FcεRI γ-Chain Negatively Modulates Dectin-1 Responses in Dendritic Cells

The inhibitory effect of immunoreceptor tyrosine-based activation motif (ITAM)-containing adapters DAP12 and FcεRI γ-chain (FcRγ) has been found in many immune functions. Herein, we have further explored the role of these adapters in C-type lectin receptors response. We identified that FcRγ, but not DAP12, could negatively regulate the Dectin-1 responses in dendritic cells (DCs). Loss of FcRγ or both DAP12 and FcRγ enhanced the maturation and cytokine production in DCs upon Dectin-1 activation compared to normal cells, whereas DCs lacking only DAP12 showed little changes. In addition, increments of T cell activation and T helper 17 polarization induced by FcRγ-deficient DCs were observed both in vitro and in vivo. Examining the Dectin-1 signaling, we revealed that the activations of several signaling molecules were augmented in FcRγ-deficient DCs stimulated with Dectin-1 ligands. Furthermore, we demonstrated that the association of phosphatases SHP-1 and PTEN with FcRγ may contribute to the negative regulation of FcRγ in Dectin-1 activation in DCs. These results extend the inhibitory effect of ITAM-containing adapters to Dectin-1 response in immune functions, even though Dectin-1 contains an ITAM-like intracellular domain. According to the role of Dectin-1 in responding to microbes and tumor cells, our finding may have applications in the development of vaccine and cancer therapy.

The inhibitory effect of immunoreceptor tyrosine-based activation motif (ITAM)containing adapters DAP12 and FcεRI γ-chain (FcRγ) has been found in many immune functions. Herein, we have further explored the role of these adapters in C-type lectin receptors response. We identified that FcRγ, but not DAP12, could negatively regulate the Dectin-1 responses in dendritic cells (DCs). Loss of FcRγ or both DAP12 and FcRγ enhanced the maturation and cytokine production in DCs upon Dectin-1 activation compared to normal cells, whereas DCs lacking only DAP12 showed little changes. In addition, increments of T cell activation and T helper 17 polarization induced by FcRγdeficient DCs were observed both in vitro and in vivo. Examining the Dectin-1 signaling, we revealed that the activations of several signaling molecules were augmented in FcRγ-deficient DCs stimulated with Dectin-1 ligands. Furthermore, we demonstrated that the association of phosphatases SHP-1 and PTEN with FcRγ may contribute to the negative regulation of FcRγ in Dectin-1 activation in DCs. These results extend the inhibitory effect of ITAM-containing adapters to Dectin-1 response in immune functions, even though Dectin-1 contains an ITAM-like intracellular domain. According to the role of Dectin-1 in responding to microbes and tumor cells, our finding may have applications in the development of vaccine and cancer therapy.
Keywords: Fcεri γ-chain, DaP12, dendritic cell, Dectin-1, immunoreceptor tyrosine-based activation motif, shP-1, PTen inTrODUcTiOn Dendritic cells (DCs) monitor danger signals in surrounding environment, capture and process antigens, migrate to secondary lymphoid organs, and activate T cells to initiate immune responses (1). DCs are heterogenous but all subsets have intrinsic and cooperative immunoregulatory functions (2). They can direct immune responses either toward to tolerance or inflammatory reactions, which include cellular and humoral immune responses (3,4). DCs become mature when they encounter pathogen-associated molecules or inflammatory mediators, a key step to determine what function they exert (5). Thus, DCs are provided with diverse receptors that sense microbial components and trigger host responses to invading pathogens. These receptors, known as pattern recognition receptors (PRRs), include toll-like receptors (TLRs), C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and RIG-like receptors (RLRs) (6). All PRRs activate specific signaling pathways that lead to expression of genes and tailor immune responses to particular microbes.
