Phospholipase Cγ2 (PLCγ2) Is Key Component in Dectin-2 Signaling Pathway, Mediating Anti-fungal Innate Immune Responses*

Background: Dectin-2 is involved in anti-fungal immunity. However, its signaling pathway is not fully characterized. Results: We showed that PLCγ2 deficiency impairs Dectin-2-induced NF-κB and MAPK activation in response to fungal infection. Conclusion: PLCγ2 is a key component in Dectin-2 signaling pathway, mediating immune responses against fungal infection. Significance: These studies reveal a potential therapeutic target for fungal infection. C-type lectin receptors (CLRs) such as Dectin-2 function as pattern recognition receptors to sense fungal infection. However, the signaling pathways induced by these receptors remain largely unknown. Previous studies suggest that the CLR-induced signaling pathway may utilize similar signaling components as the B cell receptor-induced signaling pathway. Phospholipase Cγ2 (PLCγ2) is a key component in B cell receptor signaling, but its role in other signaling pathways has not been fully characterized. Here, we show that PLCγ2 functions downstream of Dectin-2 in response to the stimulation by the hyphal form of Candida albicans, an opportunistic pathogenic fungus. Using PLCγ2- and PLCγ1-deficient macrophages, we found that the lack of PLCγ2, but not PLCγ1, impairs cytokine production in response to infection with C. albicans. PLCγ2 deficiency results in the defective activation of NF-κB and MAPK and a significantly reduced production of reactive oxygen species following fungal challenge. In addition, PLCγ2-deficient mice are defective in clearing C. albicans infection in vivo. Together, these findings demonstrate that PLCγ2 plays a critical role in CLR-induced signaling pathways, governing antifungal innate immune responses.

Invasive Candida albicans infection remains a serious clinical complication in patients with compromised immune systems. C. albicans is a dimorphic fungus, and its pathogenicity is dependent on both its yeast and filament-shaped hyphal forms, because mutants in either form are less virulent than wild-type strains (1,2). The host defense against systemic C. albicans infection primarily depends on innate immune cells, especially macrophages and neutrophils (3). To initiate an antifungal defense, the innate immune cells have to contact the fungal cell wall, which is primarily composed of carbohydrates including ␤-glucans, mannans, and chitin (3). Recognition of the yeast versus the hyphal form of C. albicans may be mediated through the engagement of different receptors on macrophages (4,5).
Previous studies suggest that C-type lectin receptors, including Dectin-1 and Dectin-2, function as the pattern recognition receptors for sensing C. albicans infection (5)(6)(7)(8)(9)(10)(11). In response to C. albicans infection, Dectin-1 recognizes the ␤-glucan in the cell wall of the yeast form of C. albicans (4,12), whereas Dectin-2 has been suggested to interact with the mannoprotein coat in the cell wall of the hyphal form of C. albicans (5,6). It has been shown that the ␤-glucan layer in the C. albicans cell wall is usually buried by the mannoprotein coat in the yeast form but exposed when they transform into the hyphae under infection conditions (13), which may interact with Dectin-1 and induce a strong immune response during an infection (14). However, recent studies on Dectin-1-deficient mice suggest that there may be other redundant receptors mediating C. albicans infection-induced immune responses (15,16). In contrast, studies on Dectin-2-deficient mice suggest that Dectin-2 may play a more important role for the innate immune response against C. albicans infection (16). Therefore, the signal transduction pathway induced by Dectin-2 is not fully characterized, and it remains to be determined whether Dectin-1 and Dectin-2 receptors share the same signaling pathway.
Although the signaling pathways induced by Dectin-2 following C. albicans infection are not fully defined, previous studies indicate that stimulation of Dectin-2 as well as Dectin-1 by the cell wall components of C. albicans can activate the spleen tyrosine kinase (Syk) (10,17,18). The activation of Syk leads to activation of multiple signaling cascades (9,11), which induces NF-B activation through a CARD9-dependent pathway (17,19). Syk is a key component in the B cell receptor signaling pathway. However, whether other components in B cell receptor signaling pathway are also involved in Dectin-2 signaling pathway remains to be determined.
