7058.Full.Pdf

The Journal of Immunology

Antiviral Antibodies Target Adenovirus to Phagolysosomes and Amplify the Innate Immune Response1

Anne K. Zaiss,2* Akosua Vilaysane,† Matthew J. Cotter,3† Sharon A. Clark,† H. Christopher Meijndert,‡ Pina Colarusso,‡ Robin M. Yates,§ Virginie Petrilli,¶ Jurg Tschopp,¶ and Daniel A. Muruve4†

Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-␬B-dependent genes, type I IFNs, and caspase-dependent IL-1␤ maturation. Immune serum amplified TLR9- independent type I IFN expression and enhanced NLRP3-dependent IL-1␤ maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state. The Journal of Immunology, 2009, 182: 7058–7068.

denoviruses are nonenveloped DNA viruses that primar- the activation of the innate immune system, suggesting that the ily cause respiratory and gastrointestinal disease in hu- intracellular innate immune system primarily mediates the host A mans, a significant problem in young and immunocom- recognition and response to these infectious agents (9, 13–15). promised patients. In addition, recombinant adenovirus vectors are Recent studies from our group and others show that the adenovirus extensively studied and developed for gene and oncolytic therapy virion and/or DNA directly activate several arms of the intracel- (1). Adenovirus activates the innate immune system, which repre- lular innate immune system in macrophages. These include the by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. sents a significant hurdle to the effective therapeutic application of NLRP3 (NALP3) inflammasome and a TLR-independent DNA these agents for somatic gene therapy, oncolytic therapy, and vac- sensor that leads to IL-1␤ maturation and type I IFN expression, cination (1–8). Macrophages play a major role in detecting ade- respectively (16, 17). Furthermore, macrophages are involved in novirus and in orchestrating the initial innate host response to in- adenovirus clearance in vivo by capturing viral particles within fection (9–12). The internalization of adenovirus is a key step in minutes of injection and transporting surface-bound virus to lymph nodes, thereby facilitating the induction of adaptive responses (11, 12, 18). TLR9, on the other hand, mediates adenovirus recognition *Department of Biochemistry and Molecular Biology, †Department of Medicine, and ‡Department of Physiology and Biophysics, Faculty of Medicine and §Department of primarily in dendritic cells (16, 17). http://classic.jimmunol.org Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, The understanding of intracellular innate receptors such as ¶ University of Calgary, Alberta, Canada; and Department of Biochemistry, Univer- TLR9 and NLRP3 is increasing. TLR9 is known to reside in en- sity of Lausanne, Epalinges, Switzerland dosomes and recent studies show that endosome acidification and Received for publication December 18, 2008. Accepted for publication April 17, 2009. maturation are required to cleave TLR9 into a functional signaling The costs of publication of this article were defrayed in part by the payment of page receptor (19). Similarly NLRP3 inflammasome activation in mac- charges. This article must therefore be hereby marked advertisement in accordance rophages has been linked to phagosome maturation and the with 18 U.S.C. Section 1734 solely to indicate this fact.

Downloaded from phagolysosome. Mycobacterium tuberculosis actively inhibits 1 This work was supported by the Alberta Heritage Foundation for Medical Research, phagosome maturation, progression to a phagolysosome, and sub- operating and group grants from the Canadian Institutes for Health Research, and infrastructure grants from the Canadian Foundation for Innovation. D.A.M. is the sequent inflammasome activation, events that increase host sus- recipient of an Alberta Heritage Foundation for Medical Reasearch Scholar Award. ceptibility to infection. Furthermore, activation of the NLRP3 in- A.K.Z. was the recipient of a Heart and Stroke Foundation of Canada Studentship. flammasome in response to silica crystals depends on phagocytosis 2 Current address: Department of Pharmacology, University of California Los Ange- and lysosomal damage (20, 21). Conversely, little is currently les, Boyer Hall, Room 329, 611 Charles E. Young Drive East, Los Angeles, CA 90095. known regarding the biology of the TLR-independent DNA-sens- 3 Current address: Department of Immunology, Moffitt Cancer Center, MRC 2 East, ing pathway. Room 2068, 12902 Magnolia Drive, Tampa, FL 33612-9416. Although macrophages, dendritic cells, and other leukocytes 4 Address correspondence and reprint requests to Dr. Daniel A. Muruve, Department play a significant role in the detection and clearance of adenovirus of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N and viruses in general, few viruses exhibit a natural tropism for 4N1 Canada. E-mail address: [email protected] these innate effector cells. In contrast to the extensive studies that Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 characterize adenovirus binding and entry in nonhematopoietic

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804269 The Journal of Immunology 7059

