VIRAL IMMUNOLOGYVolume 21, Number 1, 2008© Mary Ann Liebert, Inc.Pp. 68–77DOI: 10.1089/vim.2007.0097

Interferon-� Production by Swine Dendritic Cells Is InhibitedDuring Acute Infection with Foot-and-Mouth Disease Virus

CHARLES K. NFON, GEOFFREY S. FERMAN, FELIX N. TOKA, DOUGLAS A. GREGG, and WILLIAM T. GOLDE

ABSTRACT

Viruses have evolved multiple mechanisms to evade the innate immune response, particularly theactions of interferons (IFNs). We have previously reported that exposure of dendritic cells (DCs) tofoot-and-mouth disease virus (FMDV) in vitro yields no infection and induces a strong type I IFN(IFN-� and IFN-�) response, indicating that DCs may play a critical role in the innate response tothe virus. In vivo, FMDV induces lymphopenia and reduced T-cell proliferative responses to mitogen,viral effects that may contribute to evasion of early immune responses. In this study we analyzed thein vivo effects of FMDV infection on the IFN-� response of two populations of dendritic cells. Duringthe acute phase of infection of swine, production of IFN-� from monocyte-derived DCs (MoDCs) andskin-derived DCs (skin DCs) is inhibited. This effect occurs concurrently with rising viral titers in theblood; however, these cells are not productively infected. Interestingly, there are no changes in the ca-pability of these DCs to take up particles and process antigens, indicating that antigen-presenting cellfunction is normal. These data indicate that inhibition of the IFN-� response of dendritic cell popu-lations from blood and skin by FMDV enhances viral pathogenesis in infected animals.

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Plum Island Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York.

INTRODUCTION

THE BALANCE BETWEEN INNATE RESPONSES TO PATHOGENS

AND IMMUNE EVASION BY PATHOGENS is often easilyskewed in favor of the pathogen. Viruses in particular haveevolved very integrated mechanisms for blocking the de-velopment of immunity. Such mechanisms include the in-fection of dendritic cells (DCs) and CD4 T cells by humanimmunodeficiency virus (HIV) (25), and B cells by Epstein-Barr virus and measles virus (18,31). Other viruses, includ-ing poxviruses, block IFN-� production and/or interferewith the cellular response to IFN-� (23,33,34). In addition,bovine herpes virus (27) and pseudorabies virus (4) down-modulate major histocompatibility complex (MHC) class Iexpression, making virus-infected cells refractory to cyto-toxic T lymphocyte (CTL) induction or killing.

Infection with foot-and-mouth disease virus (FMDV)causes an acute vesicular disease of cloven-hoofed ani-mals. At the cellular level, infection is characterized by early production of the viral leader protease, whichcleaves elongation factor 4G, shutting off cap-dependenttranslation of cellular mRNA (17). Consequently host-cell protein synthesis is blocked, resulting in the inhibi-tion of IFN-� and IFN-� production in infected cells(12,14). Blocking these cytokines is likely critical to virusinfection and spread in an animal, as FMDV is very sen-sitive to IFN-� and IFN-� (12). This permits viral repli-cation, assembly, and new virus production (21). Fur-thermore, infected cells do not synthesize new MHCmolecules containing viral peptides via the endogenouspathway, compromising viral peptide presentation toMHC class I–restricted T cells (32).

We have previously reported that FMDV infection in-duces a severe lymphopenia that is transient and corre-sponds to the peak of viremia (7). This effect is not dueto infection of the lymphocytes and recovery from thelymphopenia is rapid. Furthermore, T-cell proliferativeresponses to the mitogen Con A are lost. These data sug-gest that the initiation of an immune response to FMDVis delayed during acute infection. Guzylack-Piriou et al.have shown that activation of plasmacytoid DCs requiresimmune complexes of antibody and FMDV-binding Fcreceptors for the stimulation of an IFN-� response (22).As Fc receptor binding antibody (IgG and IgA) do notappear until 10–14 d after infection, these effects arelikely more involved in recovery and clearing residualvirus rather than the initial response to virus.

