Contact, Collaboration, and Conflict: Signal Integration of Syk-Coupled C-Type Lectin Receptors

The immune system identifies invading microbial pathogens by conserved microbial motifs, known as pathogen-associated molecular patterns (PAMPs). For any given pathogen a combination of such PAMPs is recognized by pattern recognition receptors (PRRs) on innate immune cells. Detection of a pathogen by a combination of receptors ensures redundancy and results in lower likelihood for immune evasion by the pathogen and robustness against genetic diversity in the host. Furthermore, engagement of a pathogen-specific set of receptors allows tailoring of the immune response to protect the body against specific infections.

TLRs are the best studied family of PRRs expressed on innate immune cells. Ten functional TLRs are known in humans, and twelve have been described in mice. Interactions of TLRs and crosstalk of TLR signaling have been studied for almost two decades. C-type lectin receptors (CLRs) as another major group of PRRs have entered the field later, but their investigation has gained much momentum in the last decade. Many studies have been conducted on CLRs assigned to the so-called dendritic cell (DC)–associated C-type lectin (Dectin)-1 or Dectin-2 clusters, localized within the NK cell gene cluster on human chromosome 12 or mouse chromosome 6 (1–3). Several excellent reviews on the function of these CLRs in antimicrobial defense and homeostasis are available (4, 5). In this review we summarize the current knowledge about signaling downstream of the activating CLR Dectin-1 (Clec7a), Dectin-2 (human Clec6a, mouse Clec4n), macrophage-inducible C-type lectin (Mincle; Clec4e), and macrophage C-type lectin (Mcl; Clec4d) that is largely dependent on the kinase spleen tyrosine kinase (Syk). Table I provides an overview of defined ligands and microorganisms bound by this group of PRRs. In addition to microbial carbohydrate and glycolipid structures acting as PAMPs, several CLRs bind endogenous ligands such as SAP130 released by dying cells or cholesterol crystals. Thus, these CLRs are involved in homeostatic responses and inflammatory conditions (6–9), in addition to host response to pathogens and commensals (10–12). CLR-induced APC activation directs Th cell differentiation [see Geijtenbeek and Gringhuis (13) for review], and synthetic ligands for CLRs are under development as adjuvants (14, 15). Bacteria and fungi can express more than one CLR ligand, and therefore simultaneous engagement of CLRs during recognition of microbial pathogens is likely. Additionally, concurrent activation of TLRs and CLRs will occur, leading to synergistic and antagonistic responses with sometimes unexpected outcomes. For a generalized concept of signal integration in innate immunity, we refer to a recent publication by Elinav and colleagues (16). With regard to CLR signaling, there is evidence that Dectin-1, Dectin-2, Mincle, and Mcl not only act as activating PRRs, but they are particularly important for regulation and tailoring of immune responses. In this review, we discuss the interactions following simultaneous engagement of several CLRs and TLRs. Conceptually, we propose that such signal integration can occur on different levels, which are discussed in this structured review: 1) contact between receptors, with possible consequences for ligand binding, receptor stability, or localization; 2) control of receptor expression levels, adjusting the responsiveness; 3) collaborative signaling, leading to synergistic responses; and 4) conflicting signaling, tailoring the inflammatory response.

