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. Author manuscript; available in PMC: 2012 Dec 15.
Published in final edited form as: Cell Host Microbe. 2011 Dec 15;10(6):603–615. doi: 10.1016/j.chom.2011.10.009

The β-glucan receptor Dectin-1 activates the integrin Mac-1 in neutrophils via Vav protein signaling to promote Candida albicans clearance

Xun Li 1, Ahmad Utomo 1,3, Xavier Cullere 1, Myunghwan Mark Choi 2, Danny A Milner Jr 1, Deepak Venkatesh 1, Seok-Hyun Yun 2, Tanya N Mayadas 1
PMCID: PMC3244687  NIHMSID: NIHMS338313  PMID: 22177564

SUMMARY

Resistance to fungal infections is attributed to engagement of host pattern-recognition receptors, notably the β-glucan receptor Dectin-1 and the integrin Mac-1, which induce phagocytosis and antifungal immunity. However, the mechanisms by which these receptors coordinate fungal clearance are unknown. We show that upon ligand binding, Dectin-1 activates Mac-1 to also recognize fungal components and this stepwise process is critical for neutrophil cytotoxic responses. Both Mac-1 activation and Dectin-1- and Mac-1-induced neutrophil effector functions require Vav1 and Vav3, exchange factors for RhoGTPases. Mac-1- or Vav1,3-deficient mice have increased susceptibility to systemic candidiasis that is not due to impaired neutrophil recruitment but defective intracellular killing of C. albicans yeast forms, and Mac-1 or Vav1,3 reconstitution in hematopoietic cells restores resistance. Our results demonstrate that antifungal immunity depends on Dectin-1-induced activation of Mac-1 functions that is coordinated by Vav proteins, a pathway that may localize cytotoxic responses of circulating neutrophils to infected tissues.

INTRODUCTION

Infections by opportunistic pathogens are becoming increasingly prevalent due to the growing use of immunosuppressive therapies and acquired immunodeficiency. Seventy five percent of invasive fungal infections are attributed to the otherwise commensal fungi Candida albicans (C.albicans) and result in 40-50% mortality (De Rosa et al., 2009). Host resistance relies on neutrophil and macrophage mediated fungal recognition and uptake, generation of reactive oxygen species (ROS) and release of proteases that promote fungal killing. These functions, in addition to cytokines and chemokines produced by cells of adaptive and innate immunity keep fungal infections under control (Netea et al., 2008).

C.albicans recognition by neutrophils is attributed to the pattern recognition receptors Dectin-1, Toll-like receptors (TLR) and the CD18 integrin Mac-1 (Netea et al., 2008). Dectin-1 binds to β1,3-glucan, the most abundant polysaccharide in fungal pathogens and is required for fungal resistance in mice (Saijo et al., 2007; Taylor et al., 2007) and humans (Ferwerda et al., 2009). It promotes phagocytosis and ROS generation, and produces cytokines in collaborative signaling with TLRs (Netea et al., 2008). Mac-1 (CD11b/CD18, complement receptor 3), a member of the CD18 family of integrins present on neutrophils, macrophages and other leukocyte subsets, binds and phagocytoses complement-opsonized targets. It also binds β-glucan and mannose structures (Thornton et al., 1996), interacts with C.albicans and internalizes unopsonized zymosan (Ross, 2000), a particulate β-glucan and mannan rich yeast cell wall extract (Di Carlo and Fiore, 1958). Unlike Dectin-1 and TLRs, Mac-1 on circulating neutrophils requires activation via “inside-out signaling” to engage its ligands (Hynes, 2002). β-glucan has been reported to bind directly to the membrane proximal lectin domain of Mac-1 to switch its ligand binding I domain into an active state (Vetvicka et al., 1996). The tight regulation of the activity of this integrin, known to support neutrophil recruitment and trigger cytotoxic responses, serves to localize the neutrophil's responses and avoid systemic inflammation (Hynes, 2002; Ross, 2000). The relative contribution of Mac-1 to host defense against fungal pathogens is debated. In vitro, there are competing views on the importance of Dectin-1 versus Mac-1 in fungal pathogen recognition and uptake by neutrophils (van Bruggen et al., 2009). Incongruous results on Mac-1's function in fungal clearance in vivo have also been reported (Romani et al., 2004; Soloviev et al., 2011).