We have also reported on the analysis of the effect ofFMDV exposure in vitro on skin-derived DCs. Now for-mally shown to be more than 70% Langerhans cells (LCs)(Nfon et al., in press), these DCs are not infected byFMDV in vitro and produce both IFN-� and IFN-� inresponse to exposure to the virus (8). In addition, thesame study showed that the LCs constitutively expressIFN-�, blocking infection with this IFN-�–sensitivevirus. Furthermore, Chinsangaram et al. have demon-strated that IFN-� can not only block FMDV infectionin vitro (12), but also does so in vivo, when delivered inan adenovirus vector to swine (13).

Given the sensitivity of FMDV to IFN-� and the re-sults showing exogenous delivery of interferon-� protectsswine from infection with FMDV, we sought to under-stand the IFN-� response of blood and skin-derived DCsduring FMDV infection of pigs. Previously (8), wedemonstrated that the most effective stimulus to IFN-�secretion for skin DCs was an attenuated FMDV strain,and for monocyte-derived DCs (MoDCs) is the TLR 3ligand, poly I:C. We analyzed these two DC subsets dur-ing the course of FMDV infection in vivo for the capac-ity to secrete IFN-� and show that both subsets lose thiscytokine response. This effect on IFN-� production isfirst detected at times concurrent with the peak ofviremia. The inhibition of DC secretion of IFN-� sug-gests a modulation of innate immunity in favor of thevirus.

Nevertheless, MoDCs and skin DCs, including LCs,are not productively infected by the FMDV strains usedhere. In addition, there is no effect on antigen uptake orprocessing, or on expression of co-stimulatory molecules.However, epidermal keratinocytes (EKs) isolated con-currently with skin DCs are highly susceptible to FMDVin vitro, and live virus can be isolated from skin of in-fected animals, but not from isolated skin DCs. Likewise,virus can be isolated from plasma but not peripheral bloodmononuclear cells (PBMCs) (7), the source of theMoDCs studied. Collectively, these data indicate that the

FMDV INFECTION BLOCKS INTERFERON PRODUCTION IN VIVO

suppression of IFN-� responses in DCs early after in-fection skews the balance of the host-pathogen interac-tion in favor of enhanced replication of FMDV duringthe acute phase of infection.

MATERIALS AND METHODS

Animals, virus strains, and FMDV infection

All animal experiments were performed under a pro-tocol reviewed and approved by the Institutional AnimalCare and Use Committee (IACUC) of Plum Island Ani-mal Disease Center. Yorkshire pigs aged 3–8 mo werepurchased from Animal Biotech, Inc., Danboro, PA. andacclimated for 1 wk before the experiments. Older, largeranimals were required for large enough skin samples foranalysis as we previously described (9), and each animalwas analyzed individually. The strains of FMDV used inour experiments included A24 Cruzeiro and 01 Campos.In each experiment, animals were infected in the heelbulbs of both hind feet with 100 �L of 1 � 104 plaque-forming units (PFU) of virus. Animals were monitoredfor clinical signs of infection, including fever, beinglethargic, and development of vesicles on the fronthooves and snout. The pigs were euthanized at predeter-mined time points for skin DC isolation as described be-low. Pigs in concurrent experiments and those euthanizedfor skin DC isolation served as blood donors for PBMCsused for MoDC differentiation and isolation.

Blood sampling and cell preparation

Blood samples were drawn from the anterior vena cavainto heparinized and non-heparized blood collectiontubes (Becton Dickinson, Franklin Lakes, NJ) at the in-dicated time points. Heparinized blood was diluted in anequal volume of PBS and layered over a separation gra-dient (Lymphoprep™; Life Technologies, Grand Island,NY) for PBMC isolation following the manufacturer’sprotocol. Serum was obtained from the blood drawn intonon-heparinized tubes and tested for FMDV with a stan-dard plaque assay to confirm development of viremia(28).

Interferon-� ELISA

IFN-� concentration in culture supernatants was de-termined by an antigen-capture ELISA as previously de-scribed (8). Briefly, anti-porcine IFN-� monoclonal an-tibodies K9 and biotinylated F17 (PBL BiomedicalLaboratories, Piscataway, NJ) were used, respectively,for capture and detection of porcine IFN-�. Both anti-bodies were used at a concentration of 1.0 �g/mL. Stan-dard curves were generated with serial twofold dilutionsof recombinant porcine IFN-� (PBL Biomedical Labo-

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ratories), and all samples were assayed in triplicate. IFN-� concentration was expressed in picograms per mil-liliter based on the standard curve.

