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C-type lectin signaling in dendritic cells: molecular control of antifungal inflammation
Wevers, B.A.
Link to publication
Citation for published version (APA):Wevers, B. A. (2014). C-type lectin signaling in dendritic cells: molecular control of antifungal inflammation.
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Download date: 02 Jan 2021
Min
ty cle
an
Pr
om dress
after blue berry pie
four.
116 Chapter four
Mincle amplifies Th17 immunity via Mdm2-dependent modulation of cytokine transcription
four.
Brigitte A. Wevers1, Tanja M.
Kaptein1, Bart Theelen2, Teun
Boekhout2, Teunis B.H.
Geijtenbeek1 and Sonja I.
Gringhuis1
1Department of Experimental Immunology, Academic Medical
Center, University of Amsterdam, Amsterdam, NL.2Centraal bureau voor schimmelcultures, Utrecht, NL.
Manuscript submitted
Immunity conferred by T helper 1 (Th1) and IL-17-
producing Th17 subsets protects from fungal infection,
yet their activation by dendritic cells (DCs) must be
stringently controlled to avoid host tissue damage
and Th17-driven inflammatory disease. Here, we
demonstrate that fungal-sensing C-type lectin receptor
mincle amplifies DC-driven Th17 immunity through
differential modulation of cytokine responses induced
by other receptors. Triggering of mincle signaling, via Syk
and the CARD9-Bcl-10-MALT1 scaffold, synergistically
enhanced Th17-promoting cytokines IL-1β and IL-23p19,
but in parallel decreased Th17-suppresive cytokine
IL-27. We show that mincle controls transcription
of these genes through a novel, NF-κB-independent
mechanism, via the PI(3)K-PKB-Mdm2 pathway. Notably,
mincle-induced IL-27p28 mRNA suppression involved
the proteasomal degradation of transcription factor
Mincle amplifies Th17 immunity via Mdm2 117
fou
r.
IRF1; we identified Trim28 as an adaptor
protein facilitating nuclear Mdm2-IRF1
interactions, thereby relaying mincle-
Mdm2 signaling to IRF1 degradation
and suppression of IL-27p28 mRNA. PKB
and Mdm2 accounted for the mincle-
dependent regulation of Th17 immunity to
pathogenic fungi, most notably Malassezia
species, which play an exacerbating role
in Th17-associated skin disorder psoriasis.
Thus, our study establishes the Syk-PKB-
Mdm2 axis as an essential component of
the mincle-driven inflammatory response,
which could provide novel target
molecules for therapeutic intervention
in settings of antifungal inflammation
and Th17-mediated immunopathology.
AUTHOR SUMMARY
Opportunistic and virulent fungi are an emerging threat for human health. Antigen-
presenting dendritic cells are amongst the first immune cells that sense invading fun-
gal microbes and activate T helper lymphocyte populations for induction of protective
immune defense mechanisms, hence fungal elimination. For this, dendritic cells rely on
the concerted actions of pathogen recognition receptors, such as Toll-like receptors and
C-type lectin receptors. However, we are at the initial stages of understanding the complex
molecular events that allow these receptors to determine the nature and extent of T helper
activation; increased knowledge of the mechanisms behind induction of antifungal immu-
nity will greatly contribute to development of novel treatment strategies. In this study we
have uncovered a specialized or ‘modulating’ role for C-type lectin mincle upon infection
by pathogenic fungi. We have unraveled molecular processes by which mincle strongly
promotes the activation of the distinct T helper 17 subset. Further functional exploration
revealed that mincle, through this transcriptional program, has an important role in the
immune response to pathogenic Malassezia spp. Thus, we propose that pharmacological
targeting of mincle responses might be a prime strategy for treating fungal diseases.
Min
cle
am
plifi
es
Th17
imm
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via
Md
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118 Chapter four
INTRODUCTION
The human body is under continuous attack by various fungal microbes. Opportunistic
fungi pose a serious threat to immunocompromised patients1, whereas emerging virulent
strains have caused considerable morbidity and mortality in healthy individuals in most
recent years2,3, highlighting the need for new treatment approaches. By providing specificity
and memory, a T helper (Th) cell-mediated immune response is of fundamental importance
for host immunity to fungal infection. Protective antifungal immune mechanisms depend
on the activation of both Th type 1 (Th1) cells, phenotypically characterized by production
of IFN-γ, and IL-17-producing Th17 effector cell subsets4,5. Together, Th1 and Th17 effector
populations, among many other pleiotropic and/or tissue-specific functions, ensure optimal
activation of neutrophils, well-established immune effectors controlling fungal microbe
elimination6: whereas Th17 effector molecule IL-17 acts on neutrophil recruitment and
function7-9, IFN-γ confers direct activation of phagocytic effector function of neutrophils and
other phagocytes10. While being essential for host protection from fungal infection, activation
of the Th17 axis must be tightly regulated: exaggerated Th17 responses often accompanies
host tissue damage and play a major role in the onset of autoimmune diseases7,11.
Instruction of Th cell differentiation is driven by dendritic cells (DCs), which rely on
a fixed repertoire of germ-line encoded pattern-recognition receptors (PRRs), notably
C-type lectin receptors (CLRs), Toll-like receptors (TLRs) and Nod-like receptors (NLRs), for
sensing and processing of microbial products for T cell priming12. Importantly, numerous
PRRs propagate intracellular signaling upon activation; by directing a specific program of
gene expression, these receptor-mediated signals facilitate secretion of effector cytokines,
allowing DCs to polarize an ensuing T helper response13. With Th1 lineage commitment
depending on interleukin 12 (IL-12)14, human Th17 differentiation seems optimal in the
presence of DC-derived IL-6, IL-1β and IL-23. However, regulation of Th immunity extends
beyond initiation and maintenance; to prevent progression towards chronic and host
tissue-destructing inflammation, DCs quench immune effector responses via production
of anti-inflammatory cytokines15.
Figure 1. Mincle promotes human DC-induced Th17 immunity. (a-h) T helper polarization was assessed
by flow cytometry analysis (FI, fluorescence intensity) by staining for intracellular IFN-γ (Th1) and IL-17A
(Th17) expression at day 12-17 after PMA plus ionomycin restimulation (a,b,d,e,g,h) or by ELISA by mea-
suring IL-17 production in supernatants at day 5 (c,f), after coculture of memory CD4+ T cells with DCs
that were left unstimulated (iDC) or primed with curdlan, Candida albicans, Fonsecaea monophora, LPS
and/or TDB, or after mincle silencing. In (b,e,h), the percentages of IL17A-producing T cells are shown,
corresponding to the upper left and right quadrants of (a,d,g). Data are representative of at least two (a-f),
three (g,h) independent experiments (mean and s.d. of duplo measurements in b,c,e,f).
