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Syk Kinase-Coupled C-type Lectin Receptors Engage Protein Kinase C-δ to Elicit Card9 Adaptor-Mediated Innate Immunity

Summary C-type lectin receptors (CLRs) that couple with the kinase Syk are major pattern recognition receptors for the activation of innate immunity and host defense. CLRs recognize fungi and other forms of microbial or sterile danger, and they induce inflammatory responses through the adaptor protein Card9. The mechanisms relaying CLR proximal signals to the core Card9 module are unknown. Here we demonstrated that protein kinase C-δ (PKCδ) was activated upon Dectin-1-Syk signaling, mediated phosphorylation of Card9 at Thr231, and was responsible for Card9-Bcl10 complex assembly and canonical NF-κB control. Prkcd−/− dendritic cells, but not those lacking PKCα, PKCβ, or PKCθ, were defective in innate responses to Dectin-1, Dectin-2, or Mincle stimulation. Moreover, Candida albicans-induced cytokine production was blocked in Prkcd−/− cells, and Prkcd−/− mice were highly susceptible to fungal infection. Thus, PKCδ is an essential link between Syk activation and Card9 signaling for CLR-mediated innate immunity and host protection.


INTRODUCTION
The innate immune system uses germline-encoded pattern recognition receptors (PRRs) to sense microbial pathogens or other forms of danger, leading to the induction of inflammatory responses and host defense (Medzhitov, 2009). Bona fide PRRs with signaling capacities that reprogram gene expression include Toll-like receptors (TLRs), nucleotide-oligomerization domain (Nod)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like helicases (RLRs), and the recently emerging family of spleen tyrosine kinase (Syk)-coupled C-type lectin receptors (CLRs) (Kerrigan and Brown, 2011;Osorio and Reis e Sousa, 2011). Central to the ability of these PRRs to elicit inflammation and innate immunity is their capacity to activate the canonical NF-kB signaling cascade (Vallabhapurapu and Karin, 2009).
Here we used a proteomic approach to search for intracellular mediators of CLR-Syk function. With Dectin-1 signaling as a model, we report that PKCd (encoded by Prkcd) is activated upon CLR stimulation and specifically required to induce Card9-Bcl10 complex assembly for TAK1 activation and canonical NF-kB control. We found that Prkcd -/dendritic cells are defective in innate immune responses to CLR stimulation and that Prkcd -/mice are highly susceptible to fungal infection. These results identify a fundamental mechanism for inflammatory cell activation, with essential roles in CLR-mediated innate immunity.

Zymosan Stimulation Triggers Tyrosine Phosphorylation of PKCd
To gain insights into the mechanisms of Dectin-1-Syk signaling, we stimulated primary bone marrow-derived dendritic cells (BMDCs) from mice with zymosan. Zymosan is a fungal cell wall preparation composed primarily of b-glucans and functions as a strong Dectin-1 agonist. In addition, it contains ligands for Dectin-2 and TLR2 (Brown and Gordon, 2001;Gross et al., 2006;Robinson et al., 2009;Taylor et al., 2007). As expected, zymosan stimulation induced tyrosine phosphorylation of multiple target proteins ( Figure 1A). To reveal the identity of these molecules in an unbiased manner, we affinity purified the tyrosine-phosphorylated proteins and analyzed these by mass spectrometry (MS) ( Figure 1B). The 20 specifically tyrosine-phosphorylated proteins with the highest Mascot scores included four kinases: the receptor proximal Src family members Lyn and Fgr, together with Syk and PKCd (data not shown). Because we were particularly interested in putative Syk effector molecules, we focused our attention on PKCd.