Isolation of skin-derived dendritic cells

Skin DCs were isolated following a previously pub-lished protocol (9). Briefly, the skin of the entire back ofa euthanized pig was washed, disinfected, and depilatedwith Nair (Carter Wallace, Inc., NY), then surgicallyscrubbed. Skin layers (0.1–0.15 mm thick) were cut witha Zimmer Brown dermatome and collected into basal Ea-gle medium (BEM) (GIBCO-Invitrogen, Carlsbad, CA)supplemented with antibiotics (BEM-AB, 1X; GIBCO-Invitrogen). Skin layers were washed twice in BEM-ABand then cultured overnight (epidermis up) in 150-cm2

cell culture Petri dishes containing lymphocyte culturemedium (LCM; RPMI-1640 containing 0.1 mM non-es-sential amino acids, 0.3 mg/mL L-glutamine, 5.5 � 10�5

M �2-mercapthoethanol, 1 mM sodium pyruvate, 4 mMdextrose, and 10% heat-inactivated fetal bovine serum)supplemented with 10% heat-inactivated autologousporcine serum. Skin DCs that migrated from the skin dur-ing the overnight culture were recovered as previouslydescribed (9), and analyzed for function and tested forFMDV infection.

Isolation of epidermal keratinocytes

Porcine skin DCs are surrounded in situ by EKs. Epi-dermal keratinocytes were isolated from skin layers re-maining after overnight skin DC migration using a mod-ification of a previously published protocol (20). Briefly,small pieces of skin layers were digested in 0.05%trypsin-EDTA (Invitrogen) at 37°C with shaking for 90min. The cell suspension was withdrawn and trypsin-EDTA once more added to the skin layers at 37°C. Af-ter another 90 min, the reaction was stopped by adding10% FBS and the cell suspension was filtered to removeskin debris. The isolated cells were suspended in leuko-cyte-conditioned medium (LCM), and viability assessedby trypan blue exclusion. Residual (CD1�) skin DCs inthe suspension of EKs were less than 5%. The cells weresubsequently exposed to FMDV and virus replicationevaluated as described below. To check for the presenceof virus in the skin harvested from FMDV-infected ani-mals, culture supernatants from the overnight culture ofthe skin layers were tested for presence of virus after pel-leting of the cells and filtration.

In vitro induction of monocyte-derived dendritic cells

Monocytes are precursors of myeloid DCs (MDCs) invivo (19) and are thus used to generate DCs in vitro withsimilar characteristics as blood MDCs. Therefore, to in-

NFON ET AL.

vestigate how blood DCs of myeloid origin respond toand are affected by FMDV, we evaluated DCs differen-tiated from monocytes of healthy or infected pigs forfunctional and phenotypic characteristics. For the gener-ation of MoDCs, we used a cocktail of porcine IL-13 andporcine GM-CSF (MoDC medium) as previously de-scribed (10). Briefly, PBMCs were cultured in plasticflasks for 1.5 h to allow monocyte adherence. The ad-herent monocytes were then cultured in MoDC mediumfor 7 d, with replacement of 25% of the medium everyother day. Fully differentiated MoDCs became non-ad-herent and were collected by pipetting and flushing.

Interferon-� response of DC subsets to FMDV and poly I:C

We have previously reported that skin DCs constitu-tively express IFN-� and secrete this cytokine after en-countering FMDV in vitro, with the highest response be-ing to a highly attenuated strain of the virus (8), LLA12,an A12 strain attenuated by deleting the leader protease(11). Due to the robust response of skin DCs to LLA12,the IFN-� response to this strain was used to analyze skinDCs from infected animals by stimulation ex vivo with theLLA12 virus. Skin DCs at 5 � 105 cells in 1.0 mL LCMwere stimulated with either the LLA12 FMDV at MOI 2,or unstimulated as controls, and cultured at 37°C. After 24h, culture supernatants were harvested for IFN-� ELISA.

