High Mobility Group B1 Protein Suppresses the Human Plasmacytoid Dendritic Cell Response to TLR9 Agonists This information is current as Petar J. Popovic, Richard DeMarco, Michael T. Lotze, of October 3, 2021. Steven E. Winikoff, David L. Bartlett, Arthur M. Krieg, Z. Sheng Guo, Charles K. Brown, Kevin J. Tracey and Herbert J. Zeh III J Immunol 2006; 177:8701-8707; ; doi: 10.4049/jimmunol.177.12.8701 http://www.jimmunol.org/content/177/12/8701 Downloaded from References This article cites 37 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/177/12/8701.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 3, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology High Mobility Group B1 Protein Suppresses the Human Plasmacytoid Dendritic Cell Response to TLR9 Agonists1 Petar J. Popovic,* Richard DeMarco,* Michael T. Lotze,† Steven E. Winikoff,* David L. Bartlett,* Arthur M. Krieg,‡ Z. Sheng Guo,* Charles K. Brown,* Kevin J. Tracey,§ and Herbert J. Zeh III2* Plasmacytoid dendritic cells (PDC) are innate immune effector cells that are recruited to sites of chronic inflammation, where they modify the quality and nature of the adaptive immune response. PDCs modulate adaptive immunity in response to signals delivered within the local inflammatory milieu by pathogen- or damage-associated molecular pattern, molecules, and activated immune cells (including NK, T, and myeloid dendritic cells). High mobility group B1 (HMGB1) is a recently identified damage- associated molecular pattern that is released during necrotic cell death and also secreted from activated macrophages, NK cells, and mature myeloid dendritic cells. We have investigated the effect of HMGB1 on the function of PDCs. In this study, we demonstrate that HMGB1 suppresses PDC cytokine secretion and maturation in response to TLR9 agonists including the hy- Downloaded from pomethylated oligodeoxynucleotide CpG- and DNA-containing viruses. HMGB1-inhibited secretion of several proinflammatory cytokines including IFN-␣, IL-6, TNF-␣, inducible protein-10, and IL-12. In addition, HMGB1 prevented the CpG induced up-regulation of costimulatory molecules on the surface of PDC and potently suppressed their ability to drive generation of IFN-␥-secreting T cells. Our observations suggest that HMGB1 may play a critical role in regulating the immune response during chronic inflammation and tissue damage through modulation of PDC function. The Journal of Immunology, 2006, 177: 8701–8707. http://www.jimmunol.org/ lasmacytoid dendritic cells (PDCs)3 are the natural type I endogenous inflammatory mediator released by mast cells and ba- IFN-producing cells, secreting high levels of IFN-␣ in re- sophils (6, 7). Adenosine, a DAMP, released from activated or P sponse to viral infection. They serve an additional impor- stressed cells promotes migration of immature PDCs but suppresses tant role as APCs, regulating other immune cell function at sites of function of mature cells (8). Together these findings suggest that chronic inflammation. Their precise biologic role has not yet been PDCs: 1) have significant functional plasticity, 2) are capable of re- fully defined, but it is clear that microbial mediators, so-called sponding to a wide range of endogenous and exogenous danger sig- pathogen-associated molecular pattern (PAMP) molecules, tissue- nals, and 3) are able to integrate these environmental signals and mod- derived factors or damage-associated molecular pattern (DAMP) ify the quality of the evolving adaptive immune response. by guest on October 3, 2021 molecules, and cytokines present in the inflammatory microenvi- High mobility group B1 (HMGB1) is an evolutionarily ancient ronment alter the effector function of PDCs, thereby shaping the DNA-binding protein that within the nucleus enhances access to adaptive immune response (1, 2). For example, PDCs, exposed to transcriptional regulatory factors, nuclear hormones/hormone re- the CpG oligodeoxynucleotide (ODN), a TLR9 agonist, and CD40 ceptors (9), transposons, and recombinases. HMGB1 also serves as ligation secrete IL-12 and preferentially drive Th1 T cell responses an extracellular cytokine, with an acetylated form secreted by ac- (3, 4). Conversely, when exposed to CpG alone, PDCs secrete very tivated macrophages, NK cells, PDCs, and mature