To validate zymosan-induced PKCd phosphorylation, we immunoprecipitated this specific kinase from stimulated BMDCs and subsequently performed immunoblots with phospho-tyrosine antibodies. Consistent with our MS data, zymosan treatment induced strong PKCd tyrosine phosphorylation ( Figure 1C). Then, we performed analyses with a phosphospecific antibody raised against PKCd tyrosine (Tyr) 311, a residue involved in PKCd activation (Konishi et al., 2001). Robust, activation-specific PKCd Tyr311 phosphorylation was rapidly detected in response to zymosan treatment ( Figure 1D). To test whether this PKCd phosphorylation was mediated via Src and Syk signaling, we Proteins were immunopurified with p-Tyr antibodies or IgG1 control (isotype) and visualized by Coomassie staining after electrophoresis. (C) BMDCs were left untreated or stimulated with zymosan as in (A). Proteins from lysates were immunoprecipitated with PKCd antibodies or isotype control and analyzed by immunoblot with p-Tyr or PKCd antibodies. (D) Immunoblot analysis of BMDCs stimulated with zymosan for various times and probed with antibodies against phospho-PKCd (Tyr311) or b-actin. (E) BMDCs were left untreated or preincubated with Syk inhibitor R406 (0.5, 1, 2 mM), Src-kinase inhibitor PP2 (1.5, 3, 6 mM), or its inactive analog PP3 (1.5, 3, 6 mM) and stimulated for 10 min with zymosan. Lysates were analyzed by immunoblot with antibodies against phospho-PKCd, PKCd, phospho-PLCg2, or PLCg2. All results are representative of at least three independent experiments. pharmacologically blocked these receptor proximal tyrosine kinases with the inhibitors PP2 or R406, respectively, before stimulation of the cells. Both inhibitors impeded zymosaninduced PKCd Tyr311 phosphorylation ( Figure 1E). Thus, we conclude from this first set of experiments that innate recognition of zymosan activates PKCd through a Src and Syk tyrosine kinase-dependent mechanism.
( Figure 2B). These findings indicate an essential and specific role for PKCd in the Dectin-1 pathway. To characterize this function in more detail, we performed dose-response experiments with different PRR agonists. Again, we observed a critical requirement for PKCd in zymosan-or curdlan-mediated TNF and IL-10 production, but cytokine production in response to TLR4 stimulation with LPS was not affected by the deletion of PKCd ( Figure 3A).
Because Dectin-1 signaling triggers not only the synthesis of cytokines but also the phagocytosis of fungal components, as well as the production of reactive oxygen species (ROS), we studied the role of PKCd in these pathways. In contrast to cytokine production, the phagocytosis of zymosan particles was independent of PKCd, as assessed by either fluorescence microscopy or flow cytometric quantification (Figures 3B and 3C). Moreover, the generation of ROS upon zymosan treatment was not significantly impaired in Prkcd À/À BMDCs ( Figure 3D).
Next we investigated whether PKCd functions in Dectin-1independent CLR responses by stimulating Prkcd À/À cells with agonistic antibodies against Dectin-2 or with trehalose-6,6-dibehenate (TDB), a synthetic adjuvant analog of the mycobacterial cord factor that functions as a selective and specific agonist for Mincle (Schoenen et al., 2010). Similar to the Dectin-1 responses, both Dectin-2-and Mincle-induced IL-10 and TNF production were severely impaired in the absence of PKCd (Figure 3E and data not shown). These findings indicate that PKCd plays a general role in Syk-coupled CLR response pathways, where it is specifically required for the control of cytokine synthesis.

PKCd Regulates Dectin-1-Mediated NF-kB Signaling via Card9 Control
To define the molecular function of PKCd in CLR signaling, we focused again on zymosan stimulation and selective Dectin-1 triggering ( Figure 4). The overall tyrosine phosphorylation pattern, the activation of Syk, as well as the phosphorylation of the Dectin-1 signal transducer PLCg2 (Xu et al., 2009) did not differ substantially between zymosan-or curdlan-stimulated WT and Prkcd À/À cells ( Figure 4A and data not shown). Moreover, PKCd was to a large extent dispensable for Erk1 and Erk2 MAPK activation, as shown by the fact that Erk1 and Erk2 phosphorylations were only slightly reduced in Prkcd À/À cells ( Figure 4B). In sharp contrast, signaling to the canonical NF-kB pathway, a key driver of cytokine production, was almost completely blocked in zymosan-treated or Dectin-1-stimulated Prkcd À/À BMDCs, as indicated by the lack of phosphorylation in the activation loops of IKKa and IKKb ( Figure 4C).
