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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/imm.12417 This article is protected by copyright. All rights reserved.
Received Date : 23-Jun-2014
Revised Date : 17-Oct-2014
Accepted Date : 19-Oct-2014
Article type : Original Article
Title:
The presence of interleukin-27 during monocyte-derived dendritic cell differentiation
promotes improved antigen processing and stimulation of T cells
Short title:
The influence of IL-27 during DC differentiation
Authors:
Joo-Yong Jungǂ, Lawton L. Roberts*, and Cory M. Robinson§
Author’s affiliations:
Department of Biology, Briar Cliff Universityǂ, Department of Pathology, Microbiology, and
Immunology, University of South Carolina School of Medicine*, Biomedical Sciences
Department, West Virginia School of Osteopathic Medicine§
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§ Corresponding author: Cory M. Robinson
Address: Cory M. Robinson, Ph.D., Biomedical Sciences Department, West Virginia School
of Osteopathic Medicine, 400 North Lee Street, Lewisburg, WV 24901.
Telephone: 304-647-6370
Email: [email protected]
Keywords: Interleukin-27, dendritic cells (DCs), antigen presenting cell
Summary
Dendritic cells (DCs) are potent antigen presentation cells necessary to establish effective
adaptive immune responses. The cytokine environment that exists at the time of DC
differentiation may be an important but often ignored determinant in the phenotypic and
functional properties of DCs. Interleukin (IL)-27 is a unique cytokine that has both
inflammatory and immune suppressive activity. While it can both promote and oppose
activity of different T cell subsets, mostly anti-inflammatory activity has been described
toward macrophages and DCs. However, the specific effect of IL-27 during DC
differentiation and how that may change the nature of the antigen presenting cell has not been
investigated. In this report, we show that IL-27 treatment during monocyte-derived DC
differentiation enhanced the ability to process antigens and stimulate T cell activity. DCs
differentiated in the presence of IL-27 showed enhanced acidification of latex bead-
containing phagosomes that was consistent with elevated expression of vacuolar-ATPases.
This resulted in inhibition of intracellular growth of Staphylococcus aureus. In addition, the
levels of MHC class II surface expression were higher in DCs differentiated in the presence
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of IL-27. IL-12 production was also significantly increased during S. aureus infection of IL-
27-differentiated DCs. The net effect of these activities was enhanced CD4+ T cell
proliferation and Th1 cytokine production. These findings are important to a wide number of
immunological contexts and should be considered in the development of future vaccines.
Introduction
Dendritic cells (DCs) are professional antigen presenting cells that can initiate and modulate
immune responses in a variety of ways. DCs undergo unique developmental stages to
effectively direct antigen specific immune responses (1). In the immature stage, DCs are
specialized in antigen capture and consequently express high levels of intracellular MHC
class II while maintaining high endocytic and phagocytic capacity (2). DCs at this stage
express chemokine receptors such as CCR1, CCR5, and CCR6 that direct them to sites of
inflammation (2). After capturing antigen, DCs upregulate CCR7 that directs migration to
secondary lymphoid organs (3). While migrating, DCs undergo maturation that limits their
ability to capture antigen, while increasing the ability to process and present antigens. DCs at
this stage express high levels of MHC class II at the cell surface as well as additional cell
adhesion and T cell costimulatory molecules such as CD40, ICAM-1 (CD54), LFA-3 (CD58),
CD80, CD83, and CD86 (2). This repertoire of surface molecules on mature DCs promotes
interaction with lymphocytes and antigen presentation through MHC class II that allows for
the selection of antigen-specific lymphocytes (2).
Interleukin (IL)-27 is produced by antigen presenting cells in response to a variety of
activation stimuli, notably microbial-derived products (4-9). IL-27 was originally described
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as a soluble factor that promotes Th1 differentiation (10). However, IL-27 also negatively
regulates these same cells (4, 11). IL-27 is also known to inhibit differentiation of Th17 cells
and production of IL-17 by inducing Tr-1 cells that produce high levels of IL-10 (12, 13).
Similarly, immunosuppressive activity of IL-27 has been described toward a number of other
immune cell types, including B cells and myeloid cells such as macrophages and DCs. (6-9,
14-20). IL-27 is expressed by M. tuberculosis-infected human macrophages (6- 9). Recently,
we published that IL-27 negatively regulates phagosomal acidification by decreasing the
expression of vacuolar ATPases in human macrophages (9, 19). IL-27 treatment of
monocyte-derived mature DCs is immunosuppressive by increasing B7-H1 and decreasing
CD80, CD83, and CD86 (21). B7-H1 is a member of the B7 family that does not interact
with CD28, CTLA-4, or ICOS on T cells and opposes inflammatory T cell activity (22).
Similarly, DCs from IL-27 receptor-deficient mice exhibit enhanced antigen presenting
function that included prolonged expression of CD80, CD86, and enhanced IL-12 production
that increased priming of CD4+ T cells to produce IFN-γ (16).