Ligand binding to either the extracellular or endosomal ectodomain of TLRs leads to dimerization of the cytoplasmic Toll/IL-1R (TIR) domain. Dimerization can occur as both homo- or heterodimers (TLR1/TLR2 and TLR2/TLR6). Adaptor proteins are subsequently recruited by TIR–TIR interactions. Downstream signaling is induced dependent on MyD88 (engaged by all TLRs except TLR3) and/or TIR domain–containing adaptor protein inducing IFN-β (TRIF, engaged by TLR3 and TLR4). Activation of NF-κB and IFN regulatory factors (IRF) are central events downstream of MyD88 and TRIF (17) (Fig. 1). Synergistic responses have been described for combination of MyD88 and TRIF-dependent TLR ligands (18, 19). C-type lectins are a protein superfamily with >1000 members belonging to 17 subgroups based on structural and ligand-binding features (20). The receptors in the Dectin-1 and Dectin-2 cluster, of which many contribute to innate immunity, belong to the related subgroup II (asialoglycoprotein and DC receptors, Ca²⁺ binding) and subgroup V (NK cell receptors, non-Ca²⁺ binding). Ligand binding is mediated by the C-type lectin domain (CTLD) (21), often containing a QPD or EPN motif. Dectin-1 (Clec7a, CD369, Clecsf12), Dectin-2 (human CLEC6A, mouse Clec4n, Clecsf10), Mincle (Clec4e, Clecsf9), and Mcl (Clec4d, CD368, Clecsf8) are activating receptors that share signaling via ITAM and Syk. Dectin-1 recruits Syk via a hemITAM motif, whereas Mcl, Mincle, and Dectin-2 associate with the ITAM-containing FcRγ-chain. Downstream of Syk, activation of the canonical NF-κB pathway is dependent on formation of the Card9/Bcl10/Malt1 complex (4) (Fig. 1).

Consistent with the shared activation of NF-κB and MAPK by TLR and CLR, ligands for both types of PRRs induce an overlapping set of proinflammatory cytokines and chemokines. However, there is also evidence for selective and preferential target gene expression. A limited number of microarray studies have compared stimulation with CLR ligands such as β-glucans (Dectin-1) or trehalose dibehenate (TDB; Mincle) with TLR ligands such as Pam3 (TLR2), LPS (TLR4), or CpG (TLR9) (22–25). Whereas TLR9 ligation induces strong IL-12 production associated with Th1 generation, the induction of IL-1β, IL-6, and IL-23 after engagement of Syk-Card9–coupled CLRs is observed across cell types and species, promoting the differentiation of IL-17–producing CD4⁺ T cells (23, 24, 26, 27). Remarkably, to date there is only very limited information about the effects of combined stimulation of CLR and TLR pathways on global gene expression.

We concentrate in the following on reviewing the current literature on Dectin-1 and the Dectin-2, Mcl and Mincle.

Dectin-1 is the best studied receptor in the Dectin-1 family; signaling events downstream of Dectin-1 ligation are often regarded as prototypic for Syk-coupled CLRs (28, 29). Dectin-1 recognizes β-glucans in fungal and mycobacterial cell walls in a Ca²⁺-independent manner (30–33) (see Table I). β-Glucans bind to Dectin-1 homodimers, and ligand binding has been suggested to induce oligomerization (34). Whereas particulate ligands result in formation of a “phagocytic synapse,” stimulation with a soluble ligand does not induce a response (35). Dectin-1 signals via its internal hemITAM motif (single YxxL/I motif) (36), which is phosphorylated upon ligand binding. Recruitment of Syk to the phosphorylated hemITAM is pivotal for Dectin-1 responses (37) and requires a phosphatase-independent chaperone function of SHP-2 (38). Lipid raft formation has been shown to be important for Syk recruitment (39, 40). Downstream of Syk, canonical NF-κB signaling is dependent on the activation of phospholipase C (PLC)γ2 (39), phosphorylation of protein kinase C (PKC)δ (41, 42), and formation of the Card9/Bcl10/Malt-1 complex (43, 44), which involves the ubiquitin ligase Trim62 (45). Different NF-κB subunits are activated following Dectin-1 ligation through Syk and Raf-1 as reviewed in detail by Geijtenbeek and Gringhuis (46). So far, Syk-independent signaling via Raf-1 has only been described after Dectin-1 ligation in human DCs (47–49). IRF1 (50) and IRF5 (51) are further transcription factors induced. Phosphorylation of the MAPK p38 and JNK appears to be partially Syk-independent (26, 38, 52); in contrast, phosphorylation of the MAPK ERK requires Syk and is mediated by Card9 and H-Ras (53, 54). Activation of ERK is critical for reactive oxygen species (ROS) production, which has been linked to induction of autophagy (55) and to assembly of the Nlrp3 inflammasome (56–58). Assembly of a noncanonical Malt1/Caspase-8/ASC inflammasome triggered by Dectin-1 has as well been described (59–61). In addition to its requirement for assembly of the Card9/Bcl10/Malt-1 complex, PLCγ2 induces Ca²⁺ flux triggering the classical calcineurin/NFAT pathway that directly induces Egr1 expression (39, 62) and is required for antifungal defense (63).

