Cutting Edge: Antibody-Mediated TLR7-Dependent Recognition of Viral RNA Jennifer P. Wang, Damon R. Asher, Melvin Chan, Evelyn A. Kurt-Jones and Robert W. Finberg This information is current as of September 24, 2021. J Immunol 2007; 178:3363-3367; ; doi: 10.4049/jimmunol.178.6.3363 http://www.jimmunol.org/content/178/6/3363 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE

JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: Antibody-Mediated TLR7-Dependent Recognition of Viral RNA1 Jennifer P. Wang,2 Damon R. Asher, Melvin Chan, Evelyn A. Kurt-Jones, and Robert W. Finberg

TLR7 recognizes the of ssRNA such as Cox- “atypical” measles, dengue hemorrhagic fever, and enhanced sackievirus B. Because TLR7 is expressed in intracellular respiratory syncytial disease. Prior immune sensitization compartments, viral RNA must be internalized before its to such viruses can lead to overwhelming production recognition by TLR7. In this study, we define plasmacy- and influx of inflammatory cells, and IC deposition has been toid dendritic cells (pDC) as peripheral blood mononu- implicated as a major disease-causing factor (10–14). Further-

␣ Downloaded from clear immune cells that respond to Coxsackievirus. pDC more, recent studies have shown that IFN- induction by cer- activation by Coxsackievirus B requires the presence of tain viruses is enhanced in the presence of virus-specific Ig and specific antiviral Abs. We show that Fc receptors mediate is mediated by FcR (15, 16). the recognition of virus-Ab complexes and that TLR7 is We define a role for virus-specific Ig activation of TLR7 by required for human and murine pDC production of cyto- Coxsackievirus B (CVB), a nonenveloped, ssRNA picornavirus of the enterovirus group. CVB is commonly associated with

kines. These data define a pathway by which intracellular http://www.jimmunol.org/ subclinical human disease but can cause acute or chronic myo- TLR7 senses viral RNA and indicate a role for TLRs in carditis. Myocardial damage may result from either cytolytic ef- association with Abs in sustaining virus-specific fects of CVB or from immune-mediated destruction (reviewed responses. The Journal of Immunology, 2007, 178: in Ref. 17). In our studies, we generated murine bone marrow- 3363–3367. derived DC (BMDC) and found that they produce IFN-␣ and IL-12 p40 in response to CVB serotype 3 (CVB3) only in the ␣ lasmacytoid dendritic cells (pDC)3 are highly special- presence of anti-CVB Abs. Human pDC also produce IFN- ized cells that are important during viral infection, in response to CVB3 only in conjunction with Ig. We demon- where they are recruited and can produce IFN-␣ (1). strate that both TLR7 and FcR participate in the virus-induced by guest on September 24, 2021 P ␣ TLRs are crucial mediators in innate immune responses to production of IFN- by both murine and human pDC and pathogens. Of at least 11 TLR family members, TLR7 and that the presence of virus-specific Ab is essential. TLR9 are expressed in pDC (2). These endosomal pattern-rec- ognition receptors recognize viral genomic ssRNA and dsDNA, Materials and Methods respectively, and engagement leads to the production of cyto- Reagents ␣ ␣ kines including IFN- (3–5). IFN- may be protective in the CVB3 strain Nancy was prepared as described previously (18) and for some host response to many viruses, but proinflammatory experiments was UV-inactivated with 1 J/cm2 UVA light for 15 min. R-848 activated by TLRs have the potential to adversely affect was a gift from D. Golenbock (University of Massachusetts, Worcester, MA). CpG 2336 was purchased from Coley Pharmaceuticals. Human AB serum was the host. obtained from Sigma-Aldrich and Sandoglobulin intravenous immune globu- Igs play an essential role in the host defense against patho- lin (IVIG) from Novartis. Anti-CVB3 mAb and IgG1 were purchased from gens. However, they can also contribute to autoimmune dis- Chemicon International, anti-CD64 and IgG2a from eBioscience, anti-CD32 eases like systemic erythematosus, in which high levels of from Abcam, and anti-CD16 from BD Pharmingen. Immunoregulatory DNA sequence (IRS) 661 was synthesized on a phosphorothioate backbone by MWG Abs against nuclear Ags are present. Immune complexes (IC) Biotech. comprised of either small nuclear ribonucleoprotein particles or chromatin can stimulate pDC to produce high amounts of Mice ␣ Ϫ Ϫ Ϫ Ϫ IFN- via TLR7 or TLR9, a phenomenon mediated by FcR Fc␥RIII / (B6.129P2-Fcgr3tm1Sjv) mice and Fc␥RII / (B6.129-Fcgr2tm1) (6–9). IC may contribute to ssRNA viral diseases, including mice, each backcrossed 10 generations to C57BL/6, were obtained from The