The cytokine microenvironment during cellular differentiation may significantly
impact the phenotypic and functional characteristics of DCs. Pro-inflammatory properties of
IL-27 toward monocytes have been reported (23, 24). IL-27 promoted enhancement of MHC
class I and II expression in THP-1 cells (23). In primary human monocytes, IL-27 activated
STAT-1-dependent inflammatory genes, augmented TLR responses, and inhibited IL-10
production (25). Here we specifically evaluated the effect of IL-27 on the differentiation of
primary human DCs from monocyte precursors. IL-27-mediated DC differentiation is
inflammatory and increases lysosomal acidification, MHC class II expression, the production
of IL-12, and bacterial control. This is relevant and important in both acute and chronic
infections when monocytes are recruited to sites of inflammation in tissues and subsequently
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receive signals for differentiation. Consequently, these results should be considered in the
development of vaccines and immunotherapies.
Materials and Methods
Cell culture
Human buffy coats were purchased from the New York Blood Center (New York, NY).
Eligible donors were 16 years of age or older, at least 110 pounds, and in good physical
health. The donor samples were anonymous and de-identified. Peripheral blood
mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll gradient centrifugation.
Monocytes were then isolated from PBMCs by Optiprep (Sigma-Aldrich) gradient
centrifugation as described previously (26). Monocytes were adhered to plastic 60 mm
culture dishes in serum-free DMEM. Media was replaced with DC differentiation media
(RPMI supplemented with 2 mM glutamine, 0.1 mM non-essential amino acid, 1 mM sodium
pyruvate, 0.05 mM 2-mercaptoethanol, 25 mM HEPES, recombinant human IL-4 [1600
U/106 cells, Gemini], GM-CSF [290 U/106 cells, Gemini], and 10% human serum) and
incubated at 37°C with 5% CO2 for 7 days. For some cultures, IL-27 was included in DC
differentiation media on day zero. For initial experiments a range of IL-27 concentrations up
to 100 ng/mL was used and it was determined that 40 ng/mL was sufficient for results
reported here. Loosely adherent DCs were removed from the culture dish with PBS that
contained 5 mM EDTA and 4 mg/mL lidocaine. The cells were washed with PBS and plated
onto new culture dishes in culture medium (RPMI supplemented with 2 mM glutamine, 0.1
mM non-essential amino acid, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 25 mM
HEPES, and 10% human serum). These cells are routinely >95% CD11chigh, MHC IIhigh,
CD86+, CD80+, CD1b+, CD40high, DC-SIGN+ (CD 209), and CD14- .
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Bacterial infection and bafilomycin treatment
Staphylococcus aureus RN6390 was kindly provided by Dr. Mark Hart (University of North
Texas Health Science Center). The bacteria were grown overnight in Tryptic Soy broth at
37°C and washed in PBS. The bacteria were adjusted to 1×108 (colony forming units)
CFU/ml using a spectrophotometer (Optical density [OD]600 nm=0.4). DCs were left
untreated or pretreated with bafilomycin (100 nM, Sigma) for four hours to block V-ATPase-
mediated lysosomal acidification as described previously (9). Next, DCs were infected at a
multiplicity of infection (MOI) of ~10 for 1 h. Gentamycin (10 μg/ml) was then added to the
infected cultures to kill extracellular staphylococci and the infection was allowed to proceed
for an additional 2, 12, or 24 h. To enumerate intracellular bacteria, DCs were permeabilized
with a 0.1% solution of saponin in PBS followed by standard serial dilution plating.
Analysis of lysosomal acidification and immunolabeling
Human DCs cultured in 24-well plates were analyzed for the level of lysosomal acidification.
In the last hour of infection, culture supernatants were replaced with medium that contained
Lysotracker DND-99 Red (Life Technologies) (100 nM). The slides were examined using a
Zeiss Meta 510 laser confocal microscope with a plan-Apochromat 63X objective lens. A
total of 10 fields containing 5-10 DCs per field were examined in each experiment. The
mean fluorescent intensity (MFI) for each DC was calculated using Image J software. Each
cell from the image was selected and histogram analysis was performed. For immunostaining,
mouse monoclonal antibodies for V1-ATPase H (sc-166227, Santa Cruz Biotechnology)
were visualized with anti-mouse-Alexafluor 568-conjugated secondary antibody.
