Download between TLRs and FcRs Polyfunctional Th Cells by Cross

This information is current as
of June 15, 2017.
Antibody-Opsonized Bacteria Evoke an
Inflammatory Dendritic Cell Phenotype and
Polyfunctional Th Cells by Cross-Talk
between TLRs and FcRs
Jantine E. Bakema, Cornelis W. Tuk, Sandra J. van Vliet,
Sven C. Bruijns, Joost B. Vos, Sophia Letsiou, Christien D.
Dijkstra, Yvette van Kooyk, Arjan B. Brenkman and
Marjolein van Egmond
Supplementary
Material
http://www.jimmunol.org/content/suppl/2015/01/09/jimmunol.130312
6.DCSupplemental
Subscription
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Permissions
Email Alerts
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
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 © 2015 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
J Immunol published online 12 January 2015
http://www.jimmunol.org/content/early/2015/01/09/jimmun
ol.1303126
Published January 12, 2015, doi:10.4049/jimmunol.1303126
The Journal of Immunology
Antibody-Opsonized Bacteria Evoke an Inflammatory
Dendritic Cell Phenotype and Polyfunctional Th Cells by
Cross-Talk between TLRs and FcRs
Jantine E. Bakema,*,† Cornelis W. Tuk,† Sandra J. van Vliet,† Sven C. Bruijns,†
Joost B. Vos,†,‡ Sophia Letsiou,x Christien D. Dijkstra,† Yvette van Kooyk,†
Arjan B. Brenkman,x and Marjolein van Egmond†,{
P
athogen recognition receptors (PRRs), which are expressed
on dendritic cells (DCs), trigger antimicrobial defenses during
initial encounters with harmful pathogens (primary immune
response) (1–5). PRRs include TLRs, scavenger receptors, C-type
lectin receptors, nucleotide oligomerization domain–like receptors, or retinoic acid–inducible gene-I–like receptors. They recognize specific pathogen-associated molecular patterns on pathogens,
such as lipids, carbohydrates, peptides, and nucleic acid structures
(1, 3). Intact microbial pathogens are composed of a variety of these
pathogen-associated molecular patterns, which can simultaneously
activate multiple PRRs and initiate a plethora of signaling programs
that execute a first line of defense against infection (3, 6–9).
However, secondary immune responses (among others) are characterized by the presence of ample amounts of pathogen-specific Abs.
*Department of Otolaryngology – Head and Neck Surgery, VU University Medical
Center, 1007 MB Amsterdam, the Netherlands; †Department of Molecular Cell
Biology and Immunology, VU University Medical Center, 1081 BT Amsterdam, the
Netherlands; ‡Immunaffect BV, 1404 AK Bussum, the Netherlands; xDepartment of
Metabolic Diseases, Netherlands Metabolomics Centre, University Medical Centre
Utrecht, Utrecht 3584 EA, the Netherlands; and {Department of Surgery, VU University
Medical Center, 1081 BT Amsterdam, the Netherlands
Received for publication November 20, 2013. Accepted for publication December 5,
2014.
This work was supported by Netherlands Organization for Scientific Research Grant
VIDI 016.086.320.
Address correspondence and reprint requests to Dr. Jantine E. Bakema, Department
of Otolaryngology – Head and Neck Surgery, VU University Medical Center, P.O.
Box 7057, 1007 MB, de Boelelaan 1117, 1081 HV, Room PK 2Y-118, 1007 MB
Amsterdam, the Netherlands. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: AA, arachidonic acid; BSA-B, BSA-coated beads;
COX, cyclooxygenase; DC, dendritic cell; iDC, immature DC; IgG-B, IgG-coated
beads; infDC, inflammatory DC; LOD, limit of detection; LOX, lipoxygenase; LTB4,
leukotriene B4; LXA4, lipoxin A4; PGN-SA, peptidoglycan of S. aureus; PKC,
protein kinase C; PMN, polymorphonuclear cell; Poly-I:C, polyinosinicpolycytidylic acid; PRR, pathogen recognition receptor; Syk, spleen tyrosine kinase;
TXB, thromboxane B.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303126
As such, an additional class of receptors, referred to as FcRs,
becomes an important part of the immune response by binding
opsonizing Abs (10–12). FcRs are expressed on several immune
cell types. For example, DCs express several FcgR subclasses that
bind IgG, of which FcgRII (CD32) is the most prominent (11, 12).
FcgRII is subdivided into FcgRIIA, which is an activating receptor
through ITAM signaling, and FcgRIIB, which contains ITIM and,
hence, is an inhibitory receptor. FcR-induced effector functions
include phagocytosis, endocytosis, Ab-dependent cellular cytotoxicity, superoxide production, release of cytokines, calcium
mobilization, and Ag presentation (10, 13).
Intriguingly, the individual involvement of different types of PRRs
or FcRs during bacterial infection has been well established. However, combined activation of these different receptor classes during
encounter with Ab-opsonized bacteria has received little attention.
Therefore, we investigated the initiation of immune responses
after simultaneous recognition of Ab-opsonized pathogens by PRRs
and FcRs on monocyte-derived immature DCs (iDCs), because DCs
are the most efficient APCs and secrete a plethora of cytokines and
lipid mediators that direct polarization of distinct Th subsets (2).
Th1-mediated responses are induced in the presence of IL-12
(14). Th1 cells, in turn, release IFN-g, which primarily activates
and recruits cytotoxic T lymphocytes, NK cells, and macrophages.
Th2 cells that produce IL-4, IL-5, and IL-13 recruit eosinophils and
mast cells. Th17 cell differentiation is initiated upon release of IL-1b
and sustained via IL-23 (15). Th17 cells stimulate granulopoiesis,
because IL-17 induces production of granulocyte-stimulating factor,
which favors an increased presence of neutrophils at sites of infection.
Recently, it was found that GM-CSF–producing Th cell subsets are
involved in the initiation of autoimmune disease (16).
Lipid mediators, such as arachidonic acid (AA)-derived eicosanoids, regulate a variety of processes like cytokine production, Ab
formation, Th cell–polarizing ability, and Ag presentation (17–19).
We identified cross-talk between TLRs and FcRs when bacteria
were opsonized with Abs. This cross-talk induced synergistic
release of inflammatory cytokines and a unique lipid metabolite
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
During secondary immune responses, Ab-opsonized bacteria are efficiently taken up via FcRs by dendritic cells. We now demonstrate that this process induces cross-talk between FcRs and TLRs, which results in synergistic release of several inflammatory
cytokines, as well as altered lipid metabolite profiles. This altered inflammatory profile redirects Th1 polarization toward Th17
cell responses. Interestingly, GM-CSF–producing Th cells were synergistically evoked as well, which suggests the onset of polyfunctional Th17 cells. Synergistic cytokine release was dependent on activation via MyD88 and ITAM signaling pathways through
TLRs and FcRs, respectively. Cytokine regulation occurred via transcription-dependent mechanisms for TNF-a and IL-23 and
posttranscriptional mechanisms for caspase-1–dependent release of IL-1b. Furthermore, cross-talk between TLRs and FcRs was
not restricted to dendritic cells. In conclusion, our results support that bacteria alone initiate fundamentally different immune
responses compared with Ab-opsonized bacteria through the combined action of two classes of receptors and, ultimately, may
refine new therapies for inflammatory diseases. The Journal of Immunology, 2015, 194: 000–000.
2
TLR–FcR CROSS-TALK ON DCs REDIRECTS Th CELL POLARIZATION
profile. The altered inflammatory profile was regulated on both
transcriptional and posttranscriptional levels, depending on the
distinct cytokine. Importantly, release of this distinct inflammatory
mediator profile by DCs redirected DC-induced Th1 responses
toward induction of polyfunctional Th cells producing both IL-17
and GM-CSF (20). Finally, combined activation of TLRs and FcRs
on iDCs induced functional characteristics that were similar to
those described for inflammatory DCs (infDCs). This novel subset
was identified recently in synovial fluid of rheumatoid arthritis
patients, as well as in inflammatory tumor ascites (21).