Department of Medicine, University of Massachusetts, Worcester, MA 01605 3 Abbreviations used in this paper: pDC, plasmacytoid ; BMDC, bone mar- row-derived DC; CVB, Coxsackievirus B; IC, immune complex; IRS, immunoregulatory Received for publication November 10, 2006. Accepted for publication January 12, 2007. DNA sequence; MOI, multiplicity of infection; WT, wild type; IVIG, intravenous im- The costs of publication of this article were defrayed in part by the payment of page charges. mune globulin. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 1 This research support was provided by National Institutes of Health Grants R01- AI049309 and R21-AI064681 (to R.W.F.) and K08-AI053542 (to J.P.W.). 2 Address correspondence and reprint requests to Dr. Jennifer Wang, Department of Med- icine, University of Massachusetts, LRB 219, 364 Plantation, Worcester, MA 01605. E- mail address: [email protected]

www.jimmunol.org 3364 CUTTING EDGE: VIRAL RNA AND TLR7 AND Abs

Jackson Laboratory. Fc common ␥-chain-deficient (B6.129P2- in HeLa cells by nearly 100-fold (data not shown). To confirm Fcerg1tm1; backcrossed 12 generations to C57BL/6) mice were obtained from Ϫ Ϫ that in vivo infection leads to generation of specific antiviral Taconic Farms. MyD88 / (backcrossed 12 generations to C57BL/6) and TLR7Ϫ/Ϫ mice (backcrossed 7 generations to C57BL/6) were a gift from S. Abs, we infected a mouse with CVB3 i.p. and collected serum Akira (Osaka University, Osaka, Japan). Serum was obtained from whole blood 12 days later. Opsonization of CVB3 with this serum induced a of wild-type (WT) mice 12 days after infection with CVB3 (5000 PFU i.p.) or strong IL-12 p40 response from DC, whereas opsonization from uninfected control mice. with serum from a CVB3-naive, uninfected mouse did not. Un- Cell culture der these conditions, UV-treated virus did not induce cyto- Human PBMC and pDC were isolated as previously described (19). Mouse bone kines; either viral replication is required for DC activation or marrow cells were cultured for 7 days with 10 ng/ml mouse fms-related tyrosine UV treatment alters the structure of viral RNA such that it can kinase 3 ligand (R&D Systems), resulting in a population of BMDC with 30– no longer engage with TLR7. 40% pDC by FACS staining. For some experiments, DC were sorted on a Since BMDC are a mixed population, we used FACS to iso- FACSAria (BD Biosciences) to isolate pDC (CD11cϩCD11bϪCD45Rϩ). Mouse pDC purity was Ն98%. late a pure population of pDC and found that pDC produced cytokines in response to virus only in the presence of specific Cytokines Abs. Using Luminex multiplex testing, we found several cytokines Cell culture supernatants were collected at 18 h. ELISA was used to quantify to be released by pDC, including IL-6, IL-12 p40, IFN-␥-induc- human IFN-␣ (Bender MedSystems), murine IL-12 p40 (BD Biosciences), and ible 10, and MIP-1␣ (data not shown). All other tested ␣ murine IFN- (PBL Biomedical Laboratories). Luminex testing was performed cytokines were negative (see Materials and Methods). at the Baylor Luminex National Institute of Allergy and Infectious Diseases Core facility (Dallas, TX) for fibroblast growth factor basic, GM-CSF, IFN-␥, Downloaded from IL-1␣, IL-1␤, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, IFN-␥-in- Murine pDC activation by CVB3 IC requires expression of FcR ducible protein 10, KC, MCP-1, MIG, MIP-1␣, TNF-␣, and vascular endo- common ␥-chain thelial growth factor. We then asked whether opsonized CVB3 triggers cytokine re- sponses by engagement with Fc␥RI (CD64), Fc␥RII (CD32), Results and Discussion Fc␥RIII (CD16), or Fc␥RIV (reviewed in Ref. 23). We used Murine pDC activation by CVB3 requires specific anti-Coxsackievirus Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ␥ / ␥ / ␥ / http://www.jimmunol.org/ Ab BMDC from FcR chain ,Fc RII , and Fc RIII mice to establish the role of FcR in the recognition of opsonized Ϫ Ϫ We examined Ab-mediated responses to CVB3 by murine CVB3. Cells from FcR␥ chain / mice lack the ␥-chain sub- BMDC. Incubation of bone marrow cells with fms-related ty- unit of FcR, which is essential for cell surface expression of Ϫ Ϫ rosine kinase 3 ligand results in a population of cells rich in Fc␥RI, Fc␥RIII, and Fc␥RIV. FcR␥ chain / DC did not pro- ␣ pDC which secrete IL-12, IFN- , and other cytokines in re- duce any IL-12 p40 or IFN-␣ in response to CVB3 opsonized Ϫ Ϫ sponse to TLR7 and TLR9 ligands, including ssRNA viruses (3, with anti-CVB3 mAb (Fig. 2a), whereas DC from Fc␥RII / Ϫ Ϫ 5, 20–22). We found that CVB3 did not induce IL-12 p40 and Fc␥RIII / mice produced comparable amounts of cyto- production in BMDC unless opsonized with Ն1 ␮g/ml mAb kine to WT DC (Fig. 2b). Identical FcR requirements were ob- by guest on September 24, 2021 specifically targeting CVB3. This combination led to vast pro- served with virus opsonized with serum from a CVB3-infected duction of IL-12 p40 (Fig. 1). At these concentrations, mAb mouse (data not shown). Thus, either Fc␥RI or Fc␥RIV is re- neutralized CVB3, causing a decrease in viral plaque formation quired for cytokine induction by opsonized CVB3 in murine cells. This requirement is distinct from that of chromatin IC, which utilize murine Fc␥III receptors for TLR9-mediated DC production of IL-12 p70 and TNF-␣ (8).

