Targeting CBLB as a potential therapeutic approach for disseminated candidiasis.

Disseminated candidiasis has become one of the leading causes of hospital-acquired blood stream infections with high mobility and mortality. However, the molecular basis of host defense against disseminated candidiasis remains elusive, and treatment options are limited. Here we report that the E3 ubiquitin ligase CBLB directs polyubiquitination of dectin-1 and dectin-2, two key pattern-recognition receptors for sensing Candida albicans, and their downstream kinase SYK, thus inhibiting dectin-1- and dectin-2-mediated innate immune responses. CBLB deficiency or inactivation protects mice from systemic infection with a lethal dose of C. albicans, and deficiency of dectin-1, dectin-2, or both in Cblb(-/-) mice abrogates this protection. Notably, silencing the Cblb gene in vivo protects mice from lethal systemic C. albicans infection. Our data reveal that CBLB is crucial for homeostatic control of innate immune responses mediated by dectin-1 and dectin-2. Our data also indicate that CBLB represents a potential therapeutic target for protection from disseminated candidiasis.

Introduction

Candida albicans (C. albicans) infection is the most common cause of fungal infections in humans and has become one of the leading causes of hospital-acquired blood stream infections. Despite the availability of several anti-fungal drugs, invasive candidiasis still has a high mortality rate ranging from 45 to 75% 1. The high morbidity and mortality associated with disseminated candidiasis are mainly due to the lack of early and accurate diagnostic tools, limited anti-fungal drugs, and emergence of drug resistance. These factors highlight the need to further understand host-pathogen interactions and the mechanisms of immune resistance to fungal spread, and to develop immune-based strategies to combat candidemia.

The fungi-responsive C-type lectin receptors (CLRs) play a central role in the detection of Candida during bloodstream infection. In normal hosts, C. albicans is controlled by activation of innate immune cells via cell surface pattern recognition receptors (PRRs) such as Toll-like receptor 2 (TLR2) and CLRs that detect the infecting fungus. The CLRs dectin-1 and -2 recognize C. albicans yeast cells and hyphae by binding to surface β-glucans and α-mannans on the two fungal forms, respectively 2–4. Recognition of these molecules results in the release of inflammatory cytokines from innate immune cells, which is critical for anti-fungal immunity 5. However, the regulation of dectin-mediated signaling pathways, including SYK, that control the pro-inflammatory response to fungal infection, is completely unknown.

Casitas B lymphoma-b (CBLB), a member of the RING finger type E3 ubiquitin ligases that directs the ubiquitination of an array of signaling proteins 6. We and others have shown a crucial role for CBLB in T cell activation, tolerance induction, and TH2/9 cell differentiation 7–14, but its role in innate immune responses is unclear. In this study, we report that CBLB functions as a negative regulator of fungal recognition during systemic C. albicans infection by targeting dectin-1, -2, and SYK for K48-linked polyubiquitination. Negative regulation by CBLB of dectin-1- and -2-mediated signaling is crucial for restraining the magnitude of innate immune responses against C. albicans infection, but leads to suboptimal protection of the host. Systemic in vivo delivery of Cblb siRNA protects C57BL/6 mice from systemic C.albicans infection. Therefore, our data suggest that CBLB is a potential drug target for systemic candidiasis.

Results

CBLB inhibits signaling via Dectin receptors

To determine the role of CBLB in innate immune responses we stimulated WT and Cblb−/− bone marrow-derived macrophages (BMDMs) and BM-derived dendritic cells (BMDCs) with TLR 1-9 ligands or zymosan (a ligand for TLR2 and dectin-1). We found that whereas TLR ligand-induced production of TNF-α and IL-6 was comparable between WT and Cblb−/− BMDMs and BMDCs, zymosan-induced TNF-α and IL-6 production was significantly higher in Cblb−/− than WT BMDMs and BMDCs (Supplementary Fig. 1a, b). Given that zymosan activates both TLR2 and dectin-1 15, this result suggests that CBLB could regulate the dectin-1 signaling pathway. To directly test this we stimulated WT and Cblb−/− BMDMs and BMDCs with curdlan, a purified β-glucan which specifically activates dectin-1 16. Curdlan stimulation induced a significantly higher level of TNF-α and IL-6 in Cblb−/− than WT BMDMs and BMDCs (Supplementary Fig. 1a,b).

To confirm this observation, and to determine whether CBLB regulates other Dectin family members, we infected BMDMs, BMDCs and BM neutrophils from WT and Cblb−/− mice with a C. albicans yeast-only mutant (cap1; hereafter referred to as yeast), in which the adenylate cyclase-associated protein-1 gene was disrupted, causing the failure of yeast-hypha transition due to lack of cAMP 17. Dectin-1 and dectin-2 recognize the yeast and hyphal forms of C. albicans, respectively, by binding to the surface β-glucans (dectin-1) and α-mannans (dectin-2) of the two fungal forms 2–4. As shown in Fig. 1a and Supplementary Fig. 2a, CBLB deficiency resulted in increased production of TNF-α and IL-6 by BMDMs and BMDCs in response to signaling via both the yeast and hyphal forms of C. albicans infection. In contrast, Cblb−/− neutrophils produced comparable amounts of TNF-α and IL-6 compared to WT neutrophils, except for the 3 h time point after infection (Supplementary Fig. 2b), suggesting that CBLB may have a limited role in affecting the inflammatory response of neutrophils against C. albicans infection. Cblb−/− BMDMs also produced more TNF-α and IL-6 than WT BMDMs infected with A. fumigatus conidia (Fig. 1b), a prevalent fungus that causes potentially lethal infections in immunosuppressed patients 18. This finding is notable since dectin-1 is a major PRR recognizing A. fumigatus 19–21. Therefore, CBLB has the potential to regulate the dectin family of CLRs in response to some fungal pathogens. Since several studies indicate that either the NLRP3 inflammasome or a non-canonical, caspase-8-mediated inflammasome participates in host defense against C. albicans infection 22, 23, we measured IL-1β production by WT and Cblb−/− BMDMs upon C. albicans yeast and hyphal infection. Both WT and Cblb−/− BMDMs produced comparable levels of IL-1β (Fig. 1a), suggesting that CBLB does not regulate the inflammasome activation mediated by dectin-1 or -2.

Figure 1

CBLB inhibits pro-inflammatory cytokine production by macrophages upon infection with C. albicans yeast or hyphae and A. fumigatus conidia. (a) ELISA of TNF-α, IL-6, and IL-1β production in the supernatants collected from BMDMs of WT and Cblb−/− mice infected with C. albicans yeast cap1 mutant (thereafter yeast) and hyphal forms (WT strain SC5314) (MOI: 1:1) for 1 and 3 h. For preparation of hyphae, washed yeast cells were counted, re-suspended in RPMI-1640 medium, grown in 12-well plates at 37 °C for 3 h, and washed three times with PBS. (b) ELISA of TNF-α and IL-6 production in the supernatants collected from BMDMs of WT and Cblb−/− mice infected with swollen A. Fumigatus conidia (AF293) (MOI = 1:1) for 2 and 4 h. (c) ELISA of IL-1RA production in the supernatants collected from BMDMs of WT and Cblb−/− mice infected with C. albicans yeast and hyphal forms. For all ELISA experiments data are representative of three independent experiments (biological replicates). Error bars are mean ± s.d. *P < 0.05, **P < 0.01; unpaired two-tailed Student’s t test. n = 3 per group, each with three repeated wells.