A
Mincle amplifies Th17 immunity via Mdm2 119
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14.9
34.1
12.5
38.5
F. mono
14.1
34.2
14.0
37.7
C. albicans
17.1
30.2
18.5
34.1
Curdlan
IFN-γ (FI)
4.85
69.6
2.5
23.1
IL-1
7 (F
I)
iDC
IFN-γ (FI)
100 101 102 103 104 105100
101
102
103
104
1058.35
42.1
13.5
36.0
F. mono
0
10
20
30
40
50
IL-1
7 (%
pos
itive
)0
10
20
30
40
50
IL-1
7 (p
g/m
l)
iDC
CurdlanC. albicans
F. mono
100 101 102 103 104 105100
101
102
103
104
105
IL-1
7 (F
I)
2.21
85.7
2.73
9.37
Unstim
100 101 102 103 104 105100
101
102
103
104
1053.67
78.6
3.67
14.1
IFN-γ (FI)
a
IL-1
7 (F
I)
IFN-γ (FI) IFN-γ (FI)
b c
ControlsiRNA
100 101 102 103 104 105100
101
102
103
104
1055.41
45.9
7.58
41.1
IFN-γ (FI)
MinclesiRNA
Control siRNA
Mincle siRNA
0
5
10
15
20
25
IL-1
7 (%
pos
itive
)
IL-1
7 (p
g/m
l)
Control siRNA
Mincle siRNA
0
5
10
15
20
F. monoUnstim
d
e f
IL-1
7 (F
I)
iDC
100 101 102 103 104 105100
101
102
103
104
105
IL-1
7 (F
I)
4.35
57.0
9.40
29.2
LPS
100 101 102 103 104 105100
101
102
103
104
1052.55
34.3
7.31
55.8
LPS + TDB
100 101 102 103 104 105100
101
102
103
104
1053.10
24.5
25.9
46.5
TDB
100 101 102 103 104 105100
101
102
103
104
1052.18
43.0
7.62
47.2
IFN-γ (FI)IFN-γ (FI)
IL-1
7 (F
I)
IFN-γ (FI) IFN-γ (FI)
IL-1
7 (%
pos
itive
) iDCTDBLPSLPS + TDB
0
10
20
30
40h
g
Min
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Th17
imm
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Md
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120 Chapter four
Multiple PRRs act in concert to regulate the overall antifungal effector response16,17. According
to the pathogenic potential of the encountered microbe, differential PRR signaling provides
information about the magnitude and duration (quantity and quality) of the subsequent T
helper response, ensuring tailored host defense mechanisms18,19. An emerging theme in the
field of receptor-mediated signaling is that certain PRRs are incapable of driving adaptive
immunity individually, but seem to exert a specialized function and ‘fine-tune’ responses
induced by other receptors20-22. A prime example includes antifungal CLR dectin-2. It has
been demonstrated that dectin-2 in human DCs selectively drives IL-1β and IL-23p19
transcription, and thereby distinctively amplifies Th17 immunity, through Syk-MALT1-
dependent activation of NF-κB subunit c-Rel16. A similar ‘modulating’ function has been
ascribed to another fungal-sensing CLR very recently: human mincle abrogates IL-12p70
biosynthesis, and hence blocks instruction of DC-driven Th1 immunity. Mincle couples
Syk-CARD9-Bcl-10-MALT1-dependent signaling to activation of a serine/threonine protein
kinase B (PKB, also known as Akt)-Mdm2 pathway, resulting in proteasome-mediated
degradation of transcription factor interferon regulatory factor 1 (IRF1), thereby abrogating
IL-12p35 expression (Chapter 3). Interestingly, mincle is held responsible for the therapeutic
Th17 adjuvancy of mycobacterial cordfactor23,24. However, its molecular mode of action in
this context is not well defined, as well as its precise contribution to human Th17 immunity
during antifungal inflammation.
Here we demonstrate that mincle enhances the capacity of human DC to induce robust
Th17 responses, by directly acting on cytokine gene transcription -independent of tran-
scription factor NF-κB activities. Instead of activating transcriptional programs on its own,
mincle synergistically enhanced transcription of Th17-polarizing cytokines IL-23p19 and
IL-1β, but at the same time attenuated IL-27p28 mRNA, and subsequent biosynthesis of
Th17-suppresive IL-27. We found both effector mechanisms relying on activation of the PI(3)
K-PKB-Mdm2 axis, but characterized nuclear corepressor Trim28 as a molecular adaptor
segregating these divergent Mdm2 functions. Our data show that, by bridging Mdm2-IRF1
interactions, Trim28 exclusively linked mincle-PKB-Mdm2 signaling to IRF1 degradation,
resulting in IL-27p28 suppression. Notably, mincle-Mdm2 dependent transcriptional
modulation controls the human DC-driven Th17 responses to F. monophora, as well as
Malassezia infection. Thus, these data strongly indicate that mincle is an amplifier of human
Th17 immunity, and by identifying the E3 ubiquitin ligase Mdm2 as the key downstream
effector, provide molecular insight into a novel mechanism by which innate DC receptors
can dictate the magnitude of the Th17 response during fungal infection, which may have
implications for immunopathology, but also development of novel therapeutic approaches.
Mincle amplifies Th17 immunity via Mdm2 121
fou
r.
RESULTS
Mincle enhances DC-induced Th17 polarized responses. Combined activation of Th1
and IL-17-producing Th17 effector cells is key to protective antifungal immunity. A select
group of highly virulent Fonsecaea spp. trigger mincle signaling in human DCs to severely
attenuate dectin-1-induced Th1 polarization (Chapter 3). In marked contrast, we observed
that human primary DCs primed with heat-killed Fonsecaea monophora conidia (strain
CBS269.30) instructed Th17 polarization and induced production of IL-17 by CD4+ T cells,
as measured by intracellular cytokine staining (Figure 1a and b) and ELISA (Figure 1c). The
F. monophora-specific Th17 response was comparable to those induced by dectin-1 agonist
curdlan and Candida albicans (strain CBS2717; Figure 1a and b). Dectin-1 on human DCs
is co-triggered upon F. monophora infection (Chapter 3), and induces Th17 differentiation
upon activation25,26. To investigate whether mincle is involved in Th17 polarization to F.
monophora, we silenced mincle expression by RNA interference (RNAi; Figure S1) and
investigates Th17 responses. Notably, we found that mincle silencing markedly attenuated
induction of IL-17 expression in CD4+ T cells by F. monophora-primed DCs (Figure 1d and e),
which strongly suggests that mincle participates in the instruction of Th17 differentiation
to F. monophora infection.
We next investigated whether mincle-specific triggering had a similar impact on Th17
polarization. Mincle agonist trehalose-6,6-dibehenate (TDB) 23 alone did not activate IL-17-
producing CD4+ T cells (Figure 1f and g). Because TDB (also known as mycobacterial cord-
factor) is a potent Th17 cell adjuvant24, we hypothesized that mincle could affect responses
induced by other innate DC receptors. Strikingly, a profound Th17 polarized response was
detected upon co-priming of DCs with TDB and LPS (Figure 1f and g) - a TLR4 agonist also
incapable of programming Th17 immunity autonomously27. Mincle silencing by RNAi abro-
gated Th17 cell polarization induced by TDB plus LPS-stimulated DCs, which underlined
an important role for mincle. These observations strongly indicate that, although mincle
itself is incapable of driving a Th17 response, mincle promotes the Th17-polarizing capacity
of human DCs by modulating PRR-induced immune responses.
Mincle promotes IL-1β and IL-23 mRNA but impairs IL-27 expression. Along with co-stim-
ulatory molecule expression, DCs dictate the Th response via secretion of polarizing cyto-
kines28. To further clarify the mechanism by which mincle could influence Th17 expansion,
we examined expression of cytokines implicated in DC-induced Th17 cell immunity7. TDB
stimulation failed to induce IL-1β, IL-23 and IL-6 protein (Figure 2a and b) and mRNA (Figure
2c) expression by human DCs, in line with our observation that mincle triggering alone
does not induce Th17 polarization (Figure 1e and f). Notably, however, TDB co-stimulation
synergized with LPS-induced IL-1β and IL-23 protein expression (Figure 2a and b), at the
transcriptional level corresponding with up to six fold higher IL-1β and IL-23p19 mRNA
Min
cle
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Th17
imm
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via
Md
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122 Chapter four
mR
NA
expr
essi
on (r
elat
ive)
mR
NA
expr
essi
on (r
elat
ive)
IL-6
Control siRNA
Mincle siRNA
0.0
0.4
0.8
1.2
1.6
Control siRNA
Mincle siRNA
0.0
0.2
0.4
0.6
0.8
IL-6
IL-1β
Control siRNA
Mincle siRNA
0.00.20.40.60.81.01.21.4
**
Control siRNA
Mincle siRNA
0.0
1.0
2.0
3.0
4.0
5.0
**
*
IL-1β
IL-12p40
Control siRNA
Mincle siRNA
0.0
0.2
0.4
0.6
0.8
Control siRNA
Mincle siRNA
0.0
0.4
0.8
1.2
1.6 IL-12p40
IL-23p19
Control siRNA
Mincle siRNA
0.0
0.2
0.4
0.6
0.8
**
Control siRNA
Mincle siRNA
0.0
2.0
4.0
6.0
8.0
10.0
**
**IL-23p19
IL-27p28
Control siRNA
Mincle siRNA
0.0
0.2
0.4
0.6
0.8
**
Control siRNA
Mincle siRNA
0.0
0.4
0.8
1.2
1.6
****IL-27p28
IL-6
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mR
NA
expr
essi
on (r
elat
ive)
IL-1β
0.0
0.2
0.4
0.6
0.8
1.0
1.2IL-12p40
0.0
0.2
0.4
0.6
0.8
1.0
1.2IL-23p19
0.0
0.2
0.4
0.6
0.8
1.0
1.2IL-27p28
0.0
0.2
0.4
0.6
0.8
1.0
1.2UnstimTDBLPS
IL-27EBI3
0.0
0.2
0.4
0.6
1.0
1.2
0.8
Expr
essio
n (n
g/m
l)
0.0
2.5
5.0
7.5
10.0
12.5
15.0**
0.00.40.81.21.62.02.42.8
0.0
0.5
1.0
1.5
2.0
2.5
3.0*
0.0
0.5
1.0
1.5
2.0
2.5UnstimLPSLPS + TDB
ATP
IL-1β
Expr
essio
n (n
g/m
l)
Expr
essio
n (n
g/m
l)
Expr
essio
n (n
g/m
l)IL-1β IL-23 IL-12p40
>>
>>
a b
c
d
Figure 2. Mincle signaling via Syk-mediated CARD9-Bcl-10-MALT1 assembly leads to amplification of
IL-1β and IL-23 but attenuation of IL-27 production. (a,b) ELISA of IL-1β or IL-23, IL-12p40, IL-6 and IL-27
protein (b) in supernatants of DCs left unstimulated or stimulated with F. monophora, LPS or LPS plus
ATP and/or TDB. *P < 0.05; **P < 0.01 (Student’s paired t-test). (c,d) Quantitative real-time PCR of IL-1β, IL-
23p19, IL-12p40, IL-6, IL-27p28, and IL-27EBI3 mRNA in DCs stimulated with F. monophora LPS and/or
TDB, or after mincle silencing by RNA interference (siRNA). Expression is normalized to GAPDH and set
at 1 in LPS-stimulated cells. *P < 0.05; **P < 0.01 (Student’s paired t-test). (e,f) Flow cytometry analysis I
responses (Figure 2d). IL-23p19 heterodimerizes with IL-12p40 to form IL-23 protein29. TDB
triggering of mincle had no effect on LPS-induced IL-12p40 protein and mRNA (Figure 2b
and d), indicating that mincle affects IL-23 expression at the level of IL-23p19 transcription.
