LAB/NTAL Facilitates Fungal/PAMP-induced IL-12 and IFN-γ Production by Repressing β-Catenin Activation in Dendritic Cells

Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2 −/− mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2−/− DCs. Accordingly, Lat2−/− DCs directed reduced Th1 polarization in vitro and Lat2 −/− mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and β-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.


Introduction
Fungal infections with pathogens such as Candida albicans are a significant health risk for immunocompromised individuals [1]. There is a high degree of mortality in these cases even with treatment, highlighting the need for a better understanding of the immune response involved in controlling fungal infections in order to develop improved treatments [2,3]. Responses to fungal infections involve both innate and adaptive immunity [4]. The host response relies on the recognition, ingestion and elimination of C. albicans by phagocytic cells. During fungal infections, various pro-inflammatory cytokines such as TNF, IL-12p70, IL-23 and IL-6, produced by the activated leukocytes, result in the promotion of a sustained Th1 and Th17 response [5,6,7]. The requirement for these cytokines and pathways has been demonstrated by increased susceptibility of several knockout mice to C. albicans infections. For example, mice deficient in genes associated with Th1 responses such as Il12a, Ifng or Ifngr1 are more susceptible to systemic C. albicans infection [8,9]. In addition, Il18 2/2 mice display enhanced susceptibility to disseminated C. albicans due to their inability to produce sufficient IFN-c [10]. More recently, fungal responses have been shown to involve the Th17 pathway; Il23a 2/2 , Il17ra 2/2 and Il17a 2/2 mice are more susceptible to oral and/or systemic candidiasis [6,11,12]. Therefore, the level of inflammatory cytokine production in response to C. albicans infection is important in determining whether the host will eliminate or succumb to the infection.
Stimulation of host immune cells to produce pro-inflammatory cytokines occurs through the recognition of PAMPs by pathogenrecognition receptors (PRRs) [13]. Various PRRs such as the mannose receptor, TLR2/4, CR3, Dectin-1 and Dectin-2 are involved in fungal recognition and responses [14]. Dectin-1 and Dectin-2 are type II, C type lectin-like receptors expressed mainly on myeloid cells [15,16]. Fungal cell walls are mostly composed of b-glucans, chitins and mannans. Dectin-1 recognizes b-glucans in the fungal cell wall while Dectin-2 binds mannans. Mice lacking either Dectin-1 (Clec7a 2/2 ) or Dectin-2 (Clec4n 2/2 ) exhibit increased susceptibility to fungal infections supporting the role of these proteins in fungal immunity [5,6,17,18]. Furthermore, identification of a CLEC7A single nucleotide polymorphism in humans, which encodes a non-functional form of Dectin-1, confirms the role of Dectin-1 in anti-fungal responses, as carriers of this polymorphism are more susceptible to mucocutaneous infections with C. albicans, in part due to reduced IL-17 production [19,20]. Consistent with these data, polymorphisms resulting in IL-17RA and IL-17F deficiency were recently identified in some patients with chronic mucocutaneous candidiasis [21].
A wide variety of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors are centrally involved in mediating the inflammatory response. The Dectin-1 cytoplasmic domain includes an ITAM-like sequence (known as a hemiITAM, YxxxI/ Lx 7 YxxL) while Dectin-2 couples to the signaling chain FceRIc, which signals via a canonical ITAM (YxxLx 7-12 YxxL) [13]. Engagement of ITAM-associated receptors such as Dectin-2 (FceRIc), or hemiITAM-containing receptors such as Dectin-1, results in phosphorylation of the tyrosines within ITAMs or hemiITAMs. Src homology domain-2 containing protein tyrosine kinases of the Syk/Zap70 family are then recruited to the ITAM/ hemiITAM and activated, resulting in CARD9-and MAPKdependent pro-inflammatory cytokine production [4]. Surprisingly however, the biochemical adaptor(s) involved in coupling Dectin-1 or Dectin-2 proximal phosphorylation to downstream effectors have yet to be identified and despite the established importance of these receptors, little is known about the regulation of their signaling. Two closely related transmembrane adaptor proteins, the Linker for Activation of T cells (LAT/Lat1) and Linker for Activation of B cells/Non-T cell Activation Linker/Linker for Activation of T cells family member 2 (LAB/NTAL/Lat2, hereafter referred to as LAB) facilitate signaling downstream of various ITAM-coupled receptors [22]. These adaptor proteins activate signaling pathways such as the MAPK cascade and regulate production of cytokines including IL-12p40 [23]. As a consequence we hypothesized that LAT and/or LAB would mediate or regulate cytokine production during fungal responses downstream of Dectin-1 and/or Dectin-2 due to the similarities in hemiITAM and canonical ITAM signaling.
Here we demonstrate that LAB is involved in anti-fungal immunity. Mice deficient in LAB (Lat2 2/2 mice) are more susceptible to systemic C. albicans infection than WT mice. However, Lat2 2/2 neutrophil cytokine production is not impaired suggesting a defect in another cell type. We show that LAB but not LAT, is expressed by DCs and that both the M-CSF/DAP12 and mannan-Dectin-2/FceRIc pathways promote LAB phosphorylation. Conversely, the b-glucan-Dectin-1 or TLR pathways do not. We also define a novel role for LAB in suppressing b-catenin nuclear translocation, which in turn permits efficient IL-12 production from bone marrow-derived DCs (BMDCs) stimulated with a range of PAMPs. Furthermore, through this novel mechanism LAB promotes NK and T cell-mediated IFN-c production, which is deficient in vivo during C. albicans infection of Lat2 2/2 mice. Thus LAB provides a molecular bridge between b-catenin activation and the cytokine production required for fungal clearance during systemic infection.

