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Antiviral-Activated Dendritic Cells: A Paracrine-Induced Response State Antonio V. Bordería, Boris M. Hartmann, Ana Fernandez-Sesma, Thomas M. Moran and Stuart C. Sealfon This information is current as of September 24, 2021. J Immunol 2008; 181:6872-6881; ; doi: 10.4049/jimmunol.181.10.6872 http://www.jimmunol.org/content/181/10/6872 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antiviral-Activated Dendritic Cells: A Paracrine-Induced Response State1

Antonio V. Bordería,2* Boris M. Hartmann,2† Ana Fernandez-Sesma,* Thomas M. Moran,* and Stuart C. Sealfon3†‡

Infection of immature dendritic cells (DCs) by virus stimulates their maturation into APC. Infected DCs can also expose uninfected DCs to a panoply of / via . Mathematical modeling suggests that a high rate of paracrine signaling is likely to occur among DCs located in three-dimensional space. Relatively little is known about how secreted factors modify the early response to virus infection. We used a transwell experimental system that allows passage of secreted factors, but not direct contact, between virus-infected DCs and uninfected DCs to investigate paracrine signaling responses. Paracrine signaling from infected DCs induced an antiviral-primed DC state distinct from that of mature virus-infected DCs that we refer to as antiviral-activated DCs

(AVDCs). AVDCs had increased surface MHC class II and CD86 levels, but in contrast to virus-infected DCs, their MHC class I levels Downloaded from were unchanged. Imaging flow cytometry showed that AVDCs had an increased rate of phagocytosis compared with naive DCs. Experiments with IFN-␤ indicated that it may be responsible for CD86, but not MHC class II regulation in AVDCs. Both IFN-inducible and IFN-independent genes are up-regulated in AVDCs. Notably, AVDCs are relatively resistant to virus infection in comparison to naive DCs and achieve accelerated and augmented levels of costimulatory molecule expression with virus infection. AVDCs show a distinct antiviral-primed state of DC maturation mediated by DC paracrine signaling. Although further in vivo study is needed, the characteristics of the AVDC suggest that it is well suited to play a role in the early innate-adaptive transition of the immune http://www.jimmunol.org/ system. The Journal of Immunology, 2008, 181: 6872–6881.

endritic cells (DCs)4 are recognized as a key bridge be- TLR-independent intracellular viral product detectors such as RIG-I tween the innate and adaptive immune responses (1). A (8) and/or MDA5 (9, 10). Virus recognition activates a signaling cas- D key event in the development of adaptive immunity cade involving different cellular factors (IFN regulatory factor 3, NF- upon exposure to infection is the maturation of DCs into APCs that ␬B, c-jun), causing the expression of type I IFNs and other inflam- instruct to generate responses to specific Ags. Acti- matory response genes including TNF-␣ and IL-6. The first type I IFN vated DCs efficiently stimulate both innate immune cells, includ- to be produced and secreted is IFN-␤ (11), which signals either in an by guest on September 24, 2021 ing NK cells (2) and NKT cells (3) as well as key components of autocrine or paracrine manner through the IFN (IFNAR) and adaptive immunity including naive (4) and memory (5) B cells and activates the JAK-STAT pathway (12). This signaling cascade further T cells (1). Thus, DCs are important both for innate immunity as amplifies the initial response and creates an antiviral state in adjacent well as for various elements of adaptive immunity (6). cells that renders them resistant to infection. Our study focuses on DC activation by virus infection, using New- Maturation is a complex process, which includes changes in castle disease virus (NDV), a RNA paramyxovirus that has been dem- morphology, loss of endocytic/phagocytic receptors, up-regulation onstrated to be a good model for immune activation (7). DC matura- of costimulatory molecules, such as CD86, translocation of MHC tion is stimulated by detection of various pathogen-associated compartments to the surface, and secretion of cytokines and che- molecular patterns (1) that are characteristic of bacteria, fungi, proto- mokines (13) that attract, differentiate, and polarize other immune zoa, or viruses. DCs recognize virus infection either by TLRs or by effector cells (6). Secretion of chemokines occurs in coordinated waves according to the type of immune cells that need to be at- *Department of Microbiology, †Department of Neurology, and ‡Center for Transla- tracted and activated (13). One late component associated with tional Systems , Mount Sinai School of Medicine, New York, NY maturation is the migration of the DCs to the secondary lymphoid Received for publication April 2, 2008. Accepted for publication September 5, 2008. organs (14), where they interact with the naive T and B cells. This The costs of publication of this article were defrayed in part by the payment of page activation of Ag-specific T cells by mature DCs is a major aspect charges. This article must therefore be hereby marked advertisement in accordance of the initiation of adaptive immunity. with 18 U.S.C. Section 1734 solely to indicate this fact. The secretion of the different cytokines and chemokines affects 1 This work was supported by Contract HHSN266200500021C and Grant U19 AI06231 from the National Institute of Allergy and Infectious Diseases. other immune cells, including immature DCs, by paracrine signal- ing. Consequently, some DCs might be exposed to both cytokines 2 A.V.B. and B.M.H. contributed equally to this work. and microbial products (11), whereas others only to inflammatory 3 Address correspondence and reprint requests to Stuart C. Sealfon, Department of Neurology and Center for Translational Systems Biology, Mount Sinai School of cytokines. is regarded as an important mech- Medicine, 1 Gustave L. Levy Place, New York, NY 10029. E-mail address: anism for virus- triggered DC maturation. Integrodifferential mod- [email protected] eling of IFN trajectories suggests that ϳ3% of IFN-␤ interacts 4 Abbreviations used in this paper: DC, dendritic ; NDV, Newcastle disease virus; with the DCs that produce it (15). AVDC, antiviral-activated DC; MHC-I/II, MHC class I/II; Ct, crossing threshold; DiO, benzoxazolium, 3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazolylidene)-1-propenyl] We investigated the effects of paracrine signaling by DCs on the perchlorate; IP-10, IFN-␥-inducible 10; RFP, red fluorescent protein; PKR, pro- response state of DCs that are not infected by virus using NDV, tein R; OAS, 2Ј-5Ј-oligoadenylate synthetase; MxA, myxovirus resistance A. which is detected primarily through the cytosolic RIG-I molecule Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 (16). To study paracrine effects, we used a Transwell system which www.jimmunol.org The Journal of Immunology 6873 is composed of two chambers separated by a membrane that allows soluble components such as cytokines and chemokines to diffuse between chambers, but prohibits direct contact be- tween the cells placed in different chambers. DCs infected with NDV and naive noninfected DCs were placed in the upper and lower chamber, respectively. The culture was left for 18 h, al- lowing the infected DCs to initiate cytokine and secretion. We found that the naive DCs exposed to the specific cytokine/chemokine secretions released by infected DCs enter a partially activated state in which they are relatively resistant to FIGURE 1. virus infection and primed to generate a more rapid and en- Experimental setup for the generation of AVDCs. hanced response to virus infection.