In conclusion, this study reveals the distinct impact on cells of
the innate immune system, which are simultaneously activated via
TLRs and FcRs, as well as the subsequent effects on the initiation
of adaptive-immune responses. Thus, our results emphasize the
fundamental difference between the development of primary and
secondary immune responses.
Materials and Methods
Bead coating
Isolation and culture of monocyte-derived DCs
PBMCs were isolated from heparinized blood or buffy coats (Sanquin,
Amsterdam, the Netherlands) by density gradient centrifugation using
LymphoPrep (Axis-Shield, Oslo, Norway). Monocytes were obtained using
a CD14 selection step with magnetic anti-CD14 MicroBeads and MACS
system (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes were
differentiated into iDCs by culturing for 7 d in complete medium (RPMI
1640 + 2 mM L-glutamine [Life Technologies, Paisley, U.K.], 10% heatinactivated FCS, and antibiotics) supplemented with IL-4 (500 U/ml) and
GM-CSF (800 U/ml; both from Biosource, Camarillo, CA). All donors
gave informed consent, according to the guidelines of the Medical Ethical
Committee of VU University Medical Center and in accordance with the
Declaration of Helsinki.
Escherichia coli phagocytosis
E. coli strain R3 (kind gift from Dr. B. Appelmelk, VU University Medical
Center) was grown overnight in Luria-Bertani medium and labeled with
FITC prior to phagocytosis experiments. Heat-inactivated bacteria (1 h,
70˚C) were incubated with 10 mg/ml FITC (Sigma-Aldrich) in carbonate
buffer (0.1 M NaHCO3 [pH 9]) for 1 h at 37˚C, after which they were
washed extensively in PBS. FITC-labeled E. coli (5 3 107 bacteria) were
incubated with 0.05 mg purified human IgG (Sigma-Aldrich) or heatinactivated serum from healthy donors (30 min, 37˚C), with or without
pan anti-FcgRII–blocking Abs (clone AT10; Abcam), or with 2B6 Ab
(anti-FcgRIIb, own hybridoma production ATCC PTA-4591) in 24-well
plates (Greiner Bio-One, Alphen aan den Rijn, the Netherlands). iDCs
(0.5 3 106/0.5 ml) were added for the indicated times at 37˚C in complete
RPMI 1640 medium. Cells were used to determine phagocytosis of bacteria by flow cytometry (FACSCalibur; Becton Dickinson, San Diego, CA)
and imaging stream flow cytometry (ImageStreamX, Amnis, EMD Merck
Millipore, Seattle, WA). The phagocytic index was calculated as geometric
mean fluorescence intensity multiplied by the percentage of gated cells.
iDCs also were analyzed for surface markers (see Flow cytometry), in
coculture with Th cells (see Th cell–differentiation assay) or for relative
mRNA levels (see Quantitative real-time PCR). Supernatants were harvested for cytokine measurements (see Multiplex protein analysis and
cytokine ELISA) and metabolic profiling (see Lipidomics analysis of inflammatory lipids).
Imaging stream flow cytometry
Alternatively, phagocytic capacity was evaluated with imaging flow
cytometry (ImageStreamX). Briefly, bright-field and fluorescence (FITC)
images were acquired for a minimum of 5000 cells/sample. A compensation
matrix was applied to the acquired images, and properly focused single cells
were gated based on the area, aspect/ratio intensity, and bright-field gradient
RMS features using IDEAS v5.0 software (Amnis). A mask depicting only
the intracellular space was designed, and the percentage of FITC+ cells was
Flow cytometry
FcgR surface expression was determined with anti-CD64, anti-CD32, and
anti-CD16 (all from BD Biosciences) or 2B6 Ab (anti-FcgRIIb, own hybridoma production ATCC PTA-4591) for 1 h at 4˚C. After washing, cells
were incubated with rabbit F(ab9)2 anti-mouse Ig-PE (DAKO, Glostrup,
Denmark) for 30 min at 4˚C. Fluorescence was measured by flow
cytometry (FACSCalibur; Becton Dickinson), and the gate was set on live
cells. Inflammatory status of iDCs was determined, as described (21). Cell
were gated for BDCA1+, CD162, and HLA-DR+ and subsequently analyzed for CD1a-PE (BD Biosciences Pharmingen), CD14-PE (BioLegend),
CD206 (BD Biosciences Pharmingen), CD11b-PE (BD Pharmingen),
FcεRI (BD Biosciences Pharmingen), and CD209-FITC (R&D Systems).
FcaRI surface expression was determined by staining for anti-CD89
(PE conjugated; BD Biosciences Pharmingen) for 1 h at 4˚C. After
washing, fluorescence was determined by FACS.
Multiplex protein analysis and cytokine ELISA
Custom made 9-plex protein assays (Millipore), including IL-12p70,
IL-12p40, IL-6, IL-1b, IL-4, IL-10, IFN-g, TNF-a, and G-CSF, were
performed according to the manufacturer’s protocol using LUMINEX
technology (LX200; Millipore). IFN-g, IL-17, GM-CSF, IL-1b, TNF-a,
and IL-23 sandwich ELISAs (eBioscience, San Diego, CA) were performed according to the manufacturer’s instructions.
Th cell–differentiation assay
PBMCs were isolated from heparinized blood from healthy volunteers
by density gradient centrifugation using LymphoPrep (Axis-Shield).
Memory CD4+ Th cells were isolated using a Memory CD4+ Th Cell
MACS Isolation Kit (Miltenyi Biotec), according to the manufacturer’s
protocol. Memory Th cells were used for coculture with stimulated
iDCs (5 3 103 iDCs with 5 3 104 Th cells in complete RPMI 1640
medium supplemented with Staphylococcus aureus enterotoxin B
[100 pg/ml; Sigma-Aldrich]). After 5 d of coculture, supernatants
were harvested and analyzed with ELISA for IFN-g, IL-17, or GM-CSF
production to determine Th1 or Th17 subset differentiation or GM-CSF
Th cell skewing, respectively.
Lipidomics analysis of inflammatory lipids
Eicosanoid detection and quantification (∼120 inflammatory lipids) were
performed with a multiple reaction–monitoring assay, using ultrahighperformance liquid chromatography (Acquity, Ultra performance LC,
Waters) and negative-mode electrospray ionization tandem mass spectrometry (Xevo Waters), essentially as described (22), with the following
modification: sample was extracted by liquid-liquid extraction using ethyltetr-butyl ether. Chromatographic separation was achieved on a Synergi
Hydro-RP column (4 mm, 2.0 mm 3 250; Phenomenex) using a flow rate of
0.35 ml/min at 40˚C in a 30-min gradient using two solvents: H2O + 0.1%
HAc (solvent A) and 90:10 AcN/isopropanol (solvent B). A set of 17
deuterated internal standards was added prior to the extraction step to
correct for matrix effects and recovery efficiency. Calibration curves were
obtained by plotting the ratio of peak areas of eicosanoids to the internal
standards against known quantities of eicosanoids, leading to a linear regression line (r2 . +0.99). The limits of detection (LODs) were defined by
a signal-to-noise ratio of 3:1, and they were in the range of 3 pM to 1 nM,
depending on the compound. Accuracy and the precision of the method
were tested and resulted in a coefficient variation , 20% for most of the
compounds.
In vitro stimulation of iDCs with isolated TLR ligands
iDCs (5 3 105 iDCs/500 ml) were stimulated with various TLR ligands
([synthetic lipopeptide Pam3CSK4 or flagellin (Bacillus subtilis); InvivoGen, San Diego, CA] or LPS [purified by gel-filtration chromatography
and checked for impurities (,1% protein)]), peptidoglycan of S. aureus
(PGN-SA), viral synthetic agonist polyinosinic-polycytidylic acid (Poly-I:C;
Sigma-Aldrich), or synthetic R848 (InvivoGen), with or without IgG-coated
beads (IgG-B) or BSA-coated beads (BSA-B), for the indicated times.