Results
Lat2 2/2 mice display increased susceptibility to challenge with C. albicans As LAB has previously been shown to mediate/regulate signaling and/or cytokine responses downstream of ITAMcoupled receptors and TLRs [23,24], we hypothesized that LAB would play a role in anti-fungal immunity. To investigate this possibility, we systemically infected WT and Lat2 2/2 mice with C. albicans. Lat2 2/2 mice displayed increased susceptibility to high (1.5610 5 ) and low (5610 4 ) dose C. albicans infection compared to WT mice ( Fig. 1A-B). The reduced survival was paralleled by increased fungal burden in the kidneys of Lat2 2/2 mice, nine days after infection (i.v.) with C. albicans (Fig. 1C). The kidney depicted from a Lat2 2/2 mouse that succumbed to infection displays a marked proliferation of fungal hyphae within the pelvis, which was surrounded by neutrophilic inflammation, consistent with an inability to clear the infection (Fig. 1D). Together, these data demonstrate that LAB is important for the host response to C. albicans infection.

Lat2 2/2 neutrophil cytokine production is not impaired
Neutrophil function is important for anti-fungal immunity. They produce cytokines such as TNF and IL-6, both of which play a role in determining susceptibility to fungal infections [25,26]. As LAB is expressed in neutrophils [24], we examined the effect of LAB deficiency on C. albicans-recruited neutrophil cytokine production following restimulation with heat-killed C. albicans yeast (HKY). TNF and IL-6 production from C. albicans-recruited neutrophils was not impaired in Lat2 2/2 cells, in fact they were enhanced ( Fig. 2A), similar to the findings of Tessarz et al [24]. Consistent with these data, we observed enhanced TNF and IL-6 production from bone marrow purified neutrophils following stimulation with zymosan and LPS (Fig. 2B). In addition, serum TNF and IL-6 levels were enhanced in Lat2 2/2 mice systemically

Author Summary
Fungal infections are a major healthcare problem and the incidence of fungal infections has increased significantly in recent years. Mortality rates are high even with treatment, highlighting the need for a better understanding of antifungal immunity in order to develop improved therapies.
Adaptive T-helper 1 and T-helper 17 (Th1 and Th17) responses are important mediators of anti-fungal immunity. Dendritic cells express Dectin-1, Dectin-2 and Toll-like receptors, which interact with fungal pathogens to induce these adaptive immune responses. Here we identify LAB as an important facilitator of IFN-c production by regulating b-catenin activation. Susceptibility to fungal infections is increased in the absence of LAB, in association with reduced IFN-c production. b-catenin activation in dendritic cells inhibits the IL-12 production required for IFN-c production. Thus targeting b-catenin therapeutically could help to promote efficient IFN-c production in patients suffering from fungal infections. These findings are important for fungal infections and potentially for other diseases where IFN-c production is important for disease outcome.
infected with C. albicans (Fig. 2C). These data indicate that neutrophil cytokine production is not impaired in Lat2 2/2 mice suggesting that LAB plays additional roles in other cell types during systemic C. albicans infection.

LAB is downstream of zymosan and C. albicans signaling
In addition to neutrophils, DCs are important for anti-fungal immunity. In previous studies, we have shown that there is a switch from LAT to LAB expression as monocytes differentiate into macrophages and that LAB is involved in regulating ITAM-mediated signals in macrophages [23]. To extend these studies to DCs we examined the expression of LAB in human monocytes and monocyte-derived DCs and murine GM-CSFcultured BMDCs. Similar to macrophages, LAB is expressed in DCs while LAT expression is lost (Fig. 3A-B). To determine whether LAB is involved in fungal-induced signaling, we stimulated BMDCs with the yeast cell wall extract zymosan and found that many proteins were tyrosine phosphorylated following stimulation (Fig. 3C). Consistent with previous findings [5,27] we found that Syk and PLCc2 were prominently phosphorylated following zymosan stimulation (Fig. 3D). This response was also associated with the phosphorylation of LAB and one of its binding partners, c-Cbl [23] (Fig. 3D). Phosphorylation of these proteins was unaffected by the absence of LAB (Fig. S1). Accordingly, we found that activation of the Erk and NFkB pathways were unaffected by the absence of LAB (Fig. S1). Similar to our findings with zymosan, we also observed that many proteins, including LAB, were phosphorylated following stimulation with heat-killed C. albicans yeast (HKY) (Fig. 3E). The zymosan-and C. albicans-induced phosphorylation of LAB in DCs suggests that this adaptor protein may be important in regulating downstream pathways in DCs that are instrumental in combating fungal infections.