Dectin-1 signaling is initiated by phosphorylation of its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)-like sequence. Despite recent progress in understanding molecular mechanisms regulating cytokine regulation (Kerrigan and Brown, 2010), signaling pathways responsible for uptake and intracellular killing of C.albicans in macrophages are largely undefined. Even less is known about these pathways in neutrophils, the major effector cells early in systemic candidiasis in humans (Ferwerda et al., 2009; Spellberg et al., 2006) and mice (Tuite et al., 2004). In neutrophils, parallels may exist between signaling through Dectin-1 and other ITAM containing receptors such as FcγRs, receptors for IgG. Phosphorylation of the ITAM motifs of FcγRs triggers the assembly of protein complexes containing Syk and Src-family tyrosine kinases. The sequence of events thereafter involves tyrosine phosphorylation of downstream targets of these kinases including Vav, a guanine exchange factor (GEF) for Rho GTPases, phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLCγ) which triggers a Ca2+ flux and adaptor proteins (e.g. SLP-76). These events culminate in the phosphorylation of phox components of the NADPH oxidase and cytoskeletal changes required for reactive oxygen species (ROS) generation and phagocytosis (Berton et al., 2005).

Here we show that Dectin-1 induces Mac-1 activation and this is required for neutrophil cytotoxic responses, which challenges the view that Mac-1 is directly activated by zymosan (Ross, 2000). Mac-1 activation and downstream responses to zymosan required Vav1,3 proteins and their signaling pathways. The physiological importance of these players was evaluated in a murine model of systemic C. albicans blastoconidia infection. Mac-1 and Vav proteins in circulating hematopoietic cells were essential for fungal clearance after neutrophil recruitment thus placing Mac-1 proximal to actual fungal clearance. We postulate that as with G-protein coupled chemokine receptors (GPCR), which trigger integrin activation through Rap GTPases to localize neutrophil recruitment to sites of inflammation (Sanchez-Madrid and Sessa, 2010), pathogen recognition receptors such as Dectin-1 activate Mac-1 through Vav proteins to localize neutrophil cytotoxic responses towards pathogens in host defense.

RESULTS

Dectin-1 activates Mac-1 in neutrophils and both collaborate in responses to fungal components

A read-out of integrin activation is their rapid and inducible binding to ligands (Hynes, 2002). Mac-1 activation can be assayed by the inducible binding of neutrophils to red blood cells opsonized with the Mac-1 ligand complement fragment C3bi (C3-RBC) (Wright and Meyer, 1986). Murine neutrophils bound C3-RBC following treatment with phorbol myristate acetate (PMA) (Fig 1A), which globally activates integrins (Bertram and Ley, 2011). The lack of RBC rosetting in PMA-treated Mac-1-deficient (Mac-1–/–) neutrophils validated this assay as a bonafide read-out of Mac-1 activation (Fig. 1A). Zymosan treatment resulted in rapid C3-RBC rosetting in murine (Fig. 1B) and human neutrophils (Fig. S1A). In contrast, an 80% reduction in zymosan-induced RBC rosette formation was observed in Dectin-1-deficient (Dectin-1–/–) neutrophils (Fig. 1B). Zymosan led to GTP-loading of Rap1GTPase (see Fig. 3A), known to be required for “inside-out” activation of integrins such as Mac-1 (Dupuy and Caron, 2008). Together these data indicate that Dectin-1 engages intracellular signals that trigger Mac-1 activation.

Figure 1. Dectin-1 activates Mac-1 in neutrophils and both collaborate in responses to fungal components.

Figure 1

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(A) Wild-type (WT) and Mac-1-/- neutrophils were stimulated with PMA and presented with IgM (negative control) or IgM-iC3b coated RBCs (Bar= 25μm). (B) Analysis of Mac-1 activation. WT and Dectin-1-/- neutrophils were incubated with IgM-iC3b RBCs in the absence (control) or presence of zymosan. (C) Neutrophils (PMN) from Dectin-1-/-, Mac-1-/- or MyD88-/-/TRIF-/- mice and their respective wild-type counterparts were incubated with FITC-labeled zymosan ±10mM MgCl2 (Mg2+). Left panel: Cells at 37°C were evaluated for internalized fluorescent particles. Right panel: Binding of FITC-labeled zymosan was evaluated by FACS analysis in cells at 4°C. (D) Zymosan phagocytosis in Mac-1-/- and WT macrophages (MΦ) was assessed as described for neutrophils. Data represent mean (±s.e.m.) of 3-4 independent experiments in (A-D). (E) Zymosan induced ROS was analyzed in Dectin-1-/-, Mac-1-/-, MyD88-/-/TRIF-/- and WT neutrophils. A graph of the mean (±s.e.m) of the relative light units (RLU) peak value for samples normalized to WT cells is shown. (F) WT and Mac-1-/- neutrophils were treated with zymosan in the presence of dectin-1 antibody 2A11 or IgG control, and real-time ROS generation was monitored. Representative ROS profiles and a graph, plotted as described in E) is shown. The reduction in ROS in Mac-1-/- neutrophils following IgG isotype control treatment was less significant than without (E) and may result from the engagement of FcγRs by the antibody. (G) ROS generation was monitored in WT and Mac-1-/- neutrophils treated with zymosan in the presence or absence of laminarin. Data are shown as in F. *, p<0.05, **, p<0.01; ***, p<0.001 (one-way or two-way ANOVA with Bonferroni post-test (B,C,E,F,G); unpaired t-test (A,D)). See also Figure S1.