Before carrying out a similar analysis of MoDCs frominfected animals, we first tested for the stimulus that in-duces the highest IFN-� response from these cells ex vivo.We previously reported that of the toll-like receptor (TLR)agonists, LPS, CpG, and poly I:C, that poly I:C stimulatedthe highest IFN-� response from these cells (10). Dupli-cate wells of MoDCs at 5 � 105 cells in 1.0 mL LCM werestimulated with either 25 �g/mL poly I:C (Amersham Biosciences, Piscataway, NJ), or FMDV (strains A24Cruzeiro, 01 Campos, or LLA12) at MOI 2, or left un-stimulated as negative controls, and cultured. After 24 h,culture supernatants were harvested and the levels of IFN-� analyzed by ELISA as described above. Again, the mostIFN-� detected from these cells was following stimulationwith poly I:C. We therefore analyzed the IFN-� responseto this TLR agonist of MoDCs isolated from infected an-imals, and stimulated ex vivo with poly I:C to assess func-tion of these cells during infection.

Ability of skin DCs and MoDCs to take up andprocess antigen

The ability of skin DCs and MoDCs to ingest partic-ulate antigens was analyzed by measuring the level of uptake of fluorescent beads (Invitrogen MolecularProbes™, Eugene, OR) that were 2.0 and 0.02 �m in di-ameter by flow cytometry as previously reported (9). Theself-quenched DQ-Ovalbumin (DQ-OVA; Invitrogen

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Molecular Probes™) was used to evaluate the ability ofskin DCs and MoDCs to process protein antigens as pre-viously reported (10). Briefly, a cell suspension of 1 �106 cells in 500 �L of LCM was incubated on ice for 10min, followed by the addition in duplicate of either flu-orescent beads (0.02 or 2.0 �m at 2 or 5 beads/cell, re-spectively), or 2 �g/mL DQ-OVA. One set of tubes wasincubated on ice as controls and the experimental sam-ples were incubated at 37°C for 2 h. Cells were washedand resuspended in chilled FACS buffer (PBS pH 7.4,containing 0.1% sodium azide and 0.3% BSA). Data wereacquired by flow cytometry (FACS Calibur; BectonDickinson [BD] Biosciences, San Jose, CA) and analyzedwith Cell Quest Pro (BD Biosciences) for the percentageof positive cells, obtained by subtracting control values.

DC expression of cell surface molecules

The expression of the surface leukocyte antigens on skinDCs and MoDCs was analyzed by flow cytometry (9). Thefollowing antibodies were used: anti-SLA-IIDR (IgG2a, bi-otinylated) obtained from BD Pharmingen (San Diego,CA), and chimeric human CTLA4-binding CD80/86(hCTLA4-mFc, IgG2a, and PE) obtained from CoulterCorp., Miami, FL. Briefly, 1 � 106 cells were washed inchilled FACS buffer prior to being stained for 30 min onice with optimal dilutions of the above antibodies or theirrespective isotype controls. For SLA-IIDR, secondarystaining was done with streptavidin PE. Data were acquiredby flow cytometry and expressed as percentage of positivecells for each marker after correction for background stain-ing with mouse IgG isotype controls.

Analysis of FMDV replication in skin DCs, EKs, and MoDCs

Virus replication in skin DCs, EKs, and MoDCs wasanalyzed as previously described for porcine skin DCs (8).

FMDV INFECTION BLOCKS INTERFERON PRODUCTION IN VIVO

Briefly, 5 � 105 cells were incubated with FMDV at anMOI of 2 in 1 mL of LCM for 1 h for virus adsorption.Cells were then washed and subjected to a mild acid treat-ment (MES, pH 5.5, Sigma) on ice to remove non-inter-nalized virus. After further washes, the cells were culturedfor 3 and 24 h before RNA, supernatants, and/or cell lysateswere collected and analyzed as described below.

To determine if skin DCs are infected in vivo, cellsfrom pigs on day 2 post-infection were subjected to mildacid treatment, lysed in PBS by repeated freeze-thawing,and stored at �70° for virus isolation. In addition, thesecells were co-cultured with baby hamster kidney(BHK21) cells for 24 h and the BHK-21 cells checkedfor cytopathic effects. Furthermore, these BHK21 cellswere lysed and checked for virus by plaque assay. Cul-ture supernatants and cell lysates were tested for virus ina standard assay for isolating virus (28). Finally, an es-tablished RT-PCR assay (8) was used to check forFMDV-specific RNA in the isolated RNA samples.