Silencing of mincle completely abrogated the TDB-induced expression of IL-1β and IL-23p19
mRNA (Figure 2d), whereas it did not affect IL-1β and IL-23p19 mRNA induced by LPS alone
(Figure 2d). TDB co-stimulation did not affect LPS-triggered IL-6 responses (Figure 2b and
d). Further analysis revealed that mincle similarly modulated F. monophora-induced Th17
polarizing cytokines, the transcription of which requires dectin-1 signaling (Chapter 3).
Mincle amplifies Th17 immunity via Mdm2 123
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IL-27EBI3
Control siRNA
Mincle siRNA
0.0
0.2
0.4
0.6
0.8UnstimF. mono
Control siRNA
Mincle siRNA
0.0
0.4
0.8
1.2
1.6 IL-27EBI3 LPSLPS + TDB
0
100
200
300
400
500
0
20
40
60
80UnstimTDBLPSLPS + TDB
- Rec IL-270
10
20
30
40
IL-1
7 (%
pos
itive
)
CurdlanLPS
Unstim
LPS + TDB
100 101 102 103 104 105100
101
102
103
104
105
IL-1
7 (F
I)4.85
69.6
2.50
23.1
100 101 102 103 104 105100
101
102
103
104
10517.1
30.2
18.6
34.1
100 101 102 103 104 105100
101
102
103
104
10510.8
41.7
9.66
37.8
iDC Curdlan
100 101 102 103 104 105100
101
102
103
104
1054.10
50.7
3.11
42.1
Expr
essio
n (n
g/m
l)
Expr
essio
n (p
g/m
l)IL-6 IL-27p28
IL-1
7 (F
I)
IFN-γ (FI) IFN-γ (FI)
Control
RecIL-27
e
f
(FI, fluorescence intensity) of T helper polarization by staining for intracellular IFN-γ (Th1) and IL-17A
(Th17) expression, after coculture of memory CD4+ T cells with DCs that were left unstimulated (iDC) or
primed with curdlan, LPS and/or TDB, in the absence or presence of recombinant IL-27. In (f), the per-
centages of IL17A-producing T cells are shown, corresponding to the upper left and right quadrants of
(e). Data are representative of at least two (b; IL-27 ELISA, e) or three (a,b,c,d) independent experiments
(mean and s.d. in a-d; mean and s.d. of duplo measurements in f).
Induction of IL-1β and IL-23p19 transcripts, but neither IL-6 nor IL-12p40, was significantly
impaired in F. monophora-stimulated DCs after mincle silencing by RNAi (Figure 2d). These
results indicate that mincle enhances production of Th17-promoting cytokines via an
increase in IL-1β and IL-23p19 mRNA expression.
Secretion of immunosuppressive mediators that constrain Th differentiation provides
DCs with an additional means to control the T helper response. IL-27 is a known cytokine
that antagonizes Th17 polarization30-33. Addition of recombinant IL-27 to human DC-T cell
cocultures led to severely diminished Th17 responses (Figure 2d and e). We found that
Min
cle
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Th17
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via
Md
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124 Chapter four
Control siRNA
PKBsiRNA
0.00.20.40.60.81.01.21.4
mR
NA
expr
essi
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**
Control siRNA
PKBsiRNA
0.0
1.0
2.0
3.0
4.0
5.0
**
IL-1β
Control siRNA
PKBsiRNA
0.0
2.0
4.0
6.0
8.0
10.0
*
**
Control siRNA
PKBsiRNA
0.0
0.2
0.4
0.6
0.8IL-23p19
Control siRNA
PKBsiRNA
0.0
0.4
0.8
1.2
1.6
***
Control siRNA
PKBsiRNA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
*IL-27p28
Control siRNA
Syk siRNA
CARD9siRNA
Bcl-10siRNA
MALT1siRNA
0.0
1.0
2.0
3.0
4.0
5.0
mR
NA
expr
essi
on (r
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IL-1β**
*
UnstimLPSLPS + TDB
Control siRNA
Syk siRNA
CARD9siRNA
Bcl-10siRNA
MALT1siRNA
0.0
2.0
4.0
6.0
8.0
10.0
mR
NA
expr
essi
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IL-23p19
** ** **
**
**
Control siRNA
Syk siRNA
CARD9siRNA
Bcl-10siRNA
MALT1siRNA
0.0
0.4
0.8
1.2
1.6
mR
NA
expr
essi
on (r
elat
ive)
IL-27p28
** ** *** *
DN
A bi
ndin
g (A
450)
p50 p65 c-Rel RelB p52
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.5
1.0
1.5
2.0
0.0
0.2
0.4
0.6
0.8
1.0Unstim
LPS + TDBLPSTDB
a
b
mR
NA
expr
essi
on (r
elat
ive)
IL-1β IL-23p19 IL-27p28
UnstimF. mono
LPSLPS + TDB
c
LPS, but not TDB, induced strong IL-27 expression (Figure 2b). Notably, and in line with
the notion that mincle amplifies Th17 polarization, TDB coexposure fully abrogated LPS-
Mincle amplifies Th17 immunity via Mdm2 125
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r.
Figure 3. Mincle couples Syk-mediated CARD9-Bcl-10-MALT1 assembly to PI(3)K-PKB signaling for
modulation of IL-1β, IL-23p19 and IL-27p28 expression, independent of NF-κB. (a,c) Quantitative re-
al-time PCR of IL-1β, IL-23p19, and IL-27p28 mRNA in DCs stimulated with F. monophora, LPS and/or
TDB, after Syk, CARD9, Bcl-10, MALT1 or PKB silencing. Expression is normalized to GAPDH and set at
1 in LPS-stimulated cells. *P < 0.05; **P < 0.01 (Student’s paired t-test). (b) DNA binding ELISA of NF-κB
subunits p50, p65, c-Rel, RelB and p52 in nuclear extracts of DCs stimulated with LPS and/or TDB. Data
are representative of at least three (a-c) independent experiments (mean and s.d. in a-c).
mediated IL-27 biosynthesis (Figure 2b). IL-27 is the product of p28 and Epstein-Barr virus
gene 3 (EBI3) subunits34. LPS-induced IL-27p28 mRNA but not EB13 was suppressed by
TDB costimulation in a mincle-dependent manner (Figure 2d). These results indicate that
mincle antagonizes IL-27 responses at the level of IL-27p28 transcription. Mincle similarly
affected F. monophora-induced IL-27 responses; F. monophora did not induce IL-27p28
mRNA (Figure 2d), but silencing of mincle led to a strong increase in IL-27p28 expression,
whereas IL-27EB13 mRNA responses remained unaffected (Figure 2d), strongly suggesting
that mincle suppresses IL-27p28 mRNA, and hence biosynthesis of Th17-antagonizing
cytokine IL-27. Together, these results indicate that mincle promotes IL-1β and IL-23p19 but
impairs IL-27p28 expression in human DCs, allowing amplification of Th17 differentiation.