compartment to mimic the condition during AVDC generation. We incu- Materials and Methods bated cells for 18 h at 37°C. Naive DCs in the lower compartment were Differentiation of DCs then stained for the maturation markers MHC class I (MHC-I), MHC class All human research protocols for this work have been reviewed and ap- II (MHC-II), and CD86. proved by the Institutional Review Board of the Mount Sinai School of Culture of human fibroblast Medicine. -derived DCs were obtained from healthy human blood donors following a standard protocol described elsewhere (7). All Lung epithelial fibroblasts were obtained from Lonza and cultured accord- Downloaded from experiments were replicated using cells obtained from different donors. ing to the supplier’s instructions. Briefly, cells were passed when conflu- Briefly, human PBMC were isolated from buffy coats by Ficoll density ence was 70–80% using fibroblast basal medium supplemented with hu- gradient centrifugation (Histopaque; Sigma-Aldrich) at 1450 rpm and man fibroblast ␤, , FBS, and gentamicin/amphotericin ϩ CD14 were immunomagnetically purified by using a B. To perform experiments in the Transwell system, 3 ϫ 105 cells were MACS CD14 isolation kit (Miltenyi Biotec). Monocytes were then dif- seeded 8 h before treatment in the Transwell system with DC medium and ferentiated into naive DCs by 5- to 6-day incubation at 37°C and 5% left at 37°C in the incubator. Fibroblasts were infected with NDV at a CO2 in DC growth medium, which contains RPMI 1640 medium (In- multiplicity of infection of 1 and left in the incubator for 40 min. Cells were vitrogen/Life Technologies) supplemented with 10% FCS (HyClone), 2 then washed with PBS and fresh DC medium was added to the culture. http://www.jimmunol.org/ mM l-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (In- Naive DCs were added in the upper compartment and left in the Transwell vitrogen), 500 U/ml human GM-CSF (PeproTech), and 1000 U/ml hu- system for 18 h in the incubator. man IL-4 (PeproTech). IFN-␤ treatment Virus preparation and viral infection DCs were generated as described previously and IFN-␤ (PBL IFN Source) The recombinant Hitchner strain of NDV (rNDV/B1) and NDV/(GFP) was added to the culture in a concentration of 2000 U/ml. The culture was were generated in Prof. P. Palese’s laboratory (Mount Sinai School of left for 18 h in incubation as described before. After the incubation, the Medicine, New York, NY) (17, 18) and grown in 9-day embryonated cells were washed with PBS and harvested for flow cytometry analysis. chicken eggs as described previously (17). Recombinant NDV-red flu- orescent protein (RFP) was obtained from Prof. A. Garcia-Sastre’s lab- Quantitative RT-PCR by guest on September 24, 2021 oratory (Mount Sinai School of Medicine, New York, NY) (19) and grown and titrated similarly to NDV/B1 and NDV-GFP. NDV viruses Viral and host RNA expression levels were quantified by real-time RT- were titrated by immunofluorescence 18 h after infection of Vero cell PCR. RNA was isolated from cells using a Qiagen Micro RNeasy kit ac- plates using mAbs specific for NDV-HN protein generated by the cording to the manufacturer’s protocol. cDNA was synthesized from total Mount Sinai Hybridoma Core Facility followed by addition of anti- RNA with AffinityScript MultiTemp RT (Stratagene) with oligo(dT)18 as mouse IgG-FITC and visualization using fluorescent microscopy. For primer. For real-time PCR, PlatinumTaq DNA polymerase (Invitrogen) infection of naive DCs, NDV stocks were appropriately diluted in and a SYBR Green (Molecular Probes)-containing buffer were used. The DMEM and added directly