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Quantitative PCR
Human DCs (1.5x105/well) cultured in 24-well dishes were subjected to RNA isolation. At
appropriate time points, the media was removed from cultures, the cells were lysed with
PureZol® (Bio-Rad), and RNA was isolated according to commercial product protocol. First
strand cDNA synthesis was performed using iScript™ cDNA synthesis reagents (Bio-Rad)
according to protocol. Primers were synthesized by Integrated DNA Technologies, Inc. The
following primer sets were used for amplification of HLA-DR or IL-12 transcripts with
SsoFast™ EvaGreen® supermix (Bio-Rad): IL-12 p35 forward; 5’-atgctccagaaggccagac-3’
reverse; 5’-tctggaatttaggcaactctca-3’ IL-12 p40 forward; cctggagaaatggtggtcct-3’ reverse; 5’-
gcttagaacctcgcctcctt-3’ HLA-DR forward; 5’-agcagtcatcttcagcat-3’ reverse; 5’-
atgttagagtacggagcaat-3’ GAPDH forward; 5’-cagccgcatcttcttttg-3’ reverse; 5’-
gcaacaatatccactttacca-3’. Gene expression was normalized to that of GAPDH, expressed
relative to untreated controls using the 2-ΔΔCt method, and log2 transformed.
Immunoblot analysis
Whole cell lysates were prepared from human DCs (1.5x105/well) cultured in 24-well dishes.
Some of the cultures were infected with S. aureus as described above. PBS supplemented
with 1% Tx-100 (40 µl) was applied to each sample and lysates collected by scraping. They
were subsequently sonicated briefly and then stored at 4°C. Equal amounts of cell lysates
were separated on SDS-PAGE gels and transferred to nitrocellulose by standard techniques.
Primary antibodies for V-ATPase H, actin, or all forms of cathepsin D were revealed with
horse radish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies. ECL
substrate (Amersham Biosciences) was applied to visualize proteins.
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ELISA analysis
Human DCs were cultivated as indicated above. Following the indicated treatment,
supernatants were collected at indicated time points for analysis of IL-12p70 (R&D Systems),
IL-2 (Ebioscience), or IFN-γ (R&D Systems) concentrations. Standard curves were
performed in parallel.
Flow Cytometry
DCs were harvested and phenotypic analysis performed as previously described (27). Briefly,
DCs were suspended in 0.1 mL of staining buffer (0.5% BSA in PBS) and Fc receptor
blocked for 20 min. Next, cells were immunolabeled with monoclonal antibodies against the
indicated molecules for 30 min and subsequently fixed with 0.5 mL 4% PBS-buffered
paraformaldehyde. At least 5,000 cells were analyzed for each sample on a FC 500
(Beckman Coulter) flow cytometer.
T cell proliferation assays
Monocyte-derived DCs differentiated in the presence or absence of IL-27 were harvested (as
above) and seeded (5 x 104) on a 96-well plate overnight in culture medium. The next day,
DCs were infected with an overnight culture of S. aureus at a MOI ~ 10 for 1 h. The media
was then removed and replaced with medium containing gentamycin (10 μg/mL) for 3 h.
Next, CD4+ T-cells were isolated from monocyte-depleted fractions by immunomagnetic
selection using a human CD4+ T cell isolation kit (Miltenyi Biotec) according to the
instructions provided by the manufacturer. The resulting CD4+ T cells were stained with
CFSE (10 μM, Biolegend) in culture medium for 10 min at room temperature. Staining was
quenched by washing once with 5 mL of room temperature FBS. Next, 2 x 105 CFSE-stained
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T cells in culture medium were antibody stimulated with 10 μg/mL of plate-bound anti-CD3
(BD Pharmingen) and 1 μg/mL soluble anti-CD28 (BD Pharmingen), left unstimulated, or
cultured with syngeneic DCs. Cells were incubated at 37o C with 5% CO2 for 5 days.
Aliquots for analysis of secreted IL-2 and IFN-γ were collected on days 1 and 5 of five
independent experiments done with separate donors. In addition, on day 5 of three
independent experiments described above, the cells were harvested and analyzed by flow
cytometry as above. T cell proliferation was determined by CFSE dilution. CFSE-stained T
cells were distinguishable from DCs. To exclude DCs from the analysis, a gate was then
established for the population that represented CFSE-stained T cells (dot plot quadrant A4,
Fig. 5 A and C). To quantify the percent T cell proliferation, we established another gate
(histogram region E, see Fig. 5 panels D-G) using the antibody controls (Fig. 5D and E) that
represents CFSE dilution indicative of proliferation. Accordingly, the percentage of gated T
cells within region E was quantified. Ten-thousand cells were analyzed for each duplicate
treatment.
Results
DCs differentiated in the presence of IL-27 exhibit enhanced phagosomal acidification
IL-27 exhibits anti-inflammatory activity towards established macrophages and DCs (4, 19).