Where indicated, iDCs were preincubated for 2 h with the caspase-1
inhibitor Ac-YVAD-CMK (Axxora, San Diego, CA), spleen tyrosine
kinase (Syk) inhibitor (piceatannol; Tocris Bioscience, Ellisville, MO),
protein kinase C (PKC) inhibitor GF109203 (BIOMOL, Plymouth Meeting, PA), or Raf-1 inhibitor GW5074 (Sigma-Aldrich) and compared with
DMSO-treated cells as vehicle control.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
CNBr-activated Sepharose beads (GE Healthcare Life Sciences, Uppsala,
Sweden) were coupled with 3 mg purified serum IgG (Sigma-Aldrich,
St. Louis, MO) or BSA (Roche Diagnostics, Basel, Switzerland), according
to the manufacturers’ instructions. IgG purity was tested by SDS electrophoresis and was $95%.
calculated. Also, a feature was designed to count the number of bacteria/
cell. This feature (spot count) was based on the Peak mask using a spot/
background ratio of 5.75.
The Journal of Immunology
3
Neutrophil isolation
Polymorphonuclear cells (PMNs; neutrophils) were isolated from heparin
anticoagulated peripheral blood drawn from healthy volunteers by
standard LymphoPrep isolation (Axis-Shield), according to the manufacturer’s protocol. Erythrocytes were removed by hypotonic lysis buffer
(155 mM NH4Cl2, 10 mM KHCO3, and 0.1 mM EDTA) (10 min, 4˚C).
Purity of PMNs was determined by cytospin preparation and always
exceeded 95%. Cell viability of PMNs exceeded 98%, as assessed by
trypan blue staining
Quantitative real-time PCR
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
mRNA was isolated with an mRNA Capture Kit (Roche Diagnostics), and
cDNA was synthesized with the Reverse Transcription System (Promega,
Madison, WI), according to the manufacturers’ protocols. PCR amplification (ABI ViiA 7 sequence detection system; Applied Biosystems) was
performed using target-specific human primers (for housekeeping gene
EF1a: forward 59-AAGCTGGAAGATGGCCCTAAA-39 and reverse 59AAGCGACCCAAAGGTGGAT-39; for pro–IL-1b: forward 59-TTTGAGTCTGCCCAGTTCCC-39 and reverse 59-TCAGTTATATCCTGGCCGCC-39;
for TNF-a: forward 59-GCCCAGGCAGTCAGATCATC-39 and reverse 59TGGGCTACAGGCTTGTCACTC-39; and for IL-23p19: forward 59GCTTGCAAAGGATCCACCA-39 and reverse 59-TCCGATCCTAGCAGCTTCTCA-39) with the SYBR Green detection method (iQ SYBR Green
SuperMix 23; Bio-Rad). The reaction protocol was identical for all PCR
products: 2 min at 95˚C, followed by 40 cycles of 15 s at 95˚C and 1 min at
60˚C (+1 cycle for dissociation curve). For each condition the normalized
amount of target mRNA (Nt) was calculated from obtained Ct values
(number of PCR cycles for which the fluorescence signal exceeds the
detection threshold value) using the equation Nt = 2Ct(Ef1a) 2 Ct(Target).
Western blotting
Cells were collected and lysed in RIPA lysis buffer (50 mM Tris-HCl
[pH 7.5], 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate 0.1% SDS,
10 mM EDTA) for 30 min at 4˚C. The lysate was clarified by centrifugation at
15,000 rpm for 10 min at 4˚C, and the supernatant was used for Western
blotting. Alternatively, supernatants of cells were harvested and centrifuged at
15,000 rpm for 10 min at 4˚C; these supernatant samples were used for further
analysis. Samples were prepared in Laemmli sample buffer and electrophoresed on 4–20% gradient precast gel (Mini-Protean TGX; Bio-Rad,
Hercules, CA). Subsequently, the gels were transferred to polyvinylidene
difluoride membranes (Immobilon-FL; Millipore, Bedford, MA). The gels
were stained with anti–IL-1b (Abcam 2105) and Goat anti-Rabbit IRDye
800CW (red signal; Li-Cor, Lincoln, NE), according to the manufacturers’
protocol, scanned using the Odyssey system (Li-Cor), and adjusted to
grayscale images.
Statistics
Data are depicted as mean 6 SD. Statistical differences were determined
using the unpaired Student t test (two groups) or ANOVA (multiple
groups). The p values , 0.05 were considered significant.
Results
Ab-opsonized E. coli evoke distinct iDC effector functions with
subsequent altered Th cell polarization
To investigate coactivation of PRRs and FcRs, iDCs were incubated
with E. coli in the presence or absence of Ab opsonization. Subsequent iDC cellular responses, as well as Th cell polarization,
were analyzed. Ab-dependent uptake of FITC-labeled E. coli was
observed in the presence of purified human IgG or human serum
(containing specific Abs) (Fig. 1A, Supplemental Fig. 1A) (23).
Using imaging stream technology, we visualized and confirmed
enhanced phagocytosis and not merely increased binding of
opsonized bacteria by iDCs (Fig. 1B). The phagocytic index decreased after 4 h of phagocytosis, which was likely due to acidic
pH quenching of the FITC label during intracellular bacterial
processing within lysosomes.
These differentially stimulated iDCs were analyzed further for
maturation markers (data not shown). No major differences in the
expression of DC maturation markers CD80, CD83, CD86, and
HLA-DR were observed after stimulation with E. coli, IgG, or
FIGURE 1. Ab-opsonized E. coli evokes distinct iDC effector functions.
(A) iDCs were stimulated with FITC-labeled E. coli, IgG, or IgG-opsonized
FITC-labeled E. coli for the indicated times and analyzed for phagocytic
capacity by flow cytometry. (B) Uptake of FITC-labeled E. coli by iDCs after
2.5 h was visualized by imaging stream technology (original magnification
360) (Ch01: bright-field; Ch02: FITC; unstimulated iDCs served as negative
control). Bar graphs depict phagocytosis calculated as the percentage of
FITC+ cells (upper right panel) and number of internalized E. coli/iDC (lower
right panel). (C) iDCs were stimulated as described in (A) for 16 h. Indicated
cytokines were measured in supernatants by multiplex protein analysis. IL-23
was measured in a separated sandwich ELISA. (D) Stimulated iDCs, as indicated, were cocultured with memory Th cells for 5 d, after which supernatants were analyzed for IFN-g and IL-17 production as markers for Th1 and
Th17 differentiation, respectively. Data are mean 6 SD (n = 3). *p , 0.05.
IgG-opsonized E. coli, which suggests no major modulation of
DC maturation compared with individual activation of PRRs (1, 2)
and FcRs (24, 25).
4
TLR–FcR CROSS-TALK ON DCs REDIRECTS Th CELL POLARIZATION
Table I. AA-derived metabolites
COX-Dependent AA Metabolites (nM)
Unstimulated
E. coli
IgG
E. coli + IgG
Fold changec
PGF2a
PGJ2
TXB2
PGE2
ND
NDa
ND
0.489
2445
ND
0.166
ND
0.657
4.0
0.238
16.087
4.353
61.129
3.8
1.082
1.586
0.557
NDb
2834
LOX-Dependent AA Metabolites (nM)
Unstimulated
E. coli
IgG
E. coli + IgG
Fold changec
LTB4
(5-LOX)
6R-LXA4
(15-LOX)
0.068
0.044
0.355
0.476
10.8
ND
ND
ND
9.39
2608
iDCs were stimulated with E. coli in the presence or absence of IgG for 16 h.
Supernatants were harvested and analyzed for the presence of inflammatory lipids.