CLRs recognize carbohydrate structures present on the pathogens (7). Dectin-1 is a group V CLR possessing a single extracellular carbohydrate-recognition domain (CRD) and a cytoplasmic tail containing an immunoreceptor tyrosine-based activation motif (ITAM)-like domain (8). Dectin-1 is expressed on myeloid cells, including macrophages, DCs, and neutrophils, and is dedicated to the recognition of β-1,3-glucans, which make up to 50% of the fungal cell wall, and is the major receptor for these carbohydrates on leukocytes (9). Upon binding of β-glucan ligands to Dectin-1, the cytoplasmic ITAM-like motif (hemITAM) is able to mediate intracellular signaling and induces a variety of cellular responses. This results in recruitment of spleen tyrosine kinase (Syk) and caspase recruitment domain protein 9 (CARD9), and then induces DC maturation and guides T helper 17 (Th17) differentiation (10). In addition, Src, AKT, PLCγ, and MAPKs are also involved in Dectin-1 signaling pathway. Dectin-1 can recognize a wide variety of fungal species and Dectin-1-deficient mice are susceptible to Candida albicans infection, indicating a role for Dectin-1 in antifungal immunity (11). Furthermore, Chiba et al. reported that the recognition of tumor cells by Dectin-1 critically contributes to the antitumor immune responses, implying the applicability of Dectin-1 in cancer therapy (12).
ITAM is critical in signal transduction not only for activation but also inhibition in hematopoietic cells (13,14). Besides, the ITAM-associated receptors cross talk with other signaling pathways and either augment or dampen these signals (15,16). Two ITAM-containing adapters, DAP12 (TYROBP) and FcεRI γ-chain (FcRγ), have been identified in myeloid cells and NK cells. DAP12 is a disulfide-bonded homodimer and associates with various activating receptors through its negatively charged aspartic residue present in the transmembrane domain and non-covalently binds to a positively charged amino acid present in the transmembrane of the activating receptor (17). FcRγ is structurally similar to DAP12 but mainly shared by all activating multi-chain Fc receptors (FcRs). FcRs contain a ligand-binding subunit (FcRα) with different extracellular domains, which are the core structure of receptors for IgA (FcαR), IgG (FcγR and FcRn), and IgE (FcεR), and one or two ITAM-containing signaling subunits (FcRγ and FcRβ) shared by multisubunit FcRs (18). Recently, receptors associated with these adapters have been implicated in inhibition of cellular activation, and several models have been proposed (19,20). In a previous study, we have identified the negative regulation of DAP12 in TLR responses in macrophages (21). Following this finding, we reported that both DAP12 and FcRγ are required for the inhibitory effect on TLR responses in DCs (22). Since DCs are equipped with many types of PRRs, we further examined the regulatory roles of DAP12 and FcRγ in other PRR responses.
Dectin-1 contains a hemITAM for signaling, so the ITAMcontaining adapters DAP12 and FcRγ are likely not required for Dectin-1 activation; however, no prior studies have addressed the effect of DAP12 and FcRγ on Dectin-1 response. Here, we explore the involvement of these adapters in Dectin-1 activation by using gene-deficient mice and surprisingly found that FcRγ negatively modulates Dectin-1 responses in DCs.

Dc Migration
To analyze the migration of DCs, we used chemotaxis assays in transwell chambers as described previously (24). Briefly, BMDCs were treated with curdlan or dZym (10 µg/mL) on day 6, harvested after 16 h, and then seeded in the upper wells of Transwell chambers (pore size, 5 mm; Costar Corning). Culture media without or with CCL21 (200 ng/mL, 250-13, Peprotech) were loaded in the lower wells. After 3 h, the migrated cells in the lower well were collected, stained with anti-CD11c Ab, and then analyzed by flow cytometry.
real-time Pcr for ccr7 expression BM DCs were collected at day 6, treated with dZym (10 µg/mL), and then harvested at the indicated time points. The total RNAs were extracted with TRIzol reagent (Invitrogen) and converted to cDNA by Revert Aid First Strand cDNA Synthesis Kits (Thermo Fisher Scientific) according to the manufacturer's instructions. SYBR Green real-time PCR was performed with Luminaris Color HiGreen qPCR master mix (Thermo Fisher Scientific) by PikoReal System (Thermo Fisher Scientific). The primers for CCR7 (forward: 5′-AGA GGC TCA AGA CCA TGA CGG A-3′; reverse: 5′-TCC AGG ACT TGG CTT CGC TGT A-3′) and GAPDH (forward: 5′-GAC AAC TTT GGC ATT GTG G-3′; reverse: 5′-ATG CAG GGA TGA TGT TCT G-3′) were used. All mRNA levels of CCR7 were normalized with GAPDH and the fold-change represented the CCR7 expression of dZym-treated BMDCs compared to that of untreated cells.

