Response to TLR9 Agonists the Human Plasmacytoid Dendritic Cell

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

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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 © 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 inflammatory 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 dendritic 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

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 backbone (Coley Pharmaceu- considered significant tical Group). Recombinant human HMGB1 (0.1–10 ␮g/ml) was provided a p value Ͻ0.01. by K. J. Tracey (North Shore-Long Island Jewish Research Institute, Manhasset, NY) or was purchased from Sigma-Aldrich. HSV was the gift of Dr. F. Jenkins (University of Pittsburgh). Adenoviruses were the gifts of Results Dr. A. Gambatto (University of Pittsburgh). rHMGB1 inhibits type I IFN production from PDCs stimulated with the TLR9 agonist, CpG ODN HMGB1 purification from cells We hypothesized that HMGB1, an inflammatory cytokine released Cells at a density of 1 ϫ 107 cells/ml were lysed in buffer containing PBS with 1% igepal, 1 mM n-ethylmaleimide, 5 mg/ml 6-aminohexanoic acid, by activated macrophages, mature MDCs, NK cells, and necrotic 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 500 nM cells (11, 12), would modulate the immune function of PDCs. To

aprotinin, 50 ␮M leupeptin, 100 ␮M E64, 36 ␮M phosphoramidon, 40 ␮M characterize PDC function, we assessed its ability to produce Downloaded from ␮ ␮ bestatin, 1 mM benzamidine, 16 M antipain, 10 g/ml trypsin inhibitor, IFN-␣ in response to TLR9 agonists such as CpG. We first estab- and 1 mM EDTA (Sigma-Aldrich). The lysate was centrifuged for 15 min at 16,000 ϫ g to pellet nuclei and insoluble material. The supernatant was lished that CpG ODNs classes A (2336) and C (2395) could induce collected and filtered through a 0.45-␮m filter. HMGB1 polyclonal Ab was significant IFN-␣ secretion from freshly purified PDCs. Significant obtained from New Zealand White rabbits immunized with the peptide IFN-␣ production was noted with CpG concentration Ͻ1 ␮g/ml, sequence KSEAGKKGPGRPTGS corresponding to amino acids 166–181 with maximal release following stimulation with 5 ␮g/ml. As pre- of HMGB1 by Sigma-Aldrich under contract. The affinity purification of ␣ sented, CpG B failed to promote IFN- secretion from PDCs in http://www.jimmunol.org/ the polyclonal anti-HMGB1 Ab was performed following standard proce- dures. To affinity purify, HMGB1 10 ml of freshly prepared and filtered culture (Fig. 1A). The presence of HMGB1 in culture inhibited cell lysate was loaded onto the rabbit polyclonal anti-HMGB1 affinity col- CpG-induced IFN-␣ secretion from PDCs in a dose-dependent umn and recirculated for 30 min. The column was then washed with fashion (Fig. 1B). This pattern of inhibition was reproduced in over roughly 5 volumes of PBS, then the bound protein was eluted using three- 20 separate donors, and over a broad range of concentrations of column volumes of 3 M potassium thiocyanate (pH 7.0). The eluted protein ␣ was dialyzed overnight at 4°C in2LofPBSwith magnetic stirring. The CpG ODNs. In most donors, significant inhibition of IFN- secre- dialyzed protein was sterile filtered through a 0.2-␮m filter and concen- tion was observed with rHMGB1 at concentrations of 0.2 ␮g/ml trated to a final volume of 1 ml using an Amicon Ultra 15 centrifugal for CpG A and 0.5 ␮g/ml for CpG C. Complete inhibition was concentrator (10,000 molecular mass). Final protein concentration was observed with 2 ␮g/ml and 5 ␮g/ml rHMGB1 for CpG A and CpG