Statistical analysis

Differences in the IFN-� response of skin DCs andMoDCs among various sampling time points (days post-infection) were determined by two-tailed Student’s t-tests. p Values of �0.05 were considered to be statisti-cally significant.

RESULTS

Infection of swine with FMDV

Yorkshire pigs aged 3–8 mo were infected with eitherFMDV A24 Cruzeiro or 01 Campos. All infected animalsshowed clinical signs of FMD, including fever for at least2 d and vesicle formation on all four feet, the snout andthe tongue, confirming all animals in the study had dis-

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TABLE 1. VIREMIA LEVELS IN PLAQUE-FORMING UNITS PER MILLILITER OF SERUM FROM FMDV-INFECTED PIGS

A24 Cruzeiro–infected pigs 01 Campos–infected pigsDaypost-infection A B C D E F G H I J

0 0 0 0 0 0 0 0 0 0 01 ND ND 0 1.7 2.3 0 3.1 2.3 2.7 3.02 4.2 5.2 4.7 5.2 4.9 4.9 6.9 5.0 5.9 5.93 ND ND 5.2 5.2 2 0 4.0 4.8 5.1 04 0 0 0 0 0 0 0 0 2.7 05 ND ND 0 0 0 0 0 0 0 06 0 0 0 0 0 0 0 0 0 0

Peak viremia was detected 2–3 d post-infection. Serum was collected daily for 6 d post-infection with either FMDV A24 Cruzeiroor 01 Campos, and virus titers expressed as log10 (x � 1) pfu/mL, as determined by a plaque assay on baby hamster kidney cells.

Abbreviations: ND, plaque assay not done.

seminated disease. The clinical signs and the disease pro-file were similar for both serotypes of FMDV tested,strains A24 Cruzeiro and 01 Campos. Thus we mergedthe results from the FMDV strains. Peak viremia was de-tected 2–3 d post-infection and cleared by day 4 or 5 post-infection (Table 1). Vesicles resolved in 7 d and all ani-mals clinically recovered by day 14 post-infection.

Effect of FMDV infection on IFN-� responses of monocyte-derived dendritic cells

Porcine MoDCs derived from naïve animals were ex-posed to FMDV and their IFN-� response measured.MoDCs responded to wild-type strains of FMDV, strainsA24 Cruzeiro and 01 Campos, and the highly attenuatedstrain, leaderless A12 (LLA12) in vitro (Fig. 1). TheLLA12 virus has had the leader protease deleted, result-ing in no blockage of host protein synthesis and a highlyattenuated virus in vivo and in vitro (12,17,26,29). TheIFN-� response of MoDCs to poly I:C was greater thanthe response to virus. As reviewed above, we subse-quently used the MoDC response to poly I:C ex vivo toanalyze MoDC IFN-� responses during FMDV infectionin vivo.

In analyzing cells differentiated from PBMCs of in-fected pigs, we found that the IFN-� response of MoDCsdeclined on day 2, but had recovered by day 7 post-in-fection (Fig. 2). These effects were coincident withviremia and the difference between the response of cellsisolated on day 0 (preinfection), and 2 d post-infection,

NFON ET AL.

was statistically significant (p � 0.05). The differencebetween day 2 and day 7 responses did not reach statis-tical significance because of high variability observed inthe day 7 responses. However, there was a statisticallysignificant difference between the response of cells fromday 2 and cells from day 30 post-infection (p � 0.05).

PBMCs harvested from infected animals and used asa source of monocytes for the generation of these MoDCswere tested for infection with FMDV. All samples wereuniformly negative for infection as we reported previ-ously (7). In addition, all MoDCs were tested for the sup-port of FMDV infection by culturing cells with virulentvirus (A24 Cruzeiro and 01 Campos) at an MOI of 2, andthese cultures were uniformly negative for virus propa-gation.