However, inflammatory activity has been associated with IL-27 toward monocytes. Since
macrophages and DCs can differentiate from monocyte precursors, we wanted to understand
if the presence of IL-27 during differentiation from a monocyte would lead to development of
a suppressive or inflammatory phenotype. To address this, monocytes were cultured with or
without IL-27 during 7 days of differentiation to DCs. An important function of DC biology
is the uptake of antigen and processing for presentation to lymphocytes. After 7 days of
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differentiation, the DCs were subjected to treatment with fluorescently labeled latex beads (2
μm) for 6 h. We did not observe a difference in the uptake of latex beads by DCs
differentiated in the presence or absence of IL-27 (not shown). Next, we evaluated the acidic
nature of the compartment surrounding the latex beads that is important for antigen
processing. Following uptake of latex beads, lysotracker (100 nM) was added to the DC
cultures that were subsequently incubated for an additional 2 h. Surprisingly, DCs
differentiated in the presence of IL-27 demonstrated enhanced acidification of the latex bead
compartment (LBC) compared to DCs differentiated without IL-27 (Fig. 1A). The level of
increased acidification was approximately 1.7 fold (Fig. 1B). Phagosomal acidification is
mediated by the expression and localization of V-ATPases that allow for a decrease in pH
from 6.5 to 5.0 (19). This acidic environment inhibits the growth of microorganisms and
further enhances the recruitment and activity of hydrolytic enzymes allowing for enhanced
antigen processing. Therefore, we examined V-ATPase localization (Fig. 1C). V-ATPase
was strongly recruited to the LBC in DCs differentiated with IL-27 and the percent
colocalization was approximately two–fold greater (Fig. 1C and D). This is likely to be
mediated, at least in part, by the increased expression of V-ATPases since signal intensity of
V-ATPases was dramatically increased by 6 h (Fig. 1C, and E). This was further
substantiated by immunoblot analysis in which V-ATPase expression was increased in DCs
that received IL-27 by approximately 1.5-fold (Fig. 1F and G). Cathepsin D is a soluble
lysosomal endopeptidase synthesized in the endoplasmic reticulum as pre-procathepsin D and
following the removal of the signal peptide, the 52 kDa procathepsin D is localized to
endosomes, phagosomes, or lysosomes (28). Phagosomal acidification allows for cleavage of
the amino-terminal propeptide yielding a 48 kDa single chain active enzyme that can undergo
additional cleavage to generate a mature lysosomal protease (24 to 30 kDa) (28). We
investigated whether or not there was a difference in processing of cathepsin D to the mature
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form in DCs differentiated in the presence of IL-27. Immunoblot analysis using an antibody
that recognizes all three forms of cathepsin D, demonstrated that DCs differentiated in the
presence of IL-27 exhibited increased accumulation of the mature form of Cathepsin D (Fig.
1H). Overall, this data suggests that the presence of IL-27 during DC differentiation increases
the potential to process antigen.
DC differentiation in the presence of IL-27 promotes bacterial clearance in human DCs
Acidification of phagosomes and activation of cathepsin D is critical for DC-mediated control
of bacterial pathogens (29, 30). Since DCs differentiated with IL-27 exhibited increased
acidification of phagosomes and accumulation of active cathepsin D, we hypothesized that
these DCs may clear bacteria more efficiently. To address this possibility, we infected DCs
with S. aureus and enumerated survival. This bacterium was chosen for two reasons.
Recently it has been shown that S. aureus is capable of surviving and multiplying within DCs
(31). Secondly, because this bacterium is a common commensal, the pool of blood donors
used in our studies likely contains circulating lymphocytes with staphylococcal antigen
specificity important for later experiments. To directly address the requirement for V-
ATPase-mediated lysosomal acidification on bacterial clearance, DCs differentiated in the
presence or absence of IL-27 were pretreated with or without bafilomycin (100 nM) for 4 h
and then infected with S. aureus (MOI=10). After 1 h, gentamycin (10 µg/ml) was added to
kill extracellular bacteria that were not internalized. The cultures were incubated for an
additional 2, 12, or 24 h. Consistent with the enhanced lysosomal acidification and cathepsin
D activity, bacteria were cleared by 12 h in DCs differentiated in the presence of IL-27 (Fig.
2). This bacterial clearance was inhibited by bafilomycin treatment demonstrating a
dependence on lysosomal acidification. This data further suggests an influence of IL-27
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during the differentiation of monocyte-derived DCs that promotes antibacterial activity and
antigen processing capability.
DC differentiation in the presence of IL-27 enhances expression of MHC class II
Since phagosomal acidification that is important for antigen processing was more efficient in
DCs differentiated in the presence of IL-27, we wanted to investigate whether or not these
DCs would be better antigen presenters. To address this, we investigated the cell surface
expression of HLA-DR in DCs differentiated in the presence and absence of IL-27. HLA-DR
was more highly expressed at the cell surface in DCs differentiated with IL-27 (Fig. 3A). S.
aureus infection further increased surface levels on both DC treatment groups, but a similar
trend was maintained (Fig. 3A). Quantitative analysis of mean fluorescent intensity (MFI)
showed an approximate two-fold increase of HLA-DR surface expression on DCs
differentiated in the presence of IL-27 (Fig. 3B). This result was at least partly influenced by
enhanced HLA-DR gene expression in DCs differentiated in the presence of IL-27 (Fig. 3C).