A recent report showed that β-glucan of C. albicans induces a strong IL-1RA response in human peripheral blood mononuclear cells (PBMC), which is independent of dectin-1 and CR3 24. To test whether CBLB affects the release of anti-inflammatory stimuli such as IL-1RA, we measured the production of IL-1RA in BMDMs of WT and Cblb mice upon infection with live C. albicans yeast and hyphae. Our data showed that there was no significant difference in IL-1RA release between WT and Cblb BMDMs infected with both forms of C. albicans (Fig. 1c). These data suggest that CBLB does not modulate the release of IL-1RA.

To determine whether CBLB has a similar effect on human macrophages upon C. albicans infection, human monocyte-derived macrophages (MDMs) were generated 25, 26, and transfected with Cblb siRNA or scrambled siRNA. Consistent with the mouse results, we found that silencing Cblb in MDMs resulted in significantly increased production of TNF-α and IL-6 upon infection with C. albicans yeast and hyphae, with IL-6 production being the more profound (Supplementary Fig. 3a, b). These results also correlated with impaired down-modulation of dectin-1 and -2 expression (Supplementary Fig. 3d), thus indicating that our observations in mouse macrophages can be recapitulated in human macrophages.

CBLB associates with dectin-1 and -2 in macrophages upon infection with C. albicans yeast and hyphal forms

Dectin family CLRs play a major role in fungal recognition and host innate responses against fungal infection 15, 27, 28. Dectin-1’s cytoplasmic tail contains an ITAM motif that can be phosphorylated by Src family kinases. Phosphorylated dectin-1 in turn, recruits and activates SYK, thereby initiating downstream signaling via the CARD9/BCL10/MALT1 complex 15, 28. Since dectin-2 lacks this ITAM-like motif it binds FcR-γ 3 which contains ITAMs 29 that recruit SYK and transduce dectin-2 signaling 30–32. We sought to determine whether and how CBLB regulates signaling via dectin-1 and -2 during C. albicans infection. First, we determined whether CBLB physically interacts with dectin receptors or their signaling intermediates, and if so, how this occurs. To this end, we infected WT BMDMs with C. albicans yeast or hyphae for different times. We found that CBLB was inducibly associated by co-immunoprecipitation with dectin-1, dectin-2, SYK and CARD9 upon infection with C. albicans yeasts or hyphae (Fig. 2a, b).

Figure 2

CBLB associates with dectin-1 and dectin-2 in macrophages upon C. albicans yeast and hyphal infection. (a,b) Immunoblot analysis of dectin-1 or dectin-2, SYK, and CARD9 after immunoprecipitation (IP) with CBLB antibodies from lysates of BMDMs uninfected or infected with C. albicans yeast or hyphae. Images are representative of three independent experiments (biological replicates), and each IP was blotted separately. (c,d) Immunoblot analysis of dectin-1 or dectin-2, SYK and CARD9 after immunoprecipitation (IP) of proteins with CBLB antibodies from lysates of WT BMDMs with or without Syk gene silencing (c) or WT and Card9 BMDMs (d) uninfected or infected with C. albicans yeast or hyphae. Images are representative of two independent experiments (biological replicates), and each IP was blotted separately. (e) Immunoblot analysis of dectin-1 after CBLB immunoprecipitation from lysates of Clec7a BMDMs reconstituted with Flag-tagged dectin-1 or dectin-1Y15F mutant, and infected with C. albicans yeast. Images are representative of three independent experiments (biological replicates), and each IP was blotted separately. (f) Immunoblot analysis of dectin-2 after CBLB immunoprecipitation from lysates of Fcer1g BMDMs reconstituted with Flag-tagged FcR-γ or FcR-γY65F,Y76F mutant, and infected with C. albicans hyphae. Images are representative of two independent experiments (biological replicates), and each IP was blotted separately. (g) Immunoblot analysis of FcR-γ after CBLB immunoprecipitation from lysates of WT and Fcer1g BMDMs infected with C. albicans hyphae. Images are representative of three independent experiments (biological replicates), and each IP was blotted separately.

It has previously been shown that CBLB binds to SYK in B cells upon BCR stimulation 33, or CARMA1 (CARD11), a homologue of CARD9, in NK T cells 34. To determine whether SYK and CARD9 are potential binding partners of CBLB in the signaling pathways derived from dectin-1 and -2, we silenced Syk gene expression in WT BMDMs by Syk siRNA. We found that knocking down Syk expression did not affect the association of CBLB with either dectin-1 or dectin-2 (Fig. 2c). Similarly, CARD9 deficiency also did not affect CBLB-dectin-1 or CBLB-dectin-2 association (Fig. 2d). Next we wanted to determine whether phosphorylation of the ITAM within dectin-1 and the ITAMs within FcR-γ is required for CBLB association in macrophages upon C. albicans infection (yeasts and hyphae). To accomplish this, we mutated the tyrosine (Y) of the hemi-ITAM to phenylalanine (F) in dectin-1’s cytoplasmic tail (Y15F), and the tyrosines within the ITAMs of the FcR-γ to F (FcR-γY65F,Y76F), then reconstituted Clec7a−/− BMDMs and Fcerg1−/− BMDMs with these mutants, and infected them with C. albicans yeast and hyphae, respectively. Mutation of dectin-1 at Y15 or FcR-γ at Y65 and Y76 completely abrogated the binding of CBLB to dectin-1 or dectin-2 (Fig. 2e, f), indicating that phospho-Y15 of dectin-1 or phospho-Y65 and Y76 of FcR-γ is critical for their binding to CBLB. Indeed, CBLB bound to FcR-γ in WT BMDMs upon C. albicans hyphal infection (Fig. 2g).

Dectin-1, dectin-2, and SYK are targets of CBLB

To determine whether dectin-1 and dectin-2, or the downstream signaling molecules are the targets of CBLB, we first examined protein stability of dectin-1, dectin-2, SYK and CARD9 in macrophages infected with C. albicans yeast or hyphae. Interestingly, dectin-1 and -2, but not SYK or CARD9, underwent degradation in WT BMDMs upon infection with C. albicans yeasts and hyphae, but not in BMDMs lacking CBLB (Fig. 3a). These findings suggest that dectin receptors are the likely targets of CBLB. Furthermore, dectin-1 and -2 degradation was completely abrogated by pretreatment with E-64, a lysosome inhibitor, but not with MG-132, a proteasome inhibitor (Fig. 3b), suggesting that dectin-1 and -2 undergo lysosome-mediated degradation.