into pelleted DCs at a multiplicity of infec- real-time PCR was performed using a thermocycler (ABI7900HT; Applied tion of 1 (7, 20). After incubation for 40 min at 37°C, fresh DC growth Biosystems) as previously described (21). The RNA levels for the house- medium (without GM-CSF and IL-4) was added back to the infected keeping genes ribosomal protein S11 (Rps11), tubulin (Tuba), and ß- cells (1 ϫ 106 cells/ml) for the remainder of the infection. Naive non- (Actb) were also assayed in all samples to be used as an internal controls. infected DCs underwent the same experimental procedure as infected mRNA measurements were normalized using a robust global normalization DCs in the absence of virus to ensure that mechanical manipulations algorithm. All control crossing threshold (Ct) values were corrected by the could not be responsible for differences in experimental readouts. median difference in all samples from Actb. All samples were then nor- malized by the difference from the median Ct of the three corrected control Generation of antiviral-activated DCs (AVDCs) gene Ct levels in each sample, with the value converted to a nominal copy number per cell by assuming 2500 Actb mRNA molecules per cell and an AVDCs were generated by using a Transwell system. The Transwell sys- ␮ amplification efficiency of 93% for all reactions. The primer sequences tem consists of an upper and a lower chamber separated by a 0.4- m used for the assays were: NDV-HN sense, 5Ј-GACAATGCTTGATGGT polyethylene terephtalate membrane (Millipore) that allows diffusion of GAAC-3Ј and antisense, 5Ј-CAATGCTGAGACAATAGGTC-3Ј; NDV- cytokines and chemokines through the membrane but avoids the interaction NP sense, 5IFN-ß sense, 5Ј-GTCAGAGTGGAAATCCTAAG-3Ј and an- of the cells in both chambers. To generate the AVDCs, naive DCs were tisense, 5Ј-ACAGCATCTGCTGGTTGAAG-3Ј; IFN-␣1 sense, 5Ј- infected as described above. After the 40-min incubation, the cells were CTGAATGACTTGGAAGCCTG-3Ј and antisense, 5Ј-ATTTCTGCTCT washed with PBS and cultured in the Transwell system. Infected and non- GACAACCTC-3Ј; PKR sense, 5Ј-TTGTACCACAAGAGAGAGTG-3Ј infected DCs were allocated in the upper and lower chamber, respectively. and antisense, 5Ј-AGTGCTGTCCCTCAAGACTC-3Ј; OAS-1 sense, 5Ј- Two independent wells were set up with infected or naive noninfected DCs TTTGATGCCCTGGGTCAGTT-3Ј and antisense, 5Ј-GTGCTTGACTAG as positive and negative controls. The cultures were incubated at 37°C in GCGGATGA-3Ј; TNF-␣ sense, 5Ј-GAGGAAGGCCTAAGGTCCAC-3Ј 5% CO2 for 18 h. All cells were then washed in PBS and harvested for flow Ј Ј Ј Ϫ and antisense, 5 -AGTGAAGTGCTGGCAACCAC-3 ; RIG-I sense, 5 - cytometry analysis and RNA isolation. The supernatant was kept at 20°C AAAGCCTTGGCATGTTACAC-3Ј and antisense, 5Ј-GGCTTGGG for ELISA analysis of cytokines/chemokines. ATGTGGTCTACT-3Ј; RANTES sense, 5Ј-AAGCTCCTGTGAG Proteinase K treatment GGGTTGA-3Ј and antisense, 5Ј-TTGCCAGGGCTCTGTGACCA-3Ј; IL-6 sense, 5Ј-CTGAGGTGCCCATGCTACAT-3Ј and antisense, 5Ј- Supernatants derived from NDV-infected DCs for 18 h were treated with AATGCCAGCCTGCTGACGAA-3Ј; IP-10 sense, 5Ј-TCCCATCACTTC proteinase K (MP Biomedicals) for2hat37°C to digest cytokines and CCTACATG-3Ј and antisense, 5Ј-TGAAGCAGGGTCAGAACATC-3Ј; chemokines. The proteinase K was then inactivated with a brief heat treat- MXA sense, 5Ј-CGTGGTGATTTAGCAGGAAG-3Ј and antisense, 5Ј-TG ment. Proteinase K-treated and nontreated supernatants were placed in the CAAGGTGGAGCGATTCTG-3Ј; Rps11 sense, 5Ј-CGAGGGCACCTA upper compartment of the Transwell system with naive DCs in the lower CATAGACA-3Ј and antisense, 5Ј-GAGATAGTCCCGGCGGATGA-3Ј; 6874 ACTIVATED DCs