Mincle signaling via PKB modulates Th17 cytokines independent of NF-κB. Mincle trans-
mits ITAM-coupled signaling via the Syk-CARD9-Bcl-10-MALT1 axis (refs35,36 and Chapter
3). Silencing of the different signaling components abrogated TDB-mediated enhancement
of LPS-induced IL-1β, IL-23p19 as well as negative modulation of IL-27p28 mRNA (Figure
3a). Signaling through Syk and the CARD9-Bcl-10-MALT1 complex is generally confined to
modulation of cytokine gene transcription via NF-κB, with subunit c-Rel required for IL-1β
and IL-23p19 expression, and hence Th17 polarization16. Therefore, we assessed possible
NF-κB contribution. Notably, we observed that mincle-specific triggering neither affected
LPS-induced nuclear translocation and DNA binding of p50-p65 complexes, nor induced
activation of NF-κB subunits autonomously (Figure 3b). Mincle has been described recently
to couple Syk and CARD9-Bcl-10-MALT1-mediated signaling to a PI(3)K-PKB cascade that
inhibits IL12A transcription, independent of NF-κB (Chapter 3). RNAi-mediated silencing
of PKB expression (Figure S2) abrogated the mincle-induced IL-1β and IL-23p19 synergy
and allowed expression of IL-27p28 mRNA after treatment of DCs with TDB plus LPS or F.
monophora (Figure 3c), without affecting LPS-induced mRNA responses (Figure 3c). In line
with these results, mincle-mediated modulation of IL-1β, IL-23p19 and IL-27p28 in response
to TDB costimulation or F. monophora was abrogated upon treatment with PKB inhibitor
triciribine (Figure S2) as well as interference with upstream PKB effector PI(3)K (Figure S2),
indicating that PKB plays a decisive role in the modulation of cytokine responses by mincle.
These data strongly suggest that mincle signaling, through Syk- and CARD9-Bcl-10-MALT1-
@
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126 Chapter four
Control siRNA
Mdm2siRNA
0.00.20.40.60.81.01.21.4
mR
NA
expr
essi
on (r
elat
ive)
*
Control siRNA
Mdm2siRNA
0.0
1.0
2.0
3.0
4.0
5.0
*
**
Control siRNA
Mdm2siRNA
0.0
2.0
4.0
6.0
8.0
10.0
*
**
Control siRNA
Mdm2siRNA
0.0
0.4
0.8
1.2
1.6
**
IL-1β
Control siRNA
Mdm2siRNA
0.0
0.2
0.4
0.6
0.8
**
IL-23p19
Control siRNA
Mdm2siRNA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
**
IL-27p28
0.0
0.4
0.8
1.2
1.6
% in
put D
NA
IL27 ISRE UnstimCurdlanLPS
IgG IRF1IP:0.0
2.0
4.0
6.0
% in
put D
NA
** 0.0
0.5
1.0
1.5
2.0
UnstimLPS
*IgG p65 IgG p65
Control siRNA IRF1 siRNA
IgG RNAPII IgG RNAPIIIP:
Control siRNA IRF1 siRNA
Control siRNA
IRF1siRNA
0.00.20.40.60.81.01.21.4
mR
NA
expr
essi
on (r
elat
ive)
UnstimCurdlanLPS
IL-1β
Control siRNA
IRF1siRNA
0.0
0.5
1.0
1.5
2.0 IL-23p19
Control siRNA
IRF1siRNA
0.0
0.2
0.4
0.6
0.8
1.0
1.2 IL-27p28
***
mR
NA
expr
essi
on (r
elat
ive)
IL-1β IL-23p19 IL-27p28
UnstimF. mono
LPSLPS + TDB
a
b
c d
dependent PI(3)K-PKB activation affects Th17 cytokine transcription, independent of NF-κB.
Mdm2 is required for differential modulation of cytokines by mincle. Mdm2, an E3 ubiqui-
tin ligase, has been demonstrated a key regulator of mincle-PKB signaling, and suppresses
IL-12p35 mRNA responsesby targeting transcriptionally active IRF1 for proteasomal degra-
dation (Chapter 3). We addressed a possible role for Mdmd2 in the modulation of IL-1β, IL-23
and IL-27 by mincle. Notably, silencing of Mdm2 by RNAi (Figure S1) completely arrested
the mincle-mediated synergy on IL-1β and IL-23p19 mRNA (Figure 4a), and significantly
reduced IL-1β and IL-23p19 responses to F. monophora (Figure 4a), to an extent similar to
that observed after silencing of mincle and PKB (Figure 2c and 3c). Furthermore, Mdm2
silencing abrogated suppression of IL-27p28 transcripts after TDB/LPS or F. monophora
stimulation (Figure 4a). Silencing of Mdm2 expression had no effect on mRNA transcripts
induced by LPS stimulation alone (Figure 4a). These results indicate that Mdm2 has a crucial
Mincle amplifies Th17 immunity via Mdm2 127
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r.
Unstim LPS LPS
+TDB F. mono
Control siRNA (MG-132)
IP:α-IRF1
Unstim LPS LPS
+TDB F. mono
Trim28 siRNA (MG-132)
IB:α-IRF1
IB:α-Mdm2
IB:α-Trim28
**
Control siRNA
Unstim LPS LPS
+TDB F. mono
Mincle siRNA
IB:α-Trim28
Unstim LPS LPS
+TDB F. mono
IB:α-RNAP2
IB:α-Trim28
IB:α-β-actin
NE
CEIB:α-Mdm2
IB:α-Mdm2
a
b
Figure 4. Mdm2 is necessary and sufficient for modulation of IL-1β, IL-23p19 and IRF-1-dependent IL-
27p28 expression by mincle-Syk-PKB signaling. (a,b) Quantitative real-time PCR of IL-1β, IL-23p19, and
IL-27p28 mRNA in DCs stimulated with curdlan, F. monophora, LPS and/or TDB, after Mdm2 or IRF1
silencing. Expression is normalized to GAPDH and set at 1 in LPS-stimulated cells. *P < 0.05; **P < 0.01
(Student’s paired t-test). (c,d) ChIP assay of IRF1 (c), RNAPII and p65 (d) recruitment to ISRE binding site
(c) and TATA box or NF-κB binding motif (d) of the IL27 promoter in DCs stimulated with curdlan or LPS
(c,d) or after IRF1 silencing (d). IgG indicates a negative control. Levels are normalized with respect to
the ‘input DNA’ sample, which had not undergone immunoprecipitation; results are expressed as the
% input DNA. *P < 0.05; **P < 0.01 (Student’s paired t-test). Data are representative of at least three (a-d)
independent experiments (mean and s.d. in a-d).
Figure 5. Trim28 functions as an
adaptor for nuclear Mdm2 and
IRF1 binding, operating down-
stream mincle. (a,b) Immuno-
blotting of Mdm2 and Trim28
in nuclear (NE) and cytoplasmic
(CE) extracts (a) or Mdm2-IRF1-
Trim28 complexes after immuno-
precipitation (IP) with anti-IRF1
from nuclear (NE) extracts pre-
pared in the presence of protea-
some inhibitor MG-132 to block
protein degradation (b). DCs were
stimulated with LPS and/or TDB,
or F. monophora after mincle or
Trim28 silencing. RNAPII and
β-actin served as loading con-
trols for nuclear and cytoplas-
mic extracts, respectively. Data
are representative of at least two
independent experiments.
@
role in the modulation of type 17 cytokines by mincle signaling via Syk-PKB.
To determine to which extent Mdm2-depdentent IRF1 degradation was involved, we
investigated contribution of IRF1 to IL1B, IL23A and IL27 transcription. Strikingly, silencing
of IRF1 (Figure S1) did neither influence LPS- nor curdlan-induced IL-1β and IL-23p19 mRNA
(Figure 4b). These data suggest that Mdm2-dependent modulation of these cytokines occurs
independent from IRF1. In contrast, and complementing previous data37-40, we found IRF1
activity a prerequisite for transcription of IL27, as evidenced by the abolished expression
Min
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128 Chapter four
of IL-27p28 mRNA after TLR4 or dectin-1 triggering under conditions of IRF1 silencing
(Figure 4b). ChIP assays revealed that both LPS and curdlan triggered IRF1 recruitment to
the ISRE site within the proximal IL27 promoter (Figure 4c). Of note, IL-27p28 is an IL-12p35
homologue34, and analogous to what has been found for IL12A (Chapter 3), silencing of IRF1
expression coincided with abolished recruitment of RNA polymerase II (RNAPII) and NF-κB
subunit p65 to the IL27 promoter (Figure 4d). These results suggest that the crucial role
of IRF1 in IL-27p28 expression is reflected by its ability to control transcription initiation
via nucleosome remodeling. Thus, although dispensable for mincle-induced IL-1β and
IL-23p19 synergy, it seems likely that proteasomal degradation of IRF1 accounts for the
suppression of IL-27p28 mRNA in response to TDB and F. monophora. Furthermore, these
results demonstrate that Mdm2 operates via a dual mechanism of action: both dependent
and independent of its ability to suppress IRF1 abundance and transcriptional activity.