Figure 3

CBLB targets dectin-1 and dectin-2 for polyubiquitination and subsequent degradation in the lysosome. (a) Immunoblot analysis of lysates of WT and Cblb−/− BMDMs infected with C. albicans yeast and hyphal forms (MOI = 1:1) with antibodies against dectin-1, dectin-2, SYK, CBLB, and ACTIN, respectively. Images are representative of five independent experiments (biological replicates). (b) Immunoblot analysis of WT BMDMs pretreated with E-64 (10 μM), MG-132 (5 μM), or both for 30 min, then infected with C. albicans yeast or hyphae (MOI = 1:1), with antibodies against to dectin-1 and dectin-2, respectively. Images are representative of three independent experiments (biological replicates). (c, d) Immunoblot analysis of dectin-1 and dectin-2 ubiquitination of dectin-1 or dectin-2 immunoprecipitates isolated from BMDMs from WT and CblbC373A mice infected with C. albicans yeast and hyphae, respectively, by anti-ubiquitin and anti-K48 ubiquitin antibodies. Images are representative of four independent experiments (biological replicates), and each IP was blotted separately. (e) Ubiquitination of dectin-1 in Clec7a−/− BMDMs reconstituted with WT dectin-1 or dectin-1K2R, dectin-1K27R, dectin-1K34R mutant, or dectin-1K2R, K27R, K34R triple mutant infected with C. albicans yeast. Images are representative of three independent experiments (biological replicates), and each IP was blotted separately. (f) Ubiquitination of dectin-2 in Clec4n−/− BMDMs reconstituted with WT dectin-2 or dectin-2K10R infected with C. albicans hyphae. Images are representative of three independent experiments (biological replicates), and each IP was blotted separately. (g, h) ELISA of TNF-α and IL-6 production by Clec7a−/− BMDMs reconstituted with WT dectin-1 or dectin-1K2R,K27R,K34R infected with C. albicans yeast (g), or by Clec4n−/− BMDMs reconstituted with WT dectin-2 or dectin-2K10R infected with C. albicans hyphae (h). Data are representative of three independent experiments (biological replicates). Error bars are mean ± s.d. *P < 0.05, **P < 0.01; unpaired two-tailed Student’s t test. n = 3 per group, each with three repeated wells.

To further determine whether CBLB is the E3 ubiquitin ligase for dectin-1 or dectin-2, BMDMs generated from WT, Cblb or mice expressing an E3 ligase dead mutation (C373A) (CblbC373A) 35 were infected with C. albicans yeast or hyphae. The CBLB C373A mutation or deficiency abrogated ubiquitination of dectin-1 and -2 (Fig. 3c,d, upper panel; Supplementary Fig. 4a, b, upper panel). To determine whether ubiquitination of dectin-1 or -2 is K48 or K63-linked, we utilized anti-K48 ubiquitin or anti-K63 ubiquitin antibodies. We confirmed that both dectin-1 and -2 underwent K48-linked polyubiquitination, and this K48-linked polyubiquitination of dectin-1- and -2 was abrogated in BMDMs expressing the CBLB C373A mutation or lacking CBLB (Fig. 3c, d, lower panel; Supplementary Fig. 4a, b, lower panel; data not shown).

It was previously shown that CBLB targets SYK for polyubiquitination but not degradation in B cells 33. To determine whether SYK is also a potential target of CBLB in macrophages triggered by dectin-1 or -2 receptor-ligand interactions, we examined SYK ubiquitination in WT and CblbC373A BMDMs upon infection with C. albicans yeast or hyphae. Indeed, SYK underwent K48-linked polyubiquitination upon infection with both C. albicans yeast and hyphae, but this ubiquitination was greatly reduced in BMDMs expressing C373A CBLB (Supplementary Fig. 4c, d). Therefore, our data suggest that dectin-1/2 and SYK are targets of CBLB, and that CBLB keeps the expression of these CLRs in check. Consistent with these data, SYK and NF-κB were highly activated in BMDMs lacking CBLB upon C. albicans yeast and hyphal infection (Supplementary Fig. 4e).

To examine the functional relevance of CBLB-mediated ubiquitination of dectin-1 and -2 we generated single and triple K to R mutations of dectin-1K2R, dectin-1K27R, dectin-1K34R, and dectin-1K2R,K27R,K34R and dectin-2K10R by site-directed mutagenesis. We reconstituted BMDMs lacking dectin-1 (from Clec7a mice) with WT dectin-1 or dectin-1 K/R mutants and BMDMs lacking dectin-2 (from Clec4n−/− mice) with WT dectin-2 or dectin-2K10R mutant by Lipofectamine transfection, and infected them with C. albicans yeast or hyphae. Reconstituting Clec7a−/− BMDMs with WT dectin-1 or dectin-1K2R,K27R,K34R completely or partially restored Dectin-1 ubiquitination, whereas dectin-1K2R,K27R,K34R mutants were not ubiquitinated (Fig. 3e). As expected, Clec4n−/− BMDMs reconstituted with WT dectin-2 but not dectin-2K10R mutant restored ubiquitination of dectin-2 (Fig. 3f). These data indicate that dectin-1 K2, K27, and K34, and dectin-2 K10 are the sites of ubiquitination of dectin-1 and -2, respectively. Consistent with these data, Clec7a−/− BMDMs reconstituted with dectin-1K2R,K27R,K34R, or Clec4n−/− BMDMs reconstituted with dectin-2K10R, produced significantly higher amounts of TNF-α and IL-6 upon infection with C. albicans yeast or hyphae (Fig. 3g, h).

CBLB regulates the internalization of dectin-1 and -2, and their trafficking to the lysosome

Cell surface receptor internalization can occur when receptors are mono- or poly-ubiquitinated following ligand-induced activation, and subsequently sorted into endocytic vesicles for delivery to the lysosome for degradation 36–38. Internalization of dectin-1 has been shown to terminate inflammatory responses in order to keep inflammation in check 39. Thus, impaired down-modulation of dectin-1 and -2 could be due to a lack of internalization or a block in intracellular vesicle sorting to the lysosome. To determine whether CBLB is critical for this process, the cell surface and internalized expression levels of dectin-1 and dectin-2 in BMDMs from WT and Cblb mice was investigated. We found a minimal level of intracellular dectin-1 or -2 in Cblb BMDMs (Fig. 4a,b), suggesting that CBLB promotes internalization of dectin-1 or dectin-2 after infection with C. albicans yeast or hyphae.