FIGURE 2. Expression of viral GFP protein and maturation markers in in- fected DCs, AVDCs, and noninfected DCs. Histograms show fluorescence in- tensity of cells for the viral GFP-tagged protein NDV-HN and the fluorochrome- conjugated Abs for CD86, HLA-ABC (MHC-I), and HLA-DR (MHC-II). Blue histograms indicate noninfected DC con- trols. Red histograms indicate infected DCs in the Transwell system (A), AVDCs (B), and infected DCs as positive control (C). The data shown are representative of three different experiments using three

different donors that showed similar Downloaded from results. http://www.jimmunol.org/

Actb sense, 5Ј-GCCTCAACACCTCAAACCAC-3Ј and antisense, 5Ј-CCA Flow cytometry analysis CAGCTGAGAGGGAAATC-3Ј; and Tuba sense, 5Ј-AGCGCCCA ACCTACACTAAC-3Ј and antisense, 5Ј-GGGAAGTGGATGCGAGG Cells were fixed with 1.5% paraformaldehyde (Sigma-Aldrich), washed GTA-3Ј. with FACS staining buffer (Beckman Coulter), and stained with mAbs for by guest on September 24, 2021

FIGURE 3. Quantitative RT-PCR of RNA expression for virus and type I IFN genes in infected control DCs (Inf-DCs), infected DCs from the Transwell cultures, AVDCs, and noninfected naive DCs (N-DCs) genes to viral infection. The results shown are averages of three independent experiments performed using different donors. The Journal of Immunology 6875

FIGURE 4. Multiplex ELISA for IL-6, IP-10, TNF-␣, and IFN␣ levels in super- natants from the chambers containing in- fected DCs, AVDCs, and noninfected na- ive DCs (N-DCs) genes to viral infection, all in the Transwell system. The results shown are averages of three independent experiments performed using different do- nors. Inf-DCs, Infected DCs. Downloaded from http://www.jimmunol.org/ MHC-I, MHC-II, and CD86. NDV-GFP- or NDV-RFP-infected cells were were analyzed with the FlowJo software (Tree Star). To provide higher stained with Abs for MHC-I, MHC-II, and CD86 (Beckman Coulter). Cells throughput and reduce cell requirements per assay, we used a modified were assayed on an LSRII flow cytometer (Beckman Coulter) and data bar-coding method previously described by Nolan et al. (22). Briefly cells by guest on September 24, 2021

FIGURE 5. Quantitative RT-PCR of antiviral and inflammatory gene expression in infected control DCs (Inf-DCs), infected DCs from the Transwell cultures, AVDCs, and noninfected naive DCs (N-DCs) genes to viral infection. The results shown are averages of three independent experiments performed using different donors. 6876 ACTIVATED DCs

CD86 MHC-I MHC-II FIGURE 6. Flow cytometry analysis studying the in- volvement of IFN-␤ signaling in the generation of AVDCs. CD86, MHC-I, and MHC-II were assayed and the results are shown in the different columns. Blue indi- cates treatment of naive DCs with IFN-␤ cytokine, green indicates AVDCs, and red-infected DCs as control The data shown are representative of three different experi- ments using three different donors that showed similar results.