Figure 3. Vav is dispensable for zymosan induced Rap GTPase activation but is required for ITAM-based signals.

Figure 3

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Wild-type (WT) and Vav1,3-deficient (V1,3-/-) neutrophils were stimulated with zymosan for times given in minutes (min) and cell lysates were prepared. (A) Western blots of GTP-loaded Rap1 (Rap1GTP; activated Rap1) as compared with total Rap1 and β-actin. (B) Western blot analysis of phosphorylation (p-) of Syk and Src, PLCγ2, PAK1/2, p40(phox), MAPK and Pyk2. β-actin served as a loading control. (C) Dectin-1-/- and Mac-1-/- neutrophils were analyzed similarly to that described in B). For all western blots representative data from 1 of 3 experiments is shown. (D) Indo-1 preloaded wild-type and Vav1,3-/- neutrophils were stimulated with zymosan (arrow). Ca2+ flux was evaluated by measuring a ratio of fluorescence signal at 405nm to 485nm over time. Data shown is a representative profile of the percentage of responding cells as a function of time calculated. Mean (±s.e.m) of the maximum percentage of responding cells from 4 independent experiments is given. *, p<0.05 (unpaired t-test). (E) Western blot analysis as described in B) of wild-type cells stimulated with zymosan and pretreated without (-) or with an inhibitor to PLCγ (U73122) or a chelator of intracellular Ca2+ (BAPTA). See also Figure S2.

The importance of Dectin-1 and Mac-1's relationship was explored in the context of phagocytosis of zymosan. Zymosan phagocytosis was reduced by >90% in both Dectin-1–/– (Fig. 1C)(Taylor et al., 2007) and Mac-1–/–neutrophils (Fig. 1C), while MyD88/TRIF-deficient (MyD88/TRIF–/–) neutrophils, which lack signaling through all TLRs, exhibited no defect (Fig. 1C). Phagocytosis of serum-opsonized zymosan that leads to C3 deposition, a known Mac-1 ligand, was also Mac-1 dependent (Fig. S1B). Zymosan phagocytosis in Dectin-1–/– neutrophils was evaluated in the presence of high concentrations of Mg2+. This switches the integrin to an active conformation by occupying a metal ion coordinating site in the ligand binding I-domain and thus bypassing the need for “inside-out” activating signals to induce ligand binding (Humphries, 1996; Hynes, 2002). Mg2+ enhanced phagocytosis in wild-type cells and rescued phagocytosis of unopsonized zymosan in Dectin-1–/–neutrophils (Fig. 1C) indicating that defective phagocytosis in Dectin-1–/– neutrophils, is due to lack of Mac-1 activation. Mg2+ did not trigger zymosan phagocytosis in Mac-1–/– neutrophils (Fig. 1C) thus showing the utility of Mg2+ treatment in activating Mac-1. A soluble competing β-glucan reduced Mg2+ induced zymosan phagocytosis in Dectin-1-/- neutrophils (Fig. S1C) thus demonstrating that Mac-1's β-glucan binding activity contributes to zymosan uptake and is responsive to inside-out signaling. Other zymosan polysaccharides described as Mac-1 ligands (Thornton et al., 1996) may contribute to the remaining binding activity. Consistent with Dectin-1 being a major β-glucan receptor, zymosan recognition by neutrophils relied primarily on Dectin-1 as particle binding was markedly reduced only in Dectin-1-/- and not Mac-1 or MyD88/TRIF-/- neutrophils (Fig. 1C). Although a fraction of zymosan remained bound in Dectin-1-/- neutrophils (Fig. 1C) as also previously described (Taylor et al., 2007), this fraction was not internalized (Fig. 1C). On the other hand, Mg2+ induced Mac-1 activation in Dectin-1-/- neutrophils triggered uptake of this surface bound zymosan (Fig. 1C) demonstrating that Dectin-1 and Mac-1 are the two primary phagocytic receptors on neutrophils. In contrast, zymosan phagocytosis by peritoneal macrophages was unaffected by Mac-1 deficiency (Fig. 1D). The altered requirement for Mac-1 in macrophages may be attributed to the finding that besides Dectin-1, macrophages additionally express Dectin-2 (Fig. S1D).