Fold change was calculated between IgG-opsonized E. coli and E. coli–stimulated
iDCs.
a
LOD of PGF2a = 0.0002 nM.
b
LOD of PGE2 = 0.0019 nM.
c
Fold change for E. coli + IgG versus E. coli.
d
LOD of 5S,6R-LXA4 = 0.0036 nM.
with IgG-opsonized E. coli evoked IL-17 production, indicative of
polarization into Th17 cells; this was not observed in cocultures
with unstimulated, IgG-stimulated, or E. coli–stimulated iDCs.
Taken together, iDCs stimulated simultaneously with PRRs and
FcRs displayed clearly different cellular immune responses compared
with single stimulation with PRRs or FcRs, based on the inflammatory
cytokine- and lipidomic-profiles, as well as Th cell polarization.
Both FcgRII and MyD88-dependent TLRs are essential for
cross-talk
IgG Abs are the main opsonizing Ab isotype present in serum (27).
Therefore, we tested the involvement of distinct IgG FcRs
(FcgRs) present on iDCs in the cross-talk with PRRs (Fig. 2A).
iDCs did not express activating FcgRI or FcgRIII, whereas
abundant surface expression of both activating FcgRIIa and
inhibiting FcgRIIb was detected. Phagocytosis of IgG-opsonized
FITC-labeled E. coli was reduced in the presence of pan antiFcgRII–blocking Abs (AT10) (Supplemental Fig. 1B, 1C). Similarly, IL-1b and IL-23 release was reduced (Fig. 2B, 2C, respectively), which indicates the absolute requirement of FcgRII to
induce an ample amount of Th17-polarizing cytokines. Of note,
blocking FcgRIIb with the blocking Ab 2B6 resulted in higher
IL-1b release, which was likely due to activation of the FcgRIIaITAM–mediated signaling pathway without interference of an
inhibitory signal (Fig. 2D) (12, 28). To further delineate cooperation between different TLRs and FcgRs, we next used several
isolated TLR ligands and IgG-B (because activation of FcgRs
requires cross-linking). The extracellular TLR ligands Pam3CSK4
(TLR1/2), LPS (TLR4), and flagellin (TLR5) were able to induce
synergistic IL-1b and IL-23 cytokine release when iDCs were
costimulated with IgG-B (Fig. 2E, 2F). PGN-SA (TLR2) already
induced high amounts of IL-1b and IL-23, independently of FcgR
costimulation. This is likely due to coactivation via cytoplasmic
nucleotide oligomerization domain-2 receptors, as described previously (8, 29). The cooperative effect was not restricted to Th17polarizing cytokines, because costimulation with LPS and IgG-B
induced upregulation of IL-6, IL-10, IFN-g, TNF-a, and G-CSF,
as previously observed with IgG-opsonized E. coli (Supplemental
Fig. 2A, Fig. 1C, respectively). The intracellular TLR3 ligand
Poly-I:C was not able to induce IL-1b or IL-23 (Fig. 2E, 2F). In
contrast, stimulation of intracellularly located TLR7/TLR8 with
the synthetic agonist R848 and IgG-B resulted in synergistic release of IL-1b (Fig. 2E) and TNF-a (Fig. 2G).
Next, we investigated whether cross-talk is specific for FcgRII
on DCs. Prestimulation of iDCs with IFN-g induced surface expression of FcgRI (30) (Supplemental Fig. 2BI versus 2BII).
Coincubation of iDCs with LPS and IgG-B induced an augmented
synergistic release of IL-1b in FcgRI-expressing IFN-g–stimulated iDCs (Supplemental Fig. 2C). iDCs expressed FcaRI as
well, albeit at low levels (31, 32) (Supplemental Fig. 2BIII).
Nonetheless, costimulation with LPS and IgA-coated beads induced synergistic release of IL-1b (Supplemental Fig. 2D). In
contrast, neutrophils express high levels of FcaRI, which is their
most potent FcR (33, 34). We observed synergistic TNF-a release
after simultaneous triggering of FcaRI and TLR4 (Supplemental
Fig. 2BIV, 2E). Of note, TNF-a was measured because neutrophils
are poor IL-1b producers. Thus, cross-talk occurred between TLRs
and several types of FcRs and was not constrained to iDCs.
Furthermore, we investigated the effect of differentially stimulated iDCs on Th cell polarization. Th17 differentiation was induced by the extracellular TLR ligands (Pam3CSK4, LPS, or
flagellin) combined with IgG-B triggering (Fig. 3A). PGN-SA
induced Th17 differentiation independent of FcR activation, because IL-1b and IL-23 release also was produced independent of
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Next, we investigated the secretory profile of iDCs that had been
incubated with E. coli or IgG-opsonized E. coli. Cytokine release
was determined, which included the Th-polarizing cytokines
IL-12p40, IL-12p70 (Th1), IL-6, IL-1b, and IL-23 (Th17 cells),
IL-4 (Th2), IL-10 (regulatory T cells), IFN-g, TNF-a, and G-CSF
(Fig. 1C). Minimal production of IL-4 by DCs was observed under
all conditions because this cytokine is primarily secreted by Th
cells themselves and not by DCs. IgG opsonization of E. coli did
not increase secretion of IL-12p40 compared with activation via
E. coli alone. IL-12p70, IL-6, IL-10, and IFN-g were additively
released when iDCs were incubated with IgG-opsonized E. coli
compared with cytokine release in the presence of either E. coli or
IgG. Interestingly, synergistic release of TNF-a, G-CSF, and the
Th17-polarizing cytokines IL-1b and IL-23 was observed after
costimulation with E. coli and IgG, which supported cross-talk
between PRRs and FcRs.
Additionally, we used a novel quantitative mass spectrometry
lipidomics approach (22) to profile 120 inflammatory eicosanoids
(17–19, 26). iDCs stimulated with IgG-opsonized E. coli demonstrated clear synergistic differences in the secretion of cyclooxygenase (COX)-dependent and lipoxygenase (LOX)-dependent
metabolites compared with iDCs stimulated with E. coli (Table I).
This included synergistic upregulation of AA metabolites, like
PGF2a, PGJ2 (metabolic end product of PGD2), and thromboxane
B (TXB)2. Other TXB family members (TXB1 and TXB3), which
are linoleic acid–derived metabolites, were upregulated as well
(data not shown). Furthermore, increased levels of the chemoattractant leukotriene B4 (LTB4) and its counter-regulator, lipoxin
A4 (LXA4), were found. In contrast, secretion of PGE2 was reduced (beneath detection level) when iDCs were stimulated with
IgG-opsonized bacteria compared with nonopsonized bacteria.
Next, Th cell polarization was tested by coculturing of Th cells
and iDCs, which had been stimulated for 24 h with the indicated
stimuli (Fig. 1D). Release of IFN-g and IL-17 was used as a measure of Th1 and Th17 polarization, respectively. No difference in
IFN-g level was found between iDCs cocultured with Th cells and
stimulated with IgG-opsonized bacteria or nonopsonized bacteria.
However, cocultures of Th cells with iDCs that had been stimulated
The Journal of Immunology
5
FcRs. Th17 differentiation after TLR–FcR cross-talk was dependent on the release of IL-1b by iDCs, because costimulation of
LPS and IgG-B in the presence of IL-1Ra (an IL-1R antagonist)
completely abrogated Th17 differentiation (Fig. 3B).
Because IL-23R signaling and the Th17 transcription factor
RORgt drive expression of the cytokine GM-CSF in Th cells (16,
35), we additionally measured GM-CSF production in cocultures
of iDCs and Th cells. iDCs costimulated with FcgRs and TLR
ligands (Pam3CSK4, LPS, flagellin, or PGN-SA) induced GMCSF–producing Th cells (Fig. 3C). Of note, GM-CSF was not
detected in supernatants of stimulated iDCs without coculture with
Th cells (data not shown) (36).
Thus, because only TLR3, which signals via TRIF, did not show
cross-talk with FcgR, these results support that MyD88-dependent
TLRs act preferentially in concert with FcgRII on iDCs and,
thereby, modulate the inflammatory cytokine profile and Th17 cell
polarization. Furthermore, these data support that only coactivation of TLRs and FcgRII on iDCs may evoke GM-CSF–producing Th cells.