Trim28 couples Mdm2 to IRF1 degradation and IL-27p28 suppression. To gain further
molecular insight into the pathways by which Mdm2 exerts its dual effects on cytokine
expression, we attempted to identify cofactors. Tripartite-motif (Trim) proteins have critical
roles in the regulation of innate immune signaling41. Trim28 (alternatively named KAP1 or
TIF1β) is a nuclear protein42, while it has been found to interact with, amongst others, Mdm2
and IRF143,44. As already reported (Chapter 3), immunoblot analyses showed that TDB and
Control siRNA
Trim28siRNA
0.00.20.40.60.81.01.21.4
mR
NA
expr
essi
on (r
elat
ive)
Control siRNA
Trim28siRNA
0.00.20.40.60.81.01.21.4
**
Control siRNA
Trim28siRNA
0.0
1.0
2.0
3.0
4.0
5.0
**
Control siRNA
Trim28siRNA
0.0
2.0
4.0
6.0
8.0
10.0
**
Control siRNA
Trim28siRNA
0.0
0.5
1.0
1.5
2.0
**
**
IL-1β
Control siRNA
Trim28siRNA
0.0
0.2
0.4
0.6
0.8IL-23p19 IL-27p28
mR
NA
expr
essi
on (r
elat
ive)
IL-1β IL-23p19 IL-27p28
UnstimF. mono
LPSLPS + TDB
Figure 6. Trim28 is required for mincle-induced IL-27p28 suppression, but dispensable for IL-1β and
IL-23p19 modulation. (a) Quantitative real-time PCR of IL-1β, IL-23p19, and IL-27p28 mRNA in DCs
stimulated with LPS and/or TDB, or F. monophora, after Trim28 silencing. Expression is normalized to
GAPDH and set at 1 LPS-stimulated cells. **P < 0.01 (Student’s paired t-test). Data are representative of at
least three independent experiments (mean and s.d.).
Mincle amplifies Th17 immunity via Mdm2 129
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r.
F. monophora, but not LPS, triggered Mdm2 nuclear translocation in a mincle-dependent
manner (Figure 5a). However, this was not accompanied by a noticeable change in Trim28
expression or distribution; Trim28 protein was localized constitutively in the nuclear
compartment (Figure 5a). The potential effects of Trim28 on Mdm2 binding to IRF1 were
investigated by performing immunuprecipitation experiments with nuclear extracts from
DCs stimulated in the presence of proteasome inhibitor MG132 to allow accumulation
of ubiquitinated IRF1 (Chapter 3). Mdm2 coimmunoprecipitated with IRF1 from nuclear
extracts of DCs costimulated with TDB or F. monophora (Figure 5b and c). Notably, under
these conditions, we observed that both Mdm2 and IRF1 immunoprecipitated together
with Trim28 (Figure 5b and c). Trim28 was not constitutively bound to nuclear IRF1, as
0
20
40
60
80
100
Rec mincle binding (FI)
0
20
40
60
80
100
Rec mincle binding (FI)
ControlRec mincle
M. globosaM. furfur
0
20
40
60
80
100
0
20
40
60
80
100
Eve
nts
(% o
f max
)
C. albicansF. monophora
Rec mincle binding (FI) Rec mincle binding (FI)
Control siRNA
MinclesiRNA
Mdm2siRNA
0
10
20
30
40
50
60
IL-1
7 (%
pos
itive
)
IL-1
7 (p
g/m
l)
Control siRNA
MinclesiRNA
Mdm2siRNA
0
10
20
30
40UnstimM. furfurM. globasaC. albicans
Control siRNA
PKBsiRNA
Mdm2siRNA
0
10
20
30
40IL
-17
(% p
ositi
ve) Unstim
F. mono
Control siRNA
PKBsiRNA
Mdm2siRNA
0
4
8
12
16
20
24
IL-1
7 (p
g/m
l)
UnstimF. mono
a b
c
d e
Figure 7. PKB-Mdm2 signaling is instrumental for mincle-mediated antifungal Th17 amplification.
(a,b,d,e) T helper polarization was assessed by flow cytometry analysis by staining for intracellular IFN-γ
(Th1) and IL-17A (Th17) expression at day 12-17 after PMA plus ionomycin restimulation (a,d) or by ELISA
by measuring IL-17 production in supernatants at day 5 (b,e), after coculture of memory CD4+ T cells
with DCs that were left unstimulated (iDC) or primed with curdlan, C. albicans, F. monophora, Malassezia
furfur, M. globosa, LPS and/or TDB, or after mincle silencing. (c) Flow cytometry analysis (FI, fluorescence
intensity) of recombinant mincle protein (pink) binding to heat-killed C. albicans, F. monophora, M. furfur,
and M. globosa conidia. Data are representative of at least two (a-e) independent experiments (mean and
s.d. of duplo measurements in a,c).
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130 Chapter four
Trim28 did not coimmunoprecipitate with IRF1 after LPS stimulation alone (Figure 5b andc).
Thus, Trim28 is a component of the nuclear Mdm2-IRF1 complex, assembled upon mincle
signaling. Strikingly, silencing of Trim28 expression (Figure S1) prevented the association
of Mdm2 with IRF1 following mincle triggering with TDB and F. monophora (Figure 5c).
These data indicate that Trim28 acts downstream of Mdm2 to facilitate an Mdm2-IRF1
interaction within the nucleus.
E3 ligases must form stable complexes with cognate substrates to catalyze their ubiq-
uitination and degradation45, therefore we assessed the role of Trim28 in the mincle-Mdm2
pathway. Whereas IL-27p28 mRNA was restricted in response to LPS/ and TDB costimu-
lation as well as F. monophora stimulation, silencing Trim28 completely rescued IL-27p28
responses (Figure 6a), which confirmed the requirement for Trim28 in linking mincle-PKB
signaling to Mdm2-mediated degradation of IRF1. Conversely, Trim28 silencing had no
impact on the Mdm2-mediated IL-1β and IL-23p19 synergy (Figure 6b), demonstrating
specificity of Trim28 for IRF1 degradation by Mdm2. Collectively, these results indicate
that a complex of Mdm2 and the adaptor protein Trim28 mediates IRF1 degradation and
subsequent IL-27 suppression by mincle, whereas IL-1β and IL-23 cytokine responses are
modulated by mincle-PKB-Mdm2 signaling through a distinct and Trim28-independent
pathway.
Mincle dictates antifungal Th17 immunity in an Mdm2-dependent manner. Next, we
investigated whether Mdm2 transcriptional activities were specifically required for the
amplification of Th17 cell immunity upon mincle triggering. Silencing of both PKB and Mdm2
prevented the mincle-dependent induction of Th17 differentiation by F. monophora-primed
DCs (Figure 7a and b). These responses were of similar magnitude as those observed after
mincle silencing (Figure 1d and e), and demonstrate that PKB- and Mdm2-dependent
modulation of cytokine transcription underlies the Th17-promoting capacity of mincle.
Having established a major contribution of Mdm2 on the Th17 response to F. mono-
phora, we studied whether Th17 immunity to other fungal pathogens relied on min-
cle-PKB-Mdm2-dependent signaling, and screened the ability of soluble recombinant human
mincle to interact with a variety of fungi. We observed selective binding to F. monophora as
well as pathogenic species from the Malassezia genus (Figure 7c), similar to what has been
reported for its murine counterpart46. Priming of DCs with M. furfur and M. globosa led to a
profound induction of Th17 polarization (Figure 7d and e). Strikingly, Th17 polarization to
these fungi was abrogated after inhibition of mincle signaling via silencing of mincle and
Mdm2 (Figure 7d and e). Silencing of mincle and Mdm2 did not affect Th17 responses to C.
albicans conidia (strain CBS2712) (Figure 7d and e), since C. albicans did not interact with
mincle (Figure 7c). Thus, the mincle-PKB-Mdm2 axis is more broadly involved in antifungal
host defense and is instrumental to instruction of Th17 immunity in response to various
pathogenic Malassezia strains.