cells, the direct interaction between adenovirus and macrophages treated with medium containing 0.05 mM zVAD-fmk for 30 min before is poorly understood. Leukocytes lack the Coxsackievirus adenoviral infection. To block viral internalization and endosome acidification, ␮ virus receptor (CAR)5 used by adenovirus for binding to epithelial- THP-1 cells were treated with 10 g/ml cytochalasin B (CCB) or 100 nM bafilomycin A1 (Sigma-Aldrich) for 30 min before adenovirus infection. derived cells and consequently virus attachment to hematopoietic cells occurs at a much lower affinity (22). Given that adenovirus Animal studies and primary macrophage isolation internalization in leukocytes is key to the host detection of viral All studies involving animals were performed in accordance with the An- infection and the activation of intracellular innate receptors, these imal Care Committee guidelines at the University of Calgary. C57BL/6 observations suggest that other mechanisms must exist that target mice, 6–8 wk old, were purchased from Charles River Laboratories. Mice viruses to the cellular compartments necessary to mount an effi- genetically deficient in NLRP3 (NLRP3Ϫ/Ϫ), ASC (ASCϪ/Ϫ, provided by Ϫ/Ϫ cient antiviral host response. Our previous studies demonstrate that Dr. V. Dixit, Genetech, Sanfrancisco, CA), and TLR9 (TLR9 , provided by Dr. P. Kubes, University of Calgary, Calgary, Alberta, Canada) were on opsonins such as complement and antiviral Abs play a significant a C57BL/6 background as described previously (26). Mice were housed role in altering natural viral tropism and targeting the adenovirus to under single-barrier conditions and used for macrophage harvest at 8–12 neutrophils (14). Similarly, macrophages and dendritic cells effi- wk of age (25–30 g). Primary mouse bone marrow macrophages were ciently interact with and internalize adenovirus-Ab immune com- isolated from the tibia and femur of mice, followed by differentiation into plexes (10, 23). Since viral-sensing receptors are intracellular, it is macrophages for 7 days in tissue culture. Briefly, mice were euthanized, the hind legs were removed, and tibiae/femurs were carefully separated and likely that Ab-mediated viral entry in macrophages has an impact cleaned. Bone marrow stem cells were flushed from the bone marrow cav- on the innate immune response to adenovirus. ity. The cells were washed and seeded at 1.4 ϫ 106 cells/well of a 6-well In this study, we demonstrate that mice immunized with ade- plate or 2.5 ϫ 105 cells/well of 24-well plate in bone marrow medium. The novirus display enhanced innate immune responses following a plates were incubated for 5 days and the medium was replaced with fresh bone marrow medium on day 5. Bone marrow medium consists of DMEM second adenoviral challenge. Antiviral Abs increase adenovirus supplemented with 2% penicillin-streptomycin, 10% FBS, and 10% L cell internalization via FcRs in macrophages. Unlike adenovirus entry conditioned medium. L cell conditioned medium was produced by cultur- in nonhematopoietic cells that is typified by early endosomal es- ing L929 cells in T-150 tissue culture flasks at 0.24 ϫ 106 cells/flask in 50 cape, Ab-dependent viral entry in macrophages targets the adeno- ml of culture medium (DMEM supplemented with 1% penicillin-strepto- virus to the phagolysosome, an event that is associated with am- mycin and 10% FBS). After 7 and 14 days, the medium was collected, filtered, mixed 1:1, and stored at –80°C. plification of intracellular innate pathways including the NLRP3 Mouse peritoneal macrophages were isolated by peritoneal lavage 72 h inflammasome and TLR9-independent type I IFN expression. following i.p. injection of 4% thioglycolate solution. Cells were plated at the density of 5 ϫ 105 cells in 24-well dishes. Cells were cultured in RPMI Materials and Methods 1640 medium complemented with 10% FBS, sodium pyruvate, penicillin/ streptomycin, and L-glutamine. Sixteen hours after isolation, the cells were Adenovirus infected with adenovirus. For experiments involving the analysis of IL-1␤ The type 5, E1-deleted, E3-defective adenovirus encoding the GFP or the processing, infections were performed in the presence of 1 ng/ml ultrapure LacZ transgene under the control of a CMV promoter was generated using LPS (InvivoGen). an Ad-EASY system (Stratagene), propagated on human embryonic kidney In some experiments, the FBS in the culture medium was replaced with 293 cells, and purified as previously described (24). Wild-type adenovirus 5–10% mouse serum (MS). MS was obtained from naive or adenovirus- serotype 5 was propagated on 293 cells and purified as above. The particle immunized C57BL6 mice under anesthesia by cardiac puncture. For com- titer was determined by measuring the OD at 260 nm described as particle plete complement, inhibition serum was HI for 30 min at 56°C, referred to by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. per cell (part/cell). Ps-Ad* is AdGFP, which was rendered transcription as MS-HI, MS from immunized mice is referred to as MS plus Ig. ϫ 11 defective by UV/psoralen treatment as previously described (25). AdL.PB* For in vivo studies, C57BL/6 mice were injected with 2 10 particles and AdL.F* are tropism-modified E1-E3-deleted Ad5-based vectors car- of AdLacZ via the femoral vein under general anesthesia in a total volume ␮ rying the luciferase transgene in the E1 region under the control of the of 100 l (vector plus vehicle). Control animals received vehicle alone (3% CMV promoter (13). Adenovirus and vectors were labeled with the green sucrose, 150 mM NaCl, 10 mM Tris (pH 7.4), and 1 mM MgCl2). Animals fluorescent dye Cy2 as described previously (14) using an Ab labeling kit were allowed to recover and then sacrificed at 6 h and the livers and sera ϫ 11 (GE Healthcare) according to the manufacturer’s instructions. Low endo- were harvested for analysis. Some animals were immunized with 1 10 toxin tissue culture reagents and buffers were used for virus/vector pro- particles/mouse AdGFP or injected with vehicle 10 days before subsequent duction and experiments. Adenovirus and vectors were routinely tested for adenovirus administration. the presence of endotoxin using an Limulus Amebocyte Lysate Kit. All viruses/vectors contained Ͻ0.1 endotoxin units/ml. Immunoblotting and ELISA http://classic.jimmunol.org ␤ Cell culture For immunoblotting of IL-1 protein, cells were lysed with buffer (10 mM Tris (pH 7.5), 1% Nonidet P-40, 150 mM NaCl, and protease inhibitor Human embryonic kidney cells (HEK 293 cells) and HeLa cells were mixture), then proteins in cell lysate or tissue culture supernatant were maintained in DMEM containing 10% FBS and 1% penicillin-streptomy- separated on SDS-PAGE gels and transferred onto nitrocellulose mem- cin. Human THP-1 monocytic leukemia cells were grown in RPMI 1640 branes. The membranes were blocked with 5% milk proteins in 1ϫ PBS medium supplemented with 10% FBS, 1% penicillin-streptomycin, 1% and 0.5% Tween 20 and then probed with primary Abs as follows: poly- MEM, 100 mM sodium pyruvate, and 0.01% 2-ME. THP-1 cells were clonal rabbit anti-human-cleaved IL-1␤ (D116; Cell Signaling), polyclonal Downloaded from differentiated with 100 nM PMA overnight before all experiments. Cells rabbit anti-human IL-1␤ (Cell Signaling), or polyclonal sheep anti-mouse were periodically tested for Mycoplasma contamination. Viral infections IL-1␤. Appropriate HRP-conjugated secondary Abs were used and proteins were performed in 6-well plates or 24-well plates with 2 ϫ 106 cells/well were detected using ECL reagent (Amersham Biosciences). and 2 ϫ 105 cells/well, respectively. Cells were incubated for the specified IL-1␤ protein in mouse serum was quantified using an ELISA against time periods in 1 ml of medium (6-well) or 0.3 ml of medium (24-well) IL-1␤ protein as per the manufacturer’s protocol (BD Biosciences). ODs containing viruses or viral vectors. For selected experiments, the FBS in were determined spectrophotometrically at 450 nm. the cell culture medium was replaced by 5–10% pooled human serum (HS). HS was collected from 10 healthy donors, pooled, and filtered through a Microscopy and flow cytometry sterile 0.45-␮m mesh. For total inhibition of the complement system, HS To visualize GFP transgene expression and cell morphology, cells were was heat inactivated (HI) for 30 min at 56°C (referred to as HS-HI). For examined by phase contrast and fluorescent microscopy using an Olympus depletion of Ig (IgG), serum aliquots were treated with an equal volume of IX70 inverted epifluorescence microscope (Olympus). Digital images were protein G-Sepharose beads (Amersham Biosciences) (referred to as HS- obtained and analyzed using OpenLab software (Improvision). Flow cy- IgG). To inhibit caspase-dependent IL-1␤ processing, the cells were pre- tometry was performed on formalin-fixed cells using a FACScan instru- ment and analyzed with CellQuest Pro software (BD Biosciences). For the ␮ 5 Abbreviations used in this paper: CAR, Coxsackievirus adenovirus receptor; HS, FcR-blocking experiments, THP-1 cells were pretreated with 30 g/ml human serum; MS, mouse serum; HI, heat inactivated; CCB, cytochalasin B; part, anti-human CD64 (hFc␥RI) (BD Pharmingen) and/or the hFc␥RIIA-block- particle. ing Ab IV.3 (American Type Culture Collection) or isotype control Abs for 7060 Abs AND INNATE RESPONSE TO ADENOVIRUS