Figure 4

Loss of CBLB impairs dectin-1 and dectin-2 internalization and their down-regulation at the cell surface. (a,b) Cell surface and intracellular expression of dectin-1 and dectin-2 of WT and Cblb−/− BMDMs infected with C. albicans yeast or hyphae (MOI = 1:1) for times indicated by flow cytometry. For internalization of dectin-1 and dectin-2, WT and Cblb−/− BMDMs were treated with acid buffer to strip the antibodies remaining at the cell surface after infection at each time-point. Data are representative of three independent experiments (biological replicates). Error bars are mean ± s.d. *P < 0.05; unpaired two-tailed Student’s t test. n = 3 per group, each with three repeated wells. (c,d) Confocal image of dectin-1 and dectin-2 internalization and lysosome sorting of WT and Cblb BMDMs infected or uninfected with C. albicans yeast (c) or hyphae (d) (MOI = 1:1) for 30 min. Images are representative of five independent experiments (biological replicates). n = 3 per group, each with three repeated wells. Scale bar, 5 μm.

We next investigated whether retention of ligand-engaged dectin-1 or -2 in Cblb BMDMs is due to impaired sorting of endosomal vesicles to lysosomes. We compared the subcellular localization of ligand-engaged dectin-1 or -2 in WT and Cblb BMDMs by confocal microscopy. In support of impaired lysosomal degradation of dectin-1 and -2 in BMDMs lacking CBLB, intracellular trafficking of internalized dectin-1 or -2 to the lysosome was significantly reduced in the absence of CBLB (Fig. 4c, d).

CBLB negatively regulates ROS production and fungal killing but not phagocytosis of C. albicans

Neutrophils and macrophages are professional phagocytes of the innate immune system that are essential in controlling bacterial and fungal infections by phagocytosis and killing mechanisms 40. The production of highly reactive oxygen species (ROS) is one of the primary effector mechanisms used by phagocytes to control or clear microbial infections. ROS plays an important role in the initial step of fungal killing in phagosomes 41 and can be potentiated by dectin signaling. We measured ROS production by co-culturing the C. albicans yeast cap1/cap1 mutant or hyphae with WT or Cblb BMDMs. We found that Cblb and CblbC373A BMDMs produced more ROS than WT controls at MOIs of 5:1 and 2:1 (Supplementary Fig. 5a). Enhanced ROS activity in Cblb BMDMs correlated with an increase in their fungal killing potency (Supplementary Fig. 5b). Consistent with a limited role of CBLB in pro-inflammatory cytokine production by neutrophils, we did not observe a significant increase in ROS activity and fungal killing in neutrophils isolated from the BM of Cblb or CblbC373A mice compared to WT controls (Supplementary Fig. 5c). However, phagocytosis of C. albicans by Cblb BMDMs was not increased compared to WT BMDMs (Supplementary Fig. 5d).

CBLB inhibits innate immune responses against systemic C. albicans infection mediated by the dectin family of CLRs

The recognition of β-glucans and α-mannans by dectin-1 and dectin-2 respectively is thought to trigger immune responses that are primarily designed for the control of fungal pathogens 2–4. To assess the role of CBLB in anti-fungal immunity we infected WT, Cblb−/−, and CblbC373A mice with a lethal dose of C. albicans to monitor survival, and a sub-lethal dose to measure serum cytokines and fungal burden. We found that most Cblb and CblbC373A mice were protected from lethal systemic infection with C. albicans (Fig. 5a), which correlated with heightened levels of TNF-α and IL-6 in the sera of Cblb−/− and CblbC373A mice, lower fungal burden in the kidney, lung, spleen, and liver, and decreased C. albicans hyphae in the kidney on day 2 as assessed by PAS staining (Fig. 5b-d; Supplementary Fig. 6a). We also observed multifocal tubulointerstitial nephritis in WT mice infected with C. albicans, which was ameliorated in mice lacking CBLB or expressing the CBLB C373A mutation (Fig. 5c). This observation is consistent with fact that more immune cells traffic to the kidneys in WT than CblbC373A mice including macrophages, dendritic cells (DCs), and neutrophils (Supplementary Fig. 6b). Improved survival rate was also observed in Rag1 mice that lack functional adaptive immune cells (Fig. 5e), supporting a critical role of CBLB in down-regulating innate immune responses.

Figure 5

Introducing dectin-1 and dectin-2 deficiency, or double deficiency into Cblb mice renders Cblb−/− mice susceptible to systemic C. albicans infection. (a) Kaplan-Meier Survival curve of WT, Cblb−/−, and CblbC373A mice (n = 10 per group) infected with 5 × 105 CFU of C. albicans (SC5314), and monitored for 7 days for survival. Data are representative of three independent experiments (biological replicates). *P < 0.05; Log-rank test. (b) CFU assay of paired kidneys of WT, Cblb−/− and CblbC373A mice (n = 10 per group) infected with 1 × 105 CFU of C. albicans performed at day 2 after infection. Data are representative of three independent experiments (biological replicates). **P < 0.01; unpaired two-tailed Student’s t test. (c) Kidney histopathology analysis by H&E and PAS staining. Fungal burden (hyphae) in the kidneys visualized by PAS staining. Images are representative of two independent experiments (biological replicates). n = 10 per group. Scale bar, 200 μm. (d) ELISA of serum TNF-α, IL-6, and IL-1β levels of WT and Cblb−/− mice (n = 10 per group) infected with 1 × 105 CFU of C. albicans at 2, 6, 12, and 24 h after infection. Data are representative of three independent experiments (biological replicates). Error bars are mean ± s.d. *P < 0.05, **P < 0.01; unpaired two-tailed Student’s t test. Each with three repeated wells. (e) Survival rate of Rag1 and Rag1 mice (n = 8). infected with 1 × 105 CFU of C. albicans. Data are representative of three independent experiments (biological replicates). *P < 0.05, Log-rank test. (f) Survival rate of WT, Cblb, Clec7a, Clec4n, Cblb, Cblb, and Cblb mice (n = 5 per group) infected with C. albicans (3.5 × 105 CFU) by i.v. injection. Data are representative of three independent experiments (biological replicates). #P < 0.01, Cblb vs. all other groups; *P < 0.05, Cblb vs. Clec7a or Cblb vs. Clec4n; and §P < 0.05, Cblb vs Clec7a; Log-rank test.

To further determine whether monocytes, macrophages and neutrophils have a greater capacity to kill C. albicans during systemic infection, we monitored fungal burden in the blood of WT and CblbC373A mice at 2 and 6 h after infection. We found that fungal burden in the blood of CblbC373A mice was significantly lower than that of WT mice at 2 and 6 h after infection (Supplementary Fig. 7a). The lower fungal burden in the blood of CblbC373A mice correlated with enhanced fungal killing activity by PBMCs, but not by neutrophils of CblbC373A mice (Supplementary Fig. 7a). Increased fungal killing was also observed in monocytes from the spleen of CblbC373A mice (Supplementary Fig. 7b). We also monitored ROS activity in monocytes, macrophages and neutrophils from WT and CblbC373A spleens and kidneys by CellRox dye. As shown in Supplementary Figure 7c, monocytes and macrophages, but not neutrophils, displayed augmented ROS expression in C. albicans-infected CblbC373A mice when they were infected in vitro with C. albicans. Consistent with the lower fungal burden and less inflammation in CblbC373A kidneys, trafficking of CD45.2+ leukocytes, including macrophages, DCs and neutrophils to CblbC373A kidneys were significantly reduced (Supplementary Fig. 6b). Even with decreased myeloid cells in CblbC373A kidneys upon infection with C. albicans, we observed an increase in ROS expression in monocytes and macrophages, and fungal killing using CD45+ cells isolated from CblbC373A kidneys (Supplementary Fig. 7d), and increased TNF-α and IL-6 in the kidney homogenates of CblbC373A mice (Supplementary Fig. 7e).