were fixed, then stained in DMSO containing different combinations of 0, chamber during these experiments, we performed control experi- 0.3, 1, 4, or 15 ␮g/ml Pacific Blue-NHS, 0, 1.25, 5, or 20 ␮g/ml Alexa ments with a recombinant NDV containing a GFP-tagged HN pro- ␮ Fluor 350-NHS, and 0, 4, or 20 g/ml Alexa Fluor 750-NHS for 15 min at tein (17). Infection of cells was assayed by flow cytometry. When 20–25°C (see Fig. 9A). Fluorescence minus 1 controls were obtained by staining naive DCs with all fluorochromes studied excluding the fluoro- NDV-GFP-infected cells were incubated in the upper chamber for chrome of interest, conjugated Ab against CD45 (23). 18 h, GFP fluorescence was detected only in cells from the upper, but not the lower chamber, indicating that the experimental system Imaging flow cytometry analysis of phagocytosis and effectively separated infected and uninfected DCs (Fig. 2). Downloaded from morphology As a further test of the isolation of virus infection, the induction Imaging flow cytometry was used to compare the morphology and phago- of the NDV-HN and NDV-NP viral genes and virus-induced type cytosis levels of AVDCs and naive DCs. To detect phagocytosis, 1-␮m I IFN genes were compared in membrane-separated cocultured 488-nm fluorescence-labeled latex microspheres (Polysciences) at a con- cells using real-time PCR assays. Both viral genes were present in centration of 50 beads/cell were cocultured for2hat37°C with each cell type. Single-cell images were acquired using extended depth field imaging virus-infected DCs from the upper chamber and were undetectable distortion to identify beads in different focal planes within a cell. The in noninfected control DCs and in DCs coincubated in the lower numbers of beads incorporated by cells were quantified in the images cap- chamber (Fig. 3). Viral components lead to activation of type I IFN http://www.jimmunol.org/ tured using image analysis software (IDEAS Software; Amnis). The dis- via TLR activation. If uninfected cells were exposed to viral com- tributions were compared using the Kolmogorov-Smirnoff technique. To measure the cellular morphology, AVDCs, naive DCs, and in- ponents that passed through the membrane, they would be ex- fected DCs were labeled with a live membrane dye, benzoxazolium, pected to show induction of type I IFNs. Therefore, we also as- 3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazolylidene)-1-propenyl]-, sayed the expression of IFN-␤ and IFN-␣1 as a sensitive assay of perchlorate (DiO), following the manufacturer’s protocol (Invitrogen). exposure to very small amounts of virions, viral RNA, debris, or Samples were then individually acquired by imaging flow cytometry. other small particles that might cross the membrane (26). Using a To analyze the morphology, an algorithm described by Haralick et al. (24), which describes the homogeneity of an given image, was used sensitive real-time PCR assay, we found that both IFNs were up-

(IDEAS Software; Amnis). regulated in virus-infected DCs from the upper chamber, but were by guest on September 24, 2021 not induced in naive cells incubated in the lower chamber (Fig. 3). Multiplex ELISA The results of flow cytometry and real-time PCR indicate that the Four different cytokines/chemokines (IL-6, IFN-␥-inducible protein 10 (IP- culture system completely eliminates direct virus infection of un- 10), TNF-␣, and IFN␣) concentrations were assayed in the culture me- infected, membrane-isolated cocultured cells. To test further the dium. To minimize the supernatant volume to assay, a Beadlyte Human possibility that viral components such as RNA caused the surface Multiplex ELISA analysis (Millipore) was used per the manufacturer’s instructions. Briefly, 100 ␮l from each compartment per well was incu- marker up-regulation pattern observed (see below), we tested the bated in a 96-well filter polyvinylidene difluoride 1.2-␮m plate specially effect of proteinase K of supernatants from infected DCs designed to retain cytokines/chemokines, with a mixture of anti-cytokine on its capacity to affect naive DCs. Proteinase K- treated super- IgG-conjugated beads for the different cytokines/chemokines assayed. Af- natant did not up-regulate the surface markers of DCs in the other ter2hofincubation, the plate was filtered and washed three times with assay solution (PBS (pH 7.4) containing 1% BSA, 0.05% Tween 20, and Transwell chamber. Supernatants from infected DCs that were not 0.05% sodium azide). The washes were followed by a 1.5-h incubation digested with proteinase K induced a similar surface marker pat- with biotin-conjugated anti-cytokine IgG. After assay solution washing, tern to that seen in AVDCs in the Transwell system (supplemen- streptavidin-PE was added followed by addition after 30 min of stop so- tary Fig. S1).5 Thus, any responses of the uninfected DCs in the lution (0.2% (v/v) formaldehyde in PBS (pH 7.4). The plate was then lower chamber were the results solely of paracrine signaling ini- filtered and each well was resuspended in 125 ␮l of assay buffer and read in a Luminex 100 machine. tiated by infected DCs in the upper chamber. Results Effects of DC paracrine signaling on maturation markers Effectiveness of Transwell chambers to study paracrine Several cellular surface have been described as maturation signaling markers of DCs, including CD86, MHC-I, and MHC-II (6). There To isolate the effects of direct infection by virus from that of para- is an increase in the density of these markers in the cellular mem- crine signaling on naive DCs, we used a Transwell culture system. brane of the DCs upon activation and maturation. In the coculture Naive, immature DCs were infected with NDV and immediately Transwell system, upper chamber-infected DCs and the positive placed in the upper chamber. Noninfected naive DCs were placed control DCs showed a comparable up-regulation of MHC-I, MHC- in the lower chamber. The chambers were partitioned by a 0.4-␮m II, and CD86 molecules (Fig. 2, A and C), indicating the matura- pore membrane that allowed the diffusion of cytokines and che- tion of those cells. Interestingly, lower chamber DCs in the cocul- mokines (Fig. 1). After incubation, cells in each chamber were ture Transwell system also showed an up-regulation of MHC-II recovered and analyzed for their surface marker and gene expres- and CD86 molecules, while MHC-I remained at levels equivalent sion patterns. to the noninfected control DC levels (Fig. 2B). DCs do not support the productive infection of NDV (25). To ascertain whether viral particles could infect cells in the lower 5 The online version of this article contains supplemental material. The Journal of Immunology 6877