PLC␥ hydrolyzes phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate (20). In hematopoietic cells, two isoforms of PLC␥, PLC␥1 and PLC␥2, are expressed. PLC␥2 is a key component in B cell receptor signaling pathway (21), whereas PLC␥1 is the main effector in T cell receptor signaling (22). In dendritic cells, both PLC␥1 and PLC␥2 can be activated by the stimulation of ␤-glucan, zymosan, and curdlan, extracted from the cell wall of yeast (23,24). Previous studies suggest that PLC␥1 functions downstream of Syk in the Fc⑀R signaling pathway (25), whereas Syk controls PLC␥2 in the B cell receptor signaling pathway (21). Recent studies suggest that PLC␥2 is also involved in Dectin-1 signaling (23,24). However, whether PLC␥1 and PLC␥2 are involved in Dectin-2 signaling pathways in response to C. albicans infection remains unknown. In this study, we demonstrated that PLC␥2 functions downstream of Dectin-2 receptor in response to fungal infection. Using PLC␥1-and PLC␥2-deficient mice, we show that PLC␥2 but not PLC␥1 plays an essential role in the immune responses to C. albicans infection by inducing expression of cytokines and generating reactive oxygen species.
Mice-PLCg2 Ϫ/Ϫ , CARD9 Ϫ/Ϫ , and PLCg1-floxed allele mice were generated as described previously (21,22,26). All mice have been backcrossed to C57BL6 background. PLC␥2-and PLC␥1-deficient mice were maintained in the Biological Resource Center at the Medical College of Wisconsin. CARD9deficient mice were housed under specific pathogen-free conditions at the M. D. Anderson Cancer Center animal facility and were used at 8 -16 weeks of age. Bone marrow cells from Mx1Cre mice (Jackson Laboratory stock 005673). For experiments, PLCg1 ϩ/ϩ Mx1Cre mice and PLCg1 f/f Mx1Cre mice received intraperitoneal injections of 300 g of poly(I⅐C) on days 1 and 3. The mice were sacrificed 14 days after the first injection. Bone marrow cells from these mice were harvested and injected into C57B6 wild-type mice irradiated with 11 gray (Gy). For PLCg2 Ϫ/Ϫ chimera mice, similar procedures were performed. Two months after bone marrow transplantation, chimera mice were used for yeast infection experiments.
Bone Marrow-derived Macrophage Preparation-Primary cultures of bone marrow-derived macrophages (BMDMs) 2 were prepared as described previously (26 -28). Briefly, bone marrow cells were harvested from the femurs and tibias of mice. Erythrocytes were removed from cells samples by subjecting the samples to hypotonic solution. The cells were cultured for 7 days in DMEM containing 20% FBS, 55 M ␤-mercaptoethanol, streptomycin (100 g/ml), penicillin (100 units/ml), and 30% conditioned medium from L929 cells expressing macrophage colony-stimulating factor. Nonadherent cells were removed, and cells were passed every 3 days. After 1 week of cultures, flow cytometry analysis indicated that the harvested cell population contained 86 -95% CD11b ϩ F4/80 ϩ cells as assessed.
Lentivirus-encoded shRNA Knockdown of Dectin-2 in BMDMs-Lentiviral particles used for infection were prepared as described previously (28) using shRNA for mouse Dectin-2 (Sigma-Aldrich) or a nontargeting sequence. Mouse bone marrow cells were infected with virus on days 1 and 3 and selected with 2 g/ml puromycin on day 5 of culture.
C. albicans Preparation-C. albicans (strain SC5314) was kindly provided by Dr. Michael C. Lorenz (Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston). A single colony of C. albicans was grown overnight at 30°C in yeast peptone dextrose medium. The cells were washed three times with PBS and then used as live yeast. For hyphae, the washed yeast cells were resuspended in RPMI with 10% FCS, grown at 37°C for 3 h, and washed in PBS. The hyphae were then used for live stimulations. For heatinactivated yeast, yeast cells were heated at 65°C for 1 h.
In Vivo C. albicans Infection-For in vivo C. albicans infection, male mice aged at 8 -14 weeks were injected with 2 ϫ 10 5 or 1 ϫ 10 6 live C. albicans in 0.3 ml of PBS (pH 7.4). Candidal burden was determined by killing the mice 42 h after infection and harvesting and homogenizing their kidneys, lungs, livers, and spleens. Fungal colony formation units were quantified by plating serial dilutions of the homogenized organs on yeast extract peptone dextrose agar. For histological analysis, the tissue sections were cut and stained for glycogen using periodic acid-Schiff or hematoxylin and eosin.