The genes encoding these receptors are localized adjacent to each other in the Dectin-2 cluster on human chromosome 12/mouse chromosome 6 (3, 64–66). Dectin-2 and Mcl likely arose from gene duplication of Mincle (64, 67). Dectin-2, Mincle, and Mcl do not contain a cytoplasmic signaling motif, but instead they associate with the ITAM (YxxL/I,YxxL/I)-containing adaptor FcRγ-chain (6, 67–69) (Fig. 1).

Whereas ligands of Dectin-2 and Mincle are diverse and not always structurally characterized, Dectin-2, Mincle, and Mcl all recognize ligands on fungi and mycobacteria in a Ca²⁺-dependent manner (see Table I). Several studies have addressed the structural requirements for interaction of Mincle with the mycobacterial cord factor trehalose dimycolate (TDM) or synthetic trehalose esters (70–74), which have recently been summarized in excellent reviews (75–77). Mincle binds the trehalose part of the cord factor with its Ca²⁺-dependent sugar-binding pocket, and its structure revealed a hydrophobic groove that likely accommodates the lipid component of TDM or TDB. The recent interest in Mincle ligands for adjuvant development (78–80) has engendered the chemical synthesis of multiple glycolipids, which help to determine the requirements for receptor binding and macrophage activation (81–84). Different from cord factor binding, recognition of the nucleoprotein SAP130 is Ca²⁺-independent (6). Human and murine Mincle have divergent ligand specificities, for example, for glycerol monomycolates (82, 85) or cholesterol crystals (86). The CTLD of Mcl is much less conserved among species than the Mincle CTLD; consequently, Mcl appears to be a functional TDM receptor in mice (67), but not, for example, in guinea pigs (87), suggesting divergent physiological roles of Mcl between species. Mincle- or Mcl-deficient mice showed mostly moderate phenotypes in mycobacterial (88–91) or fungal infection models (92–94) compared with knockouts of the downstream Card9 (43, 95), indicating receptor redundancy. It is quite possible that double-deficient mice will show more severe phenotypes. Whereas both Dectin-2 and Mcl have phagocytic properties (66, 68), Mincle was described to be dispensable for glycolipid uptake (96), although required for cytokine production after glycolipid stimulation (97, 98). Ligand binding to Dectin-2 and Mincle leads to engagement of the FcRγ–Syk–PLCγ2–PKCδ–Card9 axis and activation of the canonical NF-κB pathway similar to Dectin-1 (6, 42, 98–100) (Fig. 1). Gringhuis et al. (101) described that Dectin-2 engagement specifically activates c-REL controlled by Malt1, in contrast to induction of all NF-κB subunits by Dectin-1 stimulation. Engagement of Dectin-2 and Mincle furthermore leads to activation of the MAPK p38, ERK, and JNK (99, 102–105). ERK phosphorylation after stimulation of Dectin-2 with Candida albicans is dependent on Syk and PLCγ2 but not Card9 (100, 103). Activation of PKB (synonym Akt) is found downstream of Mincle and Dectin-2 and is dependent on PI3K (50, 106). Both Dectin-2 and Mincle ligation can lead to production of ROS and inflammasome activation (107–112).