As mentioned above, Dectin-1-Syk signaling engages the Card9 adaptor protein for canonical IKK-dependent NF-kB activation (Gross et al., 2006;Mó csai et al., 2010). To study the potential involvement of PKCd in the Card9 signaling cascade, we first stimulated Card9 À/À BMDCs (Gross et al., 2006) with zymosan and investigated PKCd tyrosine phosphorylation. PKCd was normally activated in the absence of Card9 (Figure 5A). Then, we tested the possibility that PKCd might directly phosphorylate Card9. Indeed, in vitro kinase assays with recombinant proteins demonstrated that PKCd can phosphorylate Card9 but not bovine serum albumin ( Figure 5B). To search for putative PKCd phosphorylation sites within Card9, we performed a bioinformatic analysis applying both the NetPhosK (http:// www.cbs.dtu.dk/services/NetPhosK/) and Scansite (http:// scansite.mit.edu/) target site prediction algorithms (Blom et al., 2004;Obenauer et al., 2003). Three conserved PKC phosphorylation sites were predicted in both murine and human Card9. These were Thr95, Thr231, and Ser303 ( Figure 5C). Because Thr95 and Thr231 are within the only two conserved TxK or TxR motifs in Card9, we next performed immunoblot analysis with proteins from the PKCd-Card9 kinase reaction and a phosphoThreonine-x-Arginine (pTxR) antibody that recognizes phosphorylated Thr followed by Arg or Lys at the +2 position. Consistent with the bioinformatic prediction, PKCdphosphorylated Card9 reacted with the pTxR antibody, indicating phosphorylation at TxK motifs ( Figure 5D). To further map PKCd phosphorylation sites within Card9, we used sitedirected mutagenesis and transiently transfected expression plasmids encoding tagged forms of WT-Card9, Card9(T95A), Card9(T231A), or Card9(S303A) in HEK293 cells. We were technically unable to efficiently express the Card9(T95A) version in mammalian cells, possibly because of effects of this mutation on protein stability. Nevertheless, WT-Card9, Card9(T231A), and Card9(S303A) were equally expressed in HEK293 cells (data not shown) and were used upon purifications as substrates for in vitro PKCd kinase assays ( Figure 5E). The quantification of radio-phospho incorporation revealed that WT-Card9 and Card9(S303A) were equally phosphorylated by PKCd in vitro. In contrast, the phosphorylation of the Card9(T231A) mutant was reduced by 50% compared to the WT, demonstrating that PKCd phosphorylates Card9 at position Thr231 and presumably on additional sites.
To study the functional relevance of the Card9 Thr231 phosphorylation in CLR signaling, we next generated retroviral vectors that express WT-Card9, Card9(T231A), or Card9(S303A) to reconstitute Card9 gene-deficient BMDCs. Subsequently, we stimulated the reconstituted cells with the Dectin-1 agonist Curdlan and measured the production of IL-10 as a readout for productive signaling ( Figure 5F). Although the Card9(S303A) mutant restored the signaling defect of Card9-deficient cells to the same extent as WT-Card9, Card9(T231A) was unable to rescue Dectin-1 signaling for cytokine production. Together with the biochemical results above, these data indicate that PKCd mediates Thr231 phosphorylation of Card9 and that this phosphorylation is essential for Card9 function.
PKCd Activates TAK1 via Card9-Bcl10 Complex Formation Upon cellular stimulation, Card9 forms a signaling complex with its effector protein Bcl10 leading to canonical IKK-dependent NF-kB signaling (Gross et al., 2006;Mó csai et al., 2010). To investigate the requirement for PKCd in these events, we analyzed Card9-Bcl10 complex assembly in Prkcd À/À cells. To this end, we immunoprecipitated Bcl10 from zymosan-stimulated WT and Prkcd À/À cells and studied its association with Card9 by protein immunoblot. Card9-Bcl10 complexes assembled only in zymosan-stimulated WT BMDCs but not in cells lacking PKCd ( Figure 6A), indicating an essential function for PKCd activity in Card9-Bcl10 complex formation.