FIGURE 7. Homogeneity test to show differences in morphological shapes. A, Representative micro- graphs of naive DCs, AVDCs, and infected control DCs (Inf-DCs) captured using an imaging flow cy- tometry. B, Histogram of the homogeneity value of p Ͻ ,ءءء .naive DCs, AVDCs, and infected DCs 0.0001 by Mann-Whitney U test in comparison with naive DCs. The results shown are averages of three independent experiments performed using different donors. N-DCs, Noninfected DCs. Downloaded from

To determine whether the changes in naive DCs induced by Levels of paracrine cytokines http://www.jimmunol.org/ infected DCs were unique to infected DCs, we studied the effects We assayed cytokines that could contribute to paracrine activation of secreted factors from NDV-infected primary lung fibroblasts on of DCs. Factors secreted by DCs for which DCs expressed recep- naive DCs using the Transwell system. Our results showed that the tors include IL-6, TNF-␣, IP-10, and type I IFN. Measurement of presence of infected primary lung fibroblasts instead of infected the levels of these cytokines in upper and lower chambers by DCs in the same Transwell system did not induce a comparable ELISA during a coculture experiment indicated that high levels of surface marker expression pattern in the naive DCs (supplemen- all four cytokines were achieved in the lower chamber (Fig. 4). tary Fig. S2). Furthermore, noninfected control DCs did not up- regulate any of the surface markers, even if cells were maintained in the Transwell system for periods up to 48 h (supplementary Fig. Gene program response in AVDCs by guest on September 24, 2021 S2). These results indicate that the experimental manipulations To further understand the characteristics of the paracrine-gener- alone were not affecting maturation marker up-regulation. Our re- ated AVDC state, the regulation of a variety of genes was assayed sults suggest that paracrine signaling alone between infected and by real-time PCR in AVDCs, NDV-infected DCs, and naive DCs. uninfected DCs produces an increase of some DC maturation Paracrine and autocrine IFN acting at the type I IFN receptor ac- markers, leading to a unique DC state that differs both from naive tivates a number of genes through JAK-STAT signaling, including DCs and from fully matured virus-infected DCs. protein kinase R (PKR), 2Ј-5Ј-oligoadenylate synthetase (OAS),

A 123467 5

B FIGURE 8. Phagocytosis of microspheres. A, Repre- sentative images showing examples of cells containing between one and seven beads imaged using enhanced depth of field-equipped imaging flow cytometry. B, Dis- tribution of number of microspheres counted in naive DCs (n ϭ 2000) and AVDCs (n ϭ 2700). The distribu- tions are statistically different (p Ͻ 10Ϫ13). The data shown are representative of three different experiments using cells from three different donors that showed sim- ilar results. N-DCs, Noninfected DCs. 6878 ACTIVATED DCs

A B 250K 200K DC AVDC 150K FSC-A 100K 0 2 50K 4 0 0102 103 104 105 6 Alexa 350 8 105 105 10 4 4 10 10 12

103 103 Pacific Blue Pacific Blue

102 102

0 0

0102 103 104 105 0102 103 104 105 Alexa 750 Alexa 750

5 10 105

4 10 104

3 10 103 Pacific Blue Pacific Blue

2 10 102 2 3 4 5 010 10 10 10 0102 103 104 105 0 0

2 3 4 5 NDV-RFP 2 3 4 5 NDV-RFP 010 10 10 10 010 10 10 10 Alexa 750 Alexa 750

C Downloaded from http://www.jimmunol.org/

AVDCs

DCs by guest on September 24, 2021

FIGURE 9. AVDCs resist viral infection. A, Bar coding for flow cytometry analysis. Cells were stained with different combinations of 0, 0.3, 1, 4, or 15 ␮g/ml Pacific Blue-NHS, 0, 1.25, 5 or 20 ␮g/ml Alexa Fluor 350-NHS, and 0, 4, or 20 ␮g/ml Alexa Fluor 750-NHS to enable 60 different conditions in one FACS run. B, Time course results for NDV-RFP expression following infection in naive DCs and AVDCs. C, Time course for expression of MHC-I, MHC-II, and CD86 surface markers in the same cells studied in A and B. The results shown are representative of two independent experiments using cells from different donors.