determined using the BCA reagent (Pierce Endogen). C, respectively. We did not observe direct effects of rHMGB1 on by guest on October 3, 2021 Cytokine assays PDCs viability as assessed by trypan blue exclusion, tetrazolium salt conversion (MTT assay), or flow cytometric evaluation even at Both IFN-␣ and IL-12 levels were determined by commercially available ELISA kits according to the manufacturer’s directions. The IFN-␣ ELISA the highest concentration of rHMGB1 used (data not shown). Be- (human IFN-␣ Module Set; Bender MedSystems) detects all species of cause HMGB1 is a DNA-binding protein, we wanted to exclude human IFN-␣ at a lower detection limit of 10 pg/ml. The assay does not the possibility that the CpG ODNs were neutralized. Therefore, we recognize IFN-␤ or IFN-␥. The IL-12 ELISA (Endogen Matched Ab Pairs; first incubated PDCs with either CpG or rHMGB1 alone followed Pierce Biotechnology) detects human IL-12 p70 heterodimer at a lower by washing and reincubation. As presented (Fig. 1C), we observed detection limit of 10 pg/ml and does not cross-react with human p35 or p40 ␣ subunits. IL-6, IL-10, TNF-␣, and inducible protein-10 (IP-10) were quan- similar inhibition of IFN- secretion under these culture con- tified by Luminex immunoassay within the University of Pittsburgh Cancer ditions. Stimulation with CpG ODNs followed by washing and Institute Proteomics Core. rHMGB1 treatment demonstrated complete blocking of IFN-␣ Cell surface staining of PDCs production. Inhibition with rHMGB1 preincubation followed by washing and CpG ODN stimulation was not as complete. Com- After culture, PDCs were harvested, washed with PBS containing 2% FCS parable results were obtained when experiments were per- and 0.1% NaN3, and stained using a panel of directly conjugated mAb. mAbs used for staining were CD3-PC5, CD14-PC5, CD16-PC5, CD19- formed in the presence of polymyxin B, making contaminating PC5, CD20-PC5, CD56-PC5, HLA-DR-ECD, CD40-PE, CD54-PE, LPS as an etiology of suppression less likely (data not shown). CD58-PE, CD80-PE, CD83-PE, CD86-PE, HLA-ABC-PE (Immunotech; Beckman Coulter) and CD123-FITC (Miltenyi Biotec), TLR2-PE, Purified mammalian HMGB1 inhibits type I IFN production TLR4-PE (eBioscience), and biotinylated human receptor for advanced glycation end products (RAGE; R&D Systems). PDCs were defined as from PDCs stimulated with the TLR9 agonist, CpG ODN Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩ ϩ CD3 14 19 20 56 16 (lineage mixture negative), HLA-DR CD123 . Because it is possible that posttranslational modifications such as For surface staining, cells were light fixed with 0.5% paraformaldehyde acetylation may be critical to the function of HMGB1 in mamma- and analyzed using four-color flow cytometer (Epix XL; Beckman Coulter). lian systems, we next examined whether purified mammalian HMGB1 would result in similar inhibition of PDC function. T cell polarization assays HMGB1 was purified from human liver cell homogenates (total or After purification, PDCs were first cultured in IL-3-containing medium for nuclear fraction) or the HeLa cell line. Using PDCs from five 24 h, and then treated for an additional 48 h with CpG ODN (5 ␮g/ml), different donors, we observed the same pattern of IFN-␣ inhibition ϩ ϩ HMGB1 (5 ␮g/ml), or both. CD4 CD45RA naive T cells were isolated with recombinant and purified mammalian HMGB1 (Fig. 2). Al- from an allogeneic donor using a negative selection kit (StemSep; StemCell Technologies). T cells were then added at a 5:1 or 1:1 ratio to though the pattern of inhibition was the same, purified HMGB1 the PDCs with addition of IL-2 (100 IU/ml) and incubated for 7–10 was slightly less potent. In no instance did we observe enhance- days. After coculture, T cells were stimulated for an additional 4 h with ment of IFN-␣ production. The Journal of Immunology 8703