antigen-specific T cell responses
To determine the DC-induced antigen-specific T cell responses, we performed the DC and OT-II T cell coculture in vitro (24). Briefly, BMDCs were treated with OVA323-339 peptide (10 µg/mL, O1641, Sigma-Aldrich) for 3 h, and then CD4 + T cells, which were isolated from OT-II mice by negative selection kits (480005, Biolegend), were added into BMDC cultures at indicated ratio. For proliferation assay, cells were pulsed with [ 3 H]thymidine and harvested after 72 h. The proliferation of OT-II T cells was determined by the radioactivity of [ 3 H] thymidine incorporation. For detection of IL-17 production, supernatants were collected from DC/OT-II T cell cultures after 4 days. The amounts of IL-17 were measured by ELISA. For recall assay in vivo, C57BL/6 and FcRγ-deficient mice were immunized with OVA (50 µg, A2512, Sigma-Aldrich) and incomplete Freund's adjuvant (AR002, Sigma-Aldrich) mixed with or without curdlan/dZym (200 µg) via footpads. After 7 days, inguinal and popliteal lymph node (LN) cells were isolated and the numbers of CD3 + T lymphocyte were determined by counting and flow cytometry. For recall response measurement ex vivo, total LN cells were seeded in 96-well plate (5 × 10 5 /well) and incubated with OVA at indicated concentrations for 3 days. [ 3 H] thymidine was added 12 h before cell harvest. The proliferation of OVA-specific T cells was measured by [ 3 H] thymidine incorporation.

statistics
Significances of different Dectin-1 responses between WT and various DAP12-and FcRγ-deficient DCs were determined using a Student's t-test with two-sample equal variance with a two-tailed distribution.  (22). This finding suggests that these ITAMcontaining adapters participate in regulating innate immunity of DCs. A notable feature is that TLRs do not need an ITAM for their signaling. In contrast, a typical CLR, Dectin-1, contains a cytoplasmic hemITAM for initiating antifungus activities. Thus, it was interesting to know whether DAP12 and FcRγ have any role in the Dectin-1 responses. To study this hypothesis, we generated BMDCs from WT, Tyrobp −/− , Fcer1g −/− , and Tyrobp −/− Fce r1g −/− mice and treated these DCs with a Dectin-1-specific ligand curdlan. Because DC maturation is the key step for DC function, we firstly examined the maturation status of DCs following curdlan stimulation. As shown in Figure 1, the expression levels of MHC class II, CD40, CD80, and CD86 in     Figure 3A).

Promoted cytokine Production by
In addition, we also affirmed that FcRγ deficiency did not affect the DC phenotype in vitro as CD11b and CD11c expressions were similar between WT and FcRγ-deficient BMDCs ( Figure 3B). Next, we used another Dectin-1 ligand dZym to check whether the enhanced phenotypes of Fcer1g −/− DCs are ligandspecific. Consistent with the trend seen with curdlan stimulation, maturation ( Figure 3C) and cytokine production ( Figure 3D) were increased to a greater extent in the Fcer1g −/− DCs in comparison with WT DCs. We performed LPS stimulation as another quality control of DCs and also displayed the enhancing effects of FcRγ deficiency on LPS-induced DC maturation ( Figure 3E) and IL-10 production ( Figure 3F). Collectively, these results imply an intracellular modulation of FcRγ on the quality of Dectin-1 activation in DCs.