How the Card9-Bcl10 signalosome activates IKKs has not been well defined. However, given that Bcl10 can utilize the kinase TAK1 for T cell receptor-mediated NF-kB activation (Sun et al., 2004), we speculated that TAK1 could also be activated through the Card9-Bcl10 complex during innate immune response. To test this hypothesis, we stimulated BMDCs of the different genotypes with zymosan and investigated TAK1 phosphorylation. Indeed, zymosan-induced TAK1 phosphorylation (B) BMDCs from WT and Prkcd À/À mice were incubated for 2 hr with FITC-zymosan particles (100 mg ml À1 ). FITC-zymosan internalization was visualized by fluorescence microscopy (scale bars represent 20 mm). (C) BMDCs were incubated with FITC-zymosan as in (B) for the times indicated. The frequencies of CD11c + cells containing zymosan-FITC particles were quantified by FACS analysis. (D) BMDCs from WT and Prkcd À/À mice were incubated with Luminol and were left untreated (Medium) or stimulated with zymosan. Luminescence was assayed for 60 min in 10 min intervals and relative light units (RLU) as a measure for ROS generation integrated over time. Results represent means + SD from three independent experiments. (E) WT and Prkcd À/À BMDCs were stimulated through Dectin-1, Dectin-2, or Mincle with curdlan (20 mg ml À1 ), plate-bound Dectin-2 antibody, or TDB (100 mg ml À1 ), respectively. IL-10 concentrations in the cell culture supernatants were quantified by ELISA. Data are expressed as percent of WT + SD, derived from stimulations in triplicates. All results are representative of at least three independent experiments. was strictly dependent on Card9 ( Figure 6B). Moreover and in line with our finding that Prkcd À/À BMDCs were defective in Card9-Bcl10 activation, zymosan-induced TAK1 activation was abrogated in PKCd-deficient cells ( Figure 6C). To directly investigate the function of TAK1 in zymosan-induced NF-kB signaling, we pretreated BMDCs with the selective chemical TAK1 inhibitor (5Z)-7-Oxozeaenol before cell stimulation (Ninomiya-Tsuji et al., 2003). Although TAK1 inhibition did not block zymosan-induced PKCd activation, it prevented the activation of IKKs ( Figure 6D). Together, these findings indicate that PKCd plays an essential role upstream of the Card9-Bcl10 module, which is critical for the subsequent activation of TAK1 and the engagement of the canonical NF-kB pathway.
PKCd Is Essential for Innate Antifungal Immune Defense After uncovering the molecular functions of PKCd in CLR signaling, we were interested in its role in a pathophysiologically relevant setting. As a clinically important model pathogen we chose C. albicans because its PAMPs are recognized by Dectin-1 (Taylor et al., 2007), Dectin-2 (Robinson et al., 2009), and Mincle (Wells et al., 2008) and they drive Syk-and Card9-mediated innate immunity and host defense (Glocker et al., 2009;Gross et al., 2006;LeibundGut-Landmann et al., 2007;Robinson et al., 2009). Although C. albicans infection of WT BMDCs induced a robust and dose-dependent production of cytokines, TNF, IL-10, and IL-1b, production of these cytokines was almost completely abolished in the absence of PKCd ( Figure 7A). Also on the biochemical level we observed that, in spite of normal Syk phosphorylation, Prkcd À/À BMDCs had severe deficiencies in activating NF-kB signaling after stimulation with C. albicans hyphae, a potent agonist of Dectin-2 . In line with our results above, Prkcd À/À cells furthermore showed defects in IKK activation, as well as in IkBa phosphorylation and degradation ( Figure 7B) after C. albicans recognition. BMDCs from WT or Prkcd À/À mice were stimulated with zymosan for the indicated times. Syk and PLCg2 activation was determined by immunoblot with phospho-Syk or phospho-PLCg2 antibodies. Immunoblotting with Syk, PLCg2, and b-actin antibodies indicate equal protein loading. (B) WT or Prkcd À/À BMDCs were stimulated with zymosan as indicated. Activation of the MAP kinases Erk1 and Erk2 was determined by immunoblot with phospho-Erk1/2 antibodies. Immunoblotting with Erk1/2 and b-actin antibodies indicates equal protein loading. (C) Defective NF-kB signaling in Prkcd À/À BMDCs. Cells were stimulated with zymosan or curdlan as indicated. Lysates were analyzed by immunoblot with phospho-PKCd, phospho IKKa/b and PKCd, IKKa, IKKb, and b-actin antibodies.