FIGURE 1. Innate immune response to adenovirus in immunized mice. A, RNase protection assay of liver RNA from mice 6 h after the i.v. administration of 2 ϫ 1011 particles of AdLacZ. Mice were naive or immunized with AdGFP 10 days before. B, Quantiﬁcation of mRNA expression in mouse liver. Mean phosphor imager units Ϯ SD, n ϭ 6 (naive vs im- ␤p Ͻ 0.01). C, Serum IL-1 ,ءء ;mune levels in mice 6 h after AdLacZ administration (ELISA). Mean pg/ml Ϯ p Ͻ ,ءء ;SD, n ϭ 6 (naive vs immune 0.01). D, Real-time PCR analysis of IFN-␣4 in mouse liver. Values represent mean percent induction over vehicle-treated mice Ϯ SD, n ϭ 6 (naive vs immune, p ϭ NS). E, Liver LacZ transgene expression in naive or immunized mice at 6 h following Ad- LacZ administration. F, PCR of liver DNA probing for the AdLacZ transgene. Values represent mean AdLacZ DNA/GAPDH Ϯ SD, n ϭ 6 (naive vs .(p Ͻ 0.001 ,ءءء ;immune

10 min at 37°C. Cells were then infected with Cy2-labeled adenovirus for To assess ␤-galactosidase activity, 5-mm frozen liver sections were 90 min and analyzed by ﬂow cytometry as described above. ﬁxed in 0.2% glutaraldehyde/PBS supplemented with 0.1 M EDTA and

An analysis of the distribution of the virus within cells was performed 1 M MgCl2. After washing with PBS/1 M MgCl2/2% Nonidet P-40, sec- using an Olympus Flouview 1000 inverted confocal microscope with a tions were incubated in a solution containing 5 mM K3Fe(CN)6,5mM ϫ ϫ ␤ 63/1.42 aperture or a 60/1.20 aperture objective. Stacks in the z direc- K4Fe(CN)6, and 5-bromo-4-chloro-3-indolyl- -D-galactopyranoside for tion were acquired at 0.2-␮m intervals. Cells (THP-1 or 293) were grown 1 h at 37°C. on coverslips contained in standard 6-well plates. For live cell imaging, cells were pulsed with Alexa Fluor 594-labeled 40-kDa dextran and incu- DNA isolation and Southern blotting bated overnight. Cells were then incubated with Cy2-labeled adenovirus in

by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. the presence of 5% pooled human or FBS and incubated for 1 h followed To obtain total DNA, cells were trypsinized for 5 min and washed several by imaging. For LAMP1 staining, cells were fixed using 4% paraformal- times with PBS to remove bound virus from the cell surface. The cell pellet dehyde and treated briefly with 1% Triton X-100. Slides were blocked with or mouse liver was subjected to processing for total DNA using a DNeasy 10% goat serum and then incubated with the LAMP1 primary Ab (clone kit (Qiagen) according to the manufacturer’s protocol. For Southern anal- H4A3-c) for1hatroom temperature. Cells were then incubated with an ysis, 1–3 ␮g of DNA was digested with HindIII and separated on a 1% Alexa Fluor 555-labeled goat anti-mouse secondary Ab, stained with 4Ј,6- agarose gel. The gel was washed in 0.25 M HCl followed by another wash diamidino-2-phenylindole for 5 min (Invitrogen), and imaged. Single-la- in a 0.4 M NaOH solution. The DNA was then alkaline transferred onto a beled controls were used to check for cross-talk between the imaged chan- positive charged Hybond XL nylon membrane (Amersham Biosciences). A nels. To eliminate bleed-through of the Cy2-labeled virus, the second full-length cDNA fragment of the adenovirus 5 fiber gene (332 bp) was channel corresponding to either the red LAMP1 or the dextran signal was labeled with [32P]dCTP using a Redprime random primer labeling system limited to Ͼ580 nm using the spectral detection channel on the FV1000 (Amersham Biosciences). Hybridization was performed by using 2 ng of http://classic.jimmunol.org confocal microscope. All analysis and quantification of spatial overlap was the labeled DNA probe per 5 ml of ExpressHyb Solution (BD Clontech) at conducted using Volocity software (Improvision). 60°C for 1 h. The membrane was then washed three times for 15 min in 2ϫ

FIGURE 2. Pooled HS abrogates Downloaded from adenovirus infection and transgene expression in epithelial cells in vitro. A, GFP transgene expression in HeLa cells 24 h after infection with AdGFP in the presence of normal tissue culture medium or tissue culture medium containing 10% pooled HS. Quantiﬁ- cation based on FACS analysis. Val- p Ͻ ,ءء ;ues represent the mean Ϯ SD 0.01. B, Southern blot analysis of HeLa cells infected with AdGFP as in A, probing for the adenovirus-ﬁber gene, showing internalized vector genomes. The Journal of Immunology 7061

SSC/0.1% SDS (1ϫ SSC is 0.15 M NaCl and 0.015 M sodium citrate) at 60°C and exposed to radiographic ﬁlm for visualization. For quantiﬁcation, the blots were subjected to phosphor imager analysis by a Personal F/X Molecular Imager (Bio-Rad), and the signal intensities were analyzed with Quantity One software (Bio-Rad). To correct for DNA loading, intensity scores were normalized to the density of total DNA in the agarose gel before membrane transfer and expressed as phosphor imager units.

RNase protection assay and real-time PCR Total RNA from cells and tissue was isolated using a RNeasy kit (Qiagen) according to the manufacturer’s protocol. RNase protection assays were conducted using the RiboQuant MultiProbe RNase protection assay system and the mCK3-mCK5c-mCK2b multiprobe template sets for mouse or hCK5-hCK2b sets for human (BD Pharmingen) as per the manufacturer’s protocol. Gels were visualized by autoradiography and quantiﬁed using a Personal F/X Molecular Imager and the Quantity One software (Bio-Rad). The signal intensity of individual genes was normalized to the intensity of the housekeeping gene GAPDH within the same sample. Reverse transcription was conducted using 500 ng of RNA, random hexamers (3 ␮g/␮l), and Moloney mouse leukemia virus reverse transcrip- tase (200 U/␮l; Invitrogen) per the manufacturer’s protocol. Real-time PCR primers and probes were designed using Primer3 software to amplify sequence fragments of the murine IFN-␣4 and IFN-␤ genes. They were as follows: mIFN-␣4 forward primer, 5Ј-AAGGACAGGAAGGATTTTG GATT-3Ј and reverse primer, 5Ј-GAGCCTTCTGGATCTGTTGGTT-3Ј; probe, 6FAM-CCCTTGGAGAAGGTG-MGBNFQ; mIFN-␤ forward primer, 5Ј-CGGACTTCAAGATCCCTATGGA-3Ј and reverse primer, 5Ј- ATGGCAAAGGCAGTGTAACTCTTC-3Ј; and probe, 6FAM-TGACG GAGAAGATGC-MGBNFQ. The 20X Mouse GAPD (GAPDH) FAM/ MGB Probe (Applied Biosystems) was used as the endogenous control. Target gene reactions were formed in a total reaction volume of 25 ␮l using 12.5 ␮lof2ϫ TaqMan Universal PCR master mix (Applied Biosystems), 900 nM of each primer, 200 nM probe, 2.95 ␮l of RNase/DNase-free distilled water, and 5 ␮l of cDNA template (diluted 1/5 from original). Ampliﬁcation was performed in MicroAmp Fast Optical 96-well reaction plates (Applied Biosystems) using the 7900HT Fast Real-Time PCR Sys- tem (Applied Biosystems). Results were normalized to GAPDH transcripts and expressed as percent induction relative to vehicle-treated controls. Semiquantitative PCR was performed to probe for the ␤-galactosidase (LacZ) gene in liver DNA using forward primer, 5Ј-GATCGATC CCGTCGTTTTACAACGT-3Ј and reverse primer, 5Ј-ATCAGTTGCT by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. GTTGACTGTAGCGGC-3Ј. Results were analyzed by densitometry using the Quantity One software (Bio-Rad) and normalized to GAPDH.