Mincle amplifies Th17 immunity via Mdm2 131
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DISCUSSION
C-type lectin receptor mincle is involved in mediating antifungal responses; the mecha-
nisms by which it precisely controls human cell-mediated immunity to fungal infection are
unknown. Here, we show a specialized function for mincle in promoting Th17 polarized
responses by human DCs. Our results indicate that mincle, via Syk-CARD9-dependent
activation of the PI(3)K-PKB axis, activates two divergent molecular mechanisms for mod-
ulation of Th17 cytokine transcription: whereas one pathway abrogates production of the
Th17suppressive cytokine IL-27, a second pathway abrogates synthesis of Th17-polarizing
cytokines IL-1β and IL-23 by human DCs. The E3 ubiquitin ligase Mdm2 was found to exert a
key modulatory role: Mdm2 directly abrogates IL-27p28 transcription via Trim28-dependent
proteasomal degradation of IRF1, while also enhancing IL-1β and IL-23p19 expression.
Notably, our data indicate major contribution of mincle-Mdm2 signaling to human DC-driven
Th17 responses to Malassezia infection. Thus, by specifically modulating the abundance
of cytokines with key roles in the regulation of the human Th17 effector response, mincle
signaling amplifies host-Th17 immunity during antifungal inflammation.
The innate immune system exhibits a considerable degree of redundancy: a fungal
pathogen can be detected by more than one pattern recognition receptor16,17, usually
allowing tailored effector functions. This study provides important additional insight
into the diverse modes of molecular and functional crosstalk by which C-type lectin
receptors can modulate antifungal immunity. Our data have identified a novel pathway
by which ITAM-coupled signaling controls antifungal Th17 inflammation, independent
of transcription factor NF-κB. NF-κB is involved in the regulation of a wide range of innate
response genes47, and as such, is targeted by human CLR dectin-2 for amplification of Th17
polarization16. Here we show that mincle also enhances Th17 responses, however through
a different mechanism. Dectin-2 couples Syk signaling via the paracaspase MALT1 to
selective activation of NF-κB subunit c-Rel, leading to upregulation of IL-1β and IL-23p19
mRNA synthesis. Mincle similarly affects IL1B and IL23A transcription and pairs with the
FcRγ chain to transduce ITAM-coupled signaling35 via Syk and the complete Card9-Bcl-10-
MALT1 scaffold (refs23,36 and Chapter 3). Mincle, in contrast to dectin-2, does not induce or
modulate the activation of NF-κB subunits. How this discrepancy in signal transduction
downstream Card9-Bcl-10-MALT1 is coordinated remains unclear. Differential signaling and
effector ability is a central theme downstream immune receptor signaling, and receptors
frequently use adaptor and scaffolding proteins for controlling their effector mechanisms48.
Specifically, TRAF proteins serve as key adaptor proteins in signaling to innate response
gene transcription49. We hypothesize that adaptor proteins, such as TRAF family members,
are dictating the Syk-CARD9-Bcl-10-MALT1 (ITAM) signals emanating from dectin-2 and
mincle to facilitate flexibility of their signaling responses.
Our previous studies have shown that mincle-PI(3)K-PKB signaling suppresses IL-12A
Min
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transcription and Th1 immunity via Mdm2-dependent IRF1 proteasomal degradation
(Chapter 3). Although Mdm2 is essential for the Th17-modulating capacity of mincle, it acts
on cytokine gene transcription via two bifurcating pathways. Multimeric protein complexes
regulate the substrate specificity of Mdm250; we here identify nuclear protein Trim28 as
an adaptor protein dedicated to the IRF1-IL-27 branch of Mdm2-mediated transcriptional
modulation. Members of the Trim superfamily of proteins participate in diverse cellular
processes51,52, and are emerging immune regulators41,53. Trim28, by working together with
chromatin-modifying enzymes and transcriptional repressors, has been characterized
particularly as a nuclear corepressor54. Thus, our findings reveal an alternate mode of gene
repression by demonstrating that Trim28, by virtue of its specific interaction with Mdm2
and IRF1, selectively promotes proteasome-dependent degradation of IRF1. Trim28, like
Mdm2, contains a prototypical RING domain. Importantly, Trim28’s putative intrinsic E3
ubiqituin ligase activity has never been conclusively proven. Moreover, in association with
Mdm2, Trim28 controls the degradation of tumor suppressor protein p53, independent of
its own RING domain44. Thus, instead of functionally cooperating as an E3 ligases, Trim28
might stabilize Mdm2-IRF1 complexes in the nucleus, allowing inhibition of IRF1 transcrip-
tional activity, hence IL-27 suppression by mincle.
By contrast, the mincle-induced and Mmd2-mediated upregulation of IL-1β and IL-23
expression is independent of Trim28, and the precise underlying molecular mechanism
will require future exploration. Given that Mdm2 has a broad substrate specificity and can
target multiple proteins for proteasomal degradation50, it likely interferes with an as-yet-un-
identified protein(s) that negatively regulate(s) IL1B and IL23A transcription.
It is noteworthy that mincle modulated IL-1β expression via an additional and transcrip-
tion-independent mechanism. Mincle triggering by TDB directly induced the maturation
and release of IL-1β. The production of mature IL-1β involves a two-step process: NF-κB-
depdendent synthesis of pro-IL-1β mRNA and subsequent proteolytic maturation of pro-IL-1β
by caspase-containing multiprotein complexes. These inflammasomes can be assembled
upon receptor-mediated events. NLRP3/caspase-1 and AIM/caspase-1 inflammsome activa-
tion has been linked to Syk-dependent mechanisms55,56, while the noncononical caspase-8
inflammasome requires the activity of paracaspase MALT157. Thus, it will be of interest to
investigate further how mincle couples to inflammasome activation, and whether or not
Mdm2 might be involved.
While these findings aid in understanding the molecular mechanisms by which mincle
controls the Th17 adjuvancy of mycobacterial cordfactor (TDM) and its synthetic analogue
TDB23,24, they might also provide insight into the molecular mechanisms by which ‘sterile
inflammation’ is regulated. Apart from being typified as a microbial receptor, mincle has been
held responsible for triggering strong neutrophil influxes upon detection of damaged-self,
and binds spliceosome-associated protein 130 (SAP130) -a ribonucleoprotein released
during necrotic cell death35. It is conceivable that mincle-mediated amplification of Th17
Mincle amplifies Th17 immunity via Mdm2 133
fou
r.
polarization represents part of a physiological regulatory mechanism for generation of an
overt immune response under certain conditions of sterile inflammation.
Protective immunity to fungal infection relies on chemotaxis and activation of profes-
sional phagocytes, principally neutrophils, effector responses which require the combined
activation of Th1 and Th17 effector cell subsets4,5. Given our previous work (Chapter 3) and
the data presented here, mincle has a distinctive modulatory function in that it strongly pro-
motes the expression of Th17-polarizing cytokines, while it actively and severely represses
type 1 immunity. When uncontrolled or chronic, Th17 inflammation can be highly tissue
destructive and is a common theme in the onset of allergy and autoimmunity7,11. Beside its
known detrimental role in the immune response to virulent Fonsecaea monophora 58 (and
Chapter 3), we here demonstrate that mincle participates in immune responses to Malassezia
spp. With regard to its pathogenic potential, skin-colonizing Malassezia spp. are an exacer-
bating factor in chronic inflammatory skin disorders, such as psoriasis59 -characterized by
prolonged and pathologic Th17 inflammation7,60. Polymorphisms in the IL-23 pathway (i.e.
IL23R, IL23A, IL12B) have been associated with psoriasis susceptibility61, and elevated levels
of IL-17 and Th17 cell numbers in lesional skin and blood correlate with disease severity62;
hence, neutralization of the IL-17 pathway can be an effective and targeted therapy for
psoriasis treatment63-65. Thus, such strong ‘focusing’ of a Th17 response by mincle might
have serious implications for the pathophysiology of such chronic skin infections.
In conclusion, our study has demonstrated a specialized function for mincle in ampli-
fying host Th17 immunity. We propose that mincle-Mdm2 signaling drives the modulation
of cytokine gene transcription via two separate mechanisms: one that, via Mdm2-Trim28
controls IRF1 proteasomal degradation, and the other that modulates transcription inde-
pendent of IRF1 activity modulation. These findings not only shed light on the distinctive
function of mincle in the antifungal immune response, it also provides important addi-
tional insight into the diverse modes of molecular and functional crosstalk by which Th17
immunity can be modulated by PRRs. Therapeutic targeting of the Mincle-PKB-Mdm2 axis
may offer an ability towards resolution of a dysfunctional Th17 effector response, during
antifungal inflammation or severe autoimmune disorders.
