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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 in- creased 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 adeno- viral 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
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