Reactive Oxygen Species (ROS) Production Assay-For ROS production evaluation, 1 ϫ 10 5 BMDMs were washed with Hanks' balanced salt solution without phenol red. Then Hanks' balanced salt solution containing 100 M luminol and 5 units of horseradish peroxidase (Sigma) were added to the cells, and the cells were incubated for 10 min at 37°C. The cells were then stimulated with either hyphae (multiplicity of infection (MOI) ϭ 2) or phorbol myristate acetate. ROS production was measured every 3 min using a luminometer.
EMSA-After BMDMs were stimulated with hyphae or yeast, their nuclear extracts were prepared. The resulted nuclear protein (5 g) was incubated with 32 P-labeled NF-B or Oct-1 probe (Promega) for 15 min at 25°C and then subjected to PAGE and exposed to x-ray films.

PLC␥2 Is Involved in Dectin-2-induced Signaling Pathway-
To determine whether C. albicans can activate PLC␥2 through Dectin-2 receptor in macrophages, we examined PLC␥2 phosphorylation in BMDMs after infecting them with C. albicans hyphae. Stimulation of BMDMs with C. albicans led to an inducible phosphorylation of PLC␥2 (Fig. 1A), indicating that PLC␥2 is activated in response to C. albicans infection. Because both Dectin-1 and Dectin-2 have been shown to be involved in antifungal innate immune response, we determined whether Dectin-1 and Dectin-2 are required for C. albicans hyphaeinduced PLC␥2 activation using BMDMs treated with a specific shRNA to Dectin-2 or BMDMs from Dectin-1 knock-out mice. Consistent with previous findings that Dectin-2 but not Dectin-1 is involved in C. albicans hyphae-induced signal transduction, we found that PLC␥2 activation was dependent on Dec-tin-2 but not Dectin-1 (Fig. 1, B and C). These results indicate that although C. albicans hyphae may activate multiple receptors on macrophages, only Dectin-2 leads to activation of PLC␥2.
PLC␥2 Is Required for C. albicans Infection-induced NF-B Activation-We next sought to identify the PLC␥2-dependent signaling pathways in macrophages and determine whether these signaling pathways are redundant in response to the hyphal and yeast forms of C. albicans. Previous studies indicate that heat-inactivated yeast of C. albicans can activate Dectin-1 signaling pathway, whereas hyphae of C. albicans induces Dectin-2 pathway. Therefore, we examined the contribution of PLC␥2 in NF-B activation induced by these signaling pathways and found that NF-B activation induced by hyphae was defective in PLC␥2-deficient macrophages ( Fig. 2A), indicating that PLC␥2 is a key component in Dectin-2 signaling pathway. Consistent with recent studies (23,24), PLC␥2-deficient macrophages were also defective in NF-B activation induced by the stimulation of heat-inactivated yeast (Fig. 2B) that activates Dectin-1 signaling pathway. Together, these data indicate that PLC␥2 functions downstream of both Dectin-1 and Dectin-2 receptors and mediates NF-B activation induced by these receptors.
Earlier studies suggest that TLR2 functionally cooperates with Dectin-1 to regulate cytokine production following fungal infection (29,30). Because TLR2 can potently activate NF-B through a MyD88-dependent pathway, we examined whether the MyD88-dependent pathway might also be involved in fungal infection-induced NF-B activation. However, we have found that, unlike PLC␥2 deficiency, MyD88 deficiency does not affect C. albicans hyphae-and yeast-induced NF-B activation (Fig. 2, C and D). Therefore, our data suggest that Dectin-1and Dectin-2-induced NF-B activation are mediated through a PLC␥2-dependent but TLR/MyD88-independent pathway.