FIGURE 2. Puriﬁed mammalian HMGB1 dose dependently inhibits IFN-␣ secretion from PDCs activated with CpG. PDCs (0.2 ϫ 106/ml) were incubated in IL-3 (10 ng/ml) plus either CpG C (A)orCpGA(B)(5 Downloaded from ␮g/ml) in the presence of the indicated concentrations of puriﬁed mammalian HMGB1 (␮g/ml). Following 48 h of culture, supernatants were collected and assayed for IFN-␣ production. Data are presented as mean IFN-␣ concentration Ϯ SD from triplicates in nanograms per milliliter correspond to ء .from one representative of three independent experiments p Ͻ 0.05. http://www.jimmunol.org/

tions in Fig. 1C, as the viral-encoded CpG would be inaccessible for ex vivo recombination with our exogenously added HMGB1.

rHMGB1 suppresses IL-6, TNF-␣, and IP-10 production from PDCs stimulated with the TLR9 agonist, CpG ODN FIGURE 1. rHMGB1 dose dependently inhibits IFN-␣ secretion from Following stimulation with virus or CpG ODNs, PDCs produce PDCs activated with CpG. A, PDCs (0.2 ϫ 106/ml) were incubated in IL-3 various cytokines and chemokines (IL-6, TNF-␣, and IP-10) in (10 ng/ml) plus varying concentrations of CpG ODNs class A (2336), B by guest on October 3, 2021 ␣ (2006), and C (2395). B, PDCs (0.2 ϫ 106/ml) were incubated in IL-3 (10 addition to IFN- (2). We next examined the rHMGB1 effects on ng/ml) plus CpG ODNs (5 ␮g/ml) in the presence of the indicated CpG-induced secretion of these cytokines. As presented in Fig. 4, rHMGB1 concentrations (micrograms per milliliter). C, PDCs (0.2 ϫ 106/ CpG treatment of PDCs induced Ͼ20-fold increases in IL-6, ml) were incubated with either CpG A (5 ␮g/ml) (u) or increased doses of TNF-␣, and IP-10 production when compared with IL-3 alone. rHMGB1 (f) for 1 h, washed, and incubated for 48 h in the presence of CpG activation of PDCs alone did not result in consistent produc- either rHMGB1 (u)orCpGA(f). Following 48 h of culture, supernatants tion of either IL-10 or IL-12. The presence of rHMGB1, however, were collected and assayed for IFN-␣ production. Data are presented as decreased the induction of all three of these cytokines. The level of ␣ Ϯ mean IFN- concentration SD from triplicates in nanograms per milli- inhibition varied from ϳ35% for IL-6, 59% for IP-10, and up to -corre ء .liter from one representative of three independent experiments spond to p Ͻ 0.05.

rHMGB1 inhibits type I IFN production from PDCs stimulated with viral pathogens CpG ODNs are synthetic molecules that mimic natural viral/bacterial PAMPs. We next examined whether HMGB1 could similarly inhibit endogenous viral CpG-induced IFN-␣ release from PDCs. In preliminary experiments, we established that the effective opti- mal multiplicity of infection (MOI) for IFN-␣ production for several representative viral strains fell between 1 and 5 MOI (data not shown). We observed that HSV induced the highest level of IFN-␣ production from infected PDCs. Interestingly, the dsDNA Western ␣ Reserve vaccinia virus (WRvv) failed to elicit any IFN-␣ secretion FIGURE 3. rHMGB1 inhibits IFN- secretion from virus activated PDCs. PDCs (0.2 ϫ 106/ml) were stimulated with CpG ODNs (5 ␮g/ml) from infected puriﬁed PDCs, while the effect of ssDNA adenovirus or different viral vectors (Adeno 1, adenovirus strain 33; Adeno 2, adeno- strain 35 (Adeno 2) depended on the presence of IL-3 (Fig. 3). virus strain 35; vaccinia virus, WRvv) (the most effective MOI), with or Similarly to its effect on synthetic CpG-stimulated PDCs, we ob- without rHMGB1 (5 ␮g/ml) (A) in the presence of IL-3 (10 ng/ml) or (B) served that rHMGB1 (5 ␮g/ml) was able to signiﬁcantly inhibit without IL-3. Data are presented as a mean Ϯ SD from triplicates in pi- PDC secretion of IFN-␣ in response to the HSV and the adenoviral cograms per milliliter from one representative of three independent and .correspond to p Ͻ 0.01 ء .preparations used (Fig. 3). These data also support our observa- comparable experiments 8704 HMGB1 SUPPRESSES PDC ACTIVATION