We have confirmed the negative regulation of FcRγ deficiency in DCs. It is interesting to know whether macrophages have the same phenotype. After stimulation, the Dectin-1- (Figure 4A) and LPS- (Figure 4B) induced maturation has no significant increase in FcRγ-deficient macrophages compared to WT cells, demonstrating that FcRγ may be not involved in modulating Dectin-1 responses in macrophages. loss of Fcrγ had no effect on the Migration of Dectin-1-activated Dcs Activated DCs upregulate CCR7 expression and migrate to CCL19/21-expressing draining LNs to initiate adaptive immune responses. Therefore, we studied the effect of FcRγ deficiency on the ability of DC migration after Dectin-1 activation by chemotaxis assays using transwell chambers. Unexpectedly, the CCL21-guiding migration of Fcer1g −/− DCs was not different from that of WT cells (Figure 5A). Because CCR7 is the receptor of CCL21 on DCs, we measured the CCR7 expression of DCs after dZym stimulation. Consistently, the mRNA levels of CCR7 in Fcer1g −/− DCs were similar to that in normal cells (Figure 5B). Thus, the FcRγ deficiency does not affect the CCR7 expression and migration of Dectin-1-activated DCs.
antigen-specific T cell responses Were increased in Fcer1g −/− Mice Following migration, the mature DCs induce antigen-specific T cell activation and proliferation in draining LNs. Thus, we determined the responses of antigen-specific T cell population in vitro and ex vivo. First, WT and Fcer1g −/− BMDC were cultured with OT-II T cells at different ratio and the proliferation of OT-II T cells were detected by [ 3 H]thymidine incorporation after 3 days. As shown in Figure 6A, Fcer1g −/− BMDC could significantly induce more T cell proliferation than WT BMDC. In addition, higher production of IL-17 was detected in Fcer1g −/− BMDC/T cell cultures (Figure 6B). To further confirm the phenotype ex vivo, we did antigen-specific recall assay on WT and Fcer1g −/− mice to compare T cell proliferation. WT and Fcer1g −/− mice were immunized with OVA mixed with incomplete Freund's Adjuvant plus curdlan or dZym via foot pads. After 7 days, draining LNs were collected and the number of T cell was calculated. Significantly, the size (data not shown) and number of T cells in draining LNs of Fcer1g −/− mice were greater than that in WT mice after immunization ( Figure 6C). Furthermore, we measured OVA-specific T cell proliferation by adding OVA into the cultures of isolated LN cells for 72 h and counting incorporated [ 3 H] thymidine. As shown in Figure 6D, the proliferation of OVA-specific T cells in Fcer1g −/− LN cells was more intense than that in WT cells. The elevated activation of OVA-specific T cells in immunized Fcer1g −/− mice could be due to the higher expression levels of MHC class II and costimulators in Dectin-1-activated Fcer1g −/− DCs (Figures 1 and 3C), and supported the conclusion that loss of FcRγ causes a relative promotion in DC activation by Dectin-1 stimulation.  (Figure 3A). To explore the mechanism, we examined the signaling events of Dectin-1 in DCs after Dectin-1 activation. According to previous research, Dectin-1 ligands treatment can induce both Syk-dependent and Syk-independent signaling activation. Thus, we compared the activation status of several molecules in both Dectin-1-mediated signaling between WT and Fcer1g −/− DCs by Western blot. The phosphorylation levels of Src,   AKT, PLCγ, ERK, p38MAPK, and c-Raf were increased in Fcer1g −/− DCs compared to WT cells ( Figure 7A). Moreover, IκB degradations were accelerated in Fcer1g −/− DCs compared to WT cells, represented the enhancement of NF-κB activation ( Figure 7B). The blotting data strongly suggest that FcRγ suppresses the Dectin-1 signaling and then reduces the Dectin-1 responses in DCs. However, the phosphorylation levels of Syk were not significantly different between WT and Fcer1g −/− DCs ( Figure 7A).