Activation of TLRs results not only from homodimer formation but also from heterodimerization. TLR2 can pair with either TLR1 or TLR6, resulting in an increased ligand spectrum (113–115). Homodimeric forms of human and mouse Mincle have been described quite early (116), and recently heterodimerization of Mcl has been described with Mincle (117) and with Dectin-2 (93).

It has been controversial whether Mcl interacts directly with the adapter protein FcRγ. Mcl lacks the conserved arginine residue in the stalk region that is required for interaction of murine Mincle with FcRγ (6), and Graham et al. (92) could not find association of human Mcl with FcRγ, DAP10, or DAP12. In contrast, Miyake et al. (67) demonstrated that murine Mcl coimmunoprecipitates with FcRγ in the absence of Mincle, uniquely utilizing a hydrophilic threonine residue rather than arginine. Direct association with FcRγ was likewise found for guinea pig Mcl, which similar to human Mcl has a serine at position 38 (87). Lobato-Pascual et al. (117) showed formation of disulfide-linked Mincle–Mcl heterodimers and suggested that rat Mcl interacts with FcRγ in an indirect fashion via heterotrimer formation with Mincle. Two independent studies demonstrated that the surface expression of Mincle and Mcl on myeloid cells is interdependently stabilized by their heterodimerization (118, 119). In consequence, Mcl-deficient mice have reduced Mincle surface expression but Mcl-transgenic mice show enhanced responsiveness to TDM stimulation, and Mcl surface levels are strongly reduced in Mincle-deficient cells. The interaction of murine Mincle and Mcl requires four hydrophobic residues in the stalk region of Mincle (118). In contrast, Zhao et al. (120) neither found coimmunoprecipitation of human Mincle and Mcl coexpressed in RAW264.7 cells, nor did they observe synergistic responses. Previously, the authors had described the dimerization of human and murine Mcl with Dectin-2 and demonstrated a synergistic role of Dectin-2 and Mcl for protection in a murine C. albicans infection model (93). A phenotype in C. albicans infection had not been observed in an earlier study (92), nor was coregulation of Dectin-2 and Mcl in mice confirmed in two other reports (94, 119). Overall, there is strong evidence that Mcl is able to dimerize with related CLRs, notwithstanding some disagreement in the literature. Further studies are needed to investigate whether discrepant results can be attributed to different cell types or receptors originating from different species. Several roles for Mcl in these interactions have been suggested and are depicted in Fig. 2: 1) transcriptional regulation of Mincle expression (further discussed below); 2) posttranscriptional regulation by interdependent stabilization of Mincle surface expression (118, 119); 3) Mincle could benefit from phagocytic capacity of Mcl (117, 121); 4) enhanced ligand binding by heterodimerization with Dectin-2 or Mincle, leading to an increased response (93, 122); and 5) alteration of ligand specificity (121). It can be expected that molecular dynamics simulations based on existing crystal structures of Mincle and Mcl as well as further structural work will be instrumental in answering which of these models is correct.

Expression of PRRs is a prerequisite for recognition of a microbial ligand. However, expression of PRRs is not uniform among different innate immune cell types and can be massively regulated by cytokines and microbial stimuli. Hence, cross-regulation of expression levels is in principle a logical mechanism for crosstalk between different PRRs and their signaling pathways. Specifically, Syk-coupled CLRs show large differences in expression between different cell types and activation states. Dectin-1 mRNA can be induced by GM-CSF and IL-4, but it is downregulated by LPS, IFN-γ, and IL-10 (123). Dectin-2 protein in monocytes increases under inflammatory conditions (124). Similarly, Mincle mRNA expression is low in resting murine macrophages and DCs but strongly inducible upon stimulation with inflammatory stimuli (64, 67, 104). Matsumoto et al. (64) identified Mincle (“macrophage inducible C-type lectin”) originally in a screen for target genes of the transcription factor C/EBPβ following LPS/IFN-γ stimulation. Mincle expression is also upregulated by its ligand TDM in a feed-forward loop through Mincle itself (98, 104), or through Mcl acting as a constitutively expressed low-affinity receptor for TDM in mice (67, 120) (Figs. 2A, 3C). It is currently unclear whether such transcriptional regulation of Mincle expression is conserved in other species that express higher constitutive levels of Mincle mRNA (50, 87, 122, 125).