FIGURE 4. rHMGB1 inhibits IL-6, TNF-␣, and IP-10 secretion from CpG-stimulated PDCs. PDCs (106/ml) were cultured in IL-3 (10 ng/ml) Ϯ CpG C ODN (5 ␮g/ml) in the presence of rHMGB1 (2 ␮g/ml). After 48 h, supernatants were collected for cytokine detection. Data are presented in picograms per milliliter as a mean Ϯ SD from triplicates from one repre- .correspond to p Ͻ 0.05 ء .sentative experiment of three performed Downloaded from

75% inhibition of TNF-␣ production (Fig. 4). Such inhibition of individual cytokines suggests that rHMGB1 interferes with PDC activation and/or maturation, rather than simply with cytokine (IFN-␣) secretion alone.

rHMGB1 inhibits IL-12 production from PDCs stimulated by http://www.jimmunol.org/ TLR9 and CD40 ligation Both human and murine PDCs produce IL-12 (both p35/p40 as well as p40) in response to CpG and CD40 ligation in the presence of IL-3 (2, 14). We next examined the effect of rHMGB1 on this function. PDCs were incubated with IL-3 and CpG for 48 h. Irra- FIGURE 6. rHMGB1 diminishes expression of costimulatory and ad- diated CD40L-expressing J558 cells were then added for an addi- hesion molecules following stimulation with PDCs. A, PDCs were culti- tional 48 h and supernatants were assayed for the presence of IL- vated for 72 h in illustrated different experimental conditions or (B) in IL-3 Ϯ ␮

12. Although the observed level of IL-12 production was not as (10 ng/ml) CpG C ODN (5 g/ml) and stained for membrane expression by guest on October 3, 2021 high as in other cell types, it was consistent and reproducible in all of individual maturation molecules with or without rHMGB1 (5 ␮g/ml). ϩ ϩ tested donors. Importantly, we observed a significant decrease in Only viable (forward scatter/side scatter gate) and HLA-DR CD123 cells IL-12 production in the presence of rHMGB1 (Fig. 5). As pre- were gated and analyzed. Data are presented as mean fluorescence intensity sented, similar levels of suppression of IL-12 production were ob- (m.f.i.) and histograms fluorescence from one of five experiments with similar results. served when rHMGB1 was present at the beginning of the 4-day culture (rHMGB1 early) as well as when it was added after the first 48 h, together with the CD40L-expressing J558 cells (rHMGB1 late). The level of IL-12 secretion corresponded directly with the ever, IL-12 was not detected from PDCs following CD40 ligation level of CD40 expression on PDCs, which increased coordinately alone. This suggests that rHMGB1 could influence not only the when incubated in the presence of IL-3 and CpG (Fig. 6A). How- behavior of immature PDCs in early stages of activation but also cytokine secretion by fully stimulated and more mature cells. We also examined IL-10 production in this system. We observed consistent increase in IL-10 following CD40 ligation in all samples tested. rHMGB1 failed to mediate a consistent effect on IL-10 production (data not shown).