Statistical analysis Statistical analyses were performed using GraphPad Instat version 3.01. All data are expressed as mean Ϯ SD. The results were analyzed for statistical variance using an unpaired Student’s t test or one-way ANOVA as appropriate. Results were considered signiﬁcant when p Ͻ 0.05.

FIGURE 3. Adenovirus infection of human THP-1 cells in the presence http://classic.jimmunol.org Results Immunized mice display enhanced innate immune responses to of neutralizing Abs. A, Southern blot analysis of AdGFP-infected THP-1 adenovirus cells blotting for the adenovirus fiber gene and corresponding quantification (based on phosphor imaging). Cells were infected with AdGFP vector The majority of humans have been exposed to common adenovirus for 6 h either in the presence of normal tissue culture medium containing serotypes and carry neutralizing antiviral Abs. To determine the 10% FBS, medium containing 10% pooled HS, medium containing 10% impact of preexisting immunity on the innate immune response to HS-HI, or medium containing 10% IgG-depleted pooled HS (HS-Ig). Val- Ϯ ϭ Downloaded from adenovirus, C57BL/6 mice were immunized with AdGFP (1 ϫ ues represent mean density SD, n 3. The amount of internalized vector 1011 particles i.v.) or vehicle control 10 days before a second chal- DNA was significantly higher when the cells were infected in the presence lenge with the adenovirus vector AdLacZ (2 ϫ 1011 particles i.v.). of HS or HS-HI compared with when the cells were infected in the pres- p Ͻ 0.001). B, Cy2-Ad was incubated with ,ءءء) ence of FBS or HS-Ig At 6 h following AdLacZ administration, mice were sacrificed and THP-1 cells in the presence or absence of HS and viral internalization was p Ͻ ,ءء ;analyzed. As expected in naive mice, AdLacZ induced the expres- determined by flow cytometry (Ad vs Ad in the presence of HS sion of numerous inflammatory genes in the liver including IP-10, 0.01). C, Role of Fc␥Rs in THP-1 cell-adenovirus uptake. Cy2-Ad was MCP-1, TCA-3, and MIP-1␤ (Fig. 1, A and B). In the serum, IL-1␤ incubated with THP-1 cells in the presence of HS and blocking Fc␥R Abs levels were also elevated following AdLacZ administration as de- or isotype control. Cells were washed and analyzed by flow cytometry at termined by ELISA (Fig. 1C). In contrast, immunized mice were 90 min. Values represent mean fluorescence Ϯ SD, n ϭ 3–5. (Fc-blocking .(p Ͻ 0.001 ,ءءء ;not protected from a second adenovirus challenge and displayed Abs vs isotype control significantly higher levels of inflammatory gene expression in the liver and IL-1␤ levels in the serum (Fig. 1, A–C). Small increases in IFN-␣4 mRNA could be detected in the livers of naive mice receiving To assess the effect of increased inflammation on adenovirus- AdLacZ that was enhanced by prior immunization, although the dif- mediated LacZ expression, livers were analyzed histologically for ferences did not reach statistical significance (Fig. 1D). transgene expression. At 6 h, LacZ expression was clearly evident 7062 Abs AND INNATE RESPONSE TO ADENOVIRUS

FIGURE 4. Adenovirus activation of human THP-1 cells. A, RNase protection assay of THP-1 cell RNA 6 h following infection with AdGFP in different serum conditions. AdGFP induced expression of IP-10, MIP-1␤, MIP-1␣, MCP-1, and IL-8 mRNA is severalfold higher in the presence of 10% pooled HS compared with FBS containing normal tissue culture medium, but returns to baseline activation when the HS is IgG depleted. B, TNF-␣ and IFN-␤ mRNA is also induced in AdGFP-infected THP-1 cells in the presence of HS (RNase protection assay). C, Immunoblotting for pro-IL-1␤ in extracts and mature IL-1␤ (D116) in supernatants of THP-1 cells infected with wild-type adenovirus (Ad5) in the presence of FBS or HS. Cells were also pretreated with the pan-caspase inhibitor zVAD-fmk (50 ␮M). D, Fluorescent microscopy images of THP-1 cells 24 h after infection with AdGFP in the presence

by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. of normal tissue culture medium (FBS), medium containing 10% HS, or 10% HS-Ig. Quantitative analysis (right) of GFP transgene expression in AdGFP-infected THP-1 cells determined by FACS analysis. VH, Vehicle. Values represent mean Ϯ SD, n ϭ 3. Cells infected in the presence of 10% HS p Ͻ 0.001). Differences ,ءءء) or HS-HI showed signiﬁcantly less GFP expression compared with cells infected in the presence of FBS or 10% HS-Ig between HS and HS-HI or between FBS and HS-Ig were not signiﬁcant.

in ϳ30% of hepatocytes in naive mice. In contrast, absolutely no mented with 10% pooled HS, GFP transgene expression was al- LacZ expression could be detected at6hinimmunized mice re- most completely abrogated (Fig. 2A). Southern blot analysis of ceiving AdLacZ (Fig. 1E). The lack of transgene expression was HeLa cell DNA and blotting for the adenovirus fiber gene showed http://classic.jimmunol.org associated with a significant reduction in AdLacZ genomes in the that internalized vector genomes correlated with GFP transgene liver determined by PCR for the LacZ transgene reflecting a sig- expression; significantly less adenoviral DNA could be detected nificant alteration in viral tropism, increased viral clearance, or when the cells were infected in the presence of 10% pooled HS, both in immunized mice (Fig. 1F). Since the majority of i.v. ad- suggesting that AdGFP failed to enter the cells (Fig. 2B). These ministered adenovirus targets to the liver in naive mice, collec- results confirm that pooled HS neutralizes adenovirus entry and tively these results suggest that immunization modulates adenovi- infection in nonhematopoietic, epithelium-derived cells, consistent