Molecular mechanisms of TLR–FcgR cross-talk regulating
cytokine release
Next, we investigated the molecular mechanisms of synergistic
cytokine release via cross-talk between TLRs and FcgRs. In
contrast to other cytokines, release of active or mature IL-1b is
a two-step process. First, pro–IL-1b mRNA and pro–IL-1b protein are induced via TLR signaling (37–40). A second activation
step through so-called “danger signals” (danger-associated mo-
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 2. Both FcgRII and MyD88-dependent TLRs are essential for cross-talk. (A) FcgRI, FcgRIIa, FcgRIIb, and FcgRIII expression on iDCs
(shaded graphs). Open graphs represent isotype control. iDCs were stimulated for 16 h with FITC-labeled E. coli, IgG, or IgG-opsonized FITC-labeled
E. coli in the presence or absence of pan FcgRII (AT10)-blocking Ab (B and C) or anti-FcgRIIb (2B6) Ab (D), after which supernatants were analyzed for
production of the indicated cytokines. (E and F) iDCs were stimulated for 24 h with the TLR ligands Pam3CSK4 (Pam; 5 mg/ml), LPS (100 ng/ml), flagellin
(Flag; 5 mg/ml), PGN-SA (10 mg/ml), Poly-I:C (40 mg/ml), or R848 (2.5 mg/ml) in the presence or absence of IgG-B (to activate FcgRs) or BSA-B (as
control beads). IL-1b (Ε) or IL-23 (F) was determined in the supernatants of iDCs after the indicated stimulation. (G) TNF-a production was determined
after stimulation with R848 (2.5 mg/ml) in the presence or absence of IgG-B or BSA-B. Data are mean 6 SD (n = 3). *p , 0.05, **p , 0.01,***p , 0.001.
6
TLR–FcR CROSS-TALK ON DCs REDIRECTS Th CELL POLARIZATION
lecular patterns) is required for cleavage of pro–IL-1b protein
into mature IL-1b.
Synergistic release of mature IL-1b after incubation of iDCs
with IgG or serum-opsonized E. coli was observed over time
(Fig. 4A, Supplemental Fig. 3A, respectively), and it was reduced
by preincubation with the FcgRII-blocking Ab AT10. Additionally, activation of iDCs with LPS and IgG-B demonstrated synergy
of IL-1b release (Fig. 4B), independent of LPS concentration or
incubation times (Supplemental Fig. 3B).
As expected, IL-1b mRNA was induced in iDCs by incubation of
E. coli (Fig. 4C, Supplemental Fig. 3C). Interestingly, FcgR triggering did not alter mRNA levels of IL-1b, but it was required for
IL-1b protein release. This was further strengthened by blocking
FcgRII on iDCs, which abrogated the release of IL-1b protein but
did not decrease IL-1b mRNA levels (indicated by # in Fig. 4A, 4C,
Supplemental Fig. 3A, 3C). Similarly, IgG-B in combination with
LPS, Pam3CSK4, or PGN-SA did not alter IL-1b mRNA levels
compared with stimulation of individual TLRs (Fig. 4D, LPS;
Supplemental Fig. 3D, other TLR ligands), although flagellin–FcR
costimulation affected mRNA levels. Poly-I:C did not induce pro–
IL-1b mRNA, which correlates with its inability to initiate IL-1b
release, even in the presence of FcgR triggering. Furthermore, ex-
pression of intracellular pro–IL-1b protein (∼35 kDa) was present
after stimulation of LPS (Fig. 4E, upper panel). However, only simultaneous LPS and IgG stimulation resulted in release of mature
IL-1b (17 kDa) (Fig. 4E, lower panel). Thus, FcgR triggering did
not affect IL-1b mRNA synthesis or pro–IL-1b protein levels, but it
was involved in the process of mature IL-1b secretion as the second
step of functional IL-1b production (37, 40). This second step
requires caspase-1 activity. Therefore, we established caspase-1
dependency for functional TLR4 and FcgR coactivation. Both
IL-1b release (Fig. 5A) and subsequent Th17 cell induction (Fig.
5B) after combined TLR4 and FcgR triggering were abrogated in
the presence of the specific caspase-1 inhibitor Ac-YVAD-CMK;
thus, caspase-1 activity is required for TLR and FcgR coactivational–induced release of IL-1b production. Of note, IL-1b mRNA
levels (Fig. 5C) and pro–IL-1b protein levels (data not shown) were
not decreased after inhibition of caspase-1. Next, we investigated
how FcR ITAM signaling affects synergistic release of IL-1b. Inhibition of the downstream mediators of ITAM signaling (12), such
as Syk, PKC, or Raf-1, abrogated the synergistic release of IL-1b
(Fig. 5D, gray bars). However, neither pro–IL-1b protein levels nor
changes in caspase-1 activity were observed in the presence of these
inhibitors (data not shown).
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 3. Cross-talk between TLRs and FcgRs induces polyfunctional Th17 cells. iDCs were stimulated for 24 h with Pam3CSK4 (Pam; 5 mg/ml),
LPS (100 ng/ml), flagellin (Flag; 5 mg/ml), or PGN-SA (10 mg/ml) in the presence or absence of IgG-B (to activate FcgRs) or BSA-B (as control beads). (A
and B) Next, iDCs were cocultured with memory Th cells for 5 d, and IL-17 production was determined in supernatants. (B) iDCs also were stimulated with
LPS and IgG-B and cocultured with memory Th cells in the presence or absence of IL-1R antagonist (IL-1Ra). (C) iDCs were stimulated and cocultured
with memory Th cells, as described in (A), and GM-CSF production was determined in supernatants. Data are mean 6 SD. Experiments were repeated three
times. *p , 0.05, ***p , 0.001.
The Journal of Immunology
7
Together, these data supported the involvement of FcgRIIdependent ITAM signaling in the release of mature and functional
IL-1b rather than in the induction and/or processing of pro–IL-1b.
This was in contrast to other cytokines that were released by
simultaneous activation of TLRs and FcgRs, such as TNF-a and
IL-23. Synergistic release of TNF-a demonstrated that both
protein and mRNA were increased over time after incubation
of iDCs with IgG-opsonized E. coli (Fig. 6A, 6B; indicated
with #). Furthermore, blocking FcgRII hampered the release of
TNF-a, as well as synthesis of TNF-a mRNA, which was different compared with IL-1b mRNA levels (Fig. 6B versus
Fig. 4C, indicated by #). Stimulation of isolated TLR ligands in
combination with IgG-B enhanced both TNF-a protein and
mRNA levels (Fig. 6C, 6D). Similar results were obtained for
IL-23 (Supplemental Fig. 4A). An increase in IL-23 cytokine
release by iDCs stimulated with IgG-opsonized E. coli (Fig.
1C) correlated with increased mRNA levels (Supplemental Fig.
4A). Moreover, blocking FcgRII inhibited both cytokine production and mRNA levels, indicative of FcgRII involvement in
regulation of the expression level of transcription. TLR stimulation combined with IgG-B also linked synergistic IL-23
cytokine release with upregulation of mRNA levels, with the
exception of LPS (Supplemental Fig. 4B). These data suggest
that FcgRs can modulate TLR-initiated cytokine release via
different molecular mechanisms, which includes at both the
transcriptional and posttranscriptional levels, depending on the
specific cytokine.
Discussion
It is generally thought that clearance of pathogens during recurrent
infection is faster as the result of rapid expansion of lymphocytes and
more efficient phagocytosis because of the presence of opsonizing
Abs (5). We now demonstrate that Ab-opsonized bacteria initiate
fundamentally different immune responses compared with nonopsonized bacteria. We identified synergistic activity of TLRs and
FcRs leading to release of the Th17 polarizing cytokines IL-1b and
IL-23, as well as TNF-a, G-CSF, and a distinct metabolite profile
(both COX- and LOX-dependent eicosanoids). Notably, maturation
of DCs was not altered during FcgR and TLR cross-talk because
both independent stimuli already were capable of inducing a mature
DC phenotype (data not shown). Furthermore, in addition to IL-17–
producing Th cells, GM-CSF–producing Th cells were evoked,
which were recently described as a pathogenic subset involved in
autoimmune diseases (16). This suggests that TLR and FcR crosstalk induces polarization into polyfunctional GM-CSF–producing
Th17 cells. It was described that RORgt-mediated GM-CSF production was induced by IL-23R signaling (20).