Dendritic cells differentiated in the presence of IL-27 upregulate cell surface molecules
that facilitate activation of T cells.
Since we observed an increase in MHC class II expression when DCs were differentiated in
the presence of IL-27, we wanted to determine if the presence of IL-27 would also augment
expression of cell surface molecules known to facilitate activation of lymphocytes. To do
this, DCs were differentiated as before, harvested, and immunolabeled for a number of cell
surface molecules, and analyzed by flow cytometry. There was no significant labeling with
isotype control antibodies; this was not altered by IL-27 treatment. Consistent with increased
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MHC class II expression, numerous surface molecules were upregulated by the addition of
IL-27 (Table 1). Specifically, we observed an approximate 50% and 37% increase in the
MFI of DC-SIGN and CD40, respectively (Table 1). ICAM-1 (CD54) expression was
increased although this did not achieve statistical significance (Table 1). Additionally, the
integrin subunits CD18 and CD11b (collectively Mac-1; CR3) were also significantly
increased at the cell surface in the presence of IL-27 over four independent blood donors
(Table 1). The expression of T cell costimulatory molecule CD86 was unaffected by the
addition of IL-27 (data not shown).
Interleukin (IL)-12 production was increased in DCs differentiated in the presence of
IL-27
IL-12 production by DCs is important for effective initiation of Th1 responses (32). DCs
infected by S. aureus secrete IL-12 (31). Since we observed enhanced lysosomal
acidification, MHC class II expression, and surface levels of molecules important for
interactions with lymphocytes, we examined IL-12 production in DCs differentiated in the
presence or absence of IL-27. To address this, DCs were infected with S. aureus for 24 h and
then RNA was harvested for analysis of IL-12p35 and p40 gene expression. IL-12 p35 and
p40 gene was highly expressed in infected DCs that received IL-27 (Fig. 4A). However,
secreted IL-12p70 was only detected in culture supernatants of S. aureus-infected DCs that
were differentiated in the presence of IL-27 (Fig. 4B). We also measured levels of secreted
IL-23 that incorporates the p40 subunit, as well as IL-6, but did not observe significant
changes between treatment groups (not shown). Collectively, this data further support the
idea that DCs differentiated in the presence of IL-27 may be stronger APCs.
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Dendritic cells differentiated in the presence of IL-27 are more potent stimulators of T
cell activity
Given the augmentation of MHC class II, numerous cell surface molecules involved in T cell
interactions, and enhanced IL-12 production by the addition of IL-27 during DC
differentiation, we wanted to test the functional impact on the ability to stimulate T cells.
This was done with T cell proliferation assays that incorporated syngeneic, primary human T
cells and DCs. Considering common exposure to S. aureus in our donor pool, it is reasonable
to assume the presence of circulating antigen-specific T cells. CD4+ T cells were CFSE-
stained and co-cultured with DCs for 5 days. T cell proliferation was determined by analysis
of CFSE dilution by flow cytometry. CFSE-stained T cells were distinguished from DCs on
the basis of CFSE staining (Fig. 5 A-E). Thus, to exclude DCs from the analysis and to
quantify T cell proliferation, we gated on the CFSE-stained T cell quadrant (quadrant A4, Fig.
5C) to create histograms that express the level of T cell proliferation (Fig. 5 D-G). T cells
were either left untreated, stimulated with anti-CD3 and anti-CD28 (Ab stimulated), or
cultured with S. aureus-infected DCs that were differentiated in the absence (Med DC, Fig.
5F) or presence (IL-27 DC, Fig. 5G) of IL-27. As shown in Figure 5E, antibody stimulation
of T cells resulted in several proliferative peaks (region E), and this region was used to
quantify percent T cell proliferation. This analysis demonstrated that DCs differentiated in
the presence of IL-27 and then infected with S. aureus stimulated a 5% increase in T cell
proliferation compared to DCs not differentiated with IL- 27 (Fig. 5 F-H). This result was
consistent over three independent blood donors and reached statistical significance (p= 0.023).
Even more striking was the difference in secreted cytokine levels. T cells stimulated by
infected DCs differentiated in the presence of IL-27 increased the early production of IL-2
(Fig. 5I) and IFN-γ (Fig. 5J) over time compared to normally differentiated DCs. IL-2 levels
were increased significantly at day 1 (Fig. 5K; p= 0.035 over 5 independent donors.
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Similarly, IFN-γ secretion elicited by IL-27 differentiated DCs was significantly increased at
day 5 (Fig. 5L; p= 0.04) over 5 independent donors. The early consumption of IL-2 during
proliferative events is inversely correlated with enhanced IFN-γ production. The kinetics of
this reciprocal cytokine pattern is consistent with enhanced T cell stimulation, and further
substantiates that monocyte-derived DCs differentiated in the presence of IL-27 are stronger
stimulators of T cell activity.