To further determine whether heightened inflammatory responses caused by CBLB deficiency are mediated by dectin-1 and -2, we generated Cblb−/−Clec7a−/−, Cblb−/−Clec4n, and Cblb mice. We infected WT, Cblb−/−, Clec7a−/−, Cblb−/−Clec7a−/−, Clec4n−/−, Cblb−/−Clec4n, and Cblb mice with C. albicans. Dectin-1 or dectin-2 single deficiency rendered Cblb mice susceptible to C. albicans infection, and dectin-1 and dectin-2 double deficiency greatly increased the sensitivity of Cblb−/− mice to systemic C. albicans infection. All of the triple knockout mice died within four days after infection at a dose at which all Cblb mice survived (Fig. 5f), which correlated with significantly lower levels of TNF-α and IL-6 in their sera and fungal burden in the kidneys (Supplementary Fig. 8a,b). Therefore, our results suggest that CBLB negatively regulates both dectin-1 and -2, and that CBLB dampens inflammatory responses mediated by dectin-1 and -2 during systemic fungal infection. Notably, Cblb or CblbC373A mice at 8-12 weeks of age did not display signs of autoimmunity as revealed by comparable anti-dsDNA and anti-ssDNA antibody titers and IL-17/IFN-γ in the sera of WT and Cblb or CblbC373A mice, and no elevated IL-17 and IFN-γ in the kidneys of Cblb or CblbC373A mice compared to WT mice (Supplementary Fig. 9a-d). These data suggest that a pre-existing autoimmunity in Cblb or CblbC373A mice does not account for differences relative to WT mice after fungal infection.

We also observed that Clec7a and Clec4n mice die at a similar rate upon systemic C. albicans infection, suggesting that both dectin-1 and dectin-2 are equally important for fungal recognition (Fig. 5f). Since Cblb, and Cblb mice did not die at the same rate after infection as did Clec7a, or Clec7a mice (Fig. 5f), these results suggest that CBLB may regulate an additional CLR(s) such as the mannose receptor (MR), dectin-3 or Mincle which have been shown to be involved in host defense against C. albicans infection 4, 42–44. Indeed, loss of CBLB appeared to stabilize the protein expression of dectin-3, but not MR, Mincle and DC-SIGN (Supplementary Fig. 10).

CBLB is a potential therapeutic target for anti-fungal infection

Since CBLB down-regulates dectin family CLR signaling and host innate immune responses, decreasing CBLB expression may enhance phagocyte anti-fungal responses providing evidence for a new therapeutic approach. We performed experiments using in vivo delivery of Cblb siRNA to knock down Cblb. We first infected WT mice with C. albicans by i.v. injection, and 24 h later we injected Cblb siRNA or a nonsense siRNA via the tail vein. Mortality of the mice was monitored for 7 days. While all WT mice treated with nonsense siRNA died within 7 days after infection, 7 out of 9 WT mice treated with Cblb siRNA survived. There was a significantly higher fungal burden in the kidneys of WT mice receiving the nonsense siRNA compared to those receiving Cblb siRNA (Fig. 6). These data indicate that CBLB may serve as a potent therapeutic target for enhancing host defense against fungal infections.

Figure 6

Systemic in vivo delivery of Cblb siRNA into C57BL/6 mice protects them from lethal disseminated candidiasis. (a) Survival of C57BL/6 mice treated with in vivo grade Cblb siRNA (5’-AAAUUCUCGAAGUAUGCUCUU-3’) or a non-sense siRNA (2 mg/kg/mouse) via tail vein injection 24 h after infection with C. albicans (5 × 105 CFU). Data are representative of three independent experiments (biological replicates). *P < 0.05, Log-rank test. n = 9 per group. (b) Fungal burden in the kidneys on day 2 after infection. Data are representative of three independent experiments (biological replicates). Error bars are mean ± s.d. *P < 0.05; unpaired two-tailed Student’s t test. n = 9 per group. (c) Immunoblot analysis of spleen cells from control siRNA or Cblb siRNA-treated C57BL/6 mice with anti-CBLB and anti-actin, respectively. Data are representative of four independent experiments (biological replicates). n = 3 per group.

Discussion

The fungal cell wall consists mainly of carbohydrates, including mannose-based structures (the mannoproteins), β-glucan, and chitin. Recognition of β-glucans and α-mannans by dectin-1 and -2 is essential for anti-fungal immunity 27. However, the regulation of dectin family receptors is unknown. Here we show that CBLB functions as a negative regulator of dectin-1 and -2 CLRs which initiate innate immune responses to fungal pathogens in human and mouse macrophages. CBLB targets dectin-1 and -2, and SYK for K48-linked polyubiquitination, which inhibits dectin-1/2-mediated signaling pathways. CBLB deficiency or inactivation leads to increased pro-inflammatory responses that decrease dissemination of C. albicans and bolster host defense.

To our knowledge, our findings are the first to identify a negative regulator of dectin receptor-mediated innate immune responses. We show that dectin-1K2R, K27R, K34R and dectin-2K10R mutations, which abrogate their ubiquitination, result in increased production of TNF-α and IL-6 by macrophages infected with C. albicans yeast or hyphae (Fig. 3g,h), thus mirroring the data obtained from Cblb and CblbC373A mice. Our data therefore provide evidence that ubiquitination of dectin-1 and -2 is a key mechanism for terminating innate immune responses during fungal infection, thus avoiding excessive inflammation and subsequent tissue damage while at the same time damping optimal host defense properties.

Phagocytosis is a key cellular process, both during homeostasis and upon infection or tissue damage, and dectin-1 has been shown to be a phagocytic receptor 45. ROS production by phagocytes is associated with pathogen killing 46 and it was reported that dectin-1 activates SYK in macrophages and is important for dectin-1-stimulated ROS production, but not for phagocytosis 47. Consistent with this report, our data show that CBLB regulates both dectin-1 and -2 expression and ROS production by macrophages, but does not affect fungal phagocytosis (Supplementary Fig. 5). Our data suggest that additional receptor(s) such as Fcγ receptor family or DC-SIGN 44, 45, independent of regulation by CBLB, may be involved in controlling fungal phagocytosis.