Finally, to study the relevance of these findings for host protection in vivo, we infected Prkcd À/À mice with C. albicans. Compared to the wild-type, PKCd-deficient mice exhibited significantly greater weight loss upon infection ( Figure 7C) and had much lower survival rates ( Figure 7D). In an independent set of experiments, we sacrificed the animals 8 days after infection and assessed intravital fungal growth. Consistent with an essential role for PKCd in innate resistance in vivo, we observed massive fungal infiltration in the kidneys of Prkcd À/À mice by histopathology ( Figure 7E) and detected significantly higher titers of C. albicans in the kidneys, livers, small intestines, and spleens ( Figure 7F).

DISCUSSION
Our results defined an essential role for PKCd in the activation of CLR-mediated, Card9-dependent innate immune responses. Our findings are in line with the established functions of CLRs and Card9 in antifungal defense (Ferwerda et al., 2009;Glocker et al., 2009;Gross et al., 2006;Saijo et al., 2010;Taylor et al., 2007), and we showed an essential activity of PKCd in host resistance in vivo.
The detection of fungal particles by Dectin-1 and the subsequent activation of Syk triggers various intracellular signaling pathways (Kerrigan and Brown, 2011;Mó csai et al., 2010;Osorio and Reis e Sousa, 2011). We observed that the recognition of zymosan elicits a Src-and Syk-dependent phosphorylation of PKCd at Tyr311, indicating that PKCd activation occurs downstream of Syk. Moreover, Prkcd À/À BMDCs showed impaired Card9-Bcl10 complex assembly and NF-kB control in spite of normal Syk activation, demonstrating that PKCd acts upstream of Card9. Together, these findings indicate that PKCd operates as a missing link between Syk signaling and Card9 complex formation for the activation of innate immunity. As shown by the fact that only Prkcd À/À BMDCs but not cells lacking PKCa, PKCb, or PKCq were defective in Dectin-1-induced cytokine production, PKCd is the specific PKC isoform for signaling in the Dectin-1 pathway.
Treatment of the cells with a small molecule PKC kinase inhibitor blocked zymosan-or curdlan-induced cytokine production. This indicates that the enzymatic serine-threonine kinase activity and not merely a scaffold function of PKCd is responsible for its activity in Dectin-1 signaling. In vitro kinase assays revealed that Card9 is a direct PKCd substrate and that PKCd phosphorylates Card9 at position Thr231. PKCd is likely to phosphorylate other Card9 residues because the Card9(T231A) mutant was still substantially phosphorylated by PKCd in vitro. Another PKCd target site could be Thr95 because global proteomic approaches have demonstrated Card9 phosphorylation at Thr95 in vivo (Choudhary et al., 2009). Because we were unable to express the Card9(T95A) mutant in mammalian cells, we were not able to determine the physiological function of Thr95 phosphorylation but speculate that it might be important for protein stability. Nevertheless, our genetic experiments with Card9-deficient cells that were reconstituted with the phosphorylationdefective Card9(T231A) mutant revealed that the Thr231 residue phosphorylated by PKCd is absolutely required for downstream signaling and cytokine production. In addition, we observed that PKCd signaling is essential for Card9-Bcl10 complex assembly and for Card9-dependent TAK1 activation. Thus, we postulate a molecular model in which Syk-induced PKCd activity mediates direct Card9 phosphorylation, finally resulting in Card9-Bcl10 complex assembly for TAK1 activation. TAK1 then most probably mediates Dectin-1-induced IKK activation in a fashion similar to its mode of triggering NF-kB activity in response to stimuli from other immune receptors, such as TLRs (Vallabhapurapu and Karin, 2009). This would be consistent with the fact that pharmacological TAK1 blockage inhibited zymosaninduced IKK activation.