In addition to the sequential control of Mincle mRNA expression, Mcl also controls the surface expression of Mincle protein. As described above, Mcl was recently identified to interact with Mincle via its stalk region and to be essential for surface expression of Mincle (118, 119) (Fig. 2B). Although the molecular and kinetic details of the Mincle–Mcl interaction are not yet fully understood, it has become evident that protein interactions and protein localization are a means to control the responsiveness beyond the transcriptional level.

Many fungi and bacteria contain several different CLR ligands (see Table I) that will lead to the concurrent triggering of more than one CLR in phagocytes and DCs upon making contact with the microbes. Furthermore, scavenger receptors such as CD36, complement receptors, TLRs, and cytosolic nucleic acid sensors are engaged upon pathogen contact. Receptor crosstalk can result in synergistic or conflicting signaling, thereby modulating the immune response. Examples for experimental ligands binding to both CLRs and TLRs are nondepleted zymosan (Dectin-1–TLR2) or mannosylated O-antigens (Dectin-2–TLR4) (126, 127). Dectin-1–TLR2 crosstalk is the most extensively studied example of CLR–TLR crosstalk, mostly but not exclusively leading to synergistic responses (Fig. 3).

Dectin-1 and complement receptor 3 (CR3 or CD11b/CD18, encoded by Itgam and Itgb2) both recognize β-glucans (Fig. 3A). CR3 was described as a zymosan receptor in neutrophils (128, 129) and as a receptor for soluble β-glucan in mononuclear cells (130). The idea of Dectin-1–CR3 crosstalk is further promoted by the observation that the receptors colocalized on lipid rafts after Histoplasma capsulatum stimulation. Collaborative TNF and IL-6 responses were dependent on Syk and JNK but not NF-κB (40). CD11b can itself recruit Syk and was shown to negatively regulate TLR-mediated inflammatory responses via the E3 ubiquitin ligase Cbl-b (131, 132). Thus, CR3-Syk appears to synergize with Dectin-1 signaling, but downregulates TLR-induced responses. Very recently, Cbl-b–mediated ubiquitination and degradation of Dectin-1, Dectin-2, and Syk were demonstrated, revealing a broader role for this ubiquitin ligase in regulation of TLR and CLR signaling (133–135).

Dectin-1 and TLR2 are both required to obtain strong production of TNF and IL-12 and NF-κB activation in murine macrophages and DCs after zymosan stimulation; colocalization was observed upon stimulation (126, 136) (Fig. 3B). Results were similar after stimulation with particulate β-glucans followed by ligands for TLR2, TLR3, TLR4, TLR5, TLR7, or TLR9 (137, 138). Prolonged IκB degradation and enhanced NF-κB translocation resulted in more-than-additive production of TNF, IL-23, IL-6, and IL-10, but reduced production of IL-12 (137, 139). Syk and Card9 were required for the synergistic response (137, 140), which was similarly detected in human monocytes and macrophages (141). Of note, the synergistic signaling via Dectin-1 and TLR2 does not only result in proinflammatory cytokine production, but also in augmented secretion of anti-inflammatory IL-10 (Fig. 3B). Secretion of IL-10 is controlled by the MAPK ERK and p38, phosphorylation of mitogen- and stress-activated protein kinase 1/2, and engagement of the transcription factor CREB, consistent with induction of a regulatory phenotype and reduced activation of T cells (53, 142–144).

