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Immunobiology 216 (2011) 1184– 1191
Contents lists available at ScienceDirect
Immunobiology
jo u rn al homepage: www.elsev ier .de / imbio
eview
argeting Syk-Card9-activating C-type lectin receptors by vaccine adjuvants:indings, implications and open questions
oland Lang ∗, Hanne Schoenen, Christiane Deselnstitute of Clinical Microbiology, Immunology and Hygiene, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Wasserturmstr. 3-5,1054 Erlangen, Germany
r t i c l e i n f o
rticle history:eceived 24 April 2011ccepted 13 June 2011
eywords:-type lectin receptorsdjuvant
a b s t r a c t
Pathogen recognition by the innate immune system is essential for the induction of adaptive T cellresponses. A diverse range of pathogen-associated molecular patterns (PAMPs) are recognized by a vari-ety of pathogen recognition receptors (PRRs). Among these are the well known Toll-like receptors (TLR)and the more recently described C-type lectin receptors (CLR) which utilize distinct signaling pathwaysleading to a diverse repertoire of effector molecules produced. The composition of the inflammatory juicereleased from activated innate immune cells has a major impact on the polarization of Th cell responses.
h17yk-Card9 pathway
Defined PAMPs may therefore be used as adjuvants to direct adaptive immune responses to subunit vac-cines. Targeting CLR is an alternative or complementary strategy to TLR-triggering adjuvants that willbenefit the development of more efficient subunit vaccines for prevention of major human infectiousdiseases. In this short review, we discuss the potential of CLRs activating APC via the Syk-Card9 path-way as receptors for adjuvants that direct the development of robust Th17 and Th1 responses to subunitvaccines.
© 2011 Elsevier GmbH. All rights reserved.
ontents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184CLR as major class of transmembrane pattern recognition receptors: phagocytic and signaling CLR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185CLR signaling via the Syk-Card9 pathway generates a distinct transcriptional activation program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185The CLR triggering adjuvants Curdlan and TDB direct the development of Th17 responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1186Implications and open questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1187
Th17-induction: when is it desirable to prevent or combat infection? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1187Mechanism of Th17 induction by CLR triggering adjuvants: how is it orchestrated in vivo? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1187The more the better: should we combine adjuvants triggering TLR and CLR pathways?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188Transcriptional networks activated by CLR and TLR ligands in APC: what makes the difference? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188Translation to human vaccinology: mission possible? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189
ntroduction of how innate immune cells sense microbial ligands through pat-tern recognition receptors in the last decade has injected freshenthusiasm into the field of adjuvant research, and several ligands
Microbes and microbial components have been used as exper-mental adjuvants to enhance vaccination responses for manyecades, but there is a lack of safe and efficient adjuvants forse in humans. The tremendous progress in the understanding
Abbreviations: CLR, C-type lectin receptor; FcR�, Fc receptor gamma chain;LC�2, phospholipase Cgamma2; TLR, toll-like receptor; TB, tuberculosis; TDB,rehalose-dibehenate; TDM, trehalose-dimycolate.∗ Corresponding author. Tel.: +49 9131 8522979; fax: +49 9131 851001.
E-mail address: [email protected] (R. Lang).
171-2985/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.imbio.2011.06.005
for Toll-like receptors (TLR) have been developed as molecularlydefined adjuvants. C-type lectin receptors (CLR) are the secondmajor class of transmembrane pattern recognition receptors onantigen-presenting cells. They bind a diverse array of microbialligands with their extracellular carbohydrate recognition domain.While some CLR are purely phagocytic receptors, others activate
APC through a Syk-Card9 dependent signaling pathway. Promi-nent examples of this latter group include Dectin-1, the receptorfor �-glucans found in bacterial and yeast cell walls; Dectin-2that recognizes fungal high-mannose structures; and Mincle, the
R. Lang et al. / Immunobiology 216 (2011) 1184– 1191 1185
Table 1Signaling CLR: ligands, adapters, responses.
Other names Cell types expressing PAMP recognized ITAM/adapterprotein
Signal transduction Response type References
Dectin-1 Clec7a mDCmonocytesmacrophagesB cells
�-l,3-Glucan YxxL SykPLC�2Card9Bcl-10Malt-1Rafl
IL-l�, IL-6, IL-12 andIL23 productionThl and Thl7 inductionTNF�, CCL17 andCCL22 production
Gringhuis et al. (2009),Gross et al. (2006),LeibundGut-Landmannet al. (2007), Xu et al.(2009), Rogers et al.(2005), Brown (2006),Taylor et al. (2007)
Clec2 Cleclb Platelets neutrophils ND YxxL SykPLC�2
ND Fuller et al. (2007),Kerrigan et al. (2009)
Clec9a DNGR1 BDCA3+ DC monocytesB cells
ND YxxL Syk TNF� productionantigencross-presentation
Sancho et al. (2008,2009)
Dectin-2 Clec6a mDC pDC monocytesmacrophagesneutrophils B cells
High mannoseoligo-saccharide
FcR� SykCard9Malt-1
TNF�, IL-l�, IL-6 andIL-23 productionThl and Thl7 induction
Sato et al. (2006),Gringhuis et al. (2011),Robinson et al. (2009),Saijo et al. (2010)
Mincle Clec4e Clecsf9 mDC monocytesmacrophages
TDM �-mannoseSAP130
FcR� SykPLC�2Card9Bcl-10Malt-1
TNF�, IL-l�, IL-6, IL-12,IL-23 and IL-10productionThl and Thl7 induction
Ishikawa et al. (2009),Schoenen et al. (2010),Wells et al. (2008),Yamasaki et al. (2008,2009)
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Clec5a MDL1 Monocytesmacrophagesosteoclasts
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eceptor for the mycobacterial cord factor trehalose-dimycolate. Inhis review article, we focus on the potential of these Syk-Card9ctivating CLRs as receptors for vaccine adjuvants that direct theevelopment of robust Th17 and Th1 responses to subunit vaccinesith promising protective effects in several preclinical infectionodels for tuberculosis (TB), chlamydial infection or malaria. In
ur view, there are several major open questions regarding therchestration of immune responses following vaccination with CLRriggering adjuvant.
1) At the level of the antigen-presenting cell, the transcrip-tional networks controlling the production of cytokines drivingTh1/Th17 cell differentiation should be elucidated.
2) Furthermore, the contribution of different cell types, such asDCs, macrophages and granulocytes, and their products, forT cell priming and Th1/Th17 differentiation is incompletelyunderstood.
3) Finally, it remains to be determined whether the rules definedin mouse models for CLR expression, ligand specificity and cel-lular responses can be translated to the human system.
argeting CLR is an alternative and complementation to TLR-riggering adjuvants; elucidation of these open research questionsill therefore benefit the development of more efficient subunit
accines for prevention of major human infectious diseases.