recruitment of Phosphatases by Fcrγ after Dectin-1 activation
The ITAM domain in the FcRγ has been shown to bind Src homology 2 (SH2)-containing protein tyrosine phosphatases (PTPs) and inhibit cell activation (25,26). Thus, we examined whether any SH2-containing PTP was recruited to FcRγ after Dectin-1 activation in DCs. FcRγ was immunoprecipitated after Dectin-1 ligand treatment, and then the possible PTPs, including SH2 domain-containing phosphatase-1 (SHP-1), SHP-2, SH2 domain-containing inositol 5′-phosphatase-1 (SHIP-1), were determined by Western blot. As shown in Figure 8A, SHP-1 and SHP-2 were present in the FcRγ immunoprecipitate. However, SHIP-1 was absent. Because the enhanced AKT activation was observed in Fcer1g −/− DCs after Dectin-1 activation (Figure 7A), we were guided to examine the phosphoinositide phosphatase PTEN (phosphatase and tensin homolog), which is a suppressor for AKT activation (27), and surprisingly found the presence of PTEN in FcRγ immunoprecipitate ( Figure 8A). The results illustrate that SHP-1, SHP-2, and PTEN are recruited to FcRγ after Dectin-1 activation and probably involved in the negative modulation of FcRγ to Dectin-1 responses in DCs.  In order to support the potential functional implication of these phosphatases in the suppressive effects, we treated WT DCs with chemical inhibitors to examine whether the inhibitory effect could be reverted. It has been reported that SHP-2 can positively regulate Dectin-1-induced activation in response to fungal infection (28). Thus, we focused on SHP-1 and PTEN. As shown in Figure 8B, when we used specific inhibitors SS (for SHP-1) and SF1670 (for PTEN) for blocking phosphatase activities, both SS-and SF1670-treated BMDCs expressed higher level of CD80, CD86 and MHC-II after dZym treatment. These results confirmed that SHP-1 and PTEN could be involved in the negative regulation of FcRγ in Dectin-1 responses.

DiscUssiOn
The inhibitory effects of ITAM-containing adapters DAP12 and FcRγ have been elucidated in myeloid cells (13). In a previous study, we identified that both DAP12 and FcRγ are required for inhibition of TLR responses in DCs (22). Because ITAM is not involved in TLR signaling, we then examined the effect of DAP12 and FcRγ on Dectin-1, which contains a hemITAM, in this study. We surprisingly found that Fcer1g −/− and Tyrobp − /− Fcer1g −/− DCs, but not Tyrobp −/− DCs, showed similar hyperresponsive phenotypes, suggesting that only FcRγ plays a role in negatively regulating Dectin-1 responses in DCs. Consistently, we have observed that FcRγ most likely plays a more important role than DAP12 in some TLR activities in DCs (22). Although some activation markers already increased basally in the absence of FcRγ (Figures 1 and 3C,D), probably due to another ligand with another signaling pathway that is present in those culture conditions, the enhancement of Dectin-1 responses was really significant in Fcer1g −/− DCs both in vitro and in vivo. In contrast, the Dectin-1 responses of WT and Fcer1g −/− BM-derived macrophages were not different (Figure 4). Although the requirement of DAP12 and FcRγ for negative regulation is probably dependent on the types of cell or receptor, it seems that FcRγ is more influential than DAP12 in DCs, comparing to macrophages in which DAP12 is more important than FcRγ (21). A possibility is that the profiles of DAP12-and FcRγ-associated receptors recruited by TLR and Dectin-1 ligands are different between DC and macrophage. Further studies are required to identify the "coreceptors" in these myeloid cells.
The ITAM has been characterized in many immune receptors for initiating signal transduction. However, increasing evidences establish its negative role in immune responses. Our group first reported enhanced TLR responses in DAP12-deficient macrophages (21), and both DAP12 and FcRγ are required for the enhancement of TLR responses in DCs (22). Subsequently, the DAP12-associated triggering receptor expressed on myeloid cell-2 (TREM-2) was also reported to negatively regulate TLR responses in DCs (29). In addition, ITAM-associated receptors inhibit type I IFN (IFN-α/β) signaling in primary human macrophages (30). Suppression by ITAMs has also been found in lymphocytes. CD79a and CD79b ITAMs mediate an inhibitory signaling cascade required for B cell anergy (31). In vivo, Gmyrek et al. show that mice lacking both DAP12 and FcRγ possess a greatly enhanced monocyte-derived DC differentiation, IL-12 production, and CD8 T cell responses (32). A well-characterized function of ITAM is the inhibitory effect of the IgA receptor FcαRI. Monomeric IgA binds to FcαRI and results in partial phosphorylation of its FcRγ, which then attenuates immune activation (20). Our study identifies a new target modulated by ITAM in immune responses.