Murine DC activation by opsonized CVB3 requires TLR7 and MyD88 CVB, specifically its ssRNA genome, has been shown to inter- act with TLR7 and contribute to the production of inflamma- tory cytokines (24). We determined that TLR7 and the adapter molecule MyD88 are required for murine cytokine responses to opsonized CVB3 by using DC from mice deficient in these molecules. Induction of IL-12 p40 and IFN-␣ was absent from the cells from knockout mice (Fig. 2c); therefore, FcR-mediated delivery of viral RNA to intracellular TLR7 is critical for cyto- kine responses by pDC.

pDC are the primary responders among human PBMC to opsonized CVB3 Human PBMC demonstrate Ab-enhanced production of FIGURE 1. CVB3 opsonized with anti-CVB3 Ab induces cytokine release IFN-␣ in response to CVB (25). We examined IFN-␣ produc- from BMDC. Cells were stimulated with CVB3 (MOI, 100) opsonized with tion in human PBMC in response to opsonized CVB3, testing mAb targeting CVB3 or IgG2a isotype control (5 ␮g/ml). Cells were stimulated three sources of Ig: autologous serum, serum from human male with CVB3 opsonized with serum obtained from a WT mouse 12 days postin- blood type AB (human AB serum), and pooled IVIG. Opso- fection with CVB3 (or uninfected control mouse serum). In some conditions, virus was UV-irradiated before opsonization. LPS (100 ng/ml), R-848 (3 ␮M), nization was performed by incubating virus with an equal vol- and CpG (3 ␮g/ml) were used as control ligands for TLR4, TLR7, and TLR9. ume of serum or IVIG for 15 min at 37°C before addition to Results are representative of three independent experiments. Error bars repre- cells. Each enabled dose-dependent production of IFN-␣ in re- sent SD of triplicate samples. sponse to live, but not UV-irradiated opsonized CVB3 (Fig. The Journal of Immunology 3365