Min
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134 Chapter four
EXPERIMENTAL PROCEDURES
Cells, stimuli, inhibitors and RNA interference.
CD14+ monocytes from healthy volunteer blood
donors were isolated, cultured and differen-
tiated into immature DCs as described26 and
used at day 6 or 7 for experiments. Donors were
routinely screened for dectin-1 single nucleotide
polymorphism rs16910526 using a TaqMan
Genotyping Assay (Assay ID C_33748481_10;
Applied Biosystems); only dectin-1 wild-type
DCs were used for experiments. This study was
done in accordance with ethical guidelines of the
Academic Medical Center. DCs were stimulated
with curdlan (10 mg/ml), lipopolysaccharide
from Salmonella typhosa (10 ng/ml; both Sigma),
and/or strains from Candida albicans, Fonsecaea,
or Malassezia spp. (heat-inactivated organisms)
at a multiplicity of infection (MOI) 5, or treha-
lose-6,6-dibehenate as previously described
23 (50 mg/well; Avanti Polar Lipids). Cells were
pre-incubated with inhibitor for 2 h, with 0.5 mM
wortmannin (PI(3)K inhibitor), 5 mM triciribine
(PKB inhibitor), or 0.1 mM MG-132 (proteasome
inhibitor; all Calbiochem). DCs were transfected
with 25 nM siRNA through the use of transfec-
tion reagents DF4 (Dharmacon) and were used
for experiments 72 h after transfection. The
following SMARTpool siRNAs were used (all
from Dharmacon): Mincle (M-021374-02), Syk
(M-003176-03), CARD9 (M-004400-01), Bcl-10
(M-004381-02), MALT1 (M-005936-02), IRF1
(M-011704-01), PKB (M-003000-03), Mdm2 (M-
003279-04), Trim28 (M-005046-01) and on-tar-
geting siRNA (D-001206-13) as a control. This
protocol resulted in nearly 100% transfection effi-
ciency as determined by flow cytometry analysis
of cells transfected with siGLO-RISC free-siRNA
(D-001600-01) and did not induce IFN responses,
as determined by quantitative real-time PCR
analysis26. Silencing of expression was verified
by real-time PCR and flow cytometry for each
experiment (Figure S1, ref 16 and Chapter 3).
Fungal strains and recombinant mincle binding.
Candida strains were grown in Sabouraud dex-
trose broth and incubated for 3 d at 25 °C, while
shaking. Conidia were dislodged from slants
by gentle tapping and then were resuspended
in 0.1% (vol/vol) Tween-80 in PBS. Malassezia
strains were grown on solid mLNA medium for
3 d at 30 °C or potato dextrose agar for 3 d at 37
°C, respectively. Fonsecaea strains were grown
for 7 d at 37 °C on Oatmeal agar culture plates.
0.1% Tween-80 in PBS was used to remove and
resuspend the grown conidia. Hyphal contam-
ination was removed by straining of the cell
solutions through a glass filter. Swollen germi-
nating conidia were obtained by incubation
for 6 h at 37 °C, with shaking, in 0.1% (vol/vol)
Tween-80 in PBS. Fungi were inactivated by
being heated for 1 h at 56 °C. Binding of recom-
binant human mincle to heat-killed conidia was
determined by flow cytometry analysis: bound
purified DDK-tagged mincle (TP300244; Origene)
was labeled with anti-DDK-tag (TA50011-100;
Origene), followed by detection with Alexa Fluor
488-conjugated anti-mouse (A11029; Invitrogen).
T helper cell differentiation assays. Memory
CD4+ T cells were isolated with MACS beads
isolation as described previously66. DCs were
silenced as indicated and subsequently
activated with LPS, curdlan and/or heat-killed
fungal strains. After 48 h, cells were washed
extensively and cocultured with CD4+ T cells
(20,000 T cells/5000 DCs) in the presence of
Staphylococcus aureus enterotoxin B (10 pg/ml;
Sigma) and the absence or presence of human
recombinant IL-27 (30 ng/ml; 2526-IL-010/CF;
R&D Systems). DCs primed with LPS plus IFN-g
(1000 U/ml; U-CyTech) or curdlan were used as
positive controls for Th1 or Th17 differentiation,
respectively. After 5 days of coculture, cells were
further cultured in the presence of IL-2 (10 U/
Mincle amplifies Th17 immunity via Mdm2 135
fou
r.
ml; Chiron). Resting cells were restimulated after
12-17 days with PMA (100 ng/ml) and ionomycin
(1 mg/ml) for 6 h, the last 4 h in the presence of
brefeldin A (10 mg/ml; all Sigma). Intracellular
cytokine expression was analyzed by stain-
ing with FITC-conjugated mouse anti-IFN-γ
(25723.11) and APC-conjugated mouse anti-IL-
17A (Clone eBio64DEC17; 17-7179; eBioscience).
Cytokine production. DC and T cell culture
supernatants were harvested after 24-28
h of stimulation or after 5 d of coculture,
respectively, and concentrations of IL-6,
IL-23, IL-1β, IL-12p40 (Invitrogen), IL-12p70
(eBioscience), IL-27 (Abcam) or IL-17A (Life
Technologies) were determined by ELISA.
Quantitative real-time PCR. Isolation of mRNA
from DCs stimulated for 6 h, synthesis of cDNA
and amplification by PCR with the SYBR Green
method with the ABI 7500 Fast PCR detection
system (Applied Biosystems) were performed
as described 26. Specific primers were designed
with Primer Express 2.0 (Applied Biosystems;
Table S1). The Cycling threshold (Ct) value
was defined as the number of PCR cycles in
which the fluorescence signal exceeded the
detection threshold value. For each sample, the
normalized amount of target mRNA (Nt) was
calculated from the obtained Ct values for both
target and GAPDH mRNA with the following
equation: Nt = 2Ct of GAPDH – C
t of target. Relative
mRNA expression was obtained by setting of
Nt in LPS or curdlan-stimulated samples as 1
within one experiment and for each donor.
NF-κB DNA binding. Nuclear and cytoplas-
mic extracts of DCs were prepared after 2 h
of stimulation using the NucBuster protein
extraction kit (Novagen). NF-κB DNA binding
in nuclear extracts was determined with a
TransAM NF-κB family kit (Active Motif).
Chromatin immunoprecipitation (ChIP) assay.
The ChIP-IT Express Enzymatic kit (Active Motif)
was used for ChIP assays to determine occu-
pancy of the proximal IL27 promoter by regula-
tory proteins. After 2 h of stimulation, cells were
fixed with 1% (vol/vol) formaldehyde. Nuclei were
isolated and chromatin DNA was fragmented by
enzymatic shearing (10 min at 37 °C). Protein-
DNA complexes were immunoprecipitated over-
night at 4 °C with anti-p65 (3034; Cell Signaling),
anti-RNAPII (4H8; Active Motif), anti-IRF1 (C-20)
or normal rabbit IgG (negative control; sc-2027;
Santa Cruz) and protein G-coated magnetic
beads. Input and immunoprecipitated DNA was
purified after reversal of crosslinks. Real-time PCR
was done with primer sets spanning the ISRE,
NF-κB binding site, and the transcription initiation
site for RNAPII binding (primer sequences Table
S1). Primers spanning genomic DNA at cytoge-
netic location 12 p13.3 (Active Motif) were used
as a negative control. To normalize for DNA input,
a sample for each condition was taken along that
had not undergone immunoprecipitation (input
DNA); results are presented as the % of input DNA.
Mdm2, Trim28 and IRF1 localization and Mdm2-
IRF1-Trim28 association. Cellular localization
of total Mdm2, Trim28 or IRF1 was detected by
immunoblot analysis, with anti-Mdm2 (ab38618;
Abcam), anti-Trim28 (4123; Abcam) or anti-IRF1
(ab26109; Abcam) in cellular extracts of DCs
stimulated for 2 h. Membranes were probed with
anti-RNAPII (clone CTD4H8; 05-623; Millipore)
or anti-b-actin (sc-81178; Santa Cruz) to ensure
equal protein loading among cytoplasmic and
nuclear extracts, respectively. Primary anti-
body incubation was followed by incubation
with HRP-conjugated secondary antibody
Min
cle
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Th17
imm
unity
via
Md
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136 Chapter four
(anti-rabbit, 21230; Pierce, or anti-mouse,
P0161, DAKO) and ECL detection (Pierce). Total
nuclear levels of IRF1 were further determined
by ELISA (USCN Life Science). Detection of
Mdm2-IRF1-Trim28 association was performed
by immunoprecipitating IRF1 with anti-IRF1
(C-20; Santa Cruz) coated on protein A/G-PLUS
agarose beads (Santa Cruz) from 20 mg of
nuclear extract of DCs stimulated in the absence
or presence of MG-132. Immunoprecipitates
were resolved by SDS-PAGE, and detected
by immunoblotting as described above.