Next, the relative contribution of Dectin-1, Mac-1 and TLRs to the neutrophil respiratory burst in response to zymosan was assessed. Zymosan induces the release of extracellular and intracellular ROS (Fig. S1E). Dectin-1-/- neutrophils did not generate ROS in response to zymosan (Fig. 1E) as published (Boyle et al., 2011; Taylor et al., 2007) while Mac-1-/- neutrophils exhibited a partial reduction as did MyD88/TRIF-/- neutrophils (Fig. 1E). On the other hand, Dectin-1 functional blocking monoclonal antibody 2A11 (Fig. 1F) or laminarin (competing soluble β-glucan)(Fig. 1G) treatment of zymosan stimulated wild-type neutrophils resulted in reduced but detectable ROS. Under these conditions, ROS was abolished only when Mac-1 was also absent (Fig. 1F, G). A similar interplay between Dectin-1 and Mac-1 was shown following treatment with curdlan (Fig. S1F), a soluble polymer of β1,3-glucan and Dectin-1 agonist (Gringhuis et al., 2009). This infers that although Dectin-1 is the major receptor for ROS production under conditions of low receptor or ligand density cross-talk between Dectin-1 and Mac-1 is required for optimal ROS generation. That is, Dectin-1 that remains functional following 2A11 treatment is likely sufficient to activate Mac-1 and subsequent ROS production.

Vav proteins regulate Dectin-1 induced Mac-1 activation, phagocytosis and the respiratory burst

Vav proteins are activated downstream of integrins, immune response receptors, receptor tyrosine kinases and GPCRs. Zymosan triggered Vav-Tyr174 phosphorylation, a signature of Vav activation (Tybulewicz, 2005) in wild-type neutrophils (Fig. 2A). This did not occur in Dectin-1-/- neutrophils (Fig. 2A) while it was intact in Mac-1-/- neutrophils indicating that Vav activation by zymosan is downstream of Dectin-1. Neutrophils deficient in Vav1 and Vav3, the two major Vav proteins in neutrophils (Gakidis et al., 2004) failed to activate Mac-1 in response to zymosan as assessed in the C3-RBC rosetting assay (Fig. 2B) even though surface expression of Dectin-1 and Mac-1 was normal (Fig. 2B). Moreover, PMA promoted C3-RBC rosetting in both wild-type and Vav1,3-deficient (Vav1,3-/-) neutrophils (Fig. 2B), demonstrating that Vav is not required for C3-RBC binding per se. Next, pharmacological inhibitors were used to examine three potential pathways engaged by Dectin-1-Vav to activate Mac-1: Non-receptor tyrosine kinases, Ca2+ dependent pathways and PI3K mediated signaling. We found that Syk, Src, PLCγ and intracellular Ca2+ were essential for zymosan induced C3-RBC rosetting while PI3K and Mitogen-activated protein kinase (MAPK-ERK) were dispensable for this process (Fig. 2C), even though inhibitors of these molecules blocked zymosan induced ROS generation (data not shown). Vav1,3-/- neutrophils failed to uptake zymosan or complement opsonized zymosan (Fig. 2D) despite normal zymosan binding (Fig. 2D). Like Mac-1, Vav's role was specific for neutrophils as zymosan phagocytosis was normal in Vav 1,3-/- macrophages (Fig. 2E). Finally, Vav1,3-/- neutrophils had no detectable ROS generation in response to zymosan (Fig. 2F) or curdlan (Fig. S2A).

Figure 2. Vav activated by Dectin-1 is required for Mac-1 activation and function.

