showing: (i) lack of detectable IFNc/d bioactivity in 3

the BAL and serum of iHV68-infected mice, (ii) in situ hybridization data showing weak IFNc 4

and IFNd signals only from respiratory epithelial cells, and (ii) by previous studies showing 5

that iHV68 infects dendritic cells (18-20). 6

Our results indicate that plasmacytoid dendritic cells are the first dendritic cell population in 7

the lung to show signs of activation in response to iHV68 intra-nasal inoculation and that 8

iHV68 induces the activation of conventional dendritic cells in vitro only when external “help” 9

in the form of plasmacytoid dendritic cells is present. These data correlate with a requirement 10

for plasmacytoid dendritic cells “help” in conventional dendritic cells function during 11

cutaneous HSV infection (53). However, in a model of genital HSV-2 infection plasmacytoid 12

dendritic cells were not required to mediate Th1 immunity (29). Thus, it seems that the type of 13

herpesvirus and/or the route of infection are likely to contribute to fundamental differences in 14

the response. In addition, our data demonstrate that in response to a i-herpesvirus: (i) 15

plasmacytoid dendritic cells activation in culture is independent of IFNc/d, (ii) plasmacytoid 16

dendritic cells recruitment and activation in the lung occurs in the presence of a local and 17

systemic IFNc/d response that is below the limits of detection by bioassay, and (iii) dendritic 18

cell activation and recruitment into the lung, albeit reduced, still occurs in IFN-c/dR-/- mice. 19

Altogether, our findings suggest that type I IFN signaling is important but not an absolute 20

requirement for dendritic cells to respond to iHV68 infection. These findings contrast with the 21

observed requirement for IFNc/d signaling during the activation of conventional dendritic cells 22

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in culture (22) as well as the need for type I IFN signaling for plasmacytoid dendritic cell 1

migration and activation in response to TLR-7 and -9 ligands (2). It is possible that low levels 2

of IFNc/d below the limit of detection of our assays may contribute to the recruitment and 3

activation of dendritic cells, and that dendritic cell activation can also be induced by 4

alternative mechanisms such as CD40/CD40L interaction or membrane-bound IL-15. In 5

addition, it is likely that the response to a DNA virus infection in vivo is more complex and 6

may account for the differences observed between experimental systems. 7

The ability of plasmacytoid dendritic cells to recognize and respond to viruses is critical for 8

providing a first line of defense at mucosal surfaces. The importance of plasmacytoid 9

dendritic cells during the immune response to iHV68 is supported by our analysis of infected 10

mice. The immunohistochemistry and flow cytometry data show that plasmacytoid dendritic 11

cells are rapidly recruited to the lung after intra-nasal instillation of iHV68. This increase in 12

the number of respiratory plasmacytoid dendritic cells is accompanied by early induction of 13

an activation phenotype. The tissue distribution and migration of plasmacytoid dendritic cells 14

into the respiratory tract are less well defined in comparison to that of conventional dendritic 15

cells. Our data are consistent with human and mouse data indicating that conventional 16

dendritic cells in the lung are mainly CD11b+ or “myeloid” dendritic cells and they form a 17

contiguous subepithelial network (40, 47). Lung plasmacytoid dendritic cells have been 18

recently described in humans as BDCA2+/CD123+ cells (14) and in mice as Gr-1+/B220+ cells 19

scattered throughout the lung interstitium (12). Our analysis indicates that plasmacytoid 20

dendritic cells constitute a small fraction (2%) of pulmonary dendritic cells and they were not 21

visualized during steady state conditions by immunohistochemistry using mPDCA-1. 22

However, after respiratory iHV68 inoculation plasmacytoid dendritic cells (CD11c+B220+) are 23

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recruited into the lung, constitute 10% of lung dendritic cells, and can be visualized as 1

mPDCA-1+ cells in areas of inflammation. Thus, the distribution of plasmacytoid dendritic 2

cells in the lung is different from that of conventional CD11c+ dendritic cells. While 3

conventional dendritic cells mostly form a dense network underneath the epithelium and in 4

areas of inflammation, plasmacytoid dendritic cells appear to migrate into the lung in 5

response to an inflammatory stimulus. 6

It is becoming increasingly evident that plasmacytoid dendritic cells play an essential dual 7

role in the initiation of antiviral responses. First, as professional type I IFN producer cells, and 8

second, regulating the function of conventional dendritic cells by IFN-independent pathways 9