IFN-� response of skin-derived dendritic cellsfollowing FMDV infection

Previously, we reported that skin DCs can be freshlyisolated from skin explants and purified on density gra-dients without a requirement for exogenous cytokines (9).These skin DCs, LCs, and dermal DCs, respond to invitro FMDV exposure by secreting IFN-�, with the high-est response being to an attenuated virus strain, LLA12(8). Here we use the response of skin DCs to LLA12 tomonitor the effect of in vivo FMDV infection for the abil-ity of these cells to secrete IFN-� following stimulationex vivo. On day 2 post-infection, skin DCs lose the abil-ity to respond to in vitro stimulation (Fig. 3). The dif-

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FIG. 1. Porcine monocyte-derived DCs from naïve animalsexhibit an interferon-� response to stimulation with FMDV andpoly I:C. Dendritic cells were differentiated from monocytesfrom uninfected pigs with a cocktail of porcine IL-13 andporcine GM-CSF, exposed to FMDV (MOI 2) or stimulatedwith poly I:C (25 �g/mL) for 24 h. Supernatants from the 24-h cultures were assayed for IFN-� by a sandwich ELISA, andIFN-� concentrations are expressed in picograms per milliliterbased on standard curves included on each plate (n � 6, where“n” represents the number of animals from which MoDCs werederived). Error bars represent standard deviation of mean IFN-� response.

FIG. 2. Monocyte-derived DCs from the acute phase ofFMDV infection in pigs secrete less IFN-� than DCs from naïvepigs in response to stimulation with poly I:C. DCs differenti-ated from monocytes at the indicated time points during FMDVinfection were stimulated with poly I:C (25 �g/mL) for 24 h.Culture supernatants were assayed for IFN-� by sandwichELISA, and IFN-� concentration was expressed as picogramsper milliliter based on standard curves included on each plate(n � 9 for naïve animals, n � 5 for infected animals, where “n”represents the number of animals from which MoDCs were de-rived). Error bars represent standard deviation of mean IFN-�response.

ferences in the IFN-� response between day 0 (uninfectedcontrols), and 2 and 7 d post-infection, were statisticallysignificant (p � 0.05). However, unlike the MoDCs, thislack of IFN-� response persisted in skin DCs for up to 5wk. By day 35 post-infection the IFN-� response hadonly begun to recover, and did not fully recover until day50.

Skin DCs isolated from acutely infected animals werenot productively infected with FMDV despite the pres-ence of virus in the skin. Specifically, no virus could beisolated from supernatants of skin DCs cultured over-night, lysates of skin DCs, or co-culturing skin DCs withFMDV-susceptible cell monolayers for several days (datanot shown). However EKs, which comprise the bulk ofthe tissue structure in which skin DCs reside, can be in-fected in vitro by the strains of virus tested here (Table2). By 3 h post-exposure, progeny virus could be detectedin the supernatants and the titer had increased by 24 h.Similarly, virus was isolated from skin layers from eachof seven animals analyzed at 2 d post FMDV-infection(mean 2.1 � 106 pfu/mL; range 6.5 � 104 to 6 � 106/mLin 1 L of pooled supernatants of skin cultures).

DC function and expression of co-stimulatorymarkers in FMDV-infected animals

Results presented here indicate that the elaboration ofIFN-� by MoDCs and skin DCs isolated from pigs in-fected with FMDV is hampered. We therefore analyzedother functional aspects of these DC subsets to determine

FMDV INFECTION BLOCKS INTERFERON PRODUCTION IN VIVO

the influence of FMDV infection. Specifically, antigenuptake, antigen processing, and expression of co-stimu-latory molecules were examined. We observed no effectof FMDV infection on uptake of antigen via pinocytosisby skin DCs (Fig. 4A). Similarly, pinocytotic and phago-cytotic capacity of MoDCs prepared from PBMCs ofacutely infected animals were unaffected (Fig. 4B andC). Furthermore, antigen processing by skin DCs andMoDCs isolated from infected pigs was unchanged (Fig.4D and E). No statistically significant differences wereobserved in any case, either in percentage or intensity ofexpression (Fig. 4 A–E). The expression of cell surfaceproteins important in T-cell stimulation was also unaf-fected upon analysis of these DC populations from in-fected animals. These include porcine class II MHC(SLA-II DR) and CD 80/86 (skin DCs: Fig. 5A and C;MoDCs: Fig. 5B and D).