Downloaded from rus interaction with the innate immune system in vivo. with the presence of antiviral Abs. Macrophages play a significant role in the innate immune re- Serum Ig increases adenovirus internalization in macrophages sponse to adenovirus in vivo (9–12). To determine the role of The enhanced innate immune response to adenovirus in immu- serum components in macrophage-adenovirus interactions, THP-1 nized mice suggested that antiviral Abs may significantly alter vi- cells were infected with AdGFP in the presence of pooled HS and ral interactions with innate effector cells. To examine the mecha- the amount of intracellular vector genomes was analyzed by nism of this observation further, pooled HS (due to the high Southern blot probing for the adenovirus fiber gene (Fig. 3A). In seroprevalence to adenovirus in the human population) was used the presence of tissue culture medium supplemented with FBS, as a source of anti-adenovirus Abs for in vitro studies. First, the infection of THP-1 cells with 1 ϫ 104 part/cell of AdGFP was ability of the pooled HS to neutralize adenovirus was tested. In- generally inefficient, with small amounts of intracellular vector fection of HeLa cells with 1 ϫ 104 part/cell of an E1-deleted, genomes detectable at 6 h. In contrast to the reduction in viral entry E3-defective, serotype 5-based adenovirus vector encoding GFP in observed HeLa cells, the amount of intracellular adenoviral (AdGFP) resulted in strong expression of the transgene as deter- genomes increased up to 10-fold in THP-1 cells following infec- mined by fluorescent microscopy and FACS analysis at 24 h (Fig. tion in the presence of HS. Similarly, internalized viral genomes 2A). In contrast, when the tissue culture medium was supple- were significantly increased following AdGFP infection in the The Journal of Immunology 7063 by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd.

FIGURE 5. Adenovirus infection and activation of primary mouse macrophages. A, Increased virus vector uptake by primary mouse bone marrow macrophages in the presence of immunized MS. Southern blot analysis of internalized vector genomes 6 h after AdGFP infection of primary mouse bone marrow macrophages blotting for the adenovirus fiber gene and corresponding quantification (based on phosphor imaging). The tissue culture medium was serum-free (SF) or supplemented with 10% FBS, MS, MS-HI, or MS from mice that had been immunized with 1 ϫ 1011 part/mouse Ad-GFP 10 days earlier (referred to as immune serum, MS ϩ Ig). Values represent mean density Ϯ SD, n ϭ 3. Viral vector uptake by primary ء Ͻ ءء ϩ http://classic.jimmunol.org macrophages was significantly higher in the presence of immune MS (MS Ig) than in the presence of serum-free medium ( , p 0.01), MS ( , .p Ͻ 0.01). B, RNase protection assay of primary mouse bone marrow macrophage RNA 6 h after infection with AdGFP ,ءء) p Ͻ 0.05), or MS-HI Cells were infected in tissue culture medium supplemented with 10% MS, 10% immune MS (MS ϩ Ig), or 10% MS-HI. AdGFP induced the expression of RANTES, MIP-1␤, MIP-1␣, MIP-2, TCA-3, and IP-10 above baseline. Ad-induced chemokine and cytokine expression was highest in the presence of immune serum (MS ϩ Ig). C, Real-time PCR analysis of type I IFN expression (Ⅺ, IFN-␤; f, IFN-␣4) in bone marrow-derived murine macrophages at 6 h after infection with wild-type adenovirus. Infections were performed in the presence of 10% naive (MS) or adenovirus- ,p Ͻ 0.01). D ,ءء ;immunized MS (MS ϩ Ig). Values represent mean percent induction over vehicle-treated cells Ϯ SD, n ϭ 6 (MS vs MS ϩ Ig

Downloaded from Fluorescent microscopy of primary murine bone marrow macrophages infected with AdGFP. Cells were infected with AdGFP in the presence of either FBS containing tissue culture medium (FBS), medium containing 10% MS, or 10% immune MS (MS ϩ Ig). Immune serum abrogates transgene expression in AdGFP-infected primary mouse bone marrow macrophages. Quantification of GFP expression (left) in AdGFP-infected primary mouse bone marrow macrophages by FACS analysis. Values represent mean Ϯ SD, n ϭ 3. Cells infected in the presence of immune serum p Ͻ 0.001). E, RNase ,ءءء) MS ϩ Ig) showed significantly less GFP expression compared with cells infected in the presence of MS or MS-HI) protection assay showing chemokine mRNA expression in primary mouse bone marrow macrophages 6 h after incubation with tropism-modified adenovirus vectors in the presence or absence of immune serum. Ps-Ad, transcription-defective adenovirus vector; AdL-PB, RGD-deleted adenovirus vector; AdL.F, fiber-modified, CAR-ablated adenovirus vector. Adenovirus-induced expression of chemokine and cytokine mRNA is increased in the presence of MS containing neutralizing Abs (ϩ) compared with MS alone (Ϫ).

presence of HS-HI but not IgG-depleted HS or FBS consistent with etry (Fig. 3B). Cy2-Ad uptake by THP-1 cells was significantly en- Ab, but not complement-mediated adenovirus entry. To further con- hanced in the presence of HS compared with FBS. firm that antiviral Abs increased viral entry, THP-1 cells were infected THP-1 cells are capable of phagocytosing IgG-opsonized im- with Cy2-labeled serotype 5 adenovirus (Cy2-Ad) in the presence or mune complexes by Fc␥RI (CD64) and Fc␥RIIa (CD32) (27). absence of HS and uptake was determined at 90 min by flow cytom- To assess the contribution of THP-1 cell-expressed Fc␥Rs in 7064 Abs AND INNATE RESPONSE TO ADENOVIRUS

FIGURE 6. Ab-dependent ampliﬁcation of the NLRP3 inﬂammasome and TLR9-independent IFN pathway. A, Immunoblotting for IL-1␤ in cell extracts (ext) or supernatants (sup) from LPS-primed wild-type (WT), NLRP3Ϫ/Ϫ,orASCϪ/Ϫ peritoneal macrophages 6 h following infection with wild-type adenovirus (Ad5) in the presence of 10% MS or 10% immune MS (MS ϩ Ig). Vehicle-treated cells served as negative control. The pan-caspase inhibitor zVAD-fmk (50 ␮M) was added to cells before infection. Adenovirus-induced IL-1␤ processing is enhanced in the presence of immune serum, but completely absent in ASCϪ/Ϫ and NLRP3Ϫ/Ϫ macrophages. B, Real-time PCR analysis of type I IFN expression (IFN-␤, IFN-␣4) in TLR9ϩ/ϩ (Ⅺ) and TLR9Ϫ/Ϫ (f) bone marrow-derived murine macrophages at 6 h after infection with wild-type adenovirus. Infections were performed in the presence of 10% naive (MS) or adenovirus-immunized MS (MS ϩ Ig). Values represent mean percent induction over vehicle-treated cells Ϯ SD, n ϭ 4 (TLR9ϩ/ϩ vs TLR9Ϫ/Ϫ, p Ͼ 0.05).