Synergistic TNF and IL-10, but reduced IL-12 secretion, has similarly been described for simultaneous engagement of Mincle and TLR ligands (145, 146) (Fig. 3C). IL-10 can itself regulate IL-12 production in an autocrine manner as observed after costimulation of TLR2 and Mincle by synthetic ligands and mycobacteria (146). As mentioned above, TLR-derived signaling increases Mincle expression and can thereby enhance responsiveness to TDM (104, 147). This mechanism may also contribute to the beneficial effect of TLR ligands in Fonsecaea pedrosoi infection, a model for human chromoblastomycosis (145) (Fig. 3C).

An intriguing mechanism of synergistic action of TLR and Mincle signaling acting at the level of translation efficiency was revealed recently: combined stimulation of TLR2 and Mincle induced more-than-additive NO production, particularly at later stages of inflammation (107) (Fig. 3D). Protein expression of inducible NO synthase (iNOS) was mediated by Mincle-controlled increase in translation, which required p38-dependent hypusination of eIF5A. Importantly, the eIF5A-dependent NO production at later stages of inflammation inhibited Nlrp3-mediated IL-1β production, counteracting the synergistic induction of proIL-1β by TLR2 and Mincle. Blockade of eIF5A or iNOS deficiency resulted in exacerbating inflammation in TDM-induced lung granulomas and enhanced mortality, identifying Mincle as an important regulator of antimycobacterial immune responses at later stages of inflammation (107).

In addition to these acute synergistic effects of concurrent stimulation of CLRs and TLRs, Dectin-1 ligands can prime responses to subsequent stimulation by TLR ligands (49), an effect characterized as “training of innate immunity” by Netea et al. (148). These long-lasting effects of CLR signaling depend on Hif1α and mammalian target of rapamycin–dependent metabolic changes and epigenetic programming (149, 150) and are distinct from the collaborative effects described above.

Several mechanisms have been proposed to contribute to the negative regulation of cytokine production after CLR ligation. Eberle and Dalpke (52) demonstrated that suppressor of cytokine signaling (SOCS)1 is induced after stimulation with depleted zymosan (Dectin-1) and CpG (TLR9) in murine bone marrow macrophages and DCs (Fig. 4A). Socs1 induction is dependent on Syk, Pyk2, and ERK activation but is Ca²⁺- and NF-κB–independent. It resulted in decreased and shortened activation of NF-κB (p50 and p52) and thus reduced IL-12p40 secretion. In peritoneal macrophages, Socs1 and PIAS1 induction downstream of Dectin-1 has been described in a Ca²⁺-dependent manner to be dependent on the expression of Wnt5a, induced by the ROS/β-catenin axis. Socs1 and PIAS1 induction lead to reduced expression of IL-12, IL-1β, and TNF and abrogated TLR signaling via degradation of IL-1R–associated kinase (IRAK)1, IRAK4, and MyD88 (151). Downstream of Dectin-2, but not of Dectin-1, β-catenin stabilization in DCs occurs dependent on phosphorylation of LAB and leads to impaired IL-12 production (152).

As mentioned above, Mincle is important for recognition of F. pedrosoi, but synergistic TLR stimulation and TNF production was required to clear the infection in a mouse model of chromoblastomycosis (145) (Fig. 3C). In contrast, Mincle engagement counteracted the induction of IL-12 by Fonsecaea monophora in human DCs (Fig. 4B). F. monophora simultaneously engages Dectin-1, leading to activation of IRF1 and IL-12A (IL-12p35) transcription, and Mincle. In a PI3K-PKB–dependent manner, Mincle activates the E3 ubiquitin ligase Mdm2, leading to degradation of Dectin-1–induced IRF1, thus blocking IL-12A transcription. Degradation of TLR-induced IRF1 was similarly observed. The blockade of IL-12A resulted in a shift from a protective Th1 to a detrimental Th2 response in cocultures with T cells in vitro (50). Thus, although sensing of F. pedrosoi by Mincle is required for innate protection, the negative effect on IL-12 production may interfere with the development of protective T cell immunity. Along this line, Mincle-deficient mice showed an increased Th17 response in F. pedrosoi infection (153).