Both surface-expressed TLRs (TLR1/2, 4, 5) and intracellular
TLR 7/8 were able to cross-talk with FcgRs. TLRs and FcR
subfamily members are structurally different, which makes a
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 4. Molecular mechanisms of TLR–FcgR cross-talk regulating IL-1b cytokine release. (A) iDCs were stimulated for 2.5, 4, or 8 h with E. coli, in
the presence or absence of IgG. The pan FcgRII-blocking Ab (AT10) was added as indicated. Supernatants were analyzed for IL-1b release. (B) iDCs were
stimulated for 4 or 24 h with LPS, with or without IgG-B or BSA-B. Supernatants were analyzed for IL-1b release. (C and D) Relative IL-1b mRNA levels
were determined in cell lysates at the indicated time points, as described in (A) and (B), respectively, and normalized to EF1a. Data are mean 6 SD.
Experiments were repeated at least three times. (E) Western blots were performed to analyze pro–IL-1b and mature IL-1b protein levels (upper panel and
lower panel, respectively) of unstimulated iDCs or iDCs stimulated with LPS, with or without IgG-B. Cell lysates after a 2-h stimulation were used for pro–
IL-1b analysis (upper panel), and supernatants after 24 h of stimulation were used for detection of mature IL-1b. #denotes comparison between protein
levels in (A) versus mRNA levels in (C). ***p , 0.001.
8
TLR–FcR CROSS-TALK ON DCs REDIRECTS Th CELL POLARIZATION
physical interaction less likely. Therefore, we hypothesized that
the interacting link relied on the signaling pathways downstream
of both receptors. TLR1/2, 4, 5 and TLR 7/8 signal via MyD88,
whereas TLR3, the only TLR unable to synergistically induce
cytokine release, is dependent on TRIF. Moreover, we showed that
FcR-dependent ITAM signaling pathways were essential for IL-1b
release. Thus, both MyD88-dependent TLR signaling and FcRdependent ITAM signaling are required for functional cross-talk.
Furthermore, cross-talk was not confined to FcgRII on DCs, as
previously suggested (41). Both FcgRI and FcaRI were able to
synergize with TLR4 on DCs and neutrophils. Although infection
is linked with uptake of Ab-opsonized pathogens, we conclude that
amplified secretion of cytokines is already induced by extracellular
ligation of FcRs. This is based on our findings that stimulating cells
with isolated TLR ligands in combination with IgG-coated beads
(average size 90 mm), which were not phagocytosed by iDCs,
resulted in the release of similar cytokine profiles.
TLRs are potent inducers of pro–IL-1b transcription through
activation of MyD88, with subsequent NF-kB activation, but they
are limited in their ability to induce the second step of IL-1b
processing (4, 38, 40). After exposure to a second “so-called”
danger signal (e.g., ATP that is induced via cellular stress), assembly of the caspase-1–dependent inflammasome is initiated,
which cleaves pro–IL-1b protein into mature IL-1b cytokine.
Finally, an increase in calcium levels in the cell, which is induced
by activating FcRs (12), may provide a mechanism by which
mature IL-1b is released from the cell (38, 40). We demonstrate in
this study that TLR activation is essential for the induction of pro–
IL-1b, and coactivation with FcgRs results in the secretion of
functional and mature IL-1b. Moreover, blocking FcgRII activation either with blocking Abs or inhibitors of FcgR–ITAM signaling (Syk, PKC, and Raf) resulted in abrogated IL-1b cytokine
release, despite the presence of ample amounts of intracellular
pro–IL-1b protein and the presence of active caspase-1. In conclusion, this favors a primary role for FcRs in finalizing mature
IL-1b secretion, rather than inducing pro–IL-1b or caspase-1 activity. In contrast, FcgRII was involved in modulation of TNF-a and
IL-23 transcription, because enhanced mRNA levels were observed
when FcgRII and TLRs were activated simultaneously. Moreover,
blocking FcgRII reduced mRNA levels. Thus, FcgR can modulate
specific TLR-initiated cytokine production via different molecular
mechanisms and, in this way, alter the Th cell phenotype.
Furthermore, we found that several COX-dependent inflammatory PGs, such as PGF2a, PGJ2 (as PGD2 stable end product),
and thromboxanes (TXA2), were upregulated (42) when IgGopsonized E. coli were used as stimulus for iDCs (Table I). It is
noteworthy that IL-1b and TNF-a are regulators of COX enzymes
(18, 43). PGF2a is known to be crucial for polarization toward the
Th17 cell type (44) by binding its receptor on CD4+ Th cells; in
this way, it upregulates levels of RORgt transcription factor. In
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 5. IL-1b release is caspase-1
and FcR-ITAM–signaling dependent. (A)
iDCs were stimulated with LPS for the indicated times, with or without IgG-B or
BSA-B. The caspase-1 inhibitor Ac-YVADCMK (30 mM) was added, as shown. IL-1b
release in supernatant was measured. (B)
Subsequently, iDCs (24 h) were cocultured
with memory Th cells for 5 d, and IL-17
production was analyzed. (C) Cell lysates of
(un)stimulated iDCs described in (A) (4 h)
were analyzed for relative IL-1b mRNA,
which was normalized to EF1a. (D) IL-1b
production of iDCs stimulated (24 h) as
indicated, in the absence or presence of Syk
inhibitor (piceatannol, 50 mM), PKC inhibitor (GF109203, 1 mM), or Raf-1 inhibitor (GW5074, 2 mM). Data are mean 6
SD. One representative experiment of three
is shown. **p , 0.01, ***p , 0.001.
The Journal of Immunology
9
contrast, PGE2 can act as a negative regulator of IL-17 production
and RORgt mRNA levels in Th17 cells (45). Thus, the high levels
of PGF2a and low levels of PGE2 that we observed may contribute
to skewing toward Th17 polarization. For the LOX-dependent
class of eicosanoids, we observed increased levels of LTB4.
LTB4 is a potent inflammatory mediator and neutrophil chemoattractant and is an inducer of RORgt mRNA levels favoring
skewing into polyfunctional Th17 cells (46). The LTB4 antiinflammatory counter-regulator LXA4 also was increased. Because potent proinflammatory responses are kept under tight control
by this class of anti-inflammatory eicosanoids, it is likely that the
LXA4-feedback mechanism was already activated after 16 h.
Interestingly, the overall secretory profile of iDCs and subsequent Th cell polarization after TLR–FcR cross-talk resemble
those of the recently identified infDCs (21). This DC subset was
found in synovial fluid of arthritic joints and tumor ascites and was
designated as a novel DC subset. infDCs are particularly efficient
at inducing Th17 polarization by secreting the polarizing cytokines IL-1b and IL-23. It is clear that onset of these infDCs
resides in inflammatory microenvironments. However, the trigger
for DCs to develop into this inflammatory phenotype remains
unknown and was not dependent on ICOS/ICOS-L interaction,
which is important for Th17 polarization (21, 47). Moreover,
Pam3CSK4 (as inflammatory stimuli) activation of blood DCs
did not induce an infDC subset, because no IL-1b or IL-23 was
produced (21). Based on our findings, we postulate that cross-talk
of TLRs with FcRs on iDCs induces infDCs, because we demonstrated that this coactivation induced the release of IL-1b and
IL-23 with subsequent IL-17–producing Th17 cells. When analyzing infDC surface markers (CD1a+, CD14+, CD206+, CD11b+,
FcεRI+, and CD2092 [gated for BDCA+, HLA-DR+, and CD162
DCs]), we observed that iDCs activated with IgG-opsonized
E. coli exhibited some expression of inflammatory markers (higher
CD14 and CD206 expression, but CD1a, CD14, CD206, and CD11b
already were present on E. coli–stimulated DCs; levels of FcεRI
and CD209 on infDCs could not be confirmed in our experiments)
(Supplemental Fig. 1D). This suggests that the infDC phenotype
(based on markers) is induced by inflammatory stimuli (like TLR
stimulation). We hypothesize that functionality of infDCs (IL-1b
and IL-23 release and induction of Th17 polarization) requires
coactivation with FcRs, however.