LR as major class of transmembrane pattern recognitioneceptors: phagocytic and signaling CLR
C-type lectin receptors are characterized by the presence of Ca2+-dependent carbohydrate recognition domain which deter-ines their specificity for microbial ligands (Robinson et al. 2006).
n addition to soluble C-type lectins that function as opsonins, theouse and human genome encode for a considerable number of
ransmembrane CLR that are expressed constitutively or induciblyn different subsets of immune cells. Binding of the microbial lig-
nd to CLRs expressed on phagocytes in general induces uptake viahagocytosis, degradation of the cargo and presentation of micro-ial peptide antigens on MHC molecules. Delivering antigen viapecific CLR can therefore be employed to target certain subsets of12 ND TNF� production Bakker et al. (1999),Inui et al. (2009), Chenet al. (2008)
dendritic cells with specialized function and to distinct intracellularcompartments of antigen processing and presentation. Examplesfor successful application of this approach are targeting of the Man-nose receptor (CD206) (Burgdorf et al. 2007), the related moleculeCD205 (also known as Dec-205) and DCIR-2 (Dudziak et al. 2007);these were recently reviewed (Caminschi et al. 2009) and are notcovered here. In contrast to these purely phagocytic receptors,other CLR possess the capacity to activate antigen presenting cellsthrough the recruitment of Syk kinase either directly (e.g. Dectin-1 (Gringhuis et al. 2009; Gross et al. 2006; LeibundGut-Landmannet al. 2007; Xu et al. 2009; Rogers et al. 2005; Brown 2006; Tayloret al. 2007), Clec9a (Sancho et al. 2008, 2009) and Clec2 (Fulleret al. 2007; Kerrigan et al. 2009)) or indirectly through the adapterproteins Fc receptor gamma-chain (FcR�) (e.g. Dectin-2 (Sato et al.2006; Gringhuis et al. 2011; Robinson et al. 2009; Saijo et al. 2010),Mincle (Ishikawa et al. 2009; Schoenen et al. 2010; Wells et al. 2008;Yamasaki et al. 2008, 2009)) or Dap12 (e.g. Clec5a (Chen et al. 2008;Bakker et al. 1999; Inui et al. 2009)). Table 1 provides an overviewabout the expression and ligand specificities of these signaling CLR.
CLR signaling via the Syk-Card9 pathway generates adistinct transcriptional activation program
Syk binding to phosphorylated ITAM motifs in the intracellulardomain of the CLR or the adapter protein initiates signaling throughseveral modules (Fig. 1): (1) phospholipase C gamma 2 (PLC�2)is phosphorylated after Dectin-1 activation and induces Ca2+ flux,which in turn triggers nuclear appearance of the transcription fac-tor NFAT via calcineurin activation (Xu et al. 2009; Goodridge et al.2007). PLC�2−/− DC fail to activate MAPK, to secrete inflamma-tory cytokines and to direct Th1/Th17 polarization of T cells (Xuet al. 2009; Tassi et al. 2009). (2) The Card9-Bcl10-Malt1 complex isessential for gene expression induced by the Dectin-1 ligand Curd-lan (Gross et al. 2006) and the Mincle ligands trehalose-dibehenate(TDB) and the mycobacterial cord factor, trehalose-dimycolate(TDM) (Werninghaus et al. 2009); Card9-Bcl10-Malt1 leads to NF�B
activation (Gross et al. 2006), but may not be required for activa-tion of the MAPKs ERK1/2, JNK and p38 (Saijo et al. 2010; Haraet al. 2007). There is evidence that PLC�2 links Syk to Card9, asin PLC�2−/− DC the formation of the Card9-Bcl10-Malt1 complex
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pon Curdlan stimulation is impaired (Xu et al. 2009). (3) In the casef Dectin-1, Syk activation also activates the kinase Raf-1 leadingo phosphorylation of RelA at Ser276 and thereby directs NF�B-
ediated gene expression (Gringhuis et al. 2009).Stimulation of APC with ligands for the CLRs Dectin-1, Dectin-
or Mincle induces expression of many different inflammatoryenes, ranging from cytokines like TNF�, IL-6, IL-10, to varioushemokines and enzymes generating small molecule mediatorsike iNOS (Werninghaus et al. 2009) or Cox2 (Goodridge et al. 2007;agliardi et al. 2010). In addition, costimulatory molecules (CD80,D86) and MHC-II expression are upregulated on DC, therebynhancing T cell stimulatory capacities (LeibundGut-Landmannt al. 2007; Werninghaus et al. 2009). Many of these responsesre also observed upon stimulation of APC through TLR, as maye expected based on the shared activation of NF�B and MAPKignaling modules by both PRR families.
However, there is also evidence for distinct transcriptional acti-ation programs in response to CLR ligation, consistent with thetilization of separate signal transduction machineries and tran-cription factor activation. Early work by Ricciardi-Castagnoli’sroup on the transcriptome of DC stimulated with different classesf PAMPs uncovered the surprising expression of IL-2 (Granuccit al. 2001). IL-2 had previously been thought of as a T cell spe-ific cytokine, whose expression is induced by TCR signaling andnder the control of calcineurin-dependent activation of the tran-cription factor NFAT. While TLR stimuli also induced IL-2, yeastnd zymosan were the most efficient triggers of DC-derived IL-2elease (Granucci et al. 2003). Rogers et al. (2005) demonstratedrst that Syk is required in DC for NFAT-dependent expressionf IL-2 and IL-10 in response to Dectin-1 ligation. Since zymosanlso triggers TLR2, the relative contribution of the TLR2 versushe Dectin-1 signal to the transcriptional activation programs isifficult to judge. The �-glucan Curdlan, prepared from the cellall of Alcaligenes faecalis, is a pure Dectin-1 ligand and therefore
better tool to study CLR-induced signaling and gene expres-ion (LeibundGut-Landmann et al. 2007). Similarly, the syntheticlycolipid trehalose-dibehenate (TDB), an analog of the mycobac-
erial cord factor trehalose-dimycolate (TDM), is a pure CLR ligandhat binds to Mincle (Ishikawa et al. 2009; Schoenen et al. 2010;erninghaus et al. 2009), encoded by the Clec4e gene. Microarraynalysis of mouse macrophages stimulated with the TLR9 ligand
ig. 1. CLR activation in APC translates into Th17 induction: adapters, signaling moleculeectin-1, Dectin-2 and Mincle on innate immune cells triggers activation of the kinasectivation. The release of IL-12 enhances Th1 differentiation, and the prominent inductiriggering also induces release of substantial amounts of G-CSF from APC and recruitmen-CSF and neutrophils also contribute to Th17 induction remains to be tested.
216 (2011) 1184– 1191
CpG DNA or the CLR ligands Curdlan or TDB revealed very sim-ilar responses induced by the Dectin-1 and Mincle ligands thatwere clearly distinct from the TLR-induced activation program(Werninghaus et al. 2009). Therefore, the core innate responseprogram triggered by both PRR families is complemented by CLR-and TLR-pathway specific gene signatures – with important conse-quences for adaptive immune responses.