Finally, Miller and coworkers (154, 155) recently demonstrated an unexpected inhibition of TLR4-dependent inflammatory cytokine expression by the CLR Dectin-1 and Mincle (Fig. 4C). First, they observed that Dectin-1–deficient mice showed more hepatic fibrosis in a model of liver inflammation (154). Similarly, Mincle-deficient mice were more susceptible to endotoxic shock than were wild-type controls, resulting in higher mortality and elevated cytokine levels (155). In both studies, this enhanced susceptibility was attributed to increased levels of the TLR4 coreceptor CD14 in Dectin-1– or Mincle-deficient mice. Blockade of PKC and M-CSF abrogated the elevated CD14 expression in Dectin-1–deficient mice (154). Mincle deletion led to enhanced JNK phosphorylation but decreased p38 phosphorylation and subsequent activation of Socs1, A20, and ABIN3, which supposedly control CD14 expression, and in addition may induce degradation of Traf6 and MyD88 (155). These findings suggest that control of TLR responses by CLRs cannot only occur by transcriptional control but also by (indirect) modulation of the levels of components of the TLR signaling machinery. The nature of the ligands for Dectin-1 and Mincle in the hepatic fibrosis and LPS challenge models has not been defined. However, in the case of Mincle, the same group most recently demonstrated evidence that the endogenous Mincle ligand SAP130 (6) triggers Mincle in a mouse pancreatic tumor model, promoting tumor growth through inhibitory effects on T cell responses (156), and in a mouse model of acute liver inflammation, exacerbating disease (157). SAP130 may be induced and released during LPS- or infection-induced inflammation from dying cells and provide the trigger for inhibitory Mincle signaling.

CLRs as a group of PRRs have gained increasing attention during the last 10 y, with Dectin-1 often regarded as a prototypic receptor. Similar to Dectin-1, the related receptors Dectin-2, Mincle, and Mcl were found to signal dependent on the Syk/Card9 pathway. These CLRs have been characterized as receptors not only for various pathogens but also endogenous ligands. Consequently, their roles reach from infection and inflammatory conditions to homeostatic regulation. The number of pathways and signaling events identified downstream of CLR ligation are continuously increasing, providing us with a gradually more precise but also more complex picture of signal transduction and reprogramming triggered in innate immune cells. Further research is needed to clarify which of these pathways are universal, such as the Syk/Card9 axis, and which responses occur in certain species, certain cell types, or for certain receptors or ligands. Pathogens are recognized by multiple PRRs simultaneously, and therefore it is essential to investigate not only events dependent on a single receptor but also crosstalk between receptors or even classes of receptors. We have reviewed studies investigating the integration of signals derived from CLRs and TLRs, with examples for both synergistic and antagonistic interactions between different CLRs or with TLRs. Although there are many examples of collaborative signaling with strongly boosted responses, for example, by concurrent stimulation of TLR2 and Dectin-1, accumulating evidence shows that specific CLR signaling can attenuate or abrogate at least certain types of CLR/TLR-induced activation. Another important aspect of CLR research has been the cross-regulation of expression levels at the mRNA and protein levels, which can determine the level of responsiveness to the respective microbial ligands. A fascinating question for future research in this area will be to investigate the consequences of direct receptor interaction, such as formation of Mcl–Dectin-2/Mincle heterodimers, on the avidity and specificity of ligand binding. Thus, signaling crosstalk downstream of CLRs specifically modulates immune reactions and can control inflammatory responses. Mapping this complex signaling network will result in a new level of understanding of the role of CLRs in innate and adaptive immune responses and may open up perspectives to target these receptors for treatment and prevention of infectious and inflammatory conditions.