myxovirus resistance A (MxA), and IP-10. Consonant with the represents only part of the paracrine environment necessary for the detection of high levels of IFN-␣ in the medium by cytokine as- generation of AVDCs. says (Fig. 4), AVDCs showed robust induction of these IFN-acti- vated genes (Fig. 5). OAS and MxA showed the same levels in the Quantification of AVDC morphology and rate of phagocytosis lower chamber as in infected DCs. Although PKR and IP-10 were The morphology of AVDCs and naive DCs was compared using lower than in infected DCs, they were still significantly induced in imaging flow cytometry. Cells were stained with a live-cell mem- comparison with noninfected DCs. IFN is a key paracrine signal- brane-localized dye, DiO, and morphology was measured using ing factor during virus infection and has long been recognized to the texture analysis algorithm developed by Haralick et al. (24) as generate complex transcriptional responses (7, 26). However the implemented in IDEAS software (see Materials and Methods). By state of AVDCs appears to result from additional factors besides this method, the spatial relationships between the texture features IFN, because genes such as TNF-␣, which are not inducible by and the pixel values in an image were measured, and an H homo- IFN are up-regulated in AVDCs (Fig. 5). The hypothesis that ad- geneity mean and a SD value were obtained for each set of cells. ditional secreted factors contribute to the AVDC state was further The H homogeneity is a measure of the average shape of a cell. supported by comparing the pattern of maturation markers in Representative raw images are shown in Fig. 7A. These analyses AVDCs and in IFN-␤-treated naive DCs. The induction of the reveal that the average morphology of AVDCs is different from costimulatory molecule CD86 was similar in AVDCs and IFN- that of naive DCs. AVDCs showed an increase in textural homo- exposed DCs, suggesting that this regulatory event may result from geneity compared with naive cells that was comparable to the level IFN signaling. However, the increased expression of MHC-II was seen in infected DCs (Fig. 7B). seen only in AVDCs (Fig. 6). Overall, the gene response patterns The rates of phagocytosis of naive DCs and AVDCs were also and maturation marker analysis indicate that type I IFN signaling compared using high-resolution imaging flow cytometry. Both The Journal of Immunology 6879 cells types were incubated for 2 h with 1-␮m 488-nm fluores- induction), we demonstrated that the change in state seen in the cence-labeled latex microspheres. The number of particles taken AVDC results solely from the transmission of secreted factors and up by each cell was quantified in approximately several thousand that AVDCs do not show any signs of infection by the virus or cells in each group using automated image analysis (Fig. 8A). In exposure to viral components. three independent experiments using cells from different donors, Since the discovery of the effects of type I IFN more than 50 AVDCs showed significantly higher rates of phagocytosis. years ago, these cytokines have been well studied as inducers of a variety of antiviral responses that promote the development of DC AVDCs resist viral infection and showed enhanced maturation (28). We have shown previously that NDV infection of generation of maturation markers by infection human DCs results in a strong activation pattern, including pro- We next investigated whether AVDCs differ from naive DCs in duction of type I IFN, TNF-␣, and other proinflammatory cyto- their response to direct virus infection by NDV. For this purpose, kines as well as IFN-inducible genes (7). We find that NDV-in- AVDCs generated by coculturing with NDV-GFP-infected DCs fected DCs, located in the upper chamber of the Transwell system, for 18 h using the Transwell system and naive DCs were exposed also secrete TNF-␣, IL-6, and IP-10, which have receptors present to a recombinant NDV virus expressing RFP (NDV-RFP) (19). on DCs and are therefore also potential paracrine factors (see Fig. The level of RFP signal generated over 12 h was assayed by flow 4). Several previous studies have considered the role of uninfected cytometry, as a reflection of the generation of intracellular viral or “bystander” DCs in the response to virus infection (28–30). protein within the DCs following direct infection. To allow accu- However, the effects of paracrine signaling on these bystander rate comparison of the level of signal in different samples, a bar- cells has either not been evaluated or attributed entirely to type I coding flow cytometry approach was used (22). In this approach, IFN. Therefore, we were interested in determining the role of type Downloaded from each sample was first labeled with a characteristic pattern of three I IFN in generating the AVDC state that we have identified. We deconvolution dyes (Fig. 9A) and then the 60 mixed samples were found that only some of the characteristics of AVDCs can be at- labeled with fluorescently labeled surface marker Abs. Detection tributable to type I IFN signaling, indicating that the state of these of virus protein expression and surface marker expression in each cells reflects