myeloid den- little IL-12 but rather drive the development of CD4ϩCD25ϩ reg- dritic cells (MDCs) (9, 10). HMGB1 is also passively released by ulatory cells (5). PDCs preferentially drive Th2 T cell responses cells following unscheduled (necrotic) cell death (11, 12), linking when cultured in IL-3 alone or following exposure to histamine, an this to the subsequent recruitment of inflammatory cells. HMGB1 released in the presence of other mediators results in an inflam- matory cascade characterized by endothelial activation, recruitment † *Division of Surgical Oncology and Translational Research, Department of Surgery of inflammatory cells, mesangioblast recruitment and proliferation, and Molecular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15232; ‡Coley Pharmaceutical Group, Wellesley, MA 02481; and §Institute for Medical Re- and MDC maturation (9). HMGB1 also serves as a DAMP, delivering search, North Shore-Long Island Jewish Research Institute, Manhasset, NY 11030 signals that tissue damage has occurred possibly priming the immune Received for publication October 20, 2005. Accepted for publication September system to identify and then eradicate pathogens that might accompany 29, 2006. such tissue damage (13). The impact of HMGB1 on PDC function is The costs of publication of this article were defrayed in part by the payment of page largely unexplored. In this study, we demonstrate that HMGB1 sup- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. presses the PDC response to TLR9 agonists including viruses and 1 This work was supported in part by an American Society of Clinical Oncology CpG ODNs. HMGB1 inhibits the release of proinflammatory cyto- Career Development Award (to H.J.Z.). kines and down-regulates costimulatory and adhesion molecules on 2 Address correspondence and reprint requests to Dr. Herbert J. Zeh III, Division of PDCs stimulated with CpG ODNs. Pretreatment of PDCs with Surgical Oncology, University of Pittsburgh Cancer Institute, University of Pittsburgh HMGB1 inhibits their ability to promote a Th1 response. Medical Center Cancer Pavilion, Suite 440, 5150 Center Avenue, Pittsburgh, PA 15232. E-mail address: [email protected] 3 Abbreviations used in this paper: PDC, plasmacytoid dendritic cell; PAMP, patho- Materials and Methods gen-associated molecular pattern; DAMP, damage-associated molecular pattern; Cells and reagents ODN, oligodeoxynucleotide; HMGB1, high mobility group B1; MDC, myeloid den- dritic cell; IP-10, inducible protein-10; RAGE, the receptor for advanced glycation Human PDCs were purified from PBMC of healthy Central Blood Bank of end products; RT, room temperature; MOI, multiplicity of infection; WRvv, Western Pittsburgh donors using a direct magnetic labeling and positive selection Reserve vaccinia virus. kit for BDCA-4 (Miltenyi Biotec). PDCs were Ͼ95% pure, based upon Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 8702 HMGB1 SUPPRESSES PDC ACTIVATION positive expression of CD123, BDCA-2, and HLA-DR, and lack of ex- PMA (10 ng/ml) and ionomycin (1 ␮g/ml) in the presence of brefeldin pression of CD11c, CD3, CD19, CD14, and CD56. PDCs (0.5–5 ϫ 105/ml) A (10 ␮g/ml). Cells were washed, stained for surface Ag (CD3, 30 min were cultured in complete medium supplemented with 10 ng/ml IL-3 on ice), fixed with 2% paraformaldehyde (20 min at room temperature), (PeproTech). CD4ϩCD45RAϩ, naive T cells were obtained from PBMC permeabilized by treatment with saponin (10 min at room temperature), and of healthy donors using a human naive CD4ϩ T cell enrichment mixture stained for intracellular expression of IL-5, IL-10, or IFN-␥ (30 min at room (StemSep; StemCell Technologies). CD40L-transfected J558 cells (J558/ temperature). PDC ratio, 1:2; provided by Dr. P. Kalinski, University of Pittsburgh, Pitts- burgh, PA) were added to the culture when IL-12 production was assessed. Statistical analysis All cultures were maintained at 37°C in a humidified 5% CO2 atmosphere in flat-bottom 96-well plates (Costar). Data were analyzed using a two-tailed Student’s t test. All analyses were CpG ODNs class A (2336), B (2006), and C (2395) were created on a performed using Prism software (GraphPad Software). Differences were at(ءء) atap value Ͻ0.05 and highly significant(ء) fully or partially protected phosphorothioate