The CLR triggering adjuvants Curdlan and TDB direct thedevelopment of Th17 responses
T cell activation requires recognition of MHC-bound peptidethrough the antigen-specific T cell receptor, which is usuallyreferred to as “signal 1”. However, signal 1 alone is not suffi-cient, because naïve T cells need additional positive input for clonalexpansion and acquisition of effector functions. APC activationby PAMPs promotes the generation of T cell immune responsesthrough increased antigen presentation and costimulation (signal2) and the generation of cytokines and growth factors directing Thcell differentiation (signal 3) (Joffre et al. 2009). The composition ofthe inflammatory juice released from activated macrophages andDC has thus a major impact on the polarization of Th cell responses(Fig. 1). Specifically, high levels of IL-12p70 produced after TLR acti-vation, especially by the TLR9 ligand CpG, but also in response toLPS, lead to differentiation of IFN�-producing Th1 cells. In con-trast, APC activation by the Dectin-1 ligand Curdlan drives IL-17expression by antigen-specific CD4 T cells (LeibundGut-Landmannet al. 2007), which is correlated to production of significant lev-els of IL-23. This Th17 induction is also seen following infectionwith Candida albicans in vivo and requires the adapter proteinCard9 (LeibundGut-Landmann et al. 2007) that is central to theSyk-signaling triggered by signaling CLR (Gross et al. 2006). Inthe case of Candida infection, the development of a Th17 immuneresponse is synergistically induced by recognition of yeast andhyphal structures via Dectin-1 and Dectin-2 (Robinson et al. 2009;Saijo et al. 2010). The synthetic glycolipid TDB is an efficient adju-vant for subunit vaccination with the Mycobacterium tuberculosis
(TB) fusion protein H1 (composed of ESAT-6 and Ag85B) (Holten-Andersen et al. 2004); in comparison with CpG ODN as adjuvant,we observed a robust mixed Th1/Th17 response when TDB wasthe adjuvant, whereas CpG ODN directed strong IFN� but not IL-17s, transcription factors and cytokine output. Binding of specific ligands to the CLR Syk and signaling via the Card9-Bcl10-Malt1 complex to MAPK, NF�B and NFATon of IL-6, IL-23 and IL-1� favors a strong bias to Th17 cell differentiation. Minclet of neutrophils, which may also be attracted through the action of IL-17. Whether
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R. Lang et al. / Immunob
roduction (Werninghaus et al. 2009). The natural analog of TDB,he mycobacterial cord factor TDM, constitutes a major fraction ofhe mycobacterial cell wall. It has been known to trigger inflam-
atory and granulomatous responses for decades (Hunter et al.006). The cord factor may account for at least a fraction of thedjuvant property of the heat killed M. tuberculosis in Completereund’s Adjuvant. Of interest, TDM is also an active component inhe widely used experimental adjuvant Ribi (together with the TLR4igand monophosphoryl lipid A). Since TDB and TDM bind to Min-le and signal through the Syk-Card9 pathway in macrophages andC (Ishikawa et al. 2009; Schoenen et al. 2010; Werninghaus et al.009), the TLR-Myd88-independence of antibody induction by theibi adjuvant described by Nemazee and colleagues (Gavin et al.006) in hindsight appears not that surprising. More importantly,he Ribi adjuvant was shown by Cooper and colleagues to engender
strong Th17 response to a TB peptide vaccine; this Th17 responseas dependent on IL-23 and linked to protection to TB challenge
hrough rapid recruitment of IFN�-producing CD4 T cells to the sitef infection in the lung (Khader et al. 2007).
Of note, in addition to TDB and TDM, the host ribonucleopro-ein SAP130 (involved in splicing) has been described by Yamasakit al. (2008) as an endogenous Mincle ligand. SAP130 binding isndependent of the classical EGD motif involved in Ca2+-dependentinding of carbohydrates and the glycolipids TDB and TDM to Min-le (Ishikawa et al. 2009; Yamasaki et al. 2008). Release of SAP130rom necrotic cells can therefore signal tissue damage to Mincle-xpressing phagocytes, providing an alarm signal that leads tonflammation and repair responses. In fact, irradation triggers neu-rophil infiltration in the thymus in a Mincle-dependent manner.owever, it is unknown whether Mincle-activation by the DAMPAP130 causes the same transcriptional activation of APC as theicrobial Mincle ligands TDB/TDM or Malassezia spp. (Yamasaki
t al., 2009) and C. albicans (Wells et al., 2008), and whether SAP130as comparable Th17-inducing capacities.
mplications and open questions
h17-induction: when is it desirable to prevent or combatnfection?
As described above, fungal infection triggers developmentf Th17 responses via Dectin-1, Dectin-2 and Mincle. However,t is controversial whether this Th17 response is beneficial oretrimental to the host. On one hand, IL-17 receptor deficiencyendered mice highly susceptible to systemic challenge with C.lbicans (Saijo et al., 2010; Conti et al., 2009; Huang et al., 2004).n the other hand, Romani and colleagues have demonstrated
n a mucosal candidiasis mouse model that the IL-23-IL-17 axisenerates prolonged inflammation in response to challenge andmpairs anti-fungal resistance (Zelante et al. 2007). These contrast-ng results may be unified by the concept that IL-17-dependentnflammation is required for fighting systemic candidiasis, butontributes to pathogenesis in mucosal infection.
Data from genetic mouse models clearly show that IL-17 isequired for defence against the extracellular bacteria Staphylo-occus aureus and Citrobacter rodentium (Ishigame et al., 2009),nd that IL-17 receptor signals protect in a polymicrobial peri-onitis model (Freitas et al. 2009). IL-17 is also produced duringnfection with the intracellular parasites Trypanosoma cruzi andoxoplasma gondii, and contributes to control of protozoal repli-ation and survival (Stumhofer et al. 2006; Miyazaki et al. 2010).n mycobacterial infection, IL-17 appears to be important in the
eneration of early inflammatory responses through recruitment ofeutrophils (Umemura et al. 2007) and, later, in the formation andaintenance of the granuloma structure (Okamoto Yoshida et al.010). However, IL-23p19-deficient mice did not show increased
216 (2011) 1184– 1191 1187
susceptibility to TB infection as long as their IL-12p70 productionwas intact (Khader et al. 2005).
In vaccination approaches for these infections, it appears thatinduction of Th17 responses is desirable as it would augmenta protective host response. Concerning fungal infections, geneticmouse models showed that IL-17 is required for vaccine-inducedprotection in candidiasis (Lin et al. 2009) and systemic mycosescaused by the dimorphic fungi Histoplasma capsulatum, Coccid-ioides posadasii and Blastomyces dermatiditis (Wuthrich et al., 2011).With regard to mycobacterial infection, Wozniak et al. (2010)showed that vaccination with BCG induced and in vitro expandedIL-17 producing T cells afforded partial protection to TB chal-lenge following transfer into RAG−/− mice independent of IFN�(Wozniak et al. 2010). As described above, the use of TDB and TDMas adjuvants for recombinant or peptide subunit vaccines for TBgenerates robust Th17 immunity associated with efficient protec-tion against TB challenge (Werninghaus et al. 2009; Khader et al.2007). In the mouse model, the TDB-containing adjuvant CAF01 hasbeen employed successfully for generation of protective immunityagainst TB, malaria parasites and chlamydia challenge (Agger et al.2008; Yu et al. 2010). However, in order to show causality of IL-17-mediated vaccine protection rather than association, it remains tobe demonstrated in these mouse models that IL-17 itself or otherTh17-associated molecules are indeed required for protection bythe vaccine. This question is important to address for two reasons:first, the confirmation of IL-17 or the identification of Th17 asso-ciated factors (e.g. IL-22, defensins) as protective players wouldvalidate them as markers of protection for vaccination studies. Suchbiomarkers are urgently needed to enable the development andevaluation of preventive and therapeutic strategies against e.g. TB.Second, a proven beneficial role in vaccination responses wouldjustify the potential risks associated with strong Th17 responses.
IL-17 has a well-established pathogenic function in the inflam-mation seen in the autoimmune diseases rheumatoid arthritis andexperimental autoimmune encephalitis (Cua et al. 2003; Hueberet al. 2010). Vaccine adjuvants promoting Th17 induction maytherefore carry an increased risk of triggering flares of inflam-mation and autoimmune diseases. Even in infections, high Th17activity is not always beneficial. In addition to the report in themucosal candida models mentioned already above, increasedinflammatory responses caused by IL-17 during infection withHelicobacter pylori, Aspergillus fumigatus and Borrelia burgdorferiwere unexpectedly associated with augmented microbial growth(Burchill et al. 2003; Shi et al. 2010). While induction of Th17responses to M. tuberculosis was protective when mice werevaccinated before TB challenge (Werninghaus et al. 2009; Khaderet al. 2007), a recent publication indicated that repeated BCGvaccination of TB infected mice increased immunopathology inthe lung (the long-known Koch phenomenon) dependent onBCG-induced Th17 responses (Cruz et al. 2010).
Mechanism of Th17 induction by CLR triggering adjuvants: how isit orchestrated in vivo?
The current paradigm for Th17 cell differentiation assignspivotal function to the cytokines IL-6 and TGF� in driving theexpression of the master regulator of IL-17 expression ROR�t(encoded by the gene Rorc), whereas IL-23 is now considered astabilizer and IL-21 an amplifier of the Th17 phenotype (Bettelliet al. 2008; Stockinger and Veldhoen 2007). Production of IL-1�can strongly enhance the efficiency of Th17 induction in the human(Acosta-Rodriguez et al. 2007) and murine system (Ghoreschi et al.