DISCUSSION

The aim of this study was to further elucidate the im-munopathogenesis of FMDV in vivo by analyzing the invitro IFN-� response of two DC subsets isolated duringinfection of swine. Skin DCs and MoDCs isolated frominfected pigs lost the ability to secrete IFN-� in responseto a TLR ligand and FMDV, suggesting an inhibition ofDC innate responses. Inhibition of IFN-� production co-incided with peak viremia even though these DCs werenot infected by the virus strains used. Monocyte-derivedDCs are differentiated from PBMCs in blood that con-tains as much as 105 live virus per milliliter during thepeak of viremia. LCs and dermal DCs were isolated fromskin, where the EKs were productively infected. There-fore, these DC subsets had high viral exposure, yet wererefractory to FMDV infection.

These data indicate an inhibition of these dendriticcells to respond adequately to FMDV infection, a situa-

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FIG. 3. Skin-derived DCs from the acute phase of FMDVinfection in pigs secrete less IFN-� in response to stimulationwith LLA12 FMDV. Dendritic cells isolated from porcine skinlayers at the indicated time points during FMDV infection werestimulated with LLA12 (MOI 2) for 24 h. Culture supernatantswere assayed for IFN-� by a sandwich ELISA, and IFN-� con-centration was expressed as picograms per milliliter based onstandard curves included on each plate. n � 5 for day 0 and day50; n � 7 for day 2; and n � 4 for day 7 and day 35. “n” rep-resents the number of animals from which skin DCs were de-rived. Error bars represent standard deviation of mean IFN-�response.

TABLE 2. PRODUCTIVE INFECTION OF PORCINE EPIDERMAL

KERATINOCYTES BY FOOT-AND-MOUTH DISEASE VIRUS

Time post-infection

FMDV strain 3 h 24 h

01 Campos 130 pfu/mL 1 � 107 pfu/mLA24 Cruzeiro 5 pfu/mL 1 � 105 pfu/mLA12 400 pfu/mL 3500 pfu/mL

Epidermal keratinocytes were isolated from thin skin layersby enzyme digestion and exposed to the indicated strains ofFMDV at multiplicity of infection (MOI) 2 for 3 and 24 h, andvirus titers in culture supernatants were determined by a plaqueassay.

Abbreviations: pfu, plaque-forming units.

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FIG. 4. Antigen uptake and processing by skin DCs and MoDCs are not affected by FMDV infection. Skin DCs and MoDCsat the indicated time points during FMDV infection were tested for uptake of particulate antigens by measuring the level of in-gestion of different diameter fluorescent beads: 0.02 �m (micropinocytosis, panel A for skin DCs; and panel B for MoDCs, n �3), and 2.0 �m (phagocytosis, panel C for MoDCs; n � 3) by flow cytometry. Self-quenched DQ-Ovalbumin was used to eval-uate the ability of skin DCs (panel D) and MoDCs (panel E, n � 3) to process protein antigens. “n” represents the number ofanimals from which DCs were derived. Black bars show percentage of positive cells, and white bars show geometric mean flu-orescence intensity (MFI). Error bars represent standard deviation of means.

tion that has been reported in many other viral infections(5,6,15,16,24,30). In dengue virus infection, a blunted re-sponse of blood plasmacytoid DCs (pDCs) and reducedpDC numbers are also evident in the absence of produc-tive infection of these cells (30). Although not an acuteinfection, primary infection with HIV is also character-ized by impaired type I IFN production (24). Similarly,chronic hepatitis C virus infection results in reduced cy-tokine secretion by both pDCs and MoDCs in responseto stimulation with TLR ligands (6). Dermal DCs in wartscaused by human papillomavirus fail to respond to im-iquimod, a TLR 7 ligand (5). In addition, vaccinia virussuppresses the ability of a Langerhans cell line to secreteproinflammatory cytokines in response to stimulationwith lipopolysaccharide or poly I:C (16). Although therewas a productive infection in the LC cell line used in thisstudy, primary isolates of LCs from mouse skin were non-permissive to the vaccinia virus infection (16).