the uptake of adenovirus in the presence of HS, blocking ex- cell extracts were analyzed for processed (17 kDa) IL-1␤ by im- periments were performed with saturating amounts of anti- munoblotting. At this multiplicity of infection, adenovirus failed to Fc␥R-blocking Abs or isotype controls (Fig. 3C). The uptake of activate IL-1␤ above baseline in the absence of HS (Fig. 4C). In Cy2-Ad in the presence of HS was reduced by preincubating the presence of HS that increases viral cell entry, adenovirus ef- THP-1 cells with Fc␥RI- or Fc␥RII-blocking Abs or both, sug- ﬁciently induced IL-1␤ processing and secretion of the 17-kDa gesting a role for both receptors in serum IgG-mediated ade- cytokine at 6 h, an effect that was inhibited by the pan-caspase

by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. novirus uptake. Taken together, these results show that pooled inhibitor zVAD-fmk (Fig. 4C). These results confirm that adeno- HS contains Abs that neutralize adenovirus infection in epi- virus Abs amplify the activation of innate immune pathways in thelial cells but enhance FcR-mediated internalization in macrophages that result in the expression of NF-␬B-dependent macrophages. genes, type I IFNs, and IL-1␤ maturation. Interestingly, the increase in adenovirus internalization and amplification of the innate Abs amplify the antiviral innate immune response to adenovirus immune response in the presence of Abs was associated with a in macrophages significant decrease in virus-mediated GFP expression. This ob- Macrophages are a major source of proinflammatory cytokines af- servation is consistent with increased type I IFN expression and ter infection with adenovirus (9–12) and viral internalization is a enhancement of the antiviral state following Ab-dependent adeno- http://classic.jimmunol.org prerequisite for adenovirus induction of innate immune responses virus internalization (Fig. 4D). (9, 13). Therefore, experiments were performed to determine To confirm the findings in THP-1 cells, similar experiments whether the Ab-dependent increase in adenovirus internalization in were performed in primary bone marrow-derived murine macro- macrophages coincided with enhanced activation of the innate im- phages (Fig. 5). AdGFP uptake in primary mouse bone marrow mune system. THP-1 cells were incubated with AdGFP in different macrophages was significantly increased in the presence of serum serum conditions. Six hours following infection, total RNA was from mice immunized with adenovirus (MS plus Ig) as determined

Downloaded from harvested and analyzed for the induction of chemokine and cyto- by Southern blot, compared with naive MS alone, heat-inactivated kine expression using a RNase protection assay. In FBS, AdGFP mouse serum (MS-HI), serum-free medium, or FBS (Fig. 5A). The induced, albeit weakly, the expression of IP-10, MIP-1␤, MIP-1␣, Ab-dependent increase in AdGFP internalization in primary mac- MCP-1, and IL-8 in THP-1 cells (Fig. 4A), reflecting limited viral rophages was also associated with amplified RANTES, MIP-1␤, entry under these conditions. Viral infection in the presence of MIP-1␣, MIP-2, IP-10, and type I IFN expression (Fig. 5, B and 10% pooled HS significantly increased adenovirus-induced cyto- C). Similar to the results obtained in THP-1 cells and consistent kine/chemokine gene expression, a response that returned to base- with enhancement of the antiviral response, AdGFP infection re- line when the serum was IgG depleted (Fig. 4A). Consistent with sulted in substantially less GFP expression despite increased viral the increase in NF-␬B-dependent gene expression, TNF-␣ and uptake in the presence of adenovirus-immunized MS (Fig. 5D). IFN-␤ mRNA expression was also significantly increased in the Together, these data show that immune serum enhances adenovi- presence of immune serum (Fig. 4B). Experiments were also per- rus internalization and activation of the antiviral innate immune formed to determine whether Ab-mediated adenovirus entry en- response in primary macrophages. Again, complement inactivation hanced IL-1␤ maturation, a marker of inflammasome activation. did not affect the entry or activation of macrophages induced by Differentiated THP-1 cells were infected with 5 ϫ 103 part/cell of AdGFP, confirming that complement plays little role in mediating wild-type adenovirus and 6 h later the cell culture supernatant and adenovirus-macrophage interactions in vitro. The Journal of Immunology 7065

FIGURE 7. Phagocytosis and adenovirus activation of the innate immune response. A, CCB (10 ␮g/ml) blocks THP-1 cell-adenovirus uptake. Cy2-Ad was incubated with THP-1 cells in the presence or absence of HS and CCB. Cells were washed and analyzed by flow cytometry at 90 min. ,p Ͻ 0.001. B ,ءءء ;Values represent mean fluorescence Ϯ SD, n ϭ 3–6. CCB significantly decreases Cy2-Ad uptake in the presence of HS Immunoblotting for mature IL-1␤ (D116) in cell extracts (ext) or supernatants (sup) in PMA-differentiated THP-1 cells 6 h after infection with adenovirus. Immunoblotting of cell extracts and supernatants demonstrates increased mature IL-1␤ in the presence of HS, which is reduced in the presence of CCB, confirming that viral entry is required to activate the inflammasome. C, Immunoblotting for pro-IL-1␤ in cell extracts or mature IL-1␤ (D116) in supernatants from PMA-differentiated THP-1 cells 6 h after infection with wild-type adenovirus in the presence of HS. Increased IL-1␤ maturation is observed in the presence of HS, which is reduced in the presence of bafilomycin A1 (BafA1; 100 nM). D, Real-time PCR analysis of type I IFN expression bone marrow-derived murine macrophages at 6 h following infection with wild-type adenovirus. Infections were performed in the presence of 10% adenovirus-immunized MS with or without bafilomycin A1 (100 nM) or CCB (10 ␮g/ml). Values represent mean percent .(p Ͻ 0.01 ,ءء p Ͻ 0.05 and ,ء ;induction over vehicle-treated cells Ϯ SD, n ϭ 3 (untreated cells vs bafilomycin A1 or CCB-treated cells by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. The previous results suggest that adenovirus-macrophage in- associated molecular patterns and nonpathogen-associated danger teractions differ considerably from the classic paradigm of viral signals (28). The correlation of internalized vector genomes with ␣ entry in epithelial cells that depend on binding to CAR and V cellular activation suggests that adenovirus directly activates an integrins. To confirm that the Ab-dependent enhancement of intracellular innate receptor system, an effect that is increased by viral entry and innate activation in macrophages occurred in- antiviral Abs. TLR9, NLRP3, and a TLR-independent DNA sens- dependent of viral transcription or capsid domains that mediate ing pathway have all recently been shown to detect intracellular ␣ adenovirus binding to V integrins and CAR, experiments were adenovirus virions and/or DNA (15–17, 29). To confirm that Ab- performed using the modified vectors Ps-Ad, AdL.PB*, and dependent amplification of the innate immune response to adeno- http://classic.jimmunol.org AdL.F* (13). Ps-Ad is an AdGFP vector, which was rendered virus was mediated specifically via intracellular innate pathways, transcription defective by UV-psoralen treatment. AdL.F* and we first assessed the role of the NLRP3 inflammasome directly AdL.PB* have mutations in the fiber gene (AdL.F*) and in the using NLRP3Ϫ/Ϫ and ASCϪ/Ϫ macrophages. Consistent with the RGD peptide in the penton base (AdL.PB*), which ablates their previous findings, caspase-dependent IL-1␤ maturation was am- interaction with the adenovirus receptor CAR and coreceptor plified in LPS-primed murine peritoneal macrophages stimulated integrins for internalization, respectively (13). Tropism-modi- with adenovirus in the presence of immune serum (Fig. 6A). In