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Mincle amplifies Th17 immunity via Mdm2 141
fou
r.
SUPPLEMENTAL FIGURES
100 101 102 103 104
20
40
60
80
100
Eve
nts
(% o
f max
)
IRF1 (FI)
ControlControl siRNAIRF1 siRNA
00.0
0.2
0.4
0.6
0.8
1.0
1.2
IRF1
mR
NA
(rel
ativ
e) Control siRNAIRF1 siRNA
**
100 101 102 103 104
20
40
60
80
100
Eve
nts
(% o
f max
)
PKB (FI)
ControlControl siRNAPKB siRNA
0
100 101 102 103 104
20
40
60
80
100
Eve
nts
(% o
f max
)
Mincle (FI)
ControlControl siRNAMincle siRNA
00.0
0.2
0.4
0.6
0.8
1.0
1.2
Min
cle
mR
NA
(rel
ativ
e) Control siRNAMincle siRNA
**
100 101 102 103 104
20
40
60
80
100
Eve
nts
(% o
f max
)
MDM2 (FI)
ControlControl siRNAMdm2 siRNA
0
Isotype controlControl siRNATrim28 siRNA
Control siRNATrim28 siRNA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Trim
28 m
RN
A (r
elat
ive)
**100 101 102 103 104
20
40
60
80
100
Eve
nts
(% o
f max
)
Trim28 (FI)
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
PK
B m
RN
A (r
elat
ive) Control siRNA
PKB siRNA
**
Mdm
2 m
RN
A (r
elat
ive)
0.0
0.2
0.4
0.6
0.8
1.0
1.2Control siRNAMdm2 siRNA
a
e
g
c
i
b
dc
f
h
j
Figure S1. Silencing of mincle, PKB, Mdm2, IRF1 and Trim28 in human primary DCs by RNA interference.
(a-j) Indicated proteins were silenced using specific SMARTpools, with non-targeting siRNA as a control.
Silencing was confirmed by quantative real-time PCR (a,c,e,g,i) or by flow cytometry analysis (b,d,f,h,j)
(FI, fluorescence intensity). Antibodies used for staining include anti-mincle (clone 2D12; H00026253;
Abnova), anti-PKB (ab32505; Abcam), anti-Mdm2 (ab38618; Abcam), anti-IRF1 (clone C-20; c-497; Santa
Cruz) and anti-Trim28 (4123; Abcam). In (a,c,e,g,i), expression is normalized to GAPDH set at 1 in control
siRNA-treated cells. **P < 0.01 (Student’s paired t-test). Data are representative of at least two (e) three
(a-d,f-j) independent experiments (mean and s.d. in a,c,e,g,i).
Min
cle
am
plifi
es
Th17
imm
unity
via
Md
m2
142 Chapter four
DMSOcontrol
Wort-mannin
0.00.20.40.60.81.01.21.4
mR
NA
expr
essi
on (r
elat
ive)
*
DMSOcontrol
Wort-mannin
0.0
1.0
2.0
3.0
4.0
5.0
**
IL-1β
DMSOcontrol
Wort-mannin
0.0
0.2
0.4
0.6
0.8IL-23p19
*
DMSOcontrol
Wort-mannin
0.0
2.0
4.0
6.0
8.0
10.0
*
**
DMSOcontrol
Wort-mannin
0.0
0.4
0.8
1.2
1.6
** *
DMSOcontrol
Wort-mannin
0.0
0.2
0.4
0.6
0.8IL-27p28
*
DMSOcontrol
Trici-ribine
0.0
0.2
0.4
0.6
0.8IL-23p19
**
DMSOcontrol
Trici-ribine
0.0
2.0
4.0
6.0
8.0
10.0
*
**
DMSOcontrol
Trici-ribine
0.00.20.40.60.81.01.21.4
mR
NA
expr
essi
on (r
elat
ive)
**
DMSOcontrol
Trici-ribine
0.0
1.0
2.0
3.0
4.0
5.0
*
**
IL-1β
DMSOcontrol
Trici-ribine
0.0
0.2
0.4
0.6
0.8IL-27p28
**
DMSOcontrol
Trici-ribine
0.0
0.4
0.8
1.2
1.6
****
mR
NA
expr
essi
on (r
elat
ive)
a
IL-1β IL-23p19 IL-27p28
mR
NA
expr
essi
on (r
elat
ive)
IL-1β IL-23p19 IL-27p28
UnstimF. mono
LPSLPS + TDB
UnstimF. mono
LPSLPS + TDB
b
Figure S2. PI(3)K-PKB activity is essential for mincle-induced modulation of IL-1β, IL-23p19 and IL-
27p28 mRNA. (a-f) Quantitative real-time PCR of IL-1β, IL-23p19 and IL-27p28 in DCs stimulated with
F. monophora, LPS and/or TDB, in the absence or presence of PI(3)K inhibitor wortmannin (a-c) or PKB
inhibitor triciribine (d-f). Expression is normalized to GAPDH and set at 1 in LPS-stimulated cells. *P < 0.05;
**P < 0.01 (Student’s paired t-test). Data are representative of at least three (a-f) independent experiments
(mean and s.d. in a-f).
Mincle amplifies Th17 immunity via Mdm2 143
fou
r.
This work was supported by the Netherlands Organisation for Scientific Research (NGI 40-41009-
98-8057 to S.I.G.; VICI 918.10.619 to T.B.H.G.).
Author contributions: B.A.W. conceived ideas, designed, performed and interpreted most
experiments and prepared the manuscript. T.M.K. performed cytokine ELISAs, T cell
differentiation assays, microscopy analyses, prepared cellular extracts, and helped with
immunoblotting experiments. B.T. and TB prepared the fungal strains. T.B.H.G. supervised study
design, execution and interpretation, and manuscript preparation. S.I.G. supervised all expects of
the study.
SUPPLEMENTAL TABLE S1
Expression primer sequences
Gene product Forward primer (5’-3’) Reverse primer (5’-3’)
Bcl-10 ATGGAGCCACGAACAACCTCT TCGTGCTGGATTCTCCTTCTG
CARD9 CATGTCGGACTACGAGAACGAT CAGGTAAGGTGTGATGCGTGA
GAPDH CCATGTTCGTCATGGGTGTG GGTGCTAAGCAGTTGGTGGTG
IL-1β TTTGAGTCTGCCCAGTTCCC TCAGTTATATCCTGGCCGCC
IL-6 TGCAATAACCACCCCTGACC TGCGCAGAATGAGATGAGTTG
IL-12p40 CCAGAGCAGTGAGGTCTTAGGC TGTGAAGCAGCAGGAGCG
IL-23p19 GCTTGCAAAGGATCCACCA TCCGATCCTAGCAGCTTCTCA
IL-27EBI3 GGCTCCCTACGTGCTCAATG GGGTCGGGCTTGATGATGT
IL-27p28 GCTTTGCGGAATCTCACCTG TGAAGCGTGGTGGAGATGAAG
IRF1 TTATACAGTGCCTTGCTCGGC AGGCGCTCACACTTCCCTC
Malt1 GACCCATTCCATGGTGTTTACC AATAAATGCATCTGGAGTCCGG
Mdm2 ATCAGAACCCCCACTCACCC TGCCTCGCTCTCTTCCTACAAC
Mincle CTCACAGGAGGAGCAGGAATTC TGACCCTCGACAACCTGGTC
PKB AGAAGGACCCCAAGCAGAGG CACGATACCGGCAAAGAAGC
Syk CCAGAGACAACAACGGCTCC TGTCGATGCGATAGTGCAGC
Trim28 CAGGAAGGCTATGGCTTTGG CGTTTCACACCTGACACATGG
ChIP primer sequences
Gene target Forward primer (5’-3’) Reverse primer (5’-3’)
IL27 ISRE (ref 38) TGAGTGAACACAAAGCTGAAAGT AGCCATCTCCTGGGTAGG
IL27 NF-κB CAGTAGACCCTAGGATGATGGTGG CCTCAATGTCCCATGCTTGG