rHMGB1 inhibits the maturational phenotype of PDCs Following exposure to synthetic TLR9 agonists, PDCs undergo a maturation and differentiation program characterized by up-regulation or de novo expression of the costimulatory (CD40, CD80, CD86), adhesion (CD54, CD58), and homing (CCR7) molecules as well as maturation markers (CD83) (3, 4, 15). In preliminary FIGURE 5. rHMGB1 inhibits IL-12 production from CpG plus CD40- experiments, we observed, as have others (2), that CpG A was less 6 Ϯ stimulated PDCs. PDCs (10 /ml) were cultured in IL-3 (10 ng/ml) CpG effective than CpG C in up-regulating the relevant cell surface C ODN (5 ␮g/ml) in the presence of rHMGB1 (2 ␮g/ml). After 48 h, maturation markers and these changes were maximal at 48–72 h irradiated CD40L-transfected J558 cells were added (ratio 1:1). IL-12 production was measured after an additional 48 h of culture. rHMGB1 were (Fig. 6A). PDCs cultured in medium alone demonstrated no ap- not present (u), added at the start (f) or concurrently with J558 cells (Ⅺ). parent changes in these cell surface molecules, while PDCs cul- Data are presented in picograms per milliliter as a mean Ϯ SD from trip- tured in low-dose IL-3 alone demonstrated a small but consistent correspond up-regulation of CD40 only. As presented, the presence of IL-3 ء .licates from one representative experiment of ﬁve performed to p Ͻ 0.01. additionally increased the effect of CpGs (Fig. 6A). When The Journal of Immunology 8705

FIGURE 8. PDCs express RAGE. Freshly isolated and PDCs cultivated for 24 h in IL-3 (10 ng/ml) Ϯ CpG C ODN (5 ␮g/ml) were stained for membrane expression of RAGE. Results are representative of three independent experiments obtained with different donors.

FIGURE 7. rHMGB1 inhibits allogenic PDC-driven IFN-␥ production from naive T cells. PDCs were cultured in IL-3 (10 ng/ml) Ϯ CpG C ODN TLR2 or TLR4 on either resting or stimulated PDCs (data not (5 ␮g/ml) with or without rHMGB1 (5 ␮g/ml) for 48 h. PDCs were then shown). We, however, did observe low but consistent expression ϩ ϩ added to allogeneic naive CD4 CD45RA T cells, at 1:1 ratio. After 9 of RAGE on fresh PDCs obtained from several donors, as well as ␮ ␮ days, T cells were activated with PMA (10 g/ml) and ionomycin (1 g/ an increase in expression in culture with IL-3 and following CpG ml) in the presence of brefeldin A. Cells were then stained for CD3 and activation (Fig. 8). Interestingly, we also observed that s100␤, an-

IFN-␥. Data are presented as a percentage of IFN-␥-positive cells in CD3 Downloaded from other putative RAGE ligand, could similarly inhibit type I IFN gate. Results are representative of five independent experiments using cells obtained from five different donors. production from PDCs stimulated with the TLR9 agonist, CpG ODN (data not shown). Together, those results are consistent with the notion that HMGB1/RAGE interaction may be involved in the rHMGB1 was added to CpG C-activated PDCs, we consistently PDC suppression we observed, although additional experiments observed diminished cell surface expression of CD40, CD80, need to be done to confirm that hypothesis.