The regulation of the IKK complex in response to hyphae is likely mediated through at least two signaling events, in which a Syk-dependent pathway regulates the phosphorylation of IKK␣/␤, whereas the adaptor protein CARD9 regulates the ubiquitination of IKK␥ in a Syk-independent manner (28). We next examined whether PLC␥2 regulates the phosphorylation of IKK␣/␤ and found that the signal-induced phosphorylation of IKK␣/␤ was defective in PLC␥2 Ϫ/Ϫ but not in Card9 Ϫ/Ϫ macrophages in response to hyphae stimulation (Fig. 2E).
Because CARD9 forms a complex with Bcl10 following hyphae stimulation, we investigated whether the formation of this complex would be affected in the absence of PLC␥2. CARD9 was immunoprecipitated from WT and PLC␥2 Ϫ/Ϫ macrophages that were stimulated with or without hyphae, and then the association of CARD9 with Bcl10 was examined. Interestingly, we found that PLC␥2 deficiency did not significantly affect the formation of the CARD9-Bcl10 complex in response to the stimulation of C. albicans hyphae (Fig. 2F). These results suggest that PLC␥2 is a critical enzyme downstream of Syk and regulates the IKK complex in a CARD9-independent manner.
MAPK Signaling Is Defective in PLC␥2-deficient Macrophages-Previous studies show that stimulation of bone marrow-derived dendritic cells by C. albicans induces tyrosine phosphorylation of MAPKs (17). Therefore, we examined FIGURE 1. Dectin-2 is required for C. albicans-induced PLC␥2 activation. A, wild-type macrophages were stimulated with C. albicans hyphae (MOI ϭ 1), and the phosphorylation of PLC␥2 was examined by Western blotting. B, BMDMs were infected with lentivirus encoding Dectin-2 shRNA or GFP shRNA, then selected with puromycin, and stimulated on day 9 with C. albicans hyphae (MOI ϭ 1) at different times. Cell lysates were subjected to Western blotting analysis using indicated antibodies. C, WT and Dectin-1 Ϫ/Ϫ BMDMs were stimulated with C. albicans hyphae (MOI ϭ 1) or LPS (100 ng/ml) for the indicated times. Cell lysates were subjected to Western blotting analysis using the indicated antibodies. DECEMBER 23, 2011 • VOLUME 286 • NUMBER 51 whether MAPK activation is dependent on PLC␥2 following fungal infection. We found that the activation of ERK and JNK was completely defective in PLC␥2 Ϫ/Ϫ BMDMs following the stimulation by hyphae (Fig. 3A), whereas the activation of p38 and AKT in WT and PLC␥2 Ϫ/Ϫ BMDMs was comparable (Fig.  3A). Although C. albicans yeast could also induce ERK and JNK activation in WT cells, ERK activation was only partially defective in PLC␥2 Ϫ/Ϫ BMDMs, whereas JNK activation was significantly decreased (Fig. 3B). These results indicate that the activation of ERK and JNK in response to C. albicans hyphae is solely dependent on PLC␥2, whereas C. albicans yeast may induce ERK activation through two pathways, one of which is dependent on PLC␥2, whereas the other is PLC␥2-independent.

PLC␥2 in Dectin-2-induced NF-B MAPK Activation
Because PLC␥1, a homolog of PLC␥2, is highly expressed in macrophages, we would like to investigate whether PLC␥1 plays a role in antifungal innate immunity. To determine whether PLC␥1 is also involved in this fungal infection-induced signaling pathway, we obtained BMDMs from PLC␥1-conditional knock-out (PLC␥1 F/F /MxCre) or its control (PLC␥1 F/ϩ / MxCre) mice and examined the MAPK activation following stimulation with C. albicans. Unlike PLC␥2-deficient cells, PLC␥1-deficient cells are not defective in C. albicans hyphaeinduced ERK and IKK activation (Fig. 3C). These data indicate that PLC␥1 is not involved in Dectin-2 signaling pathway to induce anti-fungal innate immune responses.