Downloaded from fied and transcription-defective adenovirus vectors all induced a contrast, IL-1␤ processing and secretion was completely absent in similar degree of chemokine mRNA expression in primary macrophages. Not surprisingly and consistent with the previous data NLRP3- and ASC-deficient macrophages confirming that antiviral demonstrating the retargeting of adenovirus to FcRs, macro- Abs specifically enhance activation of the NLRP3 inflammasome phage activation was significantly increased in all cases by the in response to adenovirus (Fig. 6A). In macrophages, adenovirus presence of immune serum (Fig. 5E). These results show that induction of type I IFNs occurs primarily via the TLR-independent viral gene transcription, CAR-binding and capsid RGD domains DNA-sensing pathway, whereas TLR9 predominates in dendritic are dispensable for the Ab-dependent amplification of the innate cells (16, 17). The role of the TLR-independent pathway in the immune response to adenovirus. Ab-dependent amplification of type I IFNs was therefore assessed indirectly using TLR9Ϫ/Ϫ macrophages. Not unexpectedly, TLR9 Ab-dependent amplification of the innate immune response to was not required for type I IFN expression in response to adeno- adenovirus is dependent on the NLRP3 and TLR9-independent virus in the presence or absence of immune serum. Adenovirus- pathways in macrophages induced IFN-␤ and IFN-␣4 expression was similar in peritoneal Macrophages and monocytes express a wide range of intracellular macrophages (data not shown) and bone marrow-derived macro- TLR and NLR proteins that recognize a vast array of pathogen- phages isolated from TLR9Ϫ/Ϫ and wild-type mice (Fig. 6B). 7066 Abs AND INNATE RESPONSE TO ADENOVIRUS

Taken together, these data are consistent with Ab-dependent am- pliﬁcation of the TLR-independent DNA-sensing pathway following adenovirus infection in macrophages.

Antiviral Abs target the adenovirus to phagolysosomes in macrophages Recent studies show that the activation of several innate pathways is linked to phagosomes and phagosome maturation in macrophages (20, 21). In epithelial cells, Ͼ90% of adenovirus escapes the early endosome during viral entry (30). Little is known regarding viral trafficking of adenovirus in macrophages. In the presence of opsonins though, it is likely that the adenovirus is subjected to entry and trafficking based on the properties of the respective op- sonin receptor. In the context of antiviral Abs, adenovirus likely undergoes FcR-mediated phagocytosis. Given the robust activation of the innate immune response in this setting, it is possible that this mode of viral internalization targets the adenovirus to phagosomes and phagolysosomes that are implicated in the activation of the innate immune system (20, 21). To test this possibility, functional experiments were first performed using CCB and bafilomycin A1 to block phagocytosis and endosomal acidification, respectively, during adenovirus infection. At 6 h following adenovirus infection in THP-1 cells and primary mouse macrophages, both CCB and bafilomycin A1 diminished Ab-mediated amplification of type I IFN expression and IL-1␤ maturation (Fig. 7). These results suggest that phagocytosis and phagosome maturation are an important component of the Ab-mediated amplification of the innate immune response to adenovirus. Next, to determine the effect of antiviral Abs on adenovirus localization in macrophages, confocal microscopy was performed. To stain lysosomes, HEK 293 cells and differentiated THP-1 cells were pulsed with Alexa Fluor 594-labeled 40-kDa dextran and chased overnight. Cells were then infected for 1 h with 1 ϫ 104 part/cell of Cy2-labeled adenovirus and images captured in living

by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. cells. In the absence of human serum, Cy2-Ad entry in 293 cells was efficient and easily detectable (Fig. 8A) but did not colocalize significantly with dextran-containing vesicles. Similarly, in the presence of human serum and despite less viral entry, little lyso- some colocalization was found. In line with the previous studies, viral entry was found to be inefficient in THP-1 cells infected with adenovirus in the presence of FBS, as few cells demonstrated Cy2 positivity (Fig. 8B). Occasional virions however did colocalize with dextran-labeled vesicles when infection were conducted in http://classic.jimmunol.org FBS. In contrast, THP-1 cells infected with adenovirus in the presence of HS demonstrated considerable virus uptake at 1 h that was localized to dextran-labeled vesicles indicative of lysosomes (Fig. 8, B and D). To confirm that adenovirus localization was in fact with late phagosomes/phagolysosomes under these serum conditions, staining for the late phagosome marker LAMP1 was per-

Downloaded from formed. Consistent with the dextran studies, adenovirus strongly FIGURE 8. Colocalization of adenovirus to phagolysosomes. A, 293 cells colocalized with LAMP1 following infection in the presence of HS were pulsed with Alexa Fluor 594 40-kDa dextran (red) and then infected with (Fig. 8C). Importantly, quantification studies revealed that HS not Cy2-labeled adenovirus (green) for1hinthepresence or absence of HS. only increased viral entry but also the relative proportion of virions Adenovirus does not significantly colocalize with dextran-containing vesicles that colocalized to LAMP1-positive phagosomes. In the presence regardless of serum conditions. B, Differentiated THP-1 cells were pulsed with of FBS, Ͻ10% of internalized adenovirus particles were found Alexa Fluor 594 40-kDa dextran and then infected with Cy2-labeled adeno- within LAMP1-positive phagosomes compared with Ͼ50% of in- virus for1hinthepresence or absence of HS. Adenovirus entry is increased and colocalizes with dextran-containing vesicles only when infections are con- ternalized virions in the presence of HS (Fig. 8D). Therefore, these ducted in the presence of HS. In the absence of HS, adenovirus entry in THP-1 results demonstrate that Ab-dependent amplification of the innate cells is inefficient. C, Dual staining for the late phagosome marker LAMP1 immune response is associated with adenovirus targeting to (red) and Cy2-labeled adenovirus in THP-1 cells. In the presence of HS, ad- phagolysosomes in macrophages. enovirus is targeted to LAMP1-positive vesicles indicative of late phagosomes or phagolysosomes. D, Quantification of Cy2-labeled adenovirus internaliza- Discussion tion and colocalization to LAMP1 in THP-1 cells at 1 h following infection in The sensing of infectious agents is an essential component of first- the presence of FBS or HS. Mean voxels/␮m3 Ϯ SD, n ϭ 6 cells (FBS vs HS; .(p Ͻ 0.001 ,ءءء p Ͻ 0.01 and ,ءء line host defense. After recognition of viral components by host The Journal of Immunology 7067