Impaired cytokine responses in Lat2 2/2 DCs
Given that LAB is phosphorylated following zymosan stimulation in DCs, we next assessed the ability of LAB to regulate zymosan-induced cytokine responses in DCs. We found that zymosan-induced IL-12p40 production from BMDCs is partially dependent on LAB (Fig. 4A). As zymosan is a complex ligand that engages multiple receptor systems, including Dectin-1, Dectin-2, TLR2 and others, we examined the effect of LAB on various fungal and TLR ligands. WT and Lat2 2/2 BMDCs were stimulated with heat-killed C. albicans yeast, LPS, particulate bglucan (Sigma), curdlan, Pam 3 CSK 4 and CAWS (mannans). IL-12p40 production was significantly reduced in Lat2 2/2 BMDCs following stimulation with each of these ligands suggesting that LAB plays an important role in IL12-p40 production ( Fig. 4B-C). To underline the importance of these findings to fungal infection, we next stimulated BMDCs with the yeast form of live C. albicans. These results showed that induction of Il12b, Il12a and Il23a mRNA were significantly impaired in Lat2 2/2 cells compared to WT controls (Fig. 4D). Subsequently, after a twenty-four hour stimulation of BMDCs with live C. albicans, cytokine levels in the supernatants were measured. These data showed that production of IL-12p40 and IL-12p70 were both greatly reduced, and IL-23 levels were also partially suppressed, in Lat2 2/2 BMDCs following stimulation with live yeast. However, IL-10, IL-1b and TNF levels were not impaired by the absence of LAB (Fig. 4E). We next stimulated BMDCs with increasing doses of LPS. Consistent with our findings with C. albicans we demonstrated that LAB plays a significant role in mediating the production of IL-12 family cytokines downstream of LPS, while production of IL-10, IL-1b and TNF levels were not impaired by the absence of LAB (Fig. 4F). These data indicate that LAB plays an important and selective role in facilitating fungal/PAMP-induced IL-12 family cytokine production.
LAB is required to induce an efficient Th1 response in vitro C. albicans contains ligands for many receptors (including Dectin-1, Dectin-2 and TLRs 2 and 4) and systemic infection with C. albicans involves both Th1 and Th17 responses [5]. In vitro however, the T cell response to C. albicans is dominated by Th17 polarization [6]. In order to assess the capacity of Lat2 2/2 DC to direct both Th17 and Th1 responses in the context of reduced IL-12 production, heat-killed C. albicans and the alternate TLR4 selective ligand, LPS, were used respectively. Supernatants from WT and Lat2 2/2 BMDCs cultured in the presence of heat-killed C. albicans yeast or LPS were added to naïve WT CD4 + T cells stimulated with anti-CD3 and anti-CD28. IFN-c and IL-17A production were measured by flow cytometry and ELISA following 4 days of culture. As expected, conditioned media from BMDCs stimulated with heat-killed yeast induced a robust Th17 response while stimulation with LPS induced a Th1 response ( Fig. 5A-B). HKY-stimulated WT and Lat2 2/2 BMDCs induced comparable Th17 responses ( Fig. 5A-B), likely due to the production of similar levels of IL-1b and only a minimal defect in IL-23 production from Lat2 2/2 BMDCs. Interestingly, the proportion of IFN-c-secreting CD4 + T cells arising from LPS stimulated Lat2 2/2 BMDCs was significantly reduced compared to WT BMDCs ( Fig. 5A-C), which could be attributed to the significant reduction in IL-12p70 production from Lat2 2/2 BMDCs ( Fig. 4E-F). These data indicate that LAB-facilitated cytokine production is important for inducing Th1 responses but not Th17 responses in vitro. As both Th1 and Th17 responses are important for in vivo protection against C. albicans infections [6,9], our data suggests that defective Th1 responses in Lat2 2/2 mice may be responsible for the increased susceptibility of these mice to C. albicans infection.

Signaling through Dectin-2 promotes LAB phosphorylation
As LAB facilitates fungal/PAMP-induced cytokine production in DCs and subsequent Th1 polarization, we next sought to determine which signaling pathways were involved. As LAB phosphorylation is a documented feature of ITAM-coupled receptor signaling [23,28], we hypothesized that b-glucans signaling through the hemiITAM-containing Dectin-1 or mannans signaling through the ITAM-coupled Dectin-2 would induce LAB phosphorylation. After resting the cells to remove basal phosphorylation, we found, surprisingly, that stimulation of DCs with washed zymosan particles (insoluble components of zymosan such as the b-glucans) or purified particulate b-glucans does not lead to LAB phosphorylation while stimulation with zymosan extract (soluble components of zymosan such as the mannans) or purified mannans caused rapid and robust LAB phosphorylation ( Fig. 6A-B). LAB phosphorylation was not observed in response to TLR2/4 ligands Pam 3 CSK 4 or LPS ( Fig. 6A-C). These data suggest that LAB may be phosphorylated downstream of the mannan-Dectin-2/FceRIc pathway rather than via Dectin-1 or the TLR pathways. To further address this possibility, we examined LAB phosphorylation in DCs lacking Myd88 or Fcer1g. Consistent with a role for Dectin-2, LAB phosphorylation was intact in the absence of MyD88 (Fig. 6D) and ablated in Fcer1g 2/2 cells (Fig. 6E). We next stimulated RAW-264 macrophage cell lines, engineered to express either Dectin-1 or Dectin-2 [29], with zymosan and found that LAB was only phosphorylated in cells expressing Dectin-2 (Fig. 6F). This was in spite of high zymosan recognition by Dectin-1 over-expressing RAW-264 macrophages and their potential to signal via TLRs [29,30]. To confirm this selective role for Dectin-2, we conducted Dectin-2 specific shRNAknockdown experiments (Fig. 6G-H). We found that LAB phosphorylation was attenuated in DCs with partial Dectin-2 knockdown as a result of infection with shRNA against Dectin-2 compared to BMDCs infected with a scrambled shRNA control BMDCs from WT and Lat2 2/2 mice were stimulated for the indicated times with 1610 5 live C. albicans yeast. RNA was isolated, cDNA was prepared and Il12b, Il12a and Il23a mRNA transcripts were detected by real-time qPCR. mRNA levels were normalized to Hprt1. Black bars represent WT, white bars represent Lat2 2/2 . (E) BMDCs from WT and Lat2 2/2 mice were stimulated for the indicated times with live C. albicans yeast. Fungizone was added 2 h later and cytokine levels in the supernatants were measured after 24 h incubation. (F) BMDCs from WT and Lat2 2/2 mice were stimulated with 1-1000 ng/ml LPS. Cytokine levels in the supernatants were measured after 24 h incubation. For all graphical data, results are presented as means +/2 s.e.m. of three replicates and data are representative of 2-4 independent experiments. *p,0.05 **p,0.005 ***p,0.0005 (1-way ANOVA, Bonferroni's post-test or Student's t test). doi:10.1371/journal.ppat.1003357.g004 (Fig. 6H). Taken together, these data clearly demonstrate that zymosan stimulates LAB phosphorylation through a mannandriven Dectin-2-FceRIc signaling pathway and not through the bglucan-Dectin-1 or TLR pathways. The phosphorylation of LAB downstream of Dectin-2 could potentially explain the reduced IL-12p40 in response to C. albicans, zymosan and mannans. However, the reduced IL-12p40 in response to b-glucan, LPS or Pam 3 CSK 4 implies the involvement of another LAB activating pathway.