PLC␥2 but Not PLC␥1 Is Required for C. albicans-induced Cytokine Production-To determine the functional requirement of PLC␥2 in antifungal innate immune responses, we compared the levels of cytokine production in PLC␥2 Ϫ/Ϫ macrophages with those in WT macrophages in response to the hyphal (Fig. 4, A-D) or yeast (Fig. 4, E-H) forms of C. albicans. We found that the production of TNF␣, IL-10, IL-6, and IL-12p40 in PLC␥2-deficient BMDMs was significantly lower than that in WT BMDMs (Fig. 4). Because PLC␥1 is also expressed in macrophages and is activated in DCs following the stimulation by zymosan, the extract of yeast cell walls (10), we decided to examine the role of PLC␥1 in cytokine production in BMDMs in response to the stimulation with C. albicans. We found that PLC␥1 deficiency did not affect cytokine production in BMDMs following the stimulation with C. albicans (Fig. 5). Together, these data indicate that PLC␥2 but not PLC␥1 plays an essential role in antifungal immune response in macrophages.
PLC␥2 Mediates ROS Production in Macrophages in Response to C. albicans Hyphae-ROS production in phagocytes in response to C. albicans is important in antifungal host responses. In macrophages, the production of ROS in response to zymosan has been shown to be Syk-dependent (18). Aside from Syk, the signaling components that are crucial to NADPH oxidase assembly in response to C. albicans stimulation remain to be identified. Because PLC␥ enzymes were shown to link integrin-mediated adhesion to NADPH oxidase activation in neutrophils (31), we decided to examine whether PLC␥2 is involved in ROS production in response to C. albicans infection. The hyphal form of C. albicans induced robust ROS production in WT BMDMs but not in PLC␥2 Ϫ/Ϫ BMDMs (Fig.  6A), whereas phorbol myristate acetate could effectively induce ROS production in both WT and PLC␥2 Ϫ/Ϫ cells (Fig. 6B). In contrast to PLC␥2 Ϫ/Ϫ macrophages, WT and PLC␥1 Ϫ/Ϫ BMDMs showed no significant difference in ROS production following hyphae stimulation (Fig. 6C). These data indicate that PLC␥2 but not PLC␥1 is an essential signaling component for ROS production in response to C. albicans hyphae and that loss of PLC␥2, therefore, impairs the initial step of fungal killing in macrophages and increases host susceptibility to C. albicans infection.
Because activation of PLC␥2 induces diacylglycerol and inositol 1,4,5-trisphosphate, which activate PKC and increase the level of intracellular calcium, we next examined  NF-B activation induced by C. albicans hyphae and yeast is PLC␥2-dependent. A and B, WT and PLC␥2-deficient BMDMs were either untreated or treated with C. albicans hyphae (MOI ϭ 1) (A) or heat-inactivated yeast (MOI ϭ 5) (B) for 90 min. The nuclear extracts were prepared from these cells and then subjected to the electrophoretic mobility shift assay using 32 P-labeled NF-B or OCT-1 probe. C and D, WT and Myd88 Ϫ/Ϫ BMDMs were stimulated with C. albicans hyphae (MOI ϭ 1) (C) or heat-inactivated yeast (MOI ϭ 5) (D) for 90 min. The nuclear extracts were subjected to EMSA using 32 P-labeled Oct-1 or NF-B probe. E, WT, CARD9 Ϫ/Ϫ , and PLCg2 Ϫ/Ϫ BMDMs were stimulated with C. albicans hyphae for the indicated time points, and IKK␣/␤ phosphorylation was examined using anti-phospho-IKK␣ (Ser-176)/IKK␤ (Ser-180) antibody. F, WT and PLC␥2-deficient BMDMs were stimulated with C. albicans hyphae (MOI: 1) for the indicated time points, and the cell lysates were immunoprecipitated with CARD9 antibody-conjugated agarose. The immunoprecipitates were probed with BCL10 and CARD9 antibodies. IP, immunoprecipitation; IB, immunoblot.
how PLC␥2 is involved in the generation of ROS. Using the PKC inhibitor GF109203X and the calcium chelator Bapta-AM, we found that hyphae-induced ROS generation primarily depends on PKC activation, because treatment with PKC inhibitor significantly blocked ROS generation, whereas Bapta-AM only caused a modest decrease in ROS generation (supplemental Fig. S1).