2010). CLR ligands like Curdlan and the cord factor cause produc-tion of most of these factors from macrophages and DC, consistentwith the current model (Fig. 1). However, the TLR ligand CpG alsotriggers substantial production of IL-6 and IL-23 from APC with-
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ut driving Th17 induction (LeibundGut-Landmann et al. 2007;erninghaus et al. 2009). While this lack of Th17 induction by
pG has been attributed to inhibitory effects of high levels of IL-2p70 and IFN� (LeibundGut-Landmann et al. 2007), the questionemains whether additional factors controlled by CLR ligands con-ribute to Th17 responses. Release of IL-1� protein by human APCn response to Curdlan (Kankkunen et al. 2010) and murine APC inesponse to mycobacterial cord factor (Werninghaus et al. 2009) or. albicans (Gross et al., 2009) through activation of the inflamma-ome, is a distinguishing feature and may at least in part explainhe Th17 inducing properties. IL-1-induced Myd88 signaling in vivo
ay also contribute to the observed lack of adjuvanticity of TDB inyd88−/− mice but not in TLR2/3/4/7 quadruple knockout mice
Agger et al. 2008). Which other components of the cytokine mixnduced by CLR signaling in macrophages and DC could play aole in Th17 differentiation? Expression of G-CSF is very promi-ently induced by TDB and TDM but not CpG in macrophages
n vitro (Schoenen et al. 2010). Following intraperitoneal injectionf TDB-containing liposomes, we have observed high G-CSF lev-ls in the peritoneum which were accompanied by the influx of
large number of granulocytes (Fig. 1) (C. Desel, R. Lang, unpub-ished). Interestingly, very recent data from the literature suggesthat G-CSF may be a new player in Th17 differentiation: Hill et al.2010) investigated the effects of G-CSF treatment of mice on T cellerived cytokine production and observed a specific increase inhe capacity to secrete IL-17A and IL-17F which was independentf IL-6, IL-23 and IL-12p40 but required functional IL-21 signaling.t is therefore conceivable that the release of large amounts of G-SF from macrophages, and probably also infiltrating granulocytes,
ollowing administration of the cord factor amplifies Th17 gener-tion. This concept can be tested using neutralizing antibodies to-CSF in mice. In the same context, depletion of neutrophils as aotential source of high level G-CSF production will provide infor-ation about the importance of the inflammatory reaction for T
ell priming. Similar to the Th17-inducing cytokines IL-6, IL-23 andL-21, the signaling of the G-CSFR is relayed via activation of theranscription factor Stat3 (Panopoulos and Watowich 2008), whichn turn is essential for Th17 generation in mouse and man (Kornt al. 2009). At present, data about the expression of the G-CSFRn the cellular players involved is rather limited. It will thereforee important to investigate the expression of the G-CSFR as a pre-equisite for direct effects of G-CSF on resting and activated APC asell as naïve and activated T cells.
he more the better: should we combine adjuvants triggering TLRnd CLR pathways?
Combining different adjuvants in one vaccine formulation asn approach to maximize and broaden the spectrum of immuneesponses has been proposed (Coffman et al. 2010). The com-ination of adjuvants triggering CLR and TLR pathways appearsarticularly attractive for several reasons. First, as both PRR fam-
lies utilize different adapter molecules and membrane proximalinases, their combination can be expected to act in synergy ratherhan to induce cross tolerance (but see below). Second, the com-ined activation of the gene expression programs induced byoth pathways in APC would be expected to engender even moreobust induction of Th cell responses. Third, as the expressionf individual PRR is often restricted to subsets of APC, adjuvantombinations can be expected to reach a larger fraction of thevailable APC compartment and hence be more efficient in vivo.ourth, PRR expression is often inducible by PAMPs, which can
herefore mutually increase/prime responsiveness; e.g. TLR ligandsike LPS and CpG induce strong upregulation of Mincle expressionn mouse macrophages (Matsumoto et al. 1999) (H. Schoenen, R.ang, unpublished).216 (2011) 1184– 1191
On the other hand, the possibility of negative cross-regulationalso exists. While not directly involving CLR signaling, Ivashkivand colleagues showed that integrin-induced Syk-activation by fib-rinogen in human macrophages down-regulated the production ofcytokines in response to TLR stimulation (Miyazaki et al. 2010). Inaddition, strong Th1 responses can inhibit Th17 induction throughthe action of IL-12p70 and IFN� (Harrington et al. 2005; Park et al.2005). At the level of Th cell responses, combination of adjuvantsmight thus lead to partial cross-inhibition instead of a direct andlinear response amplification induced by the individual adjuvants.
Transcriptional networks activated by CLR and TLR ligands in APC:what makes the difference?
While the membrane-proximal signaling is very differentbetween the signaling CLRs and TLRs, both PRR families share manysimilarities in the activation of NF�B and MAPK signaling mod-ules required for inflammatory cytokine expression. This raises thequestion how the quite different transcriptional responses to theCLR ligands Curdlan and cord factor on one hand and the TLR ligandCpG on the other hand are brought about. Syk-dependent activa-tion of Ca2+ influx and subsequent activation of NFAT is the distinctearly event in APC treated with CLR ligands (Xu et al. 2009; Rogerset al. 2005; Ishikawa et al. 2009; Yamasaki et al. 2008; Goodridgeet al. 2007). However, the direct and indirect transcriptional tar-gets of NFAT in macrophages and DC have not been identifiedcomprehensively yet. Goodridge and Underhill have found thatyeast-induced expression of the transcription factors Egr2 and Egr3is blocked by Cyclosporin, an inhibitor of calcineurin required forNFAT activation (Goodridge et al. 2007). Egr family members areinduced much more strongly by CLR ligands compared to CpG,suggesting that they may impart some of the CLR specific geneactivation programs (Goodridge et al. 2007). We have observedthat Egr induction in TDB-treated macrophages is not inhibited byblockade of protein synthesis with cycloheximide, consistent withexpression controlled by the latent transcription factor NFAT (H.Schoenen, R. Lang, unpublished). Which other transcription fac-tors are operating in bringing about the gene expression programtypically induced by CLR ligands? The availability of genome-widetranscriptome datasets provides the possibility to easily identifytranscription factors whose expression is induced after CLR ligation.These can then be considered candidate regulators of the cytokinesand chemokines expressed at high level (e.g. IL-1�/�, IL-6, G-CSFetc.). Combining the transcriptome data with in silico analysis oftranscription factor binding sites in promoters of regulated genes,the case for a role of specific candidate regulators can be strength-ened and the number of genes of interest can be narrowed down(Ramsey et al. 2008; Weintz et al. 2010). Finally, the use of knockoutmacrophages or alternatively siRNA knockdown in primary cells,will then allow to determine which transcriptional responses arecontrolled by individual factors. Using such approaches, it shouldbe feasible to elucidate the hierarchy of transcriptional responsesinvolved in the making of the uniquely composed cytokine andchemokine output of CLR-stimulated APC that directs the adaptiveimmune response.
Translation to human vaccinology: mission possible?
The efficient induction of protective Th1/Th17 immunity to sub-unit vaccines by CLR ligands in the mouse, and the elucidation ofthe mechanisms involved in vitro, make the synthetic cord factorTDB and the �-glucan Curdlan attractive candidates as molecularly
defined adjuvants for use in humans. In fact, the TDB-containingadjuvant CAF01 has entered a phase I clinical trial for vaccinationwith the TB fusion protein H1, where it appears to be safe andimmunogenic (Ottenhoff et al. 2010). However, it cannot be taken
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or granted that the same rules as defined in the murine system wille valid in man. One of the important lessons learned in the inves-igation of TLR ligands as adjuvants was that rodents and humansiffer in the pattern of cell type-specific TLR expression (Coffmant al. 2010). For example, TLR9 is widely expressed in murine Bells, macrophages, and different DC subsets, whereas in humanshe selective expression in plasmacytoid DC may lead to quite dif-erent adjuvant properties in man and rodents. Therefore, in thease of CLR ligands, one of the challenges ahead is to determinehether indeed the same receptor specificities, tissue distribution
nd cellular responses observed in mice apply to the human CLRystem.