Discussion
The cytokine milieu is an essential determinant of effective immunity as it shapes the
development of cellular phenotypes and the nature of the response. Consequently, we wanted
to determine the impact of IL-27 on monocyte-derived DC differentiation. Since monocytes
are recruited to sites of inflammation where IL-27 is produced, this is a relevant and
important consideration in the development of DCs that are essential in the establishment and
orchestration of immune responses. To the best of our knowledge, we are the first to
examine the effects of IL-27 during the differentiation of monocyte-derived DCs. IL-27 is
known to possess dual immunological functions (11). It is well established that IL-27 exerts
anti-inflammatory activity towards macrophages (4, 6, 7, 8, 9, 11, 19). Several reports have
documented anti-inflammatory properties of IL-27 on terminally differentiated DCs, both
murine and human (16, 21). In murine DCs, using allogeneic T cell proliferation assays,
Wang found that DCs deficient for the IL-27 receptor α chain (IL-27Rα) induced greater
levels of IFN-γ and proliferation of CD4+ T cells compared to wild type DCs (16). However,
they saw no differences in IL-2 production. This suggests that IL-27R signaling suppresses
DC function as an APC. In another report, Karakhvona and colleagues found that treatment
of human, monocyte-derived DCs with IL-27 induced expression of B7-H1 and reduced their
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ability to stimulate allogeneic T cells (21). Similar to our findings, they also found no effect
of IL-27 on the T cell costimulatory molecules CD80 and CD86. In contrast, we report a
response to IL-27 during differentiation that drives monocyte-derived DCs to be more potent
antigen processing, presenting, and T cell-stimulating cells.
The acidic nature of the phagosome is important for antigen processing and can
generally reflect a suppressive or inflammatory phenotype of antigen presenting cells. The
LBC is a suitable model to study phagosomal acidification that is important for antigen
processing. We previously showed that treatment of terminally differentiated macrophages
with IL-27 significantly decreased acidification of the LBC (19). This de-acidification
correlated with reduced expression of V-ATPases and limited maturation of cathepsin D to
the active form (19). Surprisingly, in DCs differentiated in the presence of IL-27, the LBC
was significantly more acidified within 6 h. This acidification was consistent with elevated
expression of V-ATPases (Fig. 1). The number of S. aureus recovered from DCs was
significantly decreased in those that received IL-27 (Fig. 2), and this was reversed by treating
DCs with bafilomycin (a V-ATPase inhibitor). This suggests that the bacteria were internally
processed via the phagolysosomal pathway, and also demonstrates that IL-27 enhanced DC-
mediated clearance of bacteria and the cellular machinery necessary to process antigens.
These results are in contrast to the effects of IL-27 on mature macrophages that we have
observed and reported (9, 19). However, when macrophages were differentiated from
monocytes in the presence of IL-27, we did not observe an influence on phagosomal
acidification (data not shown). These studies suggest that the timing of IL-27 signaling is
important to the functionality of the cell, but also continue to emphasize the divergent nature
of IL-27 toward various cell types (11). That both macrophages and DCs can develop from
monocyte precursors suggests that the nature of the differentiation signal and interaction with
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IL-27 is important. Collectively, our data demonstrate that IL-27 elicits differential effects on
different cell types, and influences the cellular outcomes following differentiation.
In addition to the antigen processing capability, MHC class II expression on the cell
surface reflective of antigen presentation was increased in DCs differentiated in the presence
of IL-27 (Fig. 3). This is consistent with the reported proinflammatory nature of IL-27
toward monocytes that included increases in MHC class I and II and costimulatory molecules
(23, 24). In this report, we specifically demonstrate that when IL-27 is added during the
differentiation of monocytes to DCs, there is an increase in cell surface molecules that
mediate important DC biological functions, particularly T cell activation (Fig. 4).
Recognition of DC presented antigen to T cells requires the migration of DCs from antigen
capturing regions through lymphatic endothelia to draining lymph nodes, followed by the
formation of an immunological synapse; both of which are carefully orchestrated by
numerous cell-cell interactions. Of the molecules facilitating these processes, DC-SIGN has
been shown to mediate DC adhesion, antigen uptake, and activation of resting T cells (33-35).
In a similar body of work, anti-DC-SIGN antibodies inhibited DC-induced proliferation of
resting T cells (34). This suggests a fundamental role for DC-SIGN in mediating and
establishing DC-induced immune responses. In line with this, we show a significant increase
in cell surface expression of DC-SIGN on DCs differentiated in the presence of IL-27.