Intriguingly, although the Card9 signaling pathway was severely defective in Prkcd À/À BMDCs, the phagocytosis of zymosan and the production of ROS were largely intact and the activation of Erk MAPK signaling was only slightly reduced. These findings indicate that PKCd controls only specific subsets of the Dectin-1 responses. Because Dectin-1 ligation can activate the serine-threonine kinase Raf-1 through alternative mechanisms (Gringhuis et al., 2009), it is possible that Raf-1 might be responsible for Dectin-1-triggered and PKCd-independent Erk activation, consistent with the role of Raf-1 in activating MAPK pathways in numerous settings (Galabova-Kovacs et al., 2006). Interestingly, Prkcd À/À mice were similar to Card9 À/À mice (Gross et al., 2006) in being highly susceptible to fungal infections. The specific PKCd-Card9 effector response downstream of CLRs is therefore absolutely critical for host defense. The aforementioned pivotal functions of Dectin-1 and Card9 in human antifungal immunity were recently demonstrated in genetic studies (Ferwerda et al., 2009;Glocker et al., 2009). In the future it will thus be important to investigate whether genetic defects in PKCd might also cause human immunodeficiency syndromes.
Many of our experiments aimed at identifying the principle mechanisms of CLR signaling utilized zymosan stimulation or selective Dectin-1 triggering. Yet in the absence of PKCd, activation of the NF-kB pathway as well as cytokine production were also severely impaired in response to whole C. albicans cells. Recent studies have indicated that C. albicans cells, and particularly C. albicans hyphae, are potent activators of Dectin-2 signaling . Presumably the hyphae in addition also activate Mincle (Wells et al., 2008). Thus, we propose that PKCd also couples signals from those CLRs to the Card9controlled NF-kB pathway. This hypothesis is in line with our observation that Prkcd À/À BMDCs were defective in cytokine responses to selective agonists for Dectin-2 and Mincle, which formally establishes PKCd as a general integrator of CLR function.
We believe that our findings have implications beyond antifungal immunity. Mincle and Dectin-1 detect ligands on mycobacteria (Ishikawa et al., 2009;Rothfuchs et al., 2007) and Card9 À/À mice are impaired in the Mincle-induced inflammatory response to the mycobacterial cord factor (Schoenen et al., 2010;Werninghaus et al., 2009). These animals also succumb rapidly to aerosol lung infection with Mycobacterium tuberculosis (Dorhoi et al., 2010). In addition, Schistosoma mansoni activates Dectin-2 and Card9 (Ritter et al., 2010) and viruses such as the Dengue virus induce inflammatory responses through the ITAM-coupled CLR Clec5a (Chen et al., 2008). Moreover, Dectin-1 also binds to unknown endogenous structures on T cells (Ariizumi et al., 2000), the Syk-coupled CLR Clec9a recognizes ligands that are exposed upon cellular necrosis (Sancho et al., 2009), and Mincle triggers Card9-dependent inflammatory responses upon binding to SAP130 from necrotic cells under sterile conditions (Yamasaki et al., 2008). Together with our results, these data imply that PKCd might also mediate innate responses to bacteria, parasites, or viruses or could be involved in immune responses to conditions of noninfectious cell injury. These hypotheses need to be investigated.