The pivotal role of Dectin-1 and Card9 as receptor and signalingolecule, respectively, in human host defences to Candida infec-
ion was shown in two seminal publications (Ferwerda et al. 2009;locker et al. 2009). The development of chronic mucocutaneousandidiasis in patients homozygous for a mutation in Dectin-1 andwith a more severe phenotype – in humans homozygous for a
ard9 allele encoding a truncated protein was accompanied by aeduced Th17 response of PBMC. In addition, the functionality ofuman Dectin-1 and the Syk-Card9 pathway in recognition of fun-al infection has been demonstrated by Geijtenbeek and colleaguesn two recent papers (Gringhuis et al. 2009, 2011). M. tuberculosisriggers the maturation of human DC and directs the differentiationf Th17 cells in vitro in a Dectin-1-dependent manner (Zenaro et al.009). Of interest for developing Dectin-1 ligands as adjuvants forumans, Curdlan and Dectin-1-triggering antibodies induced theifferentiation of Th17 cells (Agrawal et al. 2010) and CD8 T cellesponses (Ni et al. 2010) in vitro, respectively.
In comparison to the well studied Dectin-1, there is a strikingaucity of data on the role of the other signaling CLR in human
nnate immunity and infection. Dectin-2 shows a divergent expres-ion pattern between mouse and man, with high mRNA levels inuman pDC (Gavino et al. 2005). In the case of Mincle, the humaneceptor is expressed strongly at the mRNA level in monocytes andranulocytes (K. Hofmann, R. Lang, unpublished). However, expres-ion of the Mincle protein by human APC has not been convincinglyemonstrated yet. While the sequence of mouse and human Mincle
s highly conserved, it is unclear whether they have the same lig-nd specificities for the cord factor, high-mannose carbohydratesrom fungal cell walls and the SAP130 ribonucleoprotein. In fact,espite the long history of research into the inflammatory prop-rties of the mycobacterial cord factor, the reactivity of humanacrophages and DC to TDM or the synthetic cord factor analog
DB has apparently not been studied yet. This deficit is lamentablen view of the potential relevance of cord factor recognition for theost response to M. tuberculosis. Confirmation of cross-species con-ervation of a role for Mincle in the granulomatous response to theord factor would suggest that the receptor and the associated path-ay could be targeted for modulation of immunopathology; the
lternative finding that cord factor recognition in the human sys-em does not involve Mincle would imply a potential explanationor the species difference in granuloma formation between mousend man. The recent development of the TDB containing liposomaldjuvant-delivery system CAF01 for application in human subunitaccination also creates an urgent need to investigate whether theules defined in mice for Mincle expression, interaction with TDB,ubsequent APC activation and direction of T cell responses arealid also in humans.
cknowledgments
Work in the Lang laboratory is funded by the Deutscheorschungsgemeinschaft (SFB 643, TP A10; SFB 796, TP B6; and GK660) and by the EU FP7 NEWTBVAC.
216 (2011) 1184– 1191 1189
References
Acosta-Rodriguez, E.V., Napolitani, G., Lanzavecchia, A., Sallusto, F., 2007. Inter-leukins 1beta and 6 but not transforming growth factor-beta are essential for thedifferentiation of interleukin 17-producing human T helper cells. Nat. Immunol.8, 942.
Agger, E.M., Rosenkrands, I., Hansen, J., Brahimi, K., Vandahl, B.S., Aagaard, C., Wern-inghaus, K., Kirschning, C., Lang, R., Christensen, D., Theisen, M., Follmann, F.,Andersen, P., 2008. Cationic liposomes formulated with synthetic mycobacterialcordfactor (CAF01): a versatile adjuvant for vaccines with different immunolog-ical requirements. PLoS One 3, e3116.
Agrawal, S., Gupta, S., Agrawal, A., 2010. Human dendritic cells activated via dectin-1are efficient at priming Th17, cytotoxic CD8 T and B cell responses. PLoS One 5,e13418.
Bakker, A.B., Baker, E., Sutherland, G.R., Phillips, J.H., Lanier, L.L., 1999. MyeloidDAP12-associating lectin (MDL)-1 is a cell surface receptor involved in the acti-vation of myeloid cells. Proc. Natl. Acad. Sci. U. S. A. 96, 9792.
Bettelli, E., Korn, T., Oukka, M., Kuchroo, V.K., 2008. Induction and effector functionsof T(H)17 cells. Nature 453, 1051.
Brown, G.D., 2006. Dectin-1: a signalling non-TLR pattern-recognition receptor. Nat.Rev. Immunol. 6, 33.
Burchill, M.A., Nardelli, D.T., England, D.M., DeCoster, D.J., Christopherson, J.A.,Callister, S.M., Schell, R.F., 2003. Inhibition of interleukin-17 prevents the devel-opment of arthritis in vaccinated mice challenged with Borrelia burgdorferi.Infect. Immun. 71, 3437.
Burgdorf, S., Kautz, A., Bohnert, V., Knolle, P.A., Kurts, C., 2007. Distinct pathwaysof antigen uptake and intracellular routing in CD4 and CD8 T cell activation.Science 316, 612.
Caminschi, I., Lahoud, M.H., Shortman, K., 2009. Enhancing immune responses bytargeting antigen to DC. Eur. J. Immunol. 39, 931.
Chen, S.T., Lin, Y.L., Huang, M.T., Wu, M.F., Cheng, S.C., Lei, H.Y., Lee, C.K., Chiou, T.W.,Wong, C.H., Hsieh, S.L., 2008. CLEC5A is critical for dengue-virus-induced lethaldisease. Nature 453, 672.
Coffman, R.L., Sher, A., Seder, R.A., 2010. Vaccine adjuvants: putting innate immunityto work. Immunity 33, 492.
Conti, H.R., Shen, F., Nayyar, N., Stocum, E., Sun, J.N., Lindemann, M.J., Ho, A.W.,Hai, J.H., Yu, J.J., Jung, J.W., Filler, S.G., Masso-Welch, P., Edgerton, M., Gaffen,S.L., 2009. Th17 cells and IL-17 receptor signaling are essential for mucosal hostdefense against oral candidiasis. J. Exp. Med. 206, 299.
Cruz, A., Fraga, A.G., Fountain, J.J., Rangel-Moreno, J., Torrado, E., Saraiva, M., Pereira,D.R., Randall, T.D., Pedrosa, J., Cooper, A.M., Castro, A.G., 2010. Pathological roleof interleukin 17 in mice subjected to repeated BCG vaccination after infectionwith Mycobacterium tuberculosis. J. Exp. Med. 207, 1609.
Cua, D.J., Sherlock, J., Chen, Y., Murphy, C.A., Joyce, B., Seymour, B., Lucian, L., To,W., Kwan, S., Churakova, T., Zurawski, S., Wiekowski, M., Lira, S.A., Gorman, D.,Kastelein, R.A., Sedgwick, J.D., 2003. Interleukin-23 rather than interleukin-12is the critical cytokine for autoimmune inflammation of the brain. Nature 421,744.
Dudziak, D., Kamphorst, A.O., Heidkamp, G.F., Buchholz, V.R., Trumpfheller, C.,Yamazaki, S., Cheong, C., Liu, K., Lee, H.W., Park, C.G., Steinman, R.M., Nussen-zweig, M.C., 2007. Differential antigen processing by dendritic cell subsets invivo. Science 315, 107.