Engering and colleagues have reported that DC-SIGN also functions as an antigen receptor
that efficiently targets internalized antigens to lysosomes for processing and presentation to T
cells (35). We do not specifically present data to address this link with antigen processing,
but increased expression of DC-SIGN may function similarly in our system. We also
observed a significant increase in surface levels of CD40 and Mac-1 (CD18/CD11b; CR3) on
DCs differentiated in the presence of IL-27 (Table. 1). Ligation of CD40 on DCs by its
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cognate receptor CD40L primes DCs to become more efficient APCs. Several groups have
shown that CD40:CD40L interaction favors upregulation of adhesion and costimulatory
molecules by DCs with enhanced cytokine production, including IL-12 (36, 37). The integrin
Mac-1 functions as a cell adhesion receptor and in phagocytosis of opsonized and non-
opsonized antigens, respectively (38). Concomitant with increased expression of surface
molecules that contribute to APC function (Table 1), enhanced IL-12 production (Fig. 4)
suggests an important effect of IL-27 on DC differentiation that ultimately manifests as an
APC phenotype better suited to prime T cells and promote IFN-γ production. We also
examined levels of IL-23 that shares a common p40 subunit with IL-12, as well as IL-6, and
found no difference between DCs differentiated in the presence or absence of IL-27 (not
shown). To determine the functional significance of IL-27 on DC differentiation, we tested
whether these DCs would exhibit an enhanced ability to stimulate T cell proliferation using
syngeneic T cell proliferation assays. As expected, IL-27 differentiated DCs were more
potent T cell stimulators, as determined by CFSE dilution and Th1 cytokine production (Fig.
5) in response to S. aureus infection. We report a significant increase (p= 0.023) in T cell
proliferation in the presence of DCs that were differentiated in the presence of IL-27.
Although it was expected that the donor pool would have S. aureus-responsive T cells, the
level of proliferation was unexpected and it is unlikely that all the T cell proliferation
reflected in these assays is S. aureus-specific. Certainly some antigens may be conserved
amongst a wide range of bacteria. In addition, there may be some non-specific activation, as
well as death of T cells not receiving activation signals over the five day period which could
diminish the non-proliferative fraction. Enhanced T cell proliferation was further supported
by enhanced IL-2 production in the initial days of the response and a greater than two-fold
increase in IFN-γ by day 5. Although we do not rule out a role for the influence of IL-27 on
DC differentiation having an effect on Th17 cell differentiation and function, the cytokine
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profile suggests a Th1 cell promoting activity of IL-27. Collectively, our data suggests that
IL-27 facilitates a DC phenotype capable of enhanced T cell stimulation. These mechanisms
and the major findings of this report are summarized in Figure 6. In conclusion, our findings
may have important implications for designing DC-based therapeutics and in a variety of
immunological scenarios that concern induction of adaptive immunity such as vaccination.
Acknowledgements
This work was supported by institutional funds supplied by the University of South Carolina
School of Medicine and NIH grant HL093300. Experimental results reported in this
manuscript were collected when the lead and corresponding authors were affiliated with USC.
Subsequent data analysis and manuscript preparation were also performed at Briar Cliff
University and the West Virginia School of Osteopathic Medicine.
Authorship
JYJ and LLR contributed to the design of experiments and were responsible for the
performance of experiments, data analysis and manuscript preparation. CMR and JYJ
conceived the study idea and designed experiments. CMR contributed equally to the
preparation of the manuscript.
Disclosures
The authors declare no conflict of interest.
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Table 1. Monocyte-derived DCs differentiated with IL-27 increase expression of cell surface molecules that facilitate adhesion to and activation of T-cells.
Cell surface molecule
Meda + IL-27a % increase P-Valueb
DC-SIGN 30.53±0.43 45.6±3.93 49.4 <0.05 ICAM-1 81±19.54 107.54±32.82 32.6 >0.05 CD40 31.18±2.66 42.6±4.67 36.6 <0.05 CD11b 322.3±35.39 482.8±71.18 49.8 <0.05 CD18 834.3±98.16 1062±50.02 27.3 <0.05 aHuman monocytes were differentiated to DCs without the addition of IL-27 (Med) or 50 ng/ml of IL-27 (+ IL-27) for 7 days. The mean fluorescent intensity (MFI) is reported ± standard error for four independent blood donors.
bStatistical significance was determined by a Student’s t-test.
Figure legends
Figure 1. DCs were differentiated in the presence or absence of IL-27 as indicated. (A-E)
Cells were subjected to yellow-green fluorescent labeled latex beads for 6 h. (A) Acidified
lysosomes (red) were stained with Lysotracker (200 nM) following latex bead uptake. The
images shown are from an individual experiment representative of three. (B) The MFI
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obtained from untreated or bead-treated DCs differentiated without IL-27 was set to 1.
Results obtained in the presence of IL-27 were expressed relative to this value. The values
presented here are combined results from three independent experiments. (C) DCs were
stained with anti-V-ATPase H antibody as described. Labeled proteins were detected with
anti-mouse IgG conjugated with Alexa-568 (red). Representative images from three
experiments are shown. (D) Percent colocalization was obtained as described in the Methods
section. The values presented here are combined results from three independent experiments.