PLC␥2 Controls Antifungal Immunity in Vivo-To evaluate the role of PLC␥2 in anti-fungal immunity in vivo, we sought to determine whether PLC␥2-deficient mice were highly susceptible to fungal infection. To avoid the influence of PLC␥2 deficiency in other cell types, we generated PLC␥2-deficient bone marrow chimera (PLC␥2-deficient chimera) by reconstituting ␥-irradiated wild-type mice with the bone marrow from PLC␥2-deficient mice and control mice with the bone marrow from WT mice. We then intravenously injected C. albicans into control or PLC␥2-deficient chimera mice. High dose (1 ϫ 10 6 ) injection of C. albicans resulted in the death of PLC␥2-deficient mice but not WT control mice within 24 h (data not shown). Given a low dose (2 ϫ 10 5 ) injection of C. albicans, PLC␥2deficient chimera mice were dead within 48 h, but WT mice survived for more than 5 days (Fig. 7A). Cytokine (IL-6) levels in the sera from PLC␥2-deficient mice were significantly decreased in response to C. albicans infection (Fig. 7B). To provide the quantitative assessment of C. albicans burdens, we sacrificed some of these mice 42 h after infection. The kidneys, lungs, livers, and spleens from these mice were collected. Some of these organs were homogenized, and the serial dilutions of the homogenized organs were plated on yeast extract peptone dextrose agar, and fungal colonies grown on these plates were counted and plotted (Fig. 7C). To visualize C. albicans in these organs, the tissue sections from some collected organs were stained with the periodic acid-Schiff. Compared with WT mice, PLC␥2-deficient mice had substantially higher levels of germinating hyphal C. albicans in the kidneys, lung, spleen, and liver (supplemental Fig. S2). Together, these results demonstrate that PLC␥2 plays an essential role in antifungal innate immune response in vivo.

DISCUSSION
Recent work has highlighted the role of C-type lectin receptors, Dectin-1 and Dectin-2, as pattern recognition receptors for fungal infections. So far, Syk and CARD9 are the few known signaling components downstream of Dectin-1 and Dectin-2 receptors, which initiate signal transduction cascades in response to fungal infection. In this study, we demonstrate that PLC␥2 but not PLC␥1 is an essential signaling component in the Dectin-2 signaling pathway and mediates NF-B and MAPK activation, leading to antifungal innate immune responses. Therefore, PLC␥2-deficient mice are highly susceptible to C. albicans infection.
Previous studies show that TLR signaling may collaborate with Dectin-1 signaling to induce the inflammatory response (29,30), in which they have found that cytokine expression induced by zymosan, the ␤-glucan component extracted from yeast cell wall, is dependent on TLR2 and MyD88 (29,30). These studies suggest that TLR signaling may be also involved in anti-fungal inflammation. However, in this study, we have found that the stimulation by both C. albicans hyphae and heat-inactivated yeast can effectively activate NF-B in MyD88-deficient cells but is defective in PLC␥2deficient cells, indicating that NF-B activation induced by both Dectin-1 and Dectin-2 receptors is TLR/MyD88-independent but PLC␥2-dependent. Therefore, our data indicate that previous results obtained by using zymosan are signifi-  DECEMBER 23, 2011 • VOLUME 286 • NUMBER 51

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cantly different from the results obtained by using the fungal organism itself.
In the classical NF-B pathway, the activation of IKK complex requires the signal-induced phosphorylation of IKK␣/␤ subunits, in addition to the K63-linked ubiquitination of the regulatory subunit IKK␥/NEMO for the degradation of the inhibitory IB␣ proteins and the subsequent translocation of NF-B into the nucleus (32,33). Recent work from our lab demonstrated that Syk and the adaptor protein CARD9 cooperate in activating the IKK complex with CARD9 mediating IKK ubiquitination, whereas Syk is necessary for the phosphorylation of IKK␣/␤ subunits (28). In the current study, we find that PLC␥2 Ϫ/Ϫ macrophages are defective in the activation of IKK␣/␤, whereas Card9 Ϫ/Ϫ macrophages are not. In addition, PLC␥2 deficiency did not alter the formation of the CARD9-BCL10 complex. These findings sug-gest that although PLC␥2 and CARD9 are both required for NF-B activation in response to C. albicans (34), they mediate their signaling in an independent manner. However, it remains to be determined which signaling components are required for linking CARD9 to the upstream signaling cascade.