Finally, CLR-triggered Card9 signaling not only regulates the immediate innate antimicrobial responses but also couples innate recognition to the activation of adaptive immunity (Kerrigan and Brown, 2011;Osorio and Reis e Sousa, 2011). Triggering of the Card9 pathway by Dectin-1, Dectin-2, or Mincle ligands on antigen-presenting cells controls the synthesis of distinct cytokines. This cytokine milieu instructs the development of antigen-specific Th17 cell responses and additionally induces Th1 cell immunity (LeibundGut-Landmann et al., 2007;Robinson et al., 2009;Saijo et al., 2010;Werninghaus et al., 2009). Th17 cell responses are often associated with autoimmunity and polymorphisms in Card9 are recurrently detected in human inflammatory diseases (Pointon et al., 2010;Zhernakova et al., 2008). It will therefore be important to test whether PKCd signaling in innate cells couples to Th17 cell responses and whether aberrant activity in CLR-induced PKCd-Card9 signaling contributes to human inflammatory disease. In this context, PKCd is a drugable target and PKC inhibitors are in clinical trials. Thus, our findings raise the possibility of therapeutically manipulating CLR-mediated Card9 signaling by targeting PKCd function.
For fungal burden determination, the organ homogenates were plated in dilutions on Sabouraud agar. For histological analyses, kidney sections were stained with hematoxylin and eosin or periodic acid-Schiff according to standard protocols.
Purification and Identification of Tyrosine-Phosphorylated Proteins 2 3 10 8 BMDCs were stimulated for 10 min with zymosan (300 mg ml À1 ) and lysed in RIPA buffer. The lysates were then subjected to immunopurification with 20 mg of either an antibody against phospho-tyrosine (P-Tyr-100) or mouse IgG1 as an isotype control coupled to Protein A Sepharose. Immunopurified proteins were eluted with Phenylphosphate (100 mM) in PBS, concentrated by ultrafiltration, and separated by 1-D PAGE. Whole gel lanes were cut into 23 slices. After protein digestion, MS-MS spectra of individual gel slices were obtained with a liquid chromatography coupled LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Electron). Peak lists were searched against NCBInr database with Mascot v.2.2.06 as a search engine and MudPIT for protein scoring.

Retroviral Reconstitution of Card9-Deficient BMDCs
Retroviral particles were generated by transfection of Phoenix-E cells with pMIG retroviral vectors expressing the indicated mutants of human Card9, as previously described (Glocker et al., 2009). Bone marrow cells of Card9 À/À mice were retrovirally transduced on day 1 and 2 of BMDC differentiation. Successful transduction was verified by FACS analysis of GFP expression from the bicistronic pMIG retroviral vectors and protein immunoblot analysis of Card9 expression, as previously described (Glocker et al., 2009).

Cytokine Production
Concentrations of TNF, IL-10, IL-2, and IL-1b secreted into cell culture supernatants of untreated and stimulated cells were analyzed with plate-bound ELISA-kits (ELISA Ready-SET-Go!, eBioscience or BD OptEIA, BD Biosciences PharMingen) according to manufacturers' recommendations.

Zymosan Phagocytosis
BMDCs were left untreated or incubated for 30, 120, or 180 min with FITClabeled zymosan particles (100 mg ml À1 , Molecular Probes). After washing, cells were analyzed either by microscopy for FITC fluorescence or by flow cytometry for CD11c expression and FITC fluorescence in the CD11c-positive population with a FACSCanto II (BD Biosciences) and FlowJo Software (Tree Star, Inc.) according to standard protocols. Fluorescently labeled antibodies were from eBioscience.
Reactive Oxygen Species Measurements 2 3 10 5 BMDCs were plated in 100 ml culture medium without GM-CSF into a 96-well luminometer plate (Nunc). For stimulation, 100 ml HBSS containing 100 mM Luminol and zymosan was added to each well. The luminensence was measured for 1 hr in 10 min intervals at a Mithras LB940 according to manufacturers' instructions. As a measure for ROS generation, relative light units (RLU) were integrated over time.