Ferwerda, B., Ferwerda, G., Plantinga, T.S., Willment, J.A., van Spriel, A.B., Venselaar,H., Elbers, C.C., Johnson, M.D., Cambi, A., Huysamen, C., Jacobs, L., Jansen, T.,Verheijen, K., Masthoff, L., Morre, S.A., Vriend, G., Williams, D.L., Perfect, J.R.,Joosten, L.A., Wijmenga, C., van der Meer, J.W., Adema, G.J., Kullberg, B.J., Brown,G.D., Netea, M.G., 2009. Human dectin-1 deficiency and mucocutaneous fungalinfections. N. Engl. J. Med. 361, 1760.
Freitas, A., Alves-Filho, J.C., Victoni, T., Secher, T., Lemos, H.P., Sonego, F., Cunha, F.Q.,Ryffel, B., 2009. IL-17 receptor signaling is required to control polymicrobialsepsis. J. Immunol. 182, 7846.
Fuller, G.L., Williams, J.A., Tomlinson, M.G., Eble, J.A., Hanna, S.L., Pohlmann, S.,Suzuki-Inoue, K., Ozaki, Y., Watson, S.P., Pearce, A.C., 2007. The C-type lectinreceptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signaling cascade. J. Biol. Chem. 282, 12397.
Gagliardi, M.C., Teloni, R., Mariotti, S., Bromuro, C., Chiani, P., Romagnoli, G.,Giannoni, F., Torosantucci, A., Nisini, R., 2010. Endogenous PGE2 promotesthe induction of human Th17 responses by fungal ss-glucan. J. Leukoc. Biol.88, 947.
Gavin, A.L., Hoebe, K., Duong, B., Ota, T., Martin, C., Beutler, B., Nemazee, D., 2006.Adjuvant-enhanced antibody responses in the absence of toll-like receptor sig-naling. Science 314, 1936.
Gavino, A.C., Chung, J.S., Sato, K., Ariizumi, K., Cruz Jr., P.D., 2005. Identification andexpression profiling of a human C-type lectin, structurally homologous to mousedectin-2. Exp. Dermatol. 14, 281.
Ghoreschi, K., Laurence, A., Yang, X.P., Tato, C.M., McGeachy, M.J., Konkel, J.E., Ramos,H.L., Wei, L., Davidson, T.S., Bouladoux, N., Grainger, J.R., Chen, Q., Kanno, Y.,Watford, W.T., Sun, H.W., Eberl, G., Shevach, E.M., Belkaid, Y., Cua, D.J., Chen,W., O’Shea, J.J., 2010. Generation of pathogenic T(H)17 cells in the absence ofTGF-beta signalling. Nature 467, 967.
Glocker, E.O., Hennigs, A., Nabavi, M., Schaffer, A.A., Woellner, C., Salzer, U., Pfeifer,
D., Veelken, H., Warnatz, K., Tahami, F., Jamal, S., Manguiat, A., Rezaei, N.,Amirzargar, A.A., Plebani, A., Hannesschlager, N., Gross, O., Ruland, J., Grim-bacher, B., 2009. A homozygous CARD9 mutation in a family with susceptibilityto fungal infections. N. Engl. J. Med. 361, 1727.
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190 R. Lang et al. / Immunob
oodridge, H.S., Simmons, R.M., Underhill, D.M., 2007. Dectin-1 stimulation by Can-dida albicans yeast or zymosan triggers NFAT activation in macrophages anddendritic cells. J. Immunol. 178, 3107.
ranucci, F., Vizzardelli, C., Pavelka, N., Feau, S., Persico, M., Virzi, E., Rescigno, M.,Moro, G., Ricciardi-Castagnoli, P., 2001. Inducible IL-2 production by dendriticcells revealed by global gene expression analysis. Nat. Immunol. 2, 882.
ranucci, F., Feau, S., Angeli, V., Trottein, F., Ricciardi-Castagnoli, P., 2003. Early IL-2production by mouse dendritic cells is the result of microbial-induced priming.J. Immunol. 170, 5075.
ringhuis, S.I., den Dunnen, J., Litjens, M., van der Vlist, M., Wevers, B., Bruijns, S.C.,Geijtenbeek, T.B., 2009. Dectin-1 directs T helper cell differentiation by control-ling noncanonical NF-kappaB activation through Raf-1 and Syk. Nat. Immunol.10, 203.
ringhuis, S.I., Wevers, B.A., Kaptein, T.M., van Capel, T.M., Theelen, B., Boekhout, T.,de Jong, E.C., Geijtenbeek, T.B., 2011. Selective C-Rel activation via Malt1 con-trols anti-fungal T(H)-17 immunity by Dectin-1 and Dectin-2. PLoS Pathog. 7,e1001259.
ross, O., Gewies, A., Finger, K., Schafer, M., Sparwasser, T., Peschel, C., Forster, I.,Ruland, J., 2006. Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature 442, 651.
ross, O., Poeck, H., Bscheider, M., Dostert, C., Hannesschlager, N., Endres, S., Hart-mann, G., Tardivel, A., Schweighoffer, E., Tybulewicz, V., Mocsai, A., Tschopp, J.,Ruland, J., 2009. Syk kinase signalling couples to the Nlrp3 inflammasome foranti-fungal host defence. Nature 459, 433.
ara, H., Ishihara, C., Takeuchi, A., Imanishi, T., Xue, L., Morris, S.W., Inui, M., Takai,T., Shibuya, A., Saijo, S., Iwakura, Y., Ohno, N., Koseki, H., Yoshida, H., Penninger,J.M., Saito, T., 2007. The adaptor protein CARD9 is essential for the activation ofmyeloid cells through ITAM-associated and Toll-like receptors. Nat. Immunol.8, 619.
arrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy, T.L., Murphy, K.M.,Weaver, C.T., 2005. Interleukin 17-producing CD4+ effector T cells develop via alineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6, 1123.
ill, G.R., Olver, S.D., Kuns, R.D., Varelias, A., Raffelt, N.C., Don, A.L., Markey, K.A.,Wilson, Y.A., Smyth, M.J., Iwakura, Y., Tocker, J., Clouston, A.D., Macdonald, K.P.,2010. Stem cell mobilization with G-CSF induces type 17 differentiation andpromotes scleroderma. Blood 116, 819.
olten-Andersen, L., Doherty, T.M., Korsholm, K.S., Andersen, P., 2004. Combinationof the cationic surfactant dimethyl dioctadecyl ammonium bromide and syn-thetic mycobacterial cord factor as an efficient adjuvant for tuberculosis subunitvaccines. Infect. Immun. 72, 1608.
uang, W., Na, L., Fidel, P.L., Schwarzenberger, P., 2004. Requirement of interleukin-17A for systemic anti-Candida albicans host defense in mice. J. Infect. Dis. 190,624.
ueber, A.J., Asquith, D.L., McInnes, I.B., Miller, A.M., 2010. Embracing novelcytokines in RA - complexity grows as does opportunity! Best Pract. Res. Clin.Rheumatol. 24, 479.
unter, R.L., Olsen, M., Jagannath, C., Actor, J.K., 2006. Trehalose 6,6′-dimycolate andlipid in the pathogenesis of caseating granulomas of tuberculosis in mice. Am. J.Pathol. 168, 1249.
nui, M., Kikuchi, Y., Aoki, N., Endo, S., Maeda, T., Sugahara-Tobinai, A., Fujimura,S., Nakamura, A., Kumanogoh, A., Colonna, M., Takai, T., 2009. Signal adaptorDAP10 associates with MDL-1 and triggers osteoclastogenesis in cooperationwith DAP12. Proc. Natl. Acad. Sci. U. S. A. 106, 4816.