The mechanism that couples ERK activation to upstream signaling in Candida-induced responses is not well understood. We find that ERK activation in response to hyphae is completely abrogated, whereas ERK activation in response to yeast is partially defective in PLC␥2-deficient cells. This result suggests that the ERK activation induced by hyphal form of C. albicans is through a PLC␥2-dependent pathway, whereas yeast form of C. abicans may stimulate both PLC␥2dependent and -independent pathways leading to ERK activation. Because PLC␥2 is downstream of Syk, our results are

PLC␥2 in Dectin-2-induced NF-B MAPK Activation
consistent with an earlier observation that zymosan-induced ERK activation is dependent on Syk in macrophages and DCs (34). However, a recent study using human macrophages suggests that zymosan-induced ERK activation is mediated through a PLC␥2-independent, but a Syk-CaMK-Pyk2-dependent pathway (35). These different results may be due to differences between using zymosan versus the fungal organism or to species-specific differences between human and mouse macrophages.
It is well established that the Grb2-SOS-Ras-Raf1 signaling cascade leads to a potent ERK activation. Upon receptor engagement, SOS, a Ras guanine nucleotide exchange factor, is recruited to the membrane by the adaptor protein Grb2 leading to Ras activation (36,37). Activated Ras can then activate the Raf-1 kinase, which will ultimately activate ERK1 and ERK2 kinases (38,39). Thus, it is possible that C. albicans yeast, but not hyphae, may also activate the Grb2/ SOS/Ras/Raf1 signaling cascade, which may explain the partial defect of ERK activation. We will test this hypothesis in our future studies.
In contrast to ERK activation, JNK activation is significantly reduced in PLC␥2-deficient cells following the stimulation by both yeast and hyphae, indicating that JNK is activated through the same PLC␥2-dependent pathway by the stimulation of yeast and hyphae. Because our preliminary studies found that calcium chelator Bapta-AM and PKC inhibitor could not block ELISA was used to measure the level of cytokines in these cultured media. The data are the means Ϯ S.D. of triplicate wells and are representative of three independent experiments. FIGURE 6. PLC␥2 is required for ROS production in response to C. albicans hyphae stimulation. A and B, ROS production in WT (black squares) or PLC␥2-deficient (white circles) BMDMs was measured by using a luminometer following the stimulation with C. albicans hyphae (A, MOI ϭ 2) or phorbol myristate acetate (B, 100 M). C, ROS production in MxCre/PLCg1 ϩ/ϩ (WT, black squares) and MxCre/PLCg1 fl/fl (PLC␥1 Ϫ/Ϫ , white circles) BMDMs was measured following the stimulation with hyphae (MOI ϭ 2). The data are the means Ϯ S.D. and are representative of at least three independent experiments. RU, relative units. DECEMBER 23, 2011 • VOLUME 286 • NUMBER 51 the JNK activation in response to fungal infection (data not shown), it suggests that JNK is activated through an unknown pathway that is independent of PKC and calcium. Future studies are needed to reveal this signaling cascade following fungal infection.

PLC␥2 in Dectin-2-induced NF-B MAPK Activation
The phagocytosis of ␤-glucans in macrophages can activate the production of ROS (4). Although Syk is not involved in controlling phagocytosis, it is required for ROS production following fungal infection (18). However, CARD9, another known component in the C-type lectin pathway, is only partially required for fungal infection-induced ROS production (27). Therefore, the mechanism by which fungal infection triggers ROS production remains largely unknown. In the current study, we provide evidence that PLC␥2 but not PLC␥1 is necessary for ROS production in macrophages in response to stimulation with C. albicans. Although it remains to be determined how PLC␥2 links to ROS production, our data suggest that PLC␥2-dependent PKC activation is a critical link, because PKC has been shown to regulate ROS production, and inhibition of PKC significantly blocks ROS production following fungal infection (supplemental Fig. S1). Future studies will determine which isoform(s) of PKC are involved in fungal infection-induced ROS production. In addition, ROS production in DCs has been shown to be required for C. albicans-induced inflammasome activation (40). Therefore, our findings suggest a possible role for PLC␥2 in Nlrp3 inflammasome activation. This hypothesis will be tested in the future studies. In conclusion, we dem-onstrate that PLC␥2 is a critical component of Dectin-2 signaling and mediates anti-fungal innate immune responses.