CD83, CD86, CD54, and CD58 molecules (Fig. 6B). Interestingly, http://www.jimmunol.org/ there was no change in cell surface expression of either class I or Discussion class II MHC molecules. It is important to note that the relative The impact of HMGB1 on PDC function has been largely unex- low level of HLA-DR fluorescence intensity depicted is a conse- plored. It has been shown previously that other DAMP molecules quence of compensation for multicolor flow analysis. including adenosine (8) and histamine (16) inhibit IFN-␣ production from PDCs. Here, we demonstrate for the first time that ex- rHMGB1 inhibits the generation of Th1 cells ogenous HMGB1 can inhibit TLR9-mediated IFN-␣ secretion Our findings suggest that the HMGB1 as a DAMP molecule, in from PDCs. This potent inhibitory effect was observed when the addition to altering secretion of inflammatory cytokines, also sig- activation signal was synthetic CpG ODNs or following viral by guest on October 3, 2021 nificantly alters the ability of PDCs to promote T cell activation. infection. Purified mammalian HMGB1 behaved similarly to We therefore next directly examined the effect of HMGB1 on rHMGB1 excluding the possibility that posttranslational modifi- PDC-driven maturation of naive T cells. We pretreated PDCs with cation, not available in bacterial production systems, might explain IL-3 plus CpG with or without rHMGB1 and then cocultured them the observed inhibitory effects. The biologically active range of ϩ ϩ with allogeneic naive CD4 CD45RA T lymphocytes. Following HMGB1 in our system is similar to that reported in other biologic priming and expansion in low concentrations of IL-2, T cells were assays including maturation of MDCs and activation of endothelial activated and the patterns of IL-5, IL-10, and IFN-␥ production cells and macrophages (12, 20, 21). were analyzed using intracellular staining and flow cytometry. We We also observed that rHMGB1 was able to inhibit the produc- did not observe any difference in the total number of T cells by tion of several important PDC-mediated proinflammatory cyto- trypan blue exclusion or flow cytometric evaluation between these kines. The ability to inhibit IL-6 and TNF-␣ further supports the culture conditions. PDCs treated with CpG increased the number anti-inflammatory effect of HMGB1 on PDC function. Inhibition of T cells producing IFN-␥ (Fig. 7) as previously described by of IP-10 additionally suggests that HMGB1 could prevent attrac- others (16). Pretreatment of PDCs with rHMGB1 led to potent and tion of CXCR3-bearing Th1 T cells and NK cells to the site of sometimes complete abrogation of T cell secretion of IFN-␥ (Fig. inflammation, which normally occurs in response to IP-10. To- 7). Although the effect of IL-3 was significantly weaker by itself, gether with TNF-␣, IP-10 production is an important Th1 chemo- rHMGB1 suppression was also evident in PDCs cultured in IL-3 kine secreted by PDCs in response to TLR7 agonists (22–24). alone. We did not observe significant increases in the number of T Consistent with previous reports (25), we observed that CpG cells capable of either IL-5 or IL-10 secretion (Ͻ0.5%; data not activation of PDCs alone did not result in detectable production of shown). PDCs that encountered rHMGB1 during the maturation either IL-10 or IL-12. However, CD40 ligation induced a low, but process potently inhibited generation of Th1 cells producing reproducible, IL-12 production from PDCs in our system. This IFN-␥. Overall, these findings demonstrate that rHMGB1 in addi- IL-12 production by PDC was also inhibited by rHMGB1. tion to suppressing proinflammatory cytokine production, also rHMGB1-mediated suppression of IL-12 secretion can only be modulates the ability of PDCs to communicate directly with naive partially explained by lower IFN-␣ levels in culture, or by decrease T cells. of membrane expression of CD40, as postponing addition of rHMGB1 to allow maximal IFN-␣ production and CD40 expres- PDCs express RAGE, but not TLR2 or TLR4 sion demonstrated similar inhibition. Others have observed that HMGB1 mediates its biologic effects through several receptors human and murine PDCs can produce IL-12 (p70 and p40) in including RAGE, TLR2, and TLR4 (9, 13, 17). We next tested response to CpG and CD40 ligation in the presence of IL-3 (8, 14, expression of those receptors on PDCs. Consistent with the find- 25). Some have suggested, however, that IL-12 secretion from ings of others (18, 19), we observed no significant expression of BDCA-4-purified PDCs could be from contaminating monocytes 8706 HMGB1 SUPPRESSES PDC ACTIVATION or MDCs (26). Addition of LPS to our cultures did not influence serum of patients with a variety of cancer types, as well as signif- IL-12 production, making this explanation less likely in our estima- icant release from dying tumor cells in vitro (our unpublished tion. In addition, rHMGB1 increases IL-12 production from myeloid observations). DC’s (20). Although early reports suggested that PDCs preferentially fa- Disclosures vored the development of IL-4-secreting Th2 cells, later work sug- The authors have no financial conflict of interest. gested that PDCs are more flexible in directing T cell responses, depending on the maturation stage, nature, and concentration of References the Ag (27) as well as other local factors (2). For example, virus- 1. Tough, D. F. 2004. 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