Since CBLB is critical for T cell activation, tolerance induction and TH2/9 cell differentiation 6, it is possible that the enhanced anti-fungal immunity in the absence of CBLB may result in heightened adaptive T cell responses. However, this possibility is excluded by the fact that the phenotype of Cblb mice upon C. albicans infection, which do not have T and B cells, phenocopies that of Cblb mice (Fig. 5e), supporting the notion that CBLB is crucial for controlling innate immune responses against systemic C. albicans infection. We also further demonstrate that the heightened innate immune responses observed during systemic C. albicans infection is mediated by dectin-1 and -2 because introducing dectin-1 or -2 deficiency, or both into Cblb mice abrogates these heightened responses, and renders Cblb mice susceptible to C. albicans infection (Fig. 5f; Supplementary Fig. 8a). More importantly, systemic in vivo delivery of Cblb siRNA to C57BL/6 mice protects them from lethal systemic C. albicans infection (Fig. 6). These data suggest that CBLB is a potential therapeutic target for controlling disseminated candidiasis. Of note, inhibition of CBLB may have detrimental effects due to unchecked inflammation, particularly on patients in intensive care. However, inhibition of Cblb by siRNA in vivo has a limited half-life, and dosages could be modulated to minimize the degree of inflammation. In addition, no signs of autoimmunity were observed in Cblb or CblbC373A mice (Supplementary Fig. 9). However, given that we have shown that Cblb mice develop severe airway inflammation, and an aberrant TH2 response using ovalbumin-induced asthma model 14, it would be interesting to test whether mice deficient for CBLB or expressing the CBLB C373A mutation are susceptible to allergic bronchopulmonary aspergillosis in the future.

In summary, our data provide the first evidence that CBLB plays an essential role in regulating dectin-mediated innate immune responses to fungal pathogens following inflammatory responses to fungi in immunocompetent hosts. One consequence of this dampening of inflammatory responses is the creation of a less than optimal host defense program. Targeting CBLB may therefore serve as a new therapeutic strategy in fighting fungal infections.

Online Methods

Mice

C57BL/6 mice and Rag were purchased from the Jackson Laboratory. Fcer1g mice were purchased from Taconic (Hudson, NY). Cblb−/− mice 7 were kindly provided by Dr. Josef M. Penninger (University of Toronto; Toronto, ON, Canada). CblbC373A mice and Clec7a were described previously 2, 35. Clec4n mice described previously 3 were provided by Dr. Yoichiro Iwakura (Tokyo University of Science; Chiba, Japan). Cblb mice on a C57BL/6 background were crossed onto Clec7a or Clec4n mice to generate Cblb and Cblb mice, or Cblb mice. Cblb mice were also crossed onto Rag1 strain to generate Cblb mice. The mice were used at 8-12 weeks of age, and both male and female were used in this study. The use of animals was approved by the Institutional Animal Care and Use Committees (IACUCs) of the Ohio State University and Xiangya School of Medicine, Central South University.

Reagents

Antibodies against CBLB (G-1), SYK (N-19), CARD9 (H-90), DC-SIGN (T-13), CD206 (H-300), and Ubiquitin (P4D1), were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-dectin-1 (GE2; Ab82888) was obtained from Abcam (Cambridge, MA). Anti-dectin-2 (217611) and mouse IL-1RA/IL-1F3 Quantikine ELISA Kit (MRA00) was purchased from R&D System (Minneapolis, MN). The following items were purchased from BioLegend (San Diego, CA): PE-conjugated anti-dectin-1 (144204), FITC-conjugated anti-LAMP-1 (1D4B), anti-mouse CD45.2 antibody (104), anti-mouse CD8 (53-6.7), anti-mouse CD11b (M1/70), anti-mouse F4/80 (BM8), anti-mouse CD11c (N418), anti-mouse I-A/I-E (M5/114.15.2), anti-mouse Ly6C (HK1.4), anti-mouse Ly6G (1A8), ELISA kits for mouse IL-17A (432504), IFN-γ (430805), IL-6 (431304) and TNF-α (430904). PE-conjugated anti-dectin-2 (MCA2415PE) was obtained from AbD Serotec (Raleigh, NC). Anti-Mincle (D292-3) was purchased from MBL Life Science (Woburn, MA). ELISA kits for mouse IgG (88-50400) and IgE (88-50460) were purchased from eBioscience (San Diego, CA). ELISA kits for anti-ssDNA (5310) and anti-dsDNA (total (A+G+M) (5110) were purchased from Alpha Diagnostic International Inc (San Antonio, TX). The plasmids encoding Clec7a (dectin-1) and Clec4n (dectin-2) (pCMV2-Flag) were purchased from Sino Biologicals, Inc. (Beijing, P.R. China). Anti-K48-linkage specific polyubiquitin (4289), anti-K63-linkage specific polyubiquitin (D7A11), anti-phospho-SYK (Y525/526; #2711) and anti-phospho-NF-κB p65 (S536; #3031), and anti-phospho-IκBα (Ser32/36) (5A5; #9246) were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Anti-dectin-3 was kindly provided by Dr. Xin Lin at MD Anderson Cancer Center (Houston, TX, USA). Mouse neutrophil isolation kit, monocyte isolation kit, and CD45 microbeads (mouse) were purchased from Miltenyi Biotec (San Diego, CA). Histopaque 1119 (Sigma 11191), Histopaque 1077 (Sigma 10771), and anti-Flag (M2) were obtained from Sigma-Aldrich (St. Louis, MO). Collagenase type IV (02195110) was purchased from MP Biomedicals (Santa Ana, CA). CellRox Deep Red (C10422) was purchased from ThermoFisher Scientific (Waltham, MA). The validation of the antibodies used is provided on the manufacturers’ websites.

Site-directed mutagenesis

Single and triple K to R mutations of dectin-1K2R, dectin-1K27R, dectinK34R, and dectinK2R,K27R,K34R, dectin-2K10R, dectin-1Y15F, and FcR-γY65F,Y76F were generated by site-directed mutagenesis at Mutagenex Inc. (Piscataway, NJ).

Generation of BMDMs and BMDCs and isolation of mouse BM neutrophils

BM cells were harvested from the femurs and tibias of mice. Cells were cultured in DMEM containing 10% FBS and 30% conditioned medium from L929 cells expressing M-CSF. After one week of culture, nonadherent cells were removed, and adherent cells were 80-90% F4/80+CD11b+ as determined by flow cytometric analysis. Mouse BMDCs were generated using GM-CSF and purified from bulk cultures by magnetic selection with anti-CD11c microbeads. This routinely gave purities of >98%. For isolation of BM neutrophils, total BM cells were recovered from the femurs and tibias by flushing with RPMI medium with an 18-gauge needle; erythrocytes were lysed with red blood cells (RBC) lysis buffer (eBioscience) and BM neutrophils were isolated by neutrophil isolation kit (Miltenyl), and neutrophil purity (>98%) was confirmed by flow cytometry.