pattern recognition receptors, immune cells initiate an antiviral results show that antiviral Ig has functions beyond neutralizing virus sponse that consists of cytokine and type I IFN production creating and preventing infection in target tissues such as epithelia. Anti- an antiviral state and promoting the development of adaptive im- viral Abs modulate viral tropism to nontarget FcR-bearing cells munity (28, 31). In macrophages and dendritic cells, the innate such as macrophages where innate immune mechanisms reside, machinery that recognizes adenovirus is intracellular and recently causing an up-regulation of antiviral immune responses. More- identified as TLR9, a TLR-independent DNA-sensing pathway over, the adenovirus has evolved to escape from the early endo- that activates IFN regulatory factor 3 and the NLRP3 inflammasome during cellular entry, a viral characteristic that may allow the some (15, 17, 29). However, the adenovirus lacks a specific tro- virus to evade host innate mechanisms in the initial stages of an pism for macrophages, suggesting that other host mechanisms are infection. The ability for the adaptive immune system to subvert in place to target virions to the innate immune system. In this the natural viral characteristics and rescue the antiviral capability study, we demonstrate that the adaptive immune system and anti- of the innate immune system is illustrated by our studies. Despite viral Abs in particular subvert the natural tropism of the adenovi- relatively limited adenovirus infection under naive conditions, the rus to target virions to phagolysosomes in macrophages. The Ab- adenovirus-expressed transgene was high in the face of a dimin- dependent increase in viral entry results in the specific activation utive antiviral innate response, an effect that was reversed by the of the NLRP3 inflammasome and the TLR9-independent type I presence of antiviral Abs. IFN pathway revealing a novel function for antiviral Abs in pos- Collectively, we show that the Ab- and FcR-mediated internal- itively regulating the intracellular innate immune system. ization of viruses represents a mechanism by which the intracel- Our results emphasize the importance of internalization for the lular innate immune system might be activated. Since the majority induction of the innate immune responses to adenovirus (9, 13). of viruses do not exhibit a natural tropism for effector leukocytes Therefore, it is not surprising that the immune system has evolved of the innate immune system, the ability of virus-specific Abs to an adaptive mechanism to target virions to the cellular compart- target infectious virions to the appropriate cellular compartments ments required to mount an optimal antiviral response. We show provides a mechanism to increase the efficiency of the innate im- that the Ab-dependent amplification of the innate immune re- mune system and the development of the antiviral state. This effect sponse to adenovirus was associated with viral targeting to the is illustrated by our in vivo studies where immunized mice re- phagolysosome. Although the exact mechanism of NLRP3 activa- ceived a second challenge with AdLacZ. The enhanced activation tion is only recently emerging, a link to endosomal compartments of the innate immune response in this setting was associated with has been suggested in macrophages. Recent studies demonstrate a dramatic reduction in adenovirus-mediated gene transfer to the that phagolysosome injury is a prerequisite for NLRP3 activation liver. Based on our in vitro studies that show increased targeting to in response to silica crystals (20). Furthermore, M. tuberculosis macrophage phagolysosomes, the significant decline in AdLacZ actively inhibits phagosome maturation, progression to a phagoly- genomes in the livers of immunized mice can be explained not sosome, and subsequent inflammasome activation (21). Our results only by a change in viral tropism but also by enhanced viral deg- strengthen the view that NLRP3 activation is linked to phagoly- radation. In most cases, this would provide an advantage to the sosome stress. The amplification of type I IFN expression was also host and expedite viral clearance. However, there are some in- associated with increased adenovirus internalization and localiza- stances where preexisting immunity can increase an innate im-

by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. tion to the phagolysosome; however, the response in macrophages mune response to subsequent viral infection that is detrimental. was not dependent on TLR9. Recent studies show that TLR-inde- The administration of high-dose recombinant adenovirus for pendent DNA sensing is the major mechanism of type I IFN ex- oncolytic or gene therapies to humans and other hosts with pre- pression in response to adenovirus in macrophages, while TLR9 existing immunity results in increased and sometimes fatal tox- mediates the response in plasmacytoid dendritic cells (15, 17). icity (5, 33–35). Similarly, Ab-dependent enhancement also oc- Consistent with this observation and our data, a recent study dem- curs in the setting of Dengue virus infection where heterotypic onstrates that Ab-dependent amplification of innate responses to infection with related serotypes can trigger increased viral rep- adenovirus in dendritic cells occurs via TLR9 (32). Unlike TLR9 lication and a fatal inflammatory response (36). Finally, a recent however, little is known regarding the actual receptor and cellular study has shown that Ab-dependent amplification of the innate http://classic.jimmunol.org localization of the TLR-independent DNA-sensing pathway in immune response to adenovirus vectors in dendritic cells can macrophages, although it is widely believed to be cytosolic. Al- enhance HIV replication (32). though antiviral Abs increased viral targeting to LAMP1-positive Our results shed new light on the function of antiviral Abs in phagolysosomes, our data also show that only 50% of internalized the host response to viral infection. Not only is an Ab response virions localized to these endosomes, indicating significant viral important to neutralize infection of target tissues, but they play escape into the cytosol where activation of the TLR-independent a role in targeting virions appropriate cellular compartments

Downloaded from DNA-sensing pathway could occur. This premise is consistent that enhance host defense. In this regard, our results show that with the reduction in TLR9-independent type I IFN expression the adaptive immune system has an ongoing cross-talk with the mediated by bafilomycin A1 since adenovirus requires endosomal innate immune system that positively regulates the activation of acidification to penetrate and gain entry to the cytosol. Regardless the intracellular innate immune system that is essential in the of the receptor activated, our data show that antiviral Abs specif- host response to viruses. ically target virions to intracellular compartments that facilitate the activation of the innate immune system. The diverse repertoire of Disclosures the humoral immune system therefore confers a capability for the The authors have no financial conflict of interest. host to activate intracellular innate pathways such as TLR9 and the NLRP3 inflammasome in response to a broad range of pathogens. References Our findings also suggest that the antimicrobial role of the in- 1. Kay, M. A., J. C. Glorioso, and L. Naldini. 2001. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat. Med. 7: nate immune system does not end at the onset of adaptive immu- 33–40. nity. The activation of a specific innate receptor that is facilitated 2. Crystal, R. G. 1995. Transfer of genes to humans: early lessons and obstacles to success. Science 270: 404–410. by the humoral adaptive immune system displays a novel “in- 3. Marshall, E. 2000. Gene therapy’s web of corporate connections. Science 288: verse” cross-talk between adaptive and innate immunity. Our re- 954–955. 7068 Abs AND INNATE RESPONSE TO ADENOVIRUS

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