(9, 28). The data presented here support this hypothesis using the iHV68 model of infection. 10

Due to the key role of type I IFNs in antiviral defense is not surprising that many viruses have 11

developed immune evasion mechanisms to block their production (21). Plasmacytoid 12

dendritic cells are resistant to many of these strategies either because either they cannot be 13

infected by the virus, or because the virus targets IRF-3 dependent pathways which are not 14

essential for type I IFN induction by TLR-7 and -9 (15). The exceptions to this rule include 15

some highly successful pathogens including measles virus, RSV, KSHV (21, 54), and iHV68. 16

17

Acknowledgements 18

We thank the Morphology Core at CCRI for technical help. This work was supported by NIH 19

grant AI-59603 and CCRI. 20

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Figure legends 1

2

Figure 1. Dendritic cell activation in response to iiiiHV68 infection. (A) Bone marrow-3

derived dendritic cells grown in the presence of Flt3-L (left column) or GM-CSF (right column) 4

were infected (empty histograms) or not (grey histograms) with iHV68 as described in the 5

Material and Methods. 40 h later the cells were surface stained with antibodies against 6

CD11c and the indicated activation markers to analyze their fluorescence intensity on a flow 7

cytometer. (B) Bone marrow-derived dendritic cells grown in the presence of Flt3-L were 8

infected or not with iHV68 and stimulated with CpG-ODN or LPS as described in the Material 9

and Methods. 48 h after treatment the cells were harvested and stained with antibodies 10

against the indicated cell surface markers to analyze their fluorescence intensity. Dendritic 11

cells were previously gated as plasmacytoid dendritic cells (CD11c+B220+) or conventional 12

dendritic cells (CD11c+CD11b+). The data are representative of three independent 13

experiments. The data shown in panels A and B are from two independent experiments. 14

15

Figure 2. iiiiHV68 inhibits type I IFN production by dendritic cells. (A) Type I IFN induction 16

by iHV68, RSV or NDV was measured in cell culture supernatants 40 h after infection using 17

an IFNc/d bioassay. The dendritic cells were grown in the presence of GM-CSF (left column) 18

or Flt3-L (right column). (B) The relative abilities of with iHV68 HV68, RSV or NDV to induce 19

maturation of dendritic cells were tested using bone marrow-derived dendritic cells grown in 20

the presence of GM-CSF (left column) or Flt3-L (right column). The cell cultures were 21

infected as described in the Material and Methods, and 40 h later the cells were surface 22

stained with antibodies against CD11c, CD40 and I-A. The histograms shown have been 23

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previously gated as CD11c+ cells. (C) Type I IFN induction by UV-inactivated iHV68 in 1

dendritic cell cultures supplemented with GM-CSF. The data presented are the mean and SD 2

of triplicate dendritic cell cultures. 3

4

Figure 3. Lung dendritic cell location after iiiiHV68 infection. Lungs were sampled from 5

naïve mice (A, D) or from iHV68-infected mice at 7 d.p.i (B, E). Serial sections were stained 6

with anti-CD11c (left panels) or anti-mPDCA-1 (right panels) as described in Material and 7

Methods. Objective magnification x20. Panels C and F show a detail of the tissue area 8

adjacent to the bronchi (b) or blood vessel (v) from panels B and E, respectively. One 9

representative staining out of three mice per group is shown from three independent 10

experiments. a, arteriole. 11

12

Figure 4. Location of viral antigens in the lung of iiiiHV68-infected mice. Lungs were 13

sampled from iHV68-infected mice at 7 d.p.i and sections were stained with anti-iHV68 M3 14

antiserum. A, area of inflammation in the lung parenchyma. x20, B, detail of the bronchi (b) 15

from panel A, x40. 16

17

Figure 5. Identification of dendritic cell subsets in the respiratory tract mPDCA-1+ (A) 18