Signaling through M-CSF/DAP12 promotes LAB phosphorylation
Given the broad effect of LAB deletion, we hypothesized that LAB may exert its effect prior to stimulation. Therefore, we next examined whether LAB phosphorylation occurs in DC during their expansion in vitro. For this experiment we compared WT BMDCs harvested directly from culture to those rested in cytokine/serum free conditions for 30 min with or without subsequent stimulation with zymosan for 1 min (Fig. 7A). These data demonstrate readily appreciable basal phosphorylation of LAB in WT BMDCs during culture (Fig. 7A). LAB phosphorylation in response to SCF [31] has been described previously, suggesting that a growth factor may be responsible for this basal LAB phosphorylation. Therefore, we stimulated BMDCs with GM-CSF and M-CSF. Interestingly, M-CSF induced LAB phosphorylation (Fig. 7B) and examination of the BMDC cultures demonstrated that while M-CSF is not added to these cultures, the cells are producing appreciable levels of M-CSF (Fig. 7C) during culture. Crosstalk between M-CSF and the ITAM-containing signaling chain, DAP12 has recently been described [32]. Thus, we tested whether basal LAB phosphorylation in BMDC cultures was DAP12-dependent. Basal levels of LAB phosphorylation were almost completely abolished in the absence of DAP12 (Fig. 7D). We then sought to determine whether M-CSF-induced LAB phosphorylation was dependent on Syk. Pre-treatment of WT cells with the Syk inhibitor, Piceatannol, inhibited M-CSF-and zymosan-induced LAB phosphorylation (Fig. 7E). These data indicate that LAB is phosphorylated through an M-CSF/DAP12-Syk pathway during culture. This basal M-CSF/DAP12 pathway may control IL-12p40 production in response to all of the PAMPs in this study.
LAB facilitates fungal-induced cytokine production by controlling b-catenin activation M-CSF is constitutively present in naïve mice in vivo and it is also induced during infection with C. albicans [33]. Moreover, M-CSF crosstalks with DAP12 to induce activation of b-catenin [32]. Importantly, b-catenin represses pro-inflammatory cytokine production in DCs [34,35]. Therefore we examined nuclear b-catenin levels in WT and Lat2 2/2 BMDCs. Interestingly, Lat2 2/2 BMDCs showed increased b-catenin activation as evidenced by increased nuclear localization in response to zymosan, heat-killed yeast/hyphae (HKH) or LPS (Fig. 8A-C). Importantly, basal nuclear b-catenin levels are also increased in Lat2 2/2 BMDCs (Fig. 8A-C), and the physiological relevance of this was confirmed by the increased mRNA levels of the b-catenin target genes Axin2 and Wisp1 in non-stimulated cells (Fig. 8D). We next examined whether this increasedb-catenin nuclear accumulation in Lat2 2/2 cells was responsible for the decreased cytokine production by these cells. In the absence of Wnt signaling b-catenin is phosphorylated by GSK-3b and targeted for proteasomal-medi- ated destruction [36]. We induced b-catenin stabilization by stimulating the Wnt pathway with the canonical ligand Wnt3a or with an inhibitor of GSK-3b (SB-216763). Following activation of b-catenin through Wnt3a or SB-216763, the difference in IL-12p40 production between WT and Lat2 2/2 cells was reduced (Fig. 8E-F) resulting in WT cells more closely resembling those deficient in LAB. In addition, we stimulated b-catenin degradation by stabilizing axin, a component of the destruction complex, with XAV939. This again resulted in alleviation of the difference in IL-12p40 production between WT and Lat2 2/2 cells with Lat2 2/2 cells more closely resembling WT cells (Fig. 8G). In all of the above cases, the two-way ANOVA indicates a clear interaction between the effect of the inhibitors/agonists and LAB-deficiency as predicted by the proposed role of b-catenin in LAB-facilitated IL-12p40 production. TNF levels were largely unaffected by treatment with inhibitors/activators of this pathway (Fig. 8H-I). As the basal and PAMP-induced b-catenin levels are different between WT and Lat2 2/2 BMDCs, we hypothesized that modulating b-catenin activation would normalize IL-12p40 production in response to a variety of PAMPs including those that do not directly stimulate LAB phosphorylation. Consistent with this hypothesis, activation of b-catenin through SB-216763 reduced the difference in IL-12p40 production between WT and Lat2 2/2 cells stimulated by LPS (TLR4) (Fig. 8J) or WGP (Dectin-1) (Fig. 8K). TNF levels were largely unaffected by treatment with SB-216763 in response to either LPS (Fig. 8L) or WGP (Fig. 8M). These data indicate that constitutive LAB phosphorylation maintains limited basal bcatenin nuclear accumulation, thus facilitating robust cytokine production, consistent with previous reports demonstrating that b-catenin represses pro-inflammatory cytokine production [34,35].