Isolation of mouse PBMCs and neutrophils from blood, and splenic monocytes, neutrophils, and kidney CD 45+ cells

WT and CblbC373A mice were anesthetized, and blood was collected from tail vein. The RBC were lysed using RBC lysis buffer (eBioscience). PBMCs and neutrophils were isolated by gradient centrifugation over Histopaque 1119 (density, 1.119 g/ml) and Histopaque 1077 (density, 1.077 g/ml) according to the manufacturer’s instructions at 400 × g for 30 min at 25 °C 48. PBMCs were collected from the interface between the plasma and Histopaque 1077. Neutrophils were recovered at the interface of the interface of the Histopaque 1119 and Histopaque 1077 layers, and were 80–90% pure and >95% viable as determined by flow cytometry. PBMCs and neutrophils were washed twice and were resuspended in RPMI 1640 medium supplemented with 10% FBS. Splenic monocytes and neutrophils of WT and CblbC373A mice were isolated by monocyte isolation and neutrophil isolation kits (Miltenyl), and monocyte and neutrophil purity (>98%) were confirmed by flow cytometry.

WT and CblbC373A mice were sacrificed at 48 h after infection with C. albicans by tail vein injection at a dose of 1 × 106 CFU. Kidneys were perfused, minced, and placed in 2 ml of Hank’s balanced salt solution (HBSS) (50 mM HEPES, 12 mM Dextrose, 280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, PH to 7.05) containing 2 mg/ml collagenase IV, and incubated at 37 °C for 30 min with gentle agitation. Digested kidney tissues were passed through a 40 μm Falcon™ cell strainer using the rubber end of a 1 ml syringe plunger, and a cell suspension was obtained via centrifugation at 1200 rpm for 10 min. The CD45+ cells were purified by CD45 MicroBeads (Miltenyl). The purity (~90%) was determined by flow cytometry.

In vitro infection of macrophages, dendritic cells, and neutrophils with C. albicans yeast and hyphal forms

A single colony of C. albicans strain SC5314 was grown overnight at 30 °C in yeast peptone dextrose media. The cells were washed twice with PBS before use as live yeasts. The cap1 yeast-only mutant described previously 17 was obtained from Dr. Paula Sundstrom (Dartmouth University). For the hyphal forms, the washed yeasts were resuspended at 107 cells/ml in RPMI 1640 with 10% FCS and grown 3 h at 37 °C. After washing in PBS, the hyphae were used for live stimulations. For analysis of cytokine production, 105 BMDMs, BMDCs, or neutrophils were cultured overnight in a 96-well U-bottomed plate with live C. albicans cap1 mutant or hyphae at MOI of 1 for times indicated, cytokine levels in the supernatant were measured by sandwich ELISA.

BMDM reconstitution

Clec7a BMDMs were transfected with Flag-tagged dectin-1 or dectin-1K2R, dectin-1K27R, dectinK34R, or dectin-1K2R,K27R,K34R, or dectin-1Y15F by Lipofectamine 2000. Clec4n or Fcerg1−/− BMDMs were transfected with Flag-tagged dectin-2, dectin-2K10R, FcR-γ, and FcR-γY65F,Y76F, respectively.

ROS assay, phagocytosis of C. albicans and fungal killing assay

For ROS production assay, 2 × 105 WT or BMDMs were washed with PBS twice, and replated in PBS containing 100 mM luminol and 5 units of horseradish peroxidase. The cells were incubated at 37 °C for 30 min, and then infected with C. albicans at MOI = 5:1 and 2:1, respectively. The relative amount of ROS generated by neutrophils was detected at regular intervals over 75 min by measuring the luminescence. Relative light units (RLU) were plotted as a function of time to evaluate chemiluminescence (CL) rate. To measure ROS expression in monocytes, macrophages and neutrophils in spleens and kidneys, WT and Cblb mice were infected with C. albicans by tail vein injection at a dose of 1 × 106 CFU. 48 h later mice were sacrificed, and leukocytes from spleens and kidneys were infected with C. albicans for 30 min, and stained with CellRox and cell surface markers to determine ROS expression in monocytes (Kidney: CD45.2+CD11b+ Ly6ChiLy6G−; Spleen: CD11b+Ly6C+Ly6G−), macrophages (Kidney: CD45.2+ CD11b+F4/80+Ly6CloCD11c−; Spleen: CD11b+F4/80+Ly6CloLy6G−), and neutrophils (Kidney: CD45.2+CD11b+ Ly6Clo Ly6G+; Spleen: CD11b+Ly6G+Ly6C−).

For phagocytosis of C. albicans, C. albicans yeast were labeled with Alexa Fluor 488 (Invitrogen) in 100 mM HEPES buffer (pH 7.5)(diluted to 1:500), and then co-cultured with WT or Cblb BMDMs for 45 min at 37 °C. Adherent fungal cells were quenched with trypan blue, and the rate of phagocytosis was determined by flow cytometry 49.

For in vitro fungal killing assay, WT or Cblb BMDMs (1 × 105/well) were incubated with C.albicans at MOI 1:500 for 24 h. To determine the fungal killing capacity of PBMCs, blood neutrophils, splenic monocytes and neutrophils, and kidney CD45+ cells, WT and CblbC373A mice were infected with C. albicans by tail vein injection (1 × 106 CFU). PBMCs, blood neutrophils, and splenic monocytes, neutrophils and kidney CD45+ cells were co-cultured with C.albicans form at MOI 1:10 for 24 h. After co-culture, a 100 μl suspension was spread (1:104 dilution) on YPD plates. After incubation at 37 °C for 36 h, killing was determined by counting the Candida colonies with and without indicated cells 49.

Immunopreciptation and Western blotting

For co-immunoprecipitation, WT BMDMs were infected with C. albicans yeast or hyphae (MOI = 1:1) for various times, and lysed in 0.5% NP40 lysis buffer. The cell lysates were immunoprecipitated with anti-CBLB (1:100) and blotted with anti-dectin-1 (1:1000) or anti-dectin-2 (1:5000), anti-SYK (1:1000), and anti-CARD9 (1:1000). For detection of dectin-1 or dectin-2 ubiquitination, BMDMs from WT, and Cblb or CblbC373A mice were infected with C. albicans yeast cap1 mutant or hyphae (MOI = 1:1) for various times, and lysed in RIPA buffer containing 2% SDS, and diluted to 0.5% of SDS. The cell lysates were immunoprecipitated with anti-dectin-1 (1:100) or anti-dectin-2 (1:100), and blotted with anti-ubiquitin (1:1000), anti-K48- or anti-K63-specific ubiquitin antibodies (1:1000). To assess the protein stability of dectin-1, dectin-2, dectin-3, MR, Mincle, DC-SIGN, SYK and CARD9, BMDMs from WT and Cblb mice were infected with C. albicans yeast or hyphae (MOI = 1:1) at indicated times and lysed for immunoblotting with antibodies against dectin-1 (1:1000), dectin-2 (1:1000), dectin-3 (1:1000), MR (1:1000), Mincle (1:1000), DC-SIGN (1:1000), SYK, and CARD9, respectively. To determine whether dectin-1 and dectin-2 undergo proteasome or lysosome-mediated degradation, WT BMDMs were pretreated with MG-132 (5 μM) or E64 (10 μM) for 30 min, and then infected with C. albicans yeast cap1 mutant or hyphae (MOI = 1:1) for various times, and lysed. The cell lysates were blotted with anti-dectin-1 or anti-dectin-2.