Expression of CD8c and CD11b in previously gated CD11c+ cells defines different dendritic 19

cell subsets in the lung, bone marrow and spleen of mice 5 days after iHV68 infection: 1, 20

plasmacytoid dendritic cells; 2, CD8 conventional dendritic cells; and 3, CD11b conventional 21

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dendritic cells. (B) B220 cell surface expression on mouse dendritic cell subsets as 1

previously gated in panel A. Data are representative of 3 independent experiments. 2

3

Figure 6. Dendritic cell migration into the lung and spleen in response to iiiiHV68 4

infection. (A) Time course analysis of the absolute dendritic cell numbers in bone marrow, 5

lung and spleen after iHV68 infection. The data presented are the mean and SD of three 6

independent experiments each containing 3 mice. (B) Plasmacytoid dendritic cells migrate 7

into the lung and spleen after iHV68 infection. Numbers indicate the percentage of cells 8

inside the gate. One representative of three experiments is shown. (C) Time course analysis 9

of the frequency of plasmacytoid dendritic cells in bone marrow, lung and spleen after iHV68 10

infection. The data presented are the mean and SD of three independent experiments each 11

containing 3 mice. In all the panels shown dendritic cells were analyzed as CD11c+ and 12

lineage-negative (CD3, CD19, NK1.1) non-autofluorescent cells. 13

14

Figure 7. Dendritic cell maturation and IFNc/dc/dc/dc/d production in iiiiHV68 infected mice. (A) 15

Dendritic cell subsets undergo differential maturation in the lung in response to iHV68 16

infection. Lung dendritic cells were analyzed at different time points after iHV68 infection 17

(days 0 to 7) for the level of cell surface expression of several activation markers (CD80, 18

CD86, CD40 and I-A). Histograms are previously gated as CD11c+B220+ (plasmacytoid 19

dendritic cells, first row), CD11c+CD11b+ (conventional dendritic cells, second row) or 20

CD11c+CD8a+ (conventional dendritic cells, third row). (B) Type I IFN bioactivity in BAL of 21

iHV68 and NDV infected mice at different time points after infection. (C) Type I IFN bioactivity 22

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in serum of iHV68 and NDV infected mice at different time points after infection. The data 1

presented are the mean and SD of 3-4 mice. 2

3

Figure 8. Role of type I IFN signaling in virus control and dendritic cell recruitment and 4

activation in the lung. (A) Infectious virus titers in the lung of 129SvEv or IFNc/dR-/- mice 5

were determined by plaque assay on day 5 after virus inoculation. (B) Frequency of CD11c+ 6

dendritic cells in the lung of iHV68-infected mice. (C) Representative histograms of the level 7

of expression of class II molecules on CD11c+ cells in the lung. Numbers indicate the 8

percentage of cells inside the gate. (D) Frequency of class II expression on CD11c+ cells in 9

the lung. FACS analysis was performed on naïve and iHV68-infected mice on day 7. The 10

data presented are the mean and SD of 3 individual mice. 11

12

Figure 9. Absence of type I IFN-producing cells in lung and draining lymph nodes of 13

iiiiHV68-infected mice. Lung (A-F) and MLN (G-J) tissue sections from influenza- (A-C and G-14

H) or iHV68-infected (D-F and I-J) mice (days 2, 3 and 7) or control mice were labeled with 15

riboprobes of murine IFNc4 and IFNd genes. (A) Influenza lung, day 2, IFNd probe, x20. A 16

detail is shown is shown in B, C. (B) Influenza lung, day 2, H&E, x40. (C) Influenza lung, day 17

2, IFNd probe, x40. (D) iHV68 lung, day 2, IFNd probe, x20. A detail is shown in E, F. (E) 18

iHV68 lung, day 2, H&E, x40. (F) iHV68 lung, day 2, IFNd probe, x40. (G) Influenza MLN, 19

day 3, H&E, x40. (H) Influenza MLN, day 3, IFNc4 probe, x40. (I) iHV68 MLN, day 3, H&E, 20

x40. (J) iHV68 MLN, day 3, IFNc4 probe, x40. 21

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