Lat2 2/2 mice display decreased DC and NK, T and NKT cell-mediated cytokine levels in vivo
As Lat2 2/2 mice were more susceptible to systemic C. albicans infection and Lat2 2/2 DCs displayed impaired IL-12 production and subsequent Th1 responses in vitro, we wanted to determine whether these defective cytokine responses occurred in vivo. In correlation with the reduced IL-12p40 production in vitro we observed reduced Il12b mRNA levels in the spleens of both uninfected (Fig. 9A) and C. albicans infected Lat2 2/2 mice (Fig. 9B). Additionally, Ifng mRNA levels were reduced in both uninfected and C. albicans infected Lat2 2/2 mice and Tbx21 mRNA levels were also reduced in the C. albicans infected Lat2 2/2 mice (Fig. 9A-B). In contrast Il12a, Il23a and Rorc mRNA levels were normal in the Lat2 2/2 mice and Il17 mRNA levels were undetected. Consistent with reduced RNA levels of Il12b, we also observed reduced IL-12p40 in Lat2 2/2 splenic DCs following stimulation with LPS ( Fig. 9C and Fig. S2). In order to further examine whether Lat2 2/2 mice displayed reduced IFN-c responses, we injected WT and Lat2 2/2 mice intraperitoneally with C. albicans and collected cells by peritoneal lavage 72 h post injection. Following restimulation with PMA/Ionomycin there were reduced percentages of IFN-c producing cells in Lat2 2/2 lavages ( Fig. 9D and Fig. S2). The IFN-c producing cells consisted of CD3 + NK1.1 2 CD4 + T cells, CD3 + NK1.1 2 CD4 2 T cells, CD3 2 NK1.1 + NK cells and CD3 + NK1.1 + NKT cells. Not surprisingly, IL-12, alone or in combination with IL-18, is known to induce IFN-c production in T, NK and NKT cells and it appears that each of these cell types contributes to the reduction in IFN-c production with the largest reduction in NK cell IFN-c production (Fig. 9E) [37,38,39,40]. Taken together, these data indicate that LAB facilitates IL-12 production from DCs and subsequent IFN-c production mainly from NK and T cells in vivo. These data demonstrate that LAB is important for DC and NK, T and NKT cell-mediated cytokine production during the host response to C. albicans infection.