It is likely that coactivation of TLRs and FcgRs favors recruitment, activation, and prolonged survival of neutrophils at
inflammatory sites, which will facilitate clearance of bacterial
infections, because the observed secretory profile includes IFN-g,
TNF-a, G-CSF, and LTB4 (48). Additionally, Th17 cells stimulate
production of G-CSF and granulopoiesis, thereby orchestrating
and amplifying neutrophil function (49), whereas GM-CSF is a
neutrophil growth factor as well.
However, cross-talk between PRRs and FcRs may play a detrimental role in disorders, such as rheumatoid arthritis, and in inflammatory bowel disease because of excess Ab–pathogen complexes
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 6. PRR–FcgR costimulation regulates TNF-a production on mRNA level. TNF-a production (A) and mRNA levels (B) were determined after
stimulation of iDCs for different times with E. coli in the presence or absence of IgG. The pan FcgRII-blocking Ab (AT10) was added as indicated. iDCs
were stimulated for 4 h with the indicated TLR ligands in the presence or absence of IgG-B or BSA-B, after which TNF-a protein in supernatants (C) or
relative TNF-a mRNA levels in cell lysates (D) were determined. Expression was normalized to EF1a (n = 3). Data are mean 6 SD. #denotes comparison
between protein levels in (A) versus mRNA levels in (B). ***p , 0.001.
10
TLR–FcR CROSS-TALK ON DCs REDIRECTS Th CELL POLARIZATION
Acknowledgments
We thank T. O’Toole and Dr. J.J. Garcı́a-Vallejo (Department of Molecular
Cell Biology and Immunology, VU University Medical Center) for expert
support with imaging stream flow cytometry.
Disclosures
The authors have no financial conflicts of interest
References
1. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu.
Rev. Immunol. 20: 197–216.
2. van Vliet, S. J., J. den Dunnen, S. I. Gringhuis, T. B. Geijtenbeek, and Y. van
Kooyk. 2007. Innate signaling and regulation of dendritic cell immunity. Curr.
Opin. Immunol. 19: 435–440.
3. Trinchieri, G., and A. Sher. 2007. Cooperation of Toll-like receptor signals in
innate immune defence. Nat. Rev. Immunol. 7: 179–190.
4. Newton, K., and V. M. Dixit. 2012. Signaling in innate immunity and inflammation. Cold Spring Harb. Perspect. Biol. 4: a006049.
5. Ademokun A. A, and D. Dunn-Walters. 2010. Immune responses: primary and
secondary. In: Encyclopedia of Life Sciences. Peter Delves, ed. John Wiley &
Sons Ltd, Chichester, U.K. Available at http://onlinelibrary.wiley.com/doi/
10.1002/9780470015902.a0000947.pub2/abstract.
6. Gringhuis, S. I., J. den Dunnen, M. Litjens, B. van Het Hof, Y. van Kooyk, and
T. B. Geijtenbeek. 2007. C-type lectin DC-SIGN modulates Toll-like receptor
signaling via Raf-1 kinase-dependent acetylation of transcription factor NFkappaB. Immunity 26: 605–616.
7. Geijtenbeek, T. B., and S. I. Gringhuis. 2009. Signalling through C-type lectin
receptors: shaping immune responses. Nat. Rev. Immunol. 9: 465–479.
8. Kawai, T., and S. Akira. 2011. Toll-like receptors and their crosstalk with other
innate receptors in infection and immunity. Immunity 34: 637–650.
9. Dzopalic, T., I. Rajkovic, A. Dragicevic, and M. Colic. 2012. The response of
human dendritic cells to co-ligation of pattern-recognition receptors. Immunol.
Res. 52: 20–33.
10. Daeron, M. 1997. Fc receptor biology. Annu. Rev. Immunol. 15: 203–234.
11. Nimmerjahn, F., and J. V. Ravetch. 2006. Fcgamma receptors: old friends and
new family members. Immunity 24: 19–28.
12. Nimmerjahn, F., and J. V. Ravetch. 2008. Fcgamma receptors as regulators of
immune responses. Nat. Rev. Immunol. 8: 34–47.
13. Monteiro, R. C., and J. G. Van De Winkel. 2003. IgA Fc receptors. Annu. Rev.
Immunol. 21: 177–204.
14. Liew, F. Y. 2002. T(H)1 and T(H)2 cells: a historical perspective. Nat. Rev.
Immunol. 2: 55–60.
15. Mills, K. H. 2008. Induction, function and regulation of IL-17-producing T cells.
Eur. J. Immunol. 38: 2636–2649.
16. Codarri, L., G. Gyulveszi, V. Tosevski, L. Hesske, A. Fontana, L. Magnenat,
T. Suter, and B. Becher. 2011. RORgammat drives production of the cytokine
GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation. Nat. Immunol. 12: 560–567.
17. Harizi, H., and N. Gualde. 2004. Eicosanoids: an emerging role in dendritic cell
biology. Arch. Immunol. Ther. Exp. (Warsz.) 52: 1–5.
18. Harizi, H., and N. Gualde. 2005. The impact of eicosanoids on the crosstalk
between innate and adaptive immunity: the key roles of dendritic cells. Tissue
Antigens 65: 507–514.
19. Harizi, H., J. B. Corcuff, and N. Gualde. 2008. Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology. Trends Mol. Med. 14: 461–469.
20. Griseri, T., B. S. McKenzie, C. Schiering, and F. Powrie. 2012. Dysregulated
hematopoietic stem and progenitor cell activity promotes interleukin-23-driven
chronic intestinal inflammation. Immunity 37: 1116–1129.
21. Segura, E., M. Touzot, A. Bohineust, A. Cappuccio, G. Chiocchia, A. Hosmalin,
M. Dalod, V. Soumelis, and S. Amigorena. 2013. Human inflammatory dendritic
cells induce Th17 cell differentiation. Immunity 38: 336–348.
22. Strassburg, K., A. M. Huijbrechts, K. A. Kortekaas, J. H. Lindeman,
T. L. Pedersen, A. Dane, R. Berger, A. Brenkman, T. Hankemeier, J. van
Duynhoven, et al. 2012. Quantitative profiling of oxylipins through comprehensive LC-MS/MS analysis: application in cardiac surgery. Anal. Bioanal.
Chem. 404: 1413–1426.
23. Fanger, N. A., K. Wardwell, L. Shen, T. F. Tedder, and P. M. Guyre. 1996. Type I
(CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells. J. Immunol. 157: 541–548.
24. Banki, Z., L. Kacani, B. Mullauer, D. Wilflingseder, G. Obermoser,
H. Niederegger, H. Schennach, G. M. Sprinzl, N. Sepp, A. Erdei, et al. 2003.
Cross-linking of CD32 induces maturation of human monocyte-derived dendritic
cells via NF-kappa B signaling pathway. J. Immunol. 170: 3963–3970.
25. Sedlik, C., D. Orbach, P. Veron, E. Schweighoffer, F. Colucci, R. Gamberale,
A. Ioan-Facsinay, S. Verbeek, P. Ricciardi-Castagnoli, C. Bonnerot, et al. 2003. A
critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation. J. Immunol. 170: 846–852.
26. Astudillo, A. M., D. Balgoma, M. A. Balboa, and J. Balsinde. 2012. Dynamics of
arachidonic acid mobilization by inflammatory cells. Biochim. Biophys. Acta
1821: 249–256.