An interesting question is how the FcRγ affects the "nonadapter-associated receptor" Dectin-1 in DCs. Bezbradica and Medzhitov have suggested that many immunoreceptors, all of which use a conserved ITAM-module for their signaling, can couple with members of additional classes of membrane receptors to deliver unique signal(s) to the cell (16). In studies of FcRγ, many reports have documented the inhibitory effects of FcRγ-associated FcRs. Monovalent targeting of FcαRI triggers inhibitory ITAM (ITAMi) signaling through the associated FcR γ chain (33). In addition, FcγRI engaged with immune complexes reduces the production of IFN-β and TLR4 signaling and increases the secretion of IL-10 (34). Another FcR, FcγRIIA, can ameliorate inflammation by engagement with anti-FcγRII F(ab′)2 fragments or intravenous hIgG (IVIg) treatment (35). Recently, the macrophage inducible C-type lectin (Mincle) has been found to form a complex with macrophage C-type lectin and FcRγ (36) and is a fungal receptor that can suppress Dectin-1-induced antifungal immunity (37) and Fonsecaea pedrosoi-induced Th17-cell differentiation in mice (38). Mincle can also be shifted to an ITAMi configuration by Leishmania and then impairs DC activation (39). Thus, we proposed a model that an FcRγ-associated receptor might couple with Dectin-1 after curdlan or dZym treatment, and then SHP-1 and PTEN were recruited by FcRγ to modulate Dectin-1 signaling. However, we did not know what the Dectin-1-coupled FcRγ-associated receptor is. Future studies would explore whether Mincle, a possible candidate, could be recruited to Dectin-1 after curdlan/dZym treatment in DCs in the future. We have confirmed that the inhibitory effect of FcRγ is due to modulation of Dectin-1 signaling but not reduction of Dectin-1 expression. The activities of several molecules in Dectin-1 signaling were enhanced in FcRγ-deficient DCs, including Src, AKT, PLCγ, ERK, p38MAPK, c-Raf, and NF-κB (Figure 7). Interestingly, the FcRγ deficiency did not significantly affect the Syk activation ( Figure 7A). It has been shown that Syk is required for Mincle-FcRγ-dependent inhibitory axis during Leishmania infection (39), implying that Syk could also be activated by FcRγmediated suppression after Dectin-1 stimulation. Although Syk activation was not increased in FcRγ-deficient DCs, probably due to the saturated activity after curdlan/dZym treatment, the entire activation might only trigger Dectin-1 signaling without shared by FcRγ-mediated negative signaling and resulted in the Six-day-cultured BMDCs derived from WT and Fcer1g −/− mice were collected and starved in serum-free RPMI. After 3 h, cells were treated with dZym and lysed at indicated time points. The proteins were separated by SDS-PAGE, transferred to PVDF membranes, and then analyzed by Western blot. The detection of phosphorylated and total proteins of Src, Syk, AKT, PLCγ, ERK, p38MAPK, and c-Raf (a), and total proteins of IκBα and GAPDH (B), were shown. Quantification was determined by densitometry using ImageJ software and the number represented the fold of each (phosphoprotein/total protein) value normalized to the (phosphoprotein/total protein) value of untreated WT control (WT 0 min). All data shown were representative from three to five independent experiments. defense against C. albicans infection (40). However, we did not see the effect of FcRγ deficiency on IRF5 activation and IFN-β production after Dectin-1 activation (data not shown). What is the mechanism for suppressing Dectin-1 activation by FcRγ in DCs? It has been shown that low avidity ligation of FcαRI induces the translocation of FcαRI associated with SHP-1 to membrane lipid rafts. Subsequent ligation of activating receptors results in their colocalization with FcαRI and SHP-1, and then SHP-1 exerts cell inhibition of multiple types of activation signals (25). A recent study also described that Leishmania triggers a Mincle-dependent inhibitory axis characterized by SHP-1 coupling to the FcRγ chain (39). We did observe the association of SHP-1 with FcRγ after Dectin-1 activation (Figure 8), although the indirect association could also be possible. In addition, we detected the binding of SHP-2 to FcRγ. However, SHP-2 has been reported to positively regulate Dectin-1-induced activation in response to fungal infection (28). Thus, the recruitment of SHP-2 may be irrelevant to the negative modulation of FcRγ here. In another study, the adaptor protein downstream of kinase 3 (DOK3) has been found to physically associate with the ITAM of DAP12 through its phosphotyrosine-binding domain and mediate the mitigation of LPS signaling in macrophages (41). It remains to be determined whether DOK3 is associated with FcRγ in Dectin-1 responses in DCs.