Discussion
Here we identify for the first time an important role for LAB in anti-fungal immunity. Lat2 2/2 mice displayed increased susceptibility to C. albicans infection and the fungal burden was significantly increased in these mice, similar to Clec4n 2/2 mice and Clec7a 2/2 mice [6,17]. Lat2 2/2 mice displayed reduced NK and T cell-mediated IFN-c production which reflects the attenuated production of IL-12 by Lat2 2/2 DCs, while Lat2 2/2 neutrophil cytokine responses were not impaired. Moreover, we defined a novel role for LAB in the repression of b-catenin nuclear translocation in DCs, resulting in regulated cytokine production from a range of PAMPs, which is important in mounting an effective immune response in an infectious disease model. These data also suggest that LAB will be important in other diseases where IL-12 is involved in disease outcome.
The adaptor protein LAB mediates signals downstream of multiple ITAM-coupled receptors including BCR, FcRc and TREM1/2 and the growth factor Stem Cell Factor [22]. LAB, similar to LAT, is targeted to lipid rafts where it nucleates signaling complexes [41]. Erk is activated downstream of ITAMcoupled receptors through the recruitment of a Grb2-Sos complex to LAT or LAB while PI3K is thought to be recruited through the formation of a LAB-Grb2-PI3K(p85) complex [22,23]. Activation of these signaling pathways downstream of ITAM-coupled receptors results in cytokine production. However, while LAT and LAB can mediate cytokine production by facilitating formation of these signaling complexes, LAB has also been shown to negatively regulate cytokine production in certain circumstances. In some cases, this is due to competition with LAT which signals more efficiently than LAB due to the presence of a PLCc binding site in LAT. Alternatively, in the absence of LAT, LAB binds the E3 ubiquitin ligase c-Cbl which targets multiple substrates for proteasomal degradation [23,41]. LAB mediates the production of IL-12p40 and attenuates the production of IL-8/CXCL8, TNF and IL-10 downstream of LPS or ITAM-coupled receptors in macrophages and DCs [23,24,42]. Similarly, here we demonstrate that LAB partially mediates the production of IL-12p40, IL-12p70 and IL-23 in response to multiple PAMPs. Previous reports have shown that the levels of Th1 and Th17 inducing cytokines (IL-12, IL-23 and IL-1b) downstream of Dectin-1 and Dectin-2 are regulated by different NFkB subunits [43,44]. However, while fungal and TLR-induced IL-12 family cytokine production is inhibited in the absence of LAB, IkB degradation is unaffected (Fig. S1 and data not shown). In addition, previously identified targets of LAB regulation such as Syk, c-Cbl or MAPK [23,42] are also unaffected in the absence of LAB (Fig. S1). This suggests a role for LAB in the regulation of a distinct pathway.
Otero et al [32] recently identified an exciting link between M-CSF, DAP12 and b-catenin. The authors demonstrated that the  ITAM-containing signaling chain DAP12 was required for the phosphorylation and accumulation of nuclear b-catenin during MCSF-induced signaling in macrophages. b-catenin is found in various locations in the cell. Under resting conditions, b-catenin is part of a complex found at the cell membrane bound to Ecadherin, which controls cell-cell adhesion [45]. In addition to membrane bound b-catenin, b-catenin is also found in the cytosol and cytosolic levels are tightly regulated by Wnt signaling. In the absence of a Wnt ligand b-catenin is phosphorylated by GSK-3b and CK1 and subsequently targeted for proteasomal degradation. In the presence of a Wnt ligand, GSK-3b activity is inhibited and b-catenin levels accumulate in the cytoplasm and the protein translocates to the nucleus to induce gene expression in conjunction with the TCF/LEF transcriptional activators [46]. Our data indicate that LAB is phosphorylated by two pathways, (M-CSF/DAP12 and Dectin-2) (Fig. S3), and that LAB inhibits basal b-catenin nuclear translocation mediated by the M-CSF/DAP12 pathway and also PAMP-induced b-catenin nuclear translocation. M-CSF is present in serum and organs of naïve mice and C. albicans infection further increases its levels [33] and provides the PAMPs necessary to induce these pathways in vivo. b-catenin and LAB have never before been associated and our data demonstrate that this is an important component of PAMPinduced cytokine production in DCs.
DCs expressing constitutively active b-catenin have previously been shown to display a diminished IL-12p40 response [35]. Additionally, ablation of b-catenin in DCs was recently shown to enhance the production of pro-inflammatory cytokines translating into increased Th1 and Th17 polarization [34]. Our results show that LAB plays a critical role in repression of the b-catenin pathway. In accordance with these findings, Lat2 2/2 DCs display high levels ofnuclear b-catenin accumulation and produce reduced levels of IL-12 resulting in reduced NK and T cell-mediated IFN-c production. Activation of b-catenin by Wnt3a or SB-216763 results in a reduction of IL-12p40 production in WT DCs so that they now resemble those lacking LAB. Various PAMPs (LPS, Pam 3 CSK 4 , b-glucan) do not promote LAB phosphorylation, however, basal LAB phosphorylation appears to be sufficient to inhibit b-catenin activation and to facilitate PAMP-induced IL-12 production. The Dectin-2 pathway enhances LAB phosphorylation in response to various ligands (zymosan, C. albicans, CAWS) in addition to basal M-CSF/DAP12-induced LAB phosphorylation. These data indicate that basal LAB phosphorylation is sufficient to facilitate PAMP-induced IL-12 production and subsequent IFNcproduction independently of Dectin-2. However, PAMPs that engage Dectin-2 likely play a cumulative role in promoting IL-12 production (Fig. S3). While the mechanism for LAB-mediated regulation of b-catenin activation is currently unknown, it is possible that LAB exerts its effects through regulation of the PI3K pathway. Akt phosphorylation promotes nuclear b-catenin accumulation [47] and we have previously shown in macrophages that LAB binds to the p85 subunit of PI3K, likely via Grb2 [23] suggesting that this may be a possible mechanism for LABmediated regulation of b-catenin translocation.
The LAB/b-catenin pathway in DCs exerts a specific effect on IL-12 family cytokines while minimally affecting other cytokines (Fig. 4E-F). There are some possible explanations for this specificity. Firstly, NFkB and Erk activation, critical components of IL-1b/TNF [48,49] and IL-10 production, respectively [50] are normal in Lat2 -/-DCs (Fig. S1). Secondly, b-catenin induces gene expression in conjunction with the TCF/LEF transcriptional activators [46] and examination of candidate TCF/LEF binding sites in the promoters of Il12b, IL10 and Tnf revealed interesting differences. While Il10 (21 kb) and Tnf (2300 bp) contain one candidate TCF/LEF binding site, Il12b has two such sites ,60 bp apart, ,600 bp upstream of the transcription start site. Moreover an AML-1 binding site is located between these two TCF/LEF binding sites in Il12b and AML-1 has previously been shown to cooperate with TCF in the TCRá enhancer [51]. Thirdly, Jiang et al [45] demonstrated that cluster disruption of DCs, a process involving b-catenin, selectively increased IL-12p40 production in response to LPS (other cytokines such as TNF and IL-10 were reduced in response to LPS). All of these observations indicate a definite specificity of b-catenin for IL-12 family cytokines; however, separate studies would be required to dissect this.
Here we demonstrated a novel LAB-mediated pathway for regulation of IL-12 production and subsequent IFN-c production in response to C. albicans. Collectively our data indicate the importance of this for controlling susceptibility to C. albicans infection. However, as LAB is expressed in other cell types, it may be involved in additional immune responses. For example, we have observed reduced IL-12p40 production from Lat2 2/2 macrophages (data not shown) similar to DCs suggesting that the reduced Il12b mRNA levels in Lat2 2/2 mice is likely a combined effect of DCs and macrophages. Additionally, neutrophils and associated cytokines (TNF, IL-6) are important for overcoming C. albicans infection [25,52]. Interestingly, Lat2 2/2 neutrophils display enhanced TNF and IL-6 production, demonstrating unimpaired neutrophil activation and cytokine production in Lat2 2/2 mice. High levels of TNF and IL-6 are associated with septic shock [53,54], however, the levels in C. albicans-infected Lat2 2/2 mice are much lower than those in murine sepsis models making it an unlikely cause for the increased mortality. In addition, LAB is expressed in B and activated T cells and its deficiency, similar to Lat2 2/2 neutrophils, results in ''enhanced'' rather than impaired responses. Lat2 2/2 mice have increased levels of natural antibodies and T cells from aged Lat2 2/2 mice are hyperactivated and produce more cytokine than WT T cells [55]. These data indicate that reduced DC/macrophage IL-12 production and subsequent NK and T cell-mediated IFN-c production is the predominant impairment found in Lat2 2/2 mice, however, the role of LAB in other cell-types and functions during anti-fungal immunity needs to be addressed with future studies.
In conclusion, we have shown that LAB is paramount for robust pro-inflammatory cytokine production from DCs downstream of multiple PAMPs. LAB facilitates production of these essential cytokines through novel regulation of the b-catenin pathway. LAB represses b-catenin nuclear accumulation in DCs, thereby facilitating cytokine production. Through this mechanism, LAB plays an important role in the host defense against systemic C. albicans infection by inducing NK and T cell-mediated IFN-c production. These data are the first demonstration of LAB as a prominent target for diseases dependent on IL-12 and further elucidation of these pathways may be important for the development of new therapeutics.