27. Stoop, J. W., B. J. Zegers, P. C. Sander, and R. E. Ballieux. 1969. Serum immunoglobulin levels in healthy children and adults. Clin. Exp. Immunol. 4: 101–112.
28. Boruchov, A. M., G. Heller, M. C. Veri, E. Bonvini, J. V. Ravetch, and
J. W. Young. 2005. Activating and inhibitory IgG Fc receptors on human DCs
mediate opposing functions. J. Clin. Invest. 115: 2914–2923.
29. van Beelen, A. J., Z. Zelinkova, E. W. Taanman-Kueter, F. J. Muller,
D. W. Hommes, S. A. Zaat, M. L. Kapsenberg, and E. C. de Jong. 2007. Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to promote interleukin-17 production in human memory T cells. Immunity 27: 660–669.
30. Perussia, B., E. T. Dayton, R. Lazarus, V. Fanning, and G. Trinchieri. 1983.
Immune interferon induces the receptor for monomeric IgG1 on human monocytic and myeloid cells. J. Exp. Med. 158: 1092–1113.
31. Geissmann, F., P. Launay, B. Pasquier, Y. Lepelletier, M. Leborgne, A. Lehuen,
N. Brousse, and R. C. Monteiro. 2001. A subset of human dendritic cells
expresses IgA Fc receptor (CD89), which mediates internalization and activation
upon cross-linking by IgA complexes. J. Immunol. 166: 346–352.
32. Pasquier, B., Y. Lepelletier, C. Baude, O. Hermine, and R. C. Monteiro. 2004.
Differential expression and function of IgA receptors (CD89 and CD71) during
maturation of dendritic cells. J. Leukoc. Biol. 76: 1134–1141.
33. van Egmond, M., A. B. van Spriel, H. Vermeulen, G. Huls, E. van Garderen, and
J. G. van de Winkel. 2001. Enhancement of polymorphonuclear cell-mediated
tumor cell killing on simultaneous engagement of fcgammaRI (CD64) and
fcalphaRI (CD89). Cancer Res. 61: 4055–4060.
34. Otten, M. A., E. Rudolph, M. Dechant, C. W. Tuk, R. M. Reijmers, R. H. Beelen,
J. G. van de Winkel, and M. van Egmond. 2005. Immature neutrophils mediate
tumor cell killing via IgA but not IgG Fc receptors. J. Immunol. 174: 5472–5480.
35. Zou, W., and N. P. Restifo. 2010. T(H)17 cells in tumour immunity and immunotherapy. Nat. Rev. Immunol. 10: 248–256.
36. Gasson, J. C. 1991. Molecular physiology of granulocyte-macrophage colonystimulating factor. Blood 77: 1131–1145.
37. Martinon, F., A. Mayor, and J. Tschopp. 2009. The inflammasomes: guardians of
the body. Annu. Rev. Immunol. 27: 229–265.
38. van de Veerdonk, F. L., M. G. Netea, C. A. Dinarello, and L. A. Joosten. 2011.
Inflammasome activation and IL-1beta and IL-18 processing during infection.
Trends Immunol. 32: 110–116.
39. Martinon, F., L. Agostini, E. Meylan, and J. Tschopp. 2004. Identification of
bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome. Curr. Biol. 14: 1929–1934.
40. Franchi, L., T. Eigenbrod, R. Munoz-Planillo, and G. Nunez. 2009. The
inflammasome: a caspase-1-activation platform that regulates immune responses
and disease pathogenesis. Nat. Immunol. 10: 241–247.
41. den Dunnen, J., L. T. Vogelpoel, T. Wypych, F. J. Muller, L. de Boer,
T. W. Kuijpers, S. A. Zaat, M. L. Kapsenberg, and E. C. de Jong. 2012. IgG
opsonization of bacteria promotes Th17 responses via synergy between TLRs
and FcgammaRIIa in human dendritic cells. Blood 120: 112–121.
42. Ricciotti, E., and G. A. FitzGerald. 2011. Prostaglandins and inflammation.
Arterioscler. Thromb. Vasc. Biol. 31: 986–1000.
43. Fernandez-Morata, J. C., J. Mullol, M. Fuentes, L. Pujols, J. Roca-Ferrer,
M. Perez, A. Xaubet, and C. Picado. 2000. Regulation of cyclooxygenase-1 and
-2 expression in human nasal mucosa. Effects of cytokines and dexamethasone.
Clin. Exp. Allergy 30: 1275–1284.
44. Li, H., J. A. Bradbury, R. T. Dackor, M. L. Edin, J. P. Graves, L. M. DeGraff,
P. M. Wang, C. D. Bortner, S. Maruoka, F. B. Lih, et al. 2011. Cyclooxygenase-2
regulates Th17 cell differentiation during allergic lung inflammation. Am. J.
Respir. Crit. Care Med. 184: 37–49.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
present in the gut (50). The continuous activation of both receptor
classes can result in unremitting neutrophil recruitment, which
contributes to severe tissue damage. More recently, a pathogenic role
for (IL-23–driven) GM-CSF was described in chronic inflammatory
diseases, because GM-CSF production triggered extramedullary
hematopoiesis of granulocyte-monocyte progenitors, resulting in
colitis (20). Furthermore, IL-23–dependent secretion of GM-CSF by
Th cells was shown to evoke experimental autoimmune encephalomyelitis and sustain neuroinflammation (16). Recent data suggest
that a Th17 subset is involved in (anti-)tumor immunity, although the
exact nature of this relationship is not clear (35). In patients with
head and neck squamous cell carcinoma, Th17 cells represented an
important fraction of the tumor-infiltrating lymphocytes in tumor
tissue and tumor-draining lymph nodes, which correlated with impaired proliferation and angiogenesis of head and neck squamous
cell carcinoma (51). Thus, inducing cross-talk during (therapeutic)
antitumor immune responses may boost tumor elimination.
In summary, we provide data that efficient pathogenic elimination during secondary infections induces cross-talk between
MyD88-dependent TLRs and activating FcRs, with subsequent
induction of an essentially different immune response compared
with non-Ab–mediated primary immune responses. This knowledge could be helpful in developing and refining new therapies for
inflammatory (autoimmune) diseases, as well as for Ab-mediated
treatment of cancer.
The Journal of Immunology
45. Chen, H., J. Qin, P. Wei, J. Zhang, Q. Li, L. Fu, S. Li, C. Ma, and B. Cong. 2009.
Effects of leukotriene B4 and prostaglandin E2 on the differentiation of murine
Foxp3+ T regulatory cells and Th17 cells. Prostaglandins Leukot. Essent. Fatty
Acids 80: 195–200.
46. Crooks, S. W., and R. A. Stockley. 1998. Leukotriene B4. Int. J. Biochem. Cell
Biol. 30: 173–178.
47. Paulos, C. M., C. Carpenito, G. Plesa, M. M. Suhoski, A. Varela-Rohena,
T. N. Golovina, R. G. Carroll, J. L. Riley, and C. H. June. 2010. The inducible
costimulator (ICOS) is critical for the development of human T(H)17 cells. Sci.
Transl. Med. 2: 55–78.
11
48. Mantovani, A., M. A. Cassatella, C. Costantini, and S. Jaillon. 2011. Neutrophils
in the activation and regulation of innate and adaptive immunity. Nat. Rev.
Immunol. 11: 519–531.
49. Kolls, J. K., and A. Linden. 2004. Interleukin-17 family members and inflammation. Immunity 21: 467–476.
50. Xavier, R. J., and D. K. Podolsky. 2007. Unravelling the pathogenesis of inflammatory bowel disease. Nature 448: 427–434.
51. Kesselring, R., A. Thiel, R. Pries, T. Trenkle, and B. Wollenberg. 2010. Human
Th17 cells can be induced through head and neck cancer and have a functional
impact on HNSCC development. Br. J. Cancer 103: 1245–1254.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017