In this study, we reported for the first time that PTEN was recruited to FcRγ after Dectin-1 activation (Figure 8). Although we could not exclude the indirect binding of PTEN to FcRγ, PTEN has been identified as a negative regulator of FcR signaling, an ITAM-based signaling event. For examples, PTEN abrogates FcγR-mediated phagocytosis (42,43), and the suppression of PTEN is critical for mast cell homeostasis and FcεRI-responsiveness (44). Notably, prostaglandin E2 (PGE2) enhances SHP-1 activity, resulting in increased PTEN activity. This mechanism contributes to the ability of PGE2 to abolish innate immune signaling in primary macrophages (45) and probably provides an explanation for our results (Figure 8). Serezani et al. report that PTEN is a key regulator in PGE2-mediated inhibition of phagocytosis of C. albicans (46), in agreement with our finding that FcRγ-associated PTEN might contribute to the negative regulation of antifungal responses. In addition, PTEN is responsible for the impairment of DC functions from elderly humans (47) and attenuates engulfment of apoptotic cells and apoptotic cell-induced anti-inflammatory response (48). It would be interesting to know whether PTEN-associated FcRγ also has such functions in DCs.
In the research of tumor immunology, Dectin-1 expressed on DCs and macrophages is critical to NK-mediated killing of tumor cells (12). Elucidating the molecular mechanism of Dectin-1induced signaling in immune cells is essential for the design of new therapeutic strategies against cancer (49). Recently, soluble β-glucan has been reported to induce tumor regression in synergy with TLR9 agonist via DC-mediated immunity (50). In addition, curdlan has been reported to activate DCs through Dectin-1 and TLR4 signaling and the combination of curdlan and DCs efficiently inhibit tumor growth in mice (51). Our findings about the enhancement enhancement of Dectin-1 downstream signaling. In addition to these signal molecules, the transcription factor IRF5-mediated Dectin-1-induced IFN-β production by renal DCs is crucial for In summary, we show here that FcRγ has inhibitory effects on Dectin-1 responses in DCs. Dectin-1 signaling is a key regulator of DC function in antifungal immune responses. In addition to antimicrobial host defense, Dectin-1 also plays roles in homeostasis, autoimmunity, allergy, and the recognition and response to dead and cancer cells (7). Our finding suggests that fungi and cancer cells could probably use such FcRγ-mediated negative regulation to escape the Dectin-1-induced immune responses. Thus, FcRγ could be an ideal target for strengthening the function of Dectin-1-activated DCs. Interestingly, it has been shown to improve the potency of a DC-based tumor vaccine with a small interfering RNA (siRNA) targeting PTEN (53). Therefore, removing the FcRγ-mediated inhibition of Dectin-1 signaling by siRNA, or by CRISPR technology (54), might promote immunity and potentially could be applied to vaccine development and cancer therapy.

acKnOWleDgMenTs
We thank Professor Yu-sun Chang, Mr. Chun-Nan OuYang (Chang Gung University, Taoyuan City, Taiwan), Professor Betty Wu-Hsieh (National Taiwan University, Taipei, Taiwan), and Professor Ann Chen (National Defense Medical Center, Taipei, Taiwan) for technical helps. We are also grateful to Professor Clifford Lowell (UCSF, CA, USA) for providing materials.

FUnDing
This work was supported by MOST of Taiwan under Grant NSC1012320B002023MY3 and part of Grant MOST1042320B 002050.