Mice
Lat2 2/2 mice (the product of the Lat2 locus is the adaptor protein LAB), described previously [56], have been backcrossed onto the C57BL/6 background for at least 10 generations. These mice were screened and found to be 99% C57BL/6. Lat2 2/2 , Myd88 2/2 , Fcer1g 2/2 , Tyrobp 2/2 and age, weight and gender matched control C57BL/6 mice were maintained under specific pathogen-free conditions at the NCI-Frederick, MD. Animal care was provided in accordance with the procedures in, ''A Guide for the Care and Use of Laboratory Animals''. Ethical approval for the animal experiments detailed in this manuscript was received from the Institutional Animal Care and Use Committee (Permit Number: 000386) at the NCI-Frederick.

BMDC culture
Bone marrow (BM) cells were removed from the femurs and tibiae of mice and erythrocytes were lysed in ACK buffer. Bone marrow-derived dendritic cells (BMDCs) were generated by culturing cells for 6-9 days in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, penicillin/streptomicin, HEPES, NEAA, Sodium pyruvate, 2-mercaptoethanol and 10 ng/ml GM-CSF. zymosan extract. The zymosan particles were washed 36 with endotoxin free water and used as washed zymosan.

Cell purification and stimulations
BM cells were removed from the femurs and tibiae of mice and erythrocytes were lysed in ACK buffer. Neutrophils were purified through percoll gradient or by MACS separation (Miltenyi, Auburn, CA). BMDCs were harvested and CD11c + DCs were sorted using a FACS ARIA or bead purified by MACS separation routinely giving purities of .98%. BMDCs were serum starved for 30 min at 37uC (except for Figs 7A&D). 1610 7 BMDCs were resuspended in 100 ml DPBS and stimulated at 37uC with 1 mg/ ml zymosan, zymosan extract, washed zymosan, b-glucan, mannan, Pam 3 CSK 4 , 10 ng/ml LPS, 100 ng/ml GM-CSF or M-CSF for the indicated times. Cells were lysed with Laurylmaltoside lysis buffer (1% laurylmaltoside in 20 mM Tris [pH 7.5], 100 mM NaCl, 10% glycerol, 0.4 mM Na 3 VO 4 , aprotinin, leupeptin and phenylmethylsulfonyl fluoride). Lysates were clarified by centrifugation and protein levels were normalized using a BCA protein assay. Cytosol and nuclear extracts were prepared as previously described [57]. 46 non-reducing or reducing Nupage sample buffer was added to lysates and heated for 10 min at 70uC. Lysates were separated by SDS-PAGE (Nupage, Invitrogen, Carisbad, CA), transferred to PVDF membrane (Millipore, Billerica, MA) and analyzed by Western blot.

RNA isolation and quantitative RT-PCR
Cells were resuspended in Trizol and RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was synthesized from total RNA using Superscript III First Strand Synthesis System for RT-PCR (Invitrogen). Quantitative RT-PCR was performed using ABI Taqman Primer and Probe sets and normalization was performed against Hprt1.

Lentiviral knockdown
An shRNA construct for mouse Dectin-2 (TRCN0000066785) and a scrambled shRNA control in the pLKO.1 lentiviral vector were used to infect WT and Lat2 2/2 cells. 293FT cells were transfected with the pLKO.1 construct and with the Invitrogen packaging constructs pLP1, pLP2 and pVSV-G and viral supernatants were collected at 48 and 72 h post-transfection. BM cells were plated on Day 0 at 6610 5 cells/ml in complete media containing GM-CSF. On Days 1 and 2, the media was replaced for 8 h with viral supernatants containing 10 mg/ml hexamethrine bromide. On Day 3, fresh complete media containing GM-CSF and 5 mg/ml puromycin was added to the cells. The cells were harvested 2 days later and stimulated as described. Dectin-2 surface expression was examined by flow cytometric analysis.