shigame, H., Kakuta, S., Nagai, T., Kadoki, M., Nambu, A., Komiyama, Y., Fujikado, N.,Tanahashi, Y., Akitsu, A., Kotaki, H., Sudo, K., Nakae, S., Sasakawa, C., Iwakura,Y., 2009. Differential roles of interleukin-17A and -17F in host defense againstmucoepithelial bacterial infection and allergic responses. Immunity 30, 108.
shikawa, E., Ishikawa, T., Morita, Y.S., Toyonaga, K., Yamada, H., Takeuchi, O.,Kinoshita, T., Akira, S., Yoshikai, Y., Yamasaki, S., 2009. Direct recognition of themycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle. J. Exp.Med. 206, 2879.
offre, O., Nolte, M.A., Sporri, R., Reis e Sousa, C., 2009. Inflammatory signals in den-dritic cell activation and the induction of adaptive immunity. Immunol. Rev. 227,234.
ankkunen, P., Teirila, L., Rintahaka, J., Alenius, H., Wolff, H., Matikainen, S., 2010.(1,3)-beta-glucans activate both dectin-1 and NLRP3 inflammasome in humanmacrophages. J. Immunol. 184, 6335.
errigan, A.M., Dennehy, K.M., Mourao-Sa, D., Faro-Trindade, I., Willment, J.A.,Taylor, P.R., Eble, J.A., Reis e Sousa, C., Brown, G.D., 2009. CLEC-2 is a phago-cytic activation receptor expressed on murine peripheral blood neutrophils. J.Immunol. 182, 4150.
hader, S.A., Pearl, J.E., Sakamoto, K., Gilmartin, L., Bell, G.K., Jelley-Gibbs, D.M., Ghi-lardi, N., deSauvage, F., Cooper, A.M., 2005. IL-23 compensates for the absenceof IL-12p70 and is essential for the IL-17 response during tuberculosis but is dis-pensable for protection and antigen-specific IFN-gamma responses if IL-12p70is available. J. Immunol. 175, 788.
hader, S.A., Bell, G.K., Pearl, J.E., Fountain, J.J., Rangel-Moreno, J., Cilley, G.E., Shen,F., Eaton, S.M., Gaffen, S.L., Swain, S.L., Locksley, R.M., Haynes, L., Randall, T.D.,Cooper, A.M., 2007. IL-23 and IL-17 in the establishment of protective pulmonaryCD4(+) T cell responses after vaccination and during Mycobacterium tuberculosischallenge. Nat. Immunol. 8, 369.
orn, T., Bettelli, E., Oukka, M., Kuchroo, V.K., 2009. IL-17 and Th17 Cells. Annu. Rev.Immunol. 27, 485.
eibundGut-Landmann, S., Gross, O., Robinson, M.J., Osorio, F., Slack, E.C., Tsoni, S.V.,Schweighoffer, E., Tybulewicz, V., Brown, G.D., Ruland, J., Reis e Sousa, C., 2007.
216 (2011) 1184– 1191
Syk- and CARD9-dependent coupling of innate immunity to the induction of Thelper cells that produce interleukin 17. Nat. Immunol. 8, 630.
Lin, L., Ibrahim, A.S., Xu, X., Farber, J.M., Avanesian, V., Baquir, B., Fu, Y., French,S.W., Edwards Jr., J....E., Spellberg, B., 2009. Th1- Th17 cells mediate protectiveadaptive immunity against Staphylococcus aureus and Candida albicans infectionin mice. PLoS Pathog. 5, e1000703.
Matsumoto, M., Tanaka, T., Kaisho, T., Sanjo, H., Copeland, N.G., Gilbert, D.J., Jenkins,N.A., Akira, S., 1999. A novel LPS-inducible C-type lectin is a transcriptional targetof NF-IL6 in macrophages. J. Immunol. 163, 5039.
Miyazaki, Y., Hamano, S., Wang, S., Shimanoe, Y., Iwakura, Y., Yoshida, H., 2010. IL-17is necessary for host protection against acute-phase Trypanosoma cruzi infection.J. Immunol. 185, 1150.
Ni, L., Gayet, I., Zurawski, S., Duluc, D., Flamar, A.L., Li, X.H., O’Bar, A., Clayton, S.,Palucka, A.K., Zurawski, G., Banchereau, J., Oh, S., 2010. Concomitant activationand antigen uptake via human dectin-1 results in potent antigen-specific CD8+T cell responses. J. Immunol. 185, 3504.
Okamoto Yoshida, Y., Umemura, M., Yahagi, A., O’Brien, R.L., Ikuta, K., Kishihara, K.,Hara, H., Nakae, S., Iwakura, Y., Matsuzaki, G., 2010. Essential role of IL-17A inthe formation of a mycobacterial infection-induced granuloma in the lung. J.Immunol. 184, 4414.
Ottenhoff, T.H., Doherty, T.M., Dissel, J.T., Bang, P., Lingnau, K., Kromann, I., Andersen,P., 2010. First in humans: a new molecularly defined vaccine shows excellentsafety and strong induction of long-lived Mycobacterium tuberculosis-specificTh1-cell like responses. Hum. Vaccin., 6.
Panopoulos, A.D., Watowich, S.S., 2008. Granulocyte colony-stimulating fac-tor: molecular mechanisms of action during steady state and ‘emergency’hematopoiesis. Cytokine 42, 277.
Park, H., Li, Z., Yang, X.O., Chang, S.H., Nurieva, R., Wang, Y.H., Wang, Y., Hood, L.,Zhu, Z., Tian, Q., Dong, C., 2005. A distinct lineage of CD4 T cells regulates tissueinflammation by producing interleukin 17. Nat. Immunol. 6, 1133.
Ramsey, S.A., Klemm, S.L., Zak, D.E., Kennedy, K.A., Thorsson, V., Li, B., Gilchrist,M., Gold, E.S., Johnson, C.D., Litvak, V., Navarro, G., Roach, J.C., Rosenberger,C.M., Rust, A.G., Yudkovsky, N., Aderem, A., Shmulevich, I., 2008. Uncovering amacrophage transcriptional program by integrating evidence from motif scan-ning and expression dynamics. PLoS Comput. Biol. 4, e1000021.
Robinson, M.J., Sancho, D., Slack, E.C., Leibundgut-Landmann, S., Sousa, C.R., 2006.Myeloid C-type lectins in innate immunity. Nat. Immunol. 7, 1258.
Robinson, M.J., Osorio, F., Rosas, M., Freitas, R.P., Schweighoffer, E., Gross, O., Verbeek,J.S., Ruland, J., Tybulewicz, V., Brown, G.D., Moita, L.F., Taylor, P.R., Reis e Sousa,C., 2009. Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Th17responses to fungal infection. J. Exp. Med. 206, 2037.
Rogers, N.C., Slack, E.C., Edwards, A.D., Nolte, M.A., Schulz, O., Schweighoffer, E.,Williams, D.L., Gordon, S., Tybulewicz, V.L., Brown, G.D., Reis e Sousa, C., 2005.Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recogni-tion pathway for C type lectins. Immunity 22, 507.
Saijo, S., Ikeda, S., Yamabe, K., Kakuta, S., Ishigame, H., Akitsu, A., Fujikado, N., Kusaka,T., Kubo, S., Chung, S.H., Komatsu, R., Miura, N., Adachi, Y., Ohno, N., Shibuya, K.,Yamamoto, N., Kawakami, K., Yamasaki, S., Saito, T., Akira, S., Iwakura, Y., 2010.Dectin-2 recognition of alpha-mannans and induction of Th17 cell differentia-tion is essential for host defense against Candida albicans. Immunity 32, 681.