FMDV INFECTION BLOCKS INTERFERON PRODUCTION IN VIVO

Given the ability of skin DCs and MoDCs to produceIFN-� in response to stimulation by FMDV, the modu-lation of innate DC function may be critical to the bal-ance between the animal host controlling infection andthe virus interfering with innate responses. Inhibition ofthe IFN-� response of these DC populations, initiatedearly after infection, likely skews the balance of the host-pathogen interaction in favor of the spread of FMDV dur-ing the acute phase of infection. FMDV is the most con-tagious virus ever reported (21), and it survives mainlyby rapid spread among susceptible animals. However, thevirus is eventually cleared and chronic infection is rare,especially in swine. We propose that this modulation ofthe IFN-� response by these DC subsets contributes toincreased replication of FMDV, potentially resulting inhigher levels of virus shedding. This is consistent withthe concept that pigs amplify the spread of virus duringan FMDV outbreak (1–3).

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FIG. 5. Expression of surface markers by skin DCs and MoDCs is not affected by FMDV infection. The expression of sur-face leukocyte antigens was analyzed by flow cytometry. A, SLA-IIDR on skin DCs; B, SLA-IIDR on MoDCs (n � 3); C,CD80/86 on skin DCs; D, CD80/86 on MoDCs (n � 3). “n” represents the number of animals from which DCs were derived.Black bars show percentage of positive cells, and white bars show geometric mean fluorescence intensity (MFI) after backgroundcorrection with isotype controls. Error bars represent standard deviation of means.

We have initiated studies to determine the mechanismby which FMDV can block IFN-� response in non-in-fected cells. A possible hypothesis is that this blockingof IFN-� production is mediated by a soluble factor in-duced in virus-infected cells and circulating in the in-fected animal. In addition, lysis of infected cells couldrelease viral proteins and intermediates (i.e., leader pro-tease) that bind to receptors expressed by DCs, leadingto an inhibition of cytokine secretion. Indeed, we haveshown that EKs are susceptible to FMDV and would bea potent source of such virally-derived inhibitory medi-ators for the DCs, especially in the skin where DCs arein physical contact with potentially infected EKs.

Interestingly, we show that critical characteristics en-abling DCs to function as antigen-presenting cells (anti-gen uptake, antigen processing and high expression ofMHC class II molecules) are not affected, suggesting thatantigen presentation functions are intact during the sameperiod in which IFN-� production is blocked. These cellsalso maintain expression of CD80/86, co-stimulatorymolecules on the cell surface, again indicating an ongo-ing capability of presenting FMDV antigens to T cells,and thereby initiating the adaptive immune response. In-deed FMDV infection is characterized by a strong neu-tralizing antibody response 7 to 10 d post-infection (28).This suggests an alternative hypothesis, specifically thatthe consequence of FMDV infection is to drive activa-tion of these DC populations to antigen-processing func-tion, where the more “mature” dendritic cells becomepoor sources of IFN, yet initiate an adaptive response.

CONCLUSION

In summary, the findings of this study indicate thatFMDV has evolved to either diminish the early innateimmune response in the form of IFN-� production byDCs, and/or skew the responses of these DC subsets to-wards adaptive immune responses and away from the in-nate response. This would suggest that the virus hasevolved to be opportunistic in the short time between in-fection and development of an adaptive immune re-sponse. This lends support to ongoing studies develop-ing systems to induce or deliver IFN to supplement thehost response to FMDV vaccines as an intervention strat-egy during outbreaks of FMD (13). With the informationreported here and information derived from proposedstudies, more effective and targeted approaches to inter-vention for FMD outbreaks can be developed.

ACKNOWLEDGMENTS

This work was supported by CRIS #1940-32000-048-00D from the Agricultural Research Service, U.S. De-

NFON ET AL.

partment of Agriculture (W.T.G.). C. Nfon was the re-cipient of a Plum Island Animal Disease Center ResearchParticipation Program fellowship, administered by theOak Ridge Institute for Science and Education throughan interagency agreement between the U.S. Departmentof Energy and the U.S. Department of Agriculture. Wewould like to thank the animal care staff at the Plum Is-land Animal Disease Center for their professional sup-port and assistance. Finally, we would like to thank themembers of the Foreign Animal Disease Research Unitfor their support, consultation, and discussion of thiswork.

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Address reprint requests to:Dr. William T. Golde

Plum Island Animal Disease CenterAgricultural Research Service

U.S. Department of AgricultureP.O. Box 848

Greenport, New York 11944-0848

E-mail: william.golde@ars.usda.gov

Received November 2, 2007; accepted December 31,2007

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