Detection of serum and kidney cytokines, serum IgG and IgE, and autoantibodies by ELISA

For detection of TNF-α, IL-6, IL-1β, and IL-1RA in macrophage culture supernatants, 105 BMDMs from WT, Cblb or CblbC373A mice were infected with live C. albicans cap1 mutant or hyphae at MOI 1:1 for the times indicated, and cytokine production in the supernatant was measured by ELISA. WT and Cblb BMDMs were also infected with A. fumigatus conidia (MOI = 1:1) for the times indicated, TNF-α and IL-6 in the supernatant were measured by ELISA.

For detection of serum IL-17, IFN-γ, IL-6, TNF-α and IL-1β, WT, Cblb or CblbC373A mice were infected with C. albicans (5 × 104, or 1 × 106 CFU for some experiments), sera were collected at different time-points, and subjected for ELISA analysis. The kidneys harvested at 48 h after infection were homogenized, and the supernatant was recovered following centrifugation at 15000 g for 20 min at 4 °C. The cytokines including IL-17, IFN-γ, and IL-6 in the kidney homogenates were determined by using were collected by ELISA kits according to the manufacturer’s instructions. The ELISA results were expressed as pg/g of kidney. For detection of serum IgG, IgE, and anti-ssDNA, anti-dsDNA, sera were collected from WT, Cblb or CblbC373A mice before C. albicans infection and 48 h after infection, and subjected to ELISA analysis.

Internalization of dectin-1 and dectin-2 in macrophages upon infection with C. albicans yeast and hyphae

WT and Cblb−/− BMDMs were infected with C. albicans yeast cap1 mutant (MOI: 1:1) for times indicated. The flow cytometry was then used to determine the surface expression of dectin-1 and dectin-2. For dectin-1 internalization, BMDMs from WT and Cblb−/− mice were labeled with PE-conjugated anti-dectin-1 (1:200) or anti-dectin-2 (1:200). Cells were then incubated at 37 °C for 5, 15, and 30 min. To remove uninternalized dectin-1- or dectin-2 coupled antibodies from the cell surface, half the cells from each time point were treated briefly with ice-cold acidic buffer (1% BSA at pH 3.0) and immediately neutralized in PBS containing 1% BSA and 0.5% NaN3. Both treated and untreated cells were stained with anti-F4/80 and anti-CD11b. Dectin-1 internalization was calculated with gated F4/80 and CD11b-positive cells using the formula: % of dectin-1 or dectin-2 internalization = 100 × [MFI of acid-resistant PE fluorescence (at time t)-MFI of acid-resistant PE fluorescence (at time 0)/MFI of total PE fluorescence of untreated cells].

Confocal microscopy

WT and Cblb BMDMs were attached to poly(L)lysine-coated coverslips, surface-labeled PE-conjugated anti-dectin-1 or anti-dectin-2 on ice. Labeled cells were infected with C. albicans yeast cap1 mutant or hyphae for 30 min at 37 °C to allow dectin-1 or dectin-2 internalization to occur. The cells were fixed in 1% paraformaldehyde, permeabilized in 0.05% saponin and stained with FITC-conjugated anti-LAMP-1. Imaging was performed on a Leica TCS-SP2 confocal microscope (1:100). Imaging was performed on a laser scanning confocal microscope (Flowview 1000, Olympus).

Systemic C. albicans dissemination

For survival analysis, mice were infected with C. albicans i.v. at 1-5 × 105 CFU, and monitored daily. After infection, mice were weighed and monitored daily. Mice were euthanized if they lost > 20% of their body weight. In a separate group, the kidneys were harvested 2 days after infection. The left kidneys were photographed and homogenized for enumeration of fungal burden. The right kidneys were fixed for histological analysis. The fungal burden in the kidneys, spleens, livers, and lungs was determined by CFU in kidney, spleen, liver, and lung homogenates. The fungal burden in the blood at 2 and 6 h after infection was also determined. Mice were allocated to experimental groups based upon their genotypes and randomized within their sex and age matched groups. No blinding was done in this study.

Generation of human monocyte-derived macrophages (MDM) and silencing of the Cblb gene

Human MDMs were generated as previously described 25, 26. In brief, peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from heparinized blood on Ficoll-sodium diatrizoate gradients and then cultured for 5 days in RPMI containing 20% autologous serum (2.0 × 106 mononuclear cells/ml) at 37 °C. On Day 5 human MDMs were transfected with control siRNA or Cblb siRNA (100 or 200 nM) by using Lonza nucleofector reagent and plated in RPMI 1640 containing 20% autologous serum. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from heparinized blood on Ficoll-sodium diatrizoate gradients and then cultured for 5 days in RPMI containing 20% autologous serum (2.0 × 106 mononuclear cells/ml) at 37 °C. On Day 5 human MDMs were transfected with control siRNA or Cblb siRNA (100 or 200 nM; Dharmacon RNA Technologies) by using Lonza nucleofector reagent and plated in RPMI 1640 containing 20% autologous serum. After 36 h, the MDMs were washed and infected with yeast or hyphae of C.albicans. The protocol was approved by The Ohio State University Institutional Review Board.

In vivo delivery of Cblb siRNA

WT mice were injected with treated with C. albicans i.v. at 5 × 105 CFU, and 24 h later were treated with in vivo grade Cblb siRNA (5’-AAAUUCUCGAAGUAUGCUCUU-3’) or a non-sense siRNA (2 mg/kg/mouse) (Dharmacon RNA Technologies) in In vivo-jetPEI®-FluoF (Polyplus-transfection, Inc.; New York, NY) via tail vein injection. Three days later, the spleen cells are collected, and lysed in RIPA buffer. The cell lysates were blotted with anti-CBLB and anti-ACTIN, respectively.

Data analysis and statistic analysis

Differences in concentrations of cytokines and fungal burden were analyzed using the Student’s t test. Survival data were analyzed using the Kaplan-Meyer log rank test. Differences were considered significant at a P value of < 0.05. No animals were excluded from the analysis. Mice were allocated to experimental groups based upon their genotypes and randomized within their sex and age matched groups. No statistical method was used to predetermine sample size. It was assumed that normal variance occurs between experimental groups.