Cytokine assays
BMDCs were plated at a density of 1610 5 cells/well in a 96well plate in RMPI containing 10% fetal bovine serum and 10 ng/ ml GM-CSF. BMDCs were stimulated with zymosan, zymosan extract, washed zymosan, Pam 3 CSK 4 , LPS (Escherichia coli 0111:B4), b-glucan, curdlan, yeast/hyphae or heat-killed yeast/ hyphae for 24 h. BMDCs were stimulated with CAWS for 48 h. BM neutrophils were plated at a density of 2610 5 cells/well in RPMI containing 10% fetal bovine serum and stimulated with zymosan or LPS for 24 h. Cell culture supernatants were recovered and assayed for cytokine by ELISA (eBioscience, San Diego, CA) or cytometric bead array (CBA) (BD Biosciences, San Jose, CA), according to the manufacturer's protocol. Unpurified spleen cells or CD11c + MACS bead purified spleen cells were stimulated with 100 ng/ml LPS for 6 h in the presence of Brefeldin A. IL-12p40 producing cells were determined by flow cytometry.

T cell polarization assay
CD4 + T cells were purified from WT spleens by negative selection MACS separation (Miltenyi, Auburn, CA) and naïve CD4 + T (CD4 + CD25 2 CD44 lo CD62L + ) cells were sorted using a FACS ARIA giving purities of .98% purity. Naïve CD4 + T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 4 days and supplemented with conditioned supernatants from BMDCs stimulated with heat-killed C. albicans yeast or LPS. The supernatants were collected and analyzed for IL-17A and IFN-c production by ELISA. The cells were restimulated with PMA (50 ng/ml) and Ionomycin (0.5 mg/ml) in the presence of monensin (3 mM) and IL-17A and IFN-c producing cells were determined by flow cytometry.
In vivo model of systemic candidasis C. albicans SC5314 (ATCC, Manassas, VA) was cultured for 24 h in YEPD broth, washed three times with PBS and resuspended at the required concentration in PBS. Mice were matched by gender, weight and age (10-15 weeks old) and 100 ml of C. albicans in PBS was injected i.v. Mice were monitored and weighed daily. Mice were euthanized by CO 2 asphyxiation when they were moribund or had lost 20% of their body weight. Experiments were continued for a maximum of 55 days at which point all surviving mice were euthanized. Mice were bled by cardiac puncture after CO 2 administration and kidneys and spleens were harvested. The kidneys were either placed in 10% formalin, embedded in paraffin wax blocks and stained for H&E and PAS or they were placed in PBS, dounce homogenized and serial dilutions were plated on YEPD agar containing 50 mg/ml chloramphenicol. The plates were cultured for 24 h and CFU were calculated/organ. The spleens were dounce homogenized in trizol followed by RNA and cDNA preparation. Serum samples were analyzed by CBA for cytokine levels.

Ex vivo C.albicans NK and T cell assay
Mice were injected intraperitoneally with 1610 5 live C. albicans and euthanized 72 h later. The inflammatory infiltrate was collected by peritoneal lavage with 5 ml 5 mM EDTA in RPMI. The cells were plated and restimulated with PMA (50 ng/ml) and Ionomycin (0.5 mg/ml) in the presence of monensin (3 mM) for 4 h and IL-17A and IFN-c producing cells were determined by flow cytometry.

C. albicans neutrophil recruitment and cytokine production
Mice were injected intraperitoneally with 1610 5 live C. albicans and euthanized 4 h later. The inflammatory infiltrate was collected by peritoneal lavage with 2 rounds of 5 ml 5 mM EDTA in RPMI. The cells were plated and restimulated with media or heat-killed C. albicans yeast for 12 h in the presence of monensin (3 mM). The % of TNF and IL-6 producing neutrophils were determined by flow cytometry.

Statistical methods
Data are presented as means +/2 s.e.m. and are representative of 2-3 independent experiments. Survival data was analyzed by log-rank test. One-way ANOVA followed by Bonferroni's post-test or two-way ANOVA were used for statistical analysis when multiple groups were analyzed. Student t test or Mann-Whitney test were used for statistical analysis when two groups were analyzed. When data did not follow a Gaussian distribution, it was transformed by Y = sqrt(Y+0.5) [58] and analyzed by Student's t test. If data still did not follow a Gaussian distribution after transformation, then significance was tested by Mann-Whitney test. Statistical significance was set at *p,0.05 **p,0.005 ***p,0.0005. Through crosstalk with DAP12, M-CSF promotes LAB phosphorylation. LAB inhibits b-catenin translocation to the nucleus, thereby promoting IL-12 following subsequent stimulation with PAMPs. M-CSF/DAP12-induced LAB phosphorylation is sufficient to inhibit b-catenin activation and subsequent PAMPinduced IL-12 production. In Lat2 2/2 DCs, basal b-catenin levels are increased. (2) C. albicans, zymosan or CAWS stimulate the Dectin-2 pathway, further stimulating LAB phosphorylation and inhibiting nuclear translocation of b-catenin. These fungal PAMPs also induce IL-12 production. In Lat2 2/2 DCs, IL-12 production is reduced. (3) b-glucan, LPS or Pam 3 CSK 4 stimulate IL-12 production but they do not stimulate LAB phosphorylation. In Lat2 2/2 DCs, IL-12 production is reduced demonstrating that basal M-CSF/DAP12-induced LAB phosphorylation is sufficient to control b-catenin levels and IL-12 production. (4) LABmediated IL-12 produced by DCs promotes IFN-c production from NK, T and NKT cells. IFN-c production is reduced in the absence of LAB.