Sancho, D., Mourao-Sa, D., Joffre, O.P., Schulz, O., Rogers, N.C., Pennington, D.J., Car-lyle, J.R., Reis e Sousa, C., 2008. Tumor therapy in mice via antigen targeting to anovel DC-restricted C-type lectin. J. Clin. Invest. 118, 2098.
Sancho, D., Joffre, O.P., Keller, A.M., Rogers, N.C., Martinez, D., Hernanz-Falcon, P.,Rosewell, I., Reis e Sousa, C., 2009. Identification of a dendritic cell receptor thatcouples sensing of necrosis to immunity. Nature 458, 899.
Sato, K., Yang, X.L., Yudate, T., Chung, J.S., Wu, J., Luby-Phelps, K., Kimberly, R.P.,Underhill, D., Cruz Jr., P.D., Ariizumi, K., 2006. Dectin-2 is a pattern recognitionreceptor for fungi that couples with the Fc receptor gamma chain to induceinnate immune responses. J. Biol. Chem. 281, 38854.
Schoenen, H., Bodendorfer, B., Hitchens, K., Manzanero, S., Werninghaus, K., Nim-merjahn, F., Agger, E.M., Stenger, S., Andersen, P., Ruland, J., Brown, G.D., Wells, C.,Lang, R., 2010. Cutting edge: mincle is essential for recognition and adjuvanticityof the mycobacterial cord factor and its synthetic analog trehalose-dibehenate.J Immunol 184, 2756.
Shi, Y., Liu, X.F., Zhuang, Y., Zhang, J.Y., Liu, T., Yin, Z., Wu, C., Mao, X.H., Jia, K.R., Wang,F.J., Guo, H., Flavell, R.A., Zhao, Z., Liu, K.Y., Xiao, B., Guo, Y., Zhang, W.J., Zhou, W.Y.,Guo, G., Zou, Q.M., 2010. Helicobacter pylori-induced Th17 responses modulateTh1 cell responses, benefit bacterial growth, and contribute to pathology in mice.J. Immunol. 184, 5121.
Stockinger, B., Veldhoen, M., 2007. Differentiation and function of Th17 T cells. Curr.Opin. Immunol. 19, 281.
Stumhofer, J.S., Laurence, A., Wilson, E.H., Huang, E., Tato, C.M., Johnson, L.M.,Villarino, A.V., Huang, Q., Yoshimura, A., Sehy, D., Saris, C.J., O’Shea, J.J.,Hennighausen, L., Ernst, M., Hunter, C.A., 2006. Interleukin 27 negativelyregulates the development of interleukin 17-producing T helper cells dur-ing chronic inflammation of the central nervous system. Nat. Immunol.7, 937.
Tassi, I., Cella, M., Castro, I., Gilfillan, S., Khan, W.N., Colonna, M., 2009. Require-ment of phospholipase C-gamma2 (PLCgamma2) for Dectin-1-induced antigenpresentation and induction of TH1/TH17 polarization. Eur. J. Immunol. 39, 1369.
Taylor, P.R., Tsoni, S.V., Willment, J.A., Dennehy, K.M., Rosas, M., Findon, H., Haynes,K., Steele, C., Botto, M., Gordon, S., Brown, G.D., 2007. Dectin-1 is requiredfor beta-glucan recognition and control of fungal infection. Nat. Immunol.8, 31.
iology
U
W
W
W
W
W
M., Romani, L., 2007. IL-23 and the Th17 pathway promote inflammation andimpair antifungal immune resistance. Eur. J. Immunol. 37, 2695.
R. Lang et al. / Immunob
memura, M., Yahagi, A., Hamada, S., Begum, M.D., Watanabe, H., Kawakami, K.,Suda, T., Sudo, K., Nakae, S., Iwakura, Y., Matsuzaki, G., 2007. IL-17-mediatedregulation of innate and acquired immune response against pulmonaryMycobacterium bovis bacille Calmette-Guerin infection. J. Immunol. 178, 3786.
eintz, G., Olsen, J.V., Fruhauf, K., Niedzielska, M., Amit, I., Jantsch, J., Mages, J.,Frech, C., Dolken, L., Mann, M., Lang, R., 2010. The phosphoproteome of toll-likereceptor-activated macrophages. Mol. Syst. Biol. 6, 371.
ells, C.A, Salvage-Jones, J.A., Li, X., Hitchens, K., Butcher, S., Murray, R.Z., Beckhouse,A.G., Lo, Y.L., Manzanero, S., Cobbold, C., Schroder, K., Ma, B., Orr, S., Stewart, L.,Lebus, D., Sobieszczuk, P., Hume, D.A., Stow, J., Blanchard, H., Ashman, R.B., 2008.The macrophage-inducible C-type lectin, mincle, is an essential component ofthe innate immune response to Candida albicans. J. Immunol. 180, 7404.
erninghaus, K., Babiak, A., Gross, O., Holscher, C., Dietrich, H., Agger, E.M., Mages,J., Mocsai, A., Schoenen, H., Finger, K., Nimmerjahn, F., Brown, G.D., Kirschning,C., Heit, A., Andersen, P., Wagner, H., Ruland, J., Lang, R., 2009. Adjuvanticity ofa synthetic cord factor analogue for subunit Mycobacterium tuberculosis vacci-nation requires FcRgamma-Syk-Card9-dependent innate immune activation. J.Exp. Med. 206, 89.
ozniak, T.M., Saunders, B.M., Ryan, A.A., Britton, W.J., 2010. Mycobacterium bovisBCG-specific Th17 cells confer partial protection against Mycobacterium tuber-
culosis infection in the absence of gamma interferon. Infect. Immun. 78, 4187.uthrich, M., Gern, B., Hung, C.Y., Ersland, K., Rocco, N., Pick-Jacobs, J., Galles, K.,Filutowicz, H., Warner, T., Evans, M., Cole, G., Klein, B., 2011. Vaccine-inducedprotection against 3 systemic mycoses endemic to North America requires Th17cells in mice. J. Clin. Invest. 121, 554.
216 (2011) 1184– 1191 1191
Xu, S., Huo, J., Lee, K.G., Kurosaki, T., Lam, K.P., 2009. Phospholipase Cgamma2 iscritical for Dectin-1-mediated Ca2+ flux and cytokine production in dendriticcells. J. Biol. Chem. 284, 7038.
Yamasaki, S., Ishikawa, E., Sakuma, M., Hara, H., Ogata, K., Saito, T., 2008. Mincle isan ITAM-coupled activating receptor that senses damaged cells. Nat. Immunol.9, 1179.
Yamasaki, S., Matsumoto, M., Takeuchi, O., Matsuzawa, T., Ishikawa, E., Sakuma, M.,Tateno, H., Uno, J., Hirabayashi, J., Mikami, Y., Takeda, K., Akira, S., Saito, T., 2009.C-type lectin Mincle is an activating receptor for pathogenic fungus Malassezia.Proc. Natl. Acad. Sci. U. S. A. 106, 1897.
Yu, H., Jiang, X., Shen, C., Karunakaran, K.P., Jiang, J., Rosin, N.L., Brunham, R.C.,2010. Chlamydia muridarum T-cell antigens formulated with the adjuvantDDA/TDB induce immunity against infection that correlates with a high fre-quency of gamma interferon (IFN-gamma)/tumor necrosis factor alpha andIFN-gamma/interleukin-17 double-positive CD4+T cells. Infect. Immun. 78,2272.
Zelante, T., De Luca, A., Bonifazi, P., Montagnoli, C., Bozza, S., Moretti, S., Belladonna,M.L., Vacca, C., Conte, C., Mosci, P., Bistoni, F., Puccetti, P., Kastelein, R.A., Kopf,
Zenaro, E., Donini, M., Dusi, S., 2009. Induction of Th1/Th17 immune response byMycobacterium tuberculosis: role of dectin-1 mannose receptor, and DC-SIGN. J.Leukoc. Biol. 86, 1393.
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