FIGURE 2. The FcR common ␥-chain and TLR7 are required for IL-12 p40 and IFN-␣ production by opsonized CVB3. A, BMDC from WT C57BL/6 or FcR common ␥-chain-de- ficient (FcR␥ chainϪ/Ϫ) mice were stim- ulated with CVB3 (MOI, 100) opso- nized with anti-CVB3 mAb or IgG2a isotype control at 5 ␮g/ml. B, BMDC from WT, FcR␥IIϪ/Ϫ,orFcR␥IIIϪ/Ϫ mice were similarly tested. C, BMDC from WT, MyD88Ϫ/Ϫ mice, or TLR7Ϫ/Ϫ mice were also used. Control stimuli included LPS (100 ng/ml), R-848 (10 ␮M), and CpG (3 ␮g/ml) in all experiments. Results are representa- tive of at least two independent experiments. Downloaded from

3a). Unopsonized virus did not lead to any IFN-␣ production. CVB3 at a multiplicity of infection (MOI) of 30 (42.7 Ϯ 8.8 Of note, in three independent experiments using PBMC from ng/ml), whereas CVB3 alone did not induce IFN-␣. different adult donors, virus opsonized with autologous serum induced IFN-␣. Nearly all adults are seropositive for CVB3, http://www.jimmunol.org/ accounting for the presence of specific anti-CVB IgG in serum Human pDC activation by CVB3 is mediated by CD32 and TLR7 and IVIG (26). Recent studies investigating mechanisms of TLR activation Given that pDC are strong IFN-␣ secretors, we suspected have shown that CD32a (Fc␥RIIa) is important for the activa- that pDC were responsible for the majority of the IFN-␣ pro- tion of human pDC by IC involving small nuclear ribonucleo- duced by PBMC in response to CVB3. To generate a popula- protein particle or systemic lupus erythematosus-DNA (6, 7, tion of PBMC depleted of pDC, we added anti-CD304 9). To establish the role for FcR in human pDC recognition of MicroBeads to cells and passed them through a MACS Separa- opsonized CVB3, we incubated pDC in the presence of neu- tor (Miltenyi Biotec). The resulting population of PBMC was tralizing Abs against CD16 (Fc␥RIII), CD32a (Fc␥RIIa), or by guest on September 24, 2021 largely devoid of pDC. When compared with the total PBMC CD64 (Fc␥RI) for 30 min before addition of stimuli. We found population, pDC-depleted PBMC produced negligible IFN-␣ IFN-␣ responses to be mediated by CD32a, but not CD16 or in response to opsonized CVB3 (Fig. 3b). pDC-depleted CD64; results from a representative donor are shown in Fig. 4a. PBMC had equivalent IL-6 production following stimulation In experiments using multiple donors, anti-CD32a mAb (0.5 ␮ ␣ Ϯ with the TLR2 ligand Pam3CSK4 (100 ng/ml) in comparison g/ml) blocked virus-induced IFN- production to 34 5% to total PBMC (16.1 Ϯ 1.8 ng/ml vs 12.6 Ϯ 2.7 ng/ml IL-6); compared with isotype control (n ϭ 5, p Ͻ 0.01 by Student’s t therefore, altered viability of the pDC-depleted PBMC was not test). Induction of IFN-␣ by R-848 was unaffected by the pres- a contributing factor. Of positively selected pDC, high ence of neutralizing Ab (data not shown). Mice are naturally amounts of IFN-␣ were produced in response to opsonized deficient in the CD32a isoform and only express the inhibitory

FIGURE 3. IFN-␣ production by human PBMC in response to CVB3 re- quires opsonization of virus and is pre- dominantly from the pDC population. A, CVB3 (MOI, 100, 10, or 1) was op- sonized with autologous serum, human IVIG, or human AB serum. For some conditions, CVB3 was UV-irradiated before opsonization. Results are repre- sentative of three independent experi- ments with different human donors. B, PBMC and PBMC depleted of pDC were stimulated with CVB3 opsonized with autologous serum. Controls in- cluded R-848 (20 ␮M) and CpG (10 ␮g/ml). 3366 CUTTING EDGE: VIRAL RNA AND TLR7 AND Abs

cytokines could be associated with adverse outcomes. In a model of CVB3-induced myocarditis, MyD88-deficient mice had improved survival, decreased cardiac inflammation, and decreased levels of inflammatory cytokines in comparison to WT mice (29). Further in vivo studies will define the role for FcR and TLR7 in the presence of virus-specific Ab and may identify mechanisms by which immune sensitization to virus can cause disease. Acknowledgments We thank A. Cerny for animal work and D. Libraty for helpful scientific dis- cussion, assistance with Luminex samples, and FACS. Disclosures The authors have no financial conflict of interest. References 1. Cella, M., D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, and

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