more complex, multifactor paracrine signals. The sample was then assayed in a single multispectral flow cytometry identification of which combinations and concentrations of factors run. This analysis showed that AVDCs generated much lower lev- are responsible for generating the AVDC state is an interesting http://www.jimmunol.org/ els of viral protein with infection than naive DCs (Fig. 9B). These subject for further investigation. results indicate that, in comparison to naive DCs, the AVDCs Pretreatment of immature DCs and/or DC precursors with single showed a relatively virus-resistant state. cytokines that are secreted either by DCs or other immune cells has Interestingly, AVDCs also expressed much higher levels of mat- been reported to lead to distinct cellular . The effects of uration markers than naive DCs following virus infection. Time type I IFN (31–33), thymic stromal lymphopoietin (34, 35), TNF-␣ course analysis by flow cytometry showed that the levels of CD86, (36), IL-10 (37, 38), IFN-␥ (39), and IL-15 (6, 40–42) have all MHC-I, and MHC-II were dramatically higher in infected AVDCs been reported. During infection of a host, the extracellular envi- at all time points than in infected naive DCs (Fig. 9C). Notably, ronment of the DCs in the infected tissue in vivo contains multiple this enhanced activation state included both the CD86 and MHC-II cytokines and chemokines and the response state of the uninfected by guest on September 24, 2021 markers that were increased in AVDCs without infection as well as DCs is likely to result from more than a single factor. Understand- the MHC-I signal that was unchanged in AVDCs before virus ing the mechanisms underlying the effects of the integration of infection. These results indicate that the paracrine signaling envi- multiple signals has been the object of several experimental and ronment cause the DCs to enter a state in which they are relatively computational systems biology studies (43–45). Recently, we have resistant to infection by virus and their maturation response to begun to develop models to describe paracrine and autocrine sig- infection is greatly enhanced. naling among DCs located in three-dimensional spaces (15). Un- derstanding the mechanisms by which AVDCs are generated is Discussion facilitated by studies integrating mathematical simulation with cy- These experiments reveal that paracrine signals from virus-in- tokine interaction studies. fected cultured human DCs act on naive uninfected DCs to induce Paracrine and autocrine signaling are recognized to contribute to an activated antiviral state. This AVDC state is characterized by the maturation of DCs following TLR stimulation (42, 46–49). basal up-regulation of CD86 and MHC-II cell surface markers, Spo¨rri and Reis e Sousa (49) found that paracrine signaling cannot induction of a variety of antiviral response genes, altered morphol- substitute for contact for TLR-mediated contact with pathogen ogy, and increased basal phagocytosis. The antiviral state of these components in generating fully activated DCs. To our knowledge, AVDCs is evident from their relative resistance to virus infection the investigation of the role of paracrine signaling in modulating and their enhanced rate and level of maturation marker expression the DC response to subsequent encounter with virus, a sequence following infection. These features of the AVDC are well suited to likely to occur during actual infection, has not been previously facilitate the development of adaptive immunity to virus infection. investigated. The Transwell experimental system with which we character- During the early stages of infection, only a few cells are in- ized the AVDC, while used in various studies of immune cell fected. In the case of respiratory virus, inhalation of only a few differentiation (27), has not previously been used to study para- droplets containing virus particles can be sufficient to induce a crine signaling among immune cells. The pore size of the poly- successful infection, and virus transmission usually involves few ethylene terephtalate membranes present in our experiments would viral particles (50). Thus, early in the infection, only a few DCs are not be expected to restrict virus particles or components. NDV likely to come into contact with virus or with virus-infected tissue. does not cause productive viral infection in DCs (24). Neverthe- Therefore, we hypothesize that paracrine signaling events capable less, we tested whether the Transwell culture system might effec- of inducing the generation of AVDCs are likely to play an impor- tively separate the paracrine-stimulated naive DCs from the effects tant role in the early response stages of the immune system to virus occurring with direct encounter with virus. Using sensitive assays infection (Fig. 10). Infection of primary lung fibroblasts was not for expression of fluorescent viral protein and stimulation of virus able to generate AVDCs (supplementary Fig. S2), indicating that infection-dependent gene expression (viral proteins and type I IFN the AVDC is not generated by exposure to secreted factors from 6880 ACTIVATED DCs

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