(E) MFI was obtained as described in the Methods section. The values presented here are
combined results from three independent experiments. (F) Cell lysates were prepared from
DCs differentiated with or without IL-27 for immunoblot analysis of V-ATPase expression.
An image representative of four experiments is shown. (G) The ratio of V-ATPase/actin
band intensity was expressed relative to medium alone for four combined experiments. (B, D,
E, G) A Student’s t test was used to compare ratios from the IL-27-treated group with the
control (MED) at each time point in the 95% confidence interval. (H) Cell lysates were
prepared from DCs differentiated with or without IL-27 for immunoblot analysis of cathepsin
D. Pro; procathepsin D (52kDa), Pre; Precathepsin D (48kDa), mature; mature cathepsin D
(approximately 24 kDa). An image representative of three experiments is shown.
Figure 2. DC differentiation in the presence of IL-27 promotes clearance of Staphylococcus
aureus. DCs were either differentiated with or without IL-27 as indicated. DCs were then
left untreated (Med, IL-27) or pretreated with bafilomycin (100 nM) for four hours, and then
infected with S. aureus for 1 h. Next, gentamycin was added and the DCs were incubated for
an additional 2, 12, or 24 h. Data are represented as the mean CFU recovered from infected
DCs ± SE. Results representative of two independent experiments are shown. Statistical
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significance was determined by a two-way ANOVA and Tukey’s post hoc test for multiple
comparisons. * indicates that the IL-27 group is different from all other treatments; p< 0.05.
Figure 3. HLA-DR expression is enhanced in DCs differentiated in the presence of IL-27.
DCs were differentiated in the presence or absence of IL-27 as indicated. DCs were then
infected with S. aureus for 1 h, treated with gentamycin then incubated an additional 23 h.
(A) Surface labeling of HLA-DR was performed as indicated in the Methods section.
Surface-labeled DCs were analyzed by flow cytometry and representative dot plots are shown.
(B) Quantitative analysis of mean fluorescent intensity (MFI) from of three independent
experiments done with separate blood donors is shown. (C) Quantitative analysis of HLA-DR
gene expression is presented as mean relative change in gene expression of duplicate samples
± SE for two combined experiments. Gene expression was normalized to that of GAPDH and
expressed relative to untreated controls. (B, C)* indicates p<0.001.
Figure 4. DCs differentiated in the presence of IL-27 increased production of IL-12 during S.
aureus infection. DCs were either differentiated with or without IL-27. DCs were then
infected with S. aureus for 1 h, treated with gentamycin and incubated an additional 23 h. (A)
Quantitative analysis of IL-12 p35 and p40 gene expression is presented as mean relative
change in gene expression of duplicate samples ± SE for three combined experiments. Gene
expression was normalized to that of GAPDH and expressed relative to untreated controls. (B)
Supernatants were analyzed for secreted IL-12 p70 by ELISA. Data combining three
independent experiments are shown. * indicates p<0.001.
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Figure 5. DCs differentiated in the presence of IL-27 stimulate enhanced T cell activity.
CFSE-stained T cells are distinguishable from DCs (A-C). Therefore, the CFSE-stained T
cell quadrant (A4) was gated on to exclude DCs from all subsequent analyses. To establish a
T cell proliferative region via CFSE dilution, T cells alone were either left unstimulated (D)
or antibody stimulated (Ab stimulated, E) with plate-bound anti-CD3 and soluble anti-CD28.
Region E (D-G) was then used to enumerate T cell proliferation. DCs differentiated in the
presence (IL-27 DC, G) or absence of IL-27 (Med DC, F), were co-cultured with syngeneic
CD4+ T cells for 5 days. Ten-thousand cells were analyzed for each T cell proliferation assay,
and each assay was performed in duplicate per donor. One representative donor is shown,
and this was repeated with three independent blood donors showing similar results (A-E).
Region E was then used to quantify percent T cell proliferation for three independent blood
donors (H). Statistical significance was determined by a Student’s t-test (H). (I-L)
Supernatants from T cell proliferation assays were analyzed for the production of IL-2 (I, K)
and IFN-γ (J, L). Data from a representative donor time course (I, J), or five combined
donors on day 1 (K) or day 5 (L) are shown. Statistical significance was determined by a
repeated measures paired t test.
Figure 6. Hypothetical model. Exposure to IL-27 during DC differentiation leads to a
number of functional changes. Increased lysosomal acidification is mediated by a greater
expression of V-ATPase and promotes improved antigen processing. MHC class II
expression is increased allowing for enhanced presentation of antigen to T cells. DC-SIGN,
CD40, and MAC-1 surface expression is increased strengthening the interaction with T cells.
These changes combined with augmented IL-12 production enhance T cell proliferation and
effector cytokine production.
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