Figure 2

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(A) Lysates of WT, Dectin-1-/- and Mac-1-/- neutrophils stimulated with zymosan for the times in minutes were analyzed for Vav-Tyr174 phosphorylation. (B) iC3b-RBC rosetting in wild-type and Vav1,3-/- neutrophils following treatment with vehicle control, zymosan or PMA. Representative pictures and a graph of the mean (±s.e.m) of 3 experiments are shown. FACS analysis of Dectin-1 and Mac-1 expression in wild-type (solid line) and Vav1,3-/- (dotted line) neutrophils compared to IgG isotype control is shown (C) C3-RBC rosetting was analyzed in unstimulated (-) and zymosan-stimulated wild-type cells treated with vehicle control (DMSO) or inhibitors of Syk (Piceatannol), Src (PP2), PLCγ (U73122), PI3K (PI-103, LY294002) or ERK (U0126) or a chelator of intracellular Ca2+ (BAPTA). Mean (±s.e.m) of results is given. n.d.= non-detectable (D) Upper left and bottom panel: Phagocytosis of FITC-labeled unopsonized or iC3b opsonized zymosan by WT and Vav1,3-/- neutrophils (PMN). Representative pictures are shown for unopsonized zymosan (Bar= 25μm). Upper right panel: Binding of FITC-labeled zymosan was evaluated by FACS analysis. Mean (±s.e.m) of results is given. (E) Zymosan phagocytosis in WT and Vav1,3-/- macrophages (MΦ). Mean (±s.e.m) of results is given. (F) Respiratory burst of WT and Vav1,3-/- neutrophils in response to zymosan. Representative ROS profiles and graph of the mean (±s.e.m) of the relative light units (RLU) peak value for samples (inset) is shown. n=5 independent experiments. ***, p<0.001 (one-way or two-way ANOVA with Bonferroni post-test (B,C,D upper left panel); one sample t-test (F); unpaired t-test (D upper right panel)). See also Figure S2.

Fungal components activate Vav-dependent signals in neutrophils

We sought to establish signaling events downstream of Dectin-1 and Mac-1 that are Vav dependent. As Vav was required for Dectin-1 mediated integrin activation we first evaluated whether zymosan induced Rap GTPase activation and whether this required Vav proteins. Zymosan led to GTP-loading of Rap GTPase which was similar in wild-type and Vav1,3-/- neutrophils (Fig. 3A) indicating that Vav is not essential for this step.

Zymosan induced phosphorylation of Src and Syk kinases, events proximal to Dectin-1 and Mac-1 receptors (Lowell, 2010) was largely intact in Vav1,3-/- neutrophils (Fig. 3B). Recruitment and activation of Vav downstream of classical ITAM receptors in neutrophils, is associated with activation of phospholipase C (PLCγ) to inositol triphosphate (IP3)-induced Ca2+ release, Rac GTPase to p21-activated kinase (PAK) activation, phosphorylation and membrane localization of NADPH oxidase components such as p40(phox), and activation of MAPK (Jakus et al., 2009; Utomo et al., 2006). Engagement of Dectin-1 by zymosan led to the activation of PAK, p40(phox) and the MAPK-ERK as well as pyk-2/RAFTK, a tyrosine kinase related to focal adhesion kinase. Notably, Vav 1,3-/- neutrophils were profoundly defective in each of these responses (Fig. 3B), as were Dectin-1-/- and Mac-1-/- neutrophils (Fig. 3C), suggesting that Mac-1 and Dectin-1 are upstream of Vav. Further analysis revealed that the Ca2+ flux in Vav 1,3-/- cells in response to zymosan was significantly impaired (Fig. 3D), consistent with a defect in PLCγ activation. Notably, activation of the Ca2+-sensitive tyrosine kinase pyk-2, placed downstream of Mac-1-dependent adhesive events (Fuortes et al., 1999), was also Vav1,3 (Fig. 3B) and PLCγ-Ca2+ (Fig. 3E) dependent. In summary, the Vav-PLCγ-Ca2+ axis may represent a key hub in integrating signaling pathways through Dectin-1 and Mac-1. Consistent with this, inhibition of PLCγ abolished zymosan induced ROS (Fig. S2B).

Protection against C.albicans requires Mac-1

The physiological role of Mac-1 in clearance of C.albicans blastoconidia was assessed as this process is Dectin-1 dependent(Taylor et al., 2007). Infection with 5×104 colony forming units (CFU)(Fig. 4A) of C.albicans yeast forms resulted in increased mortality in Mac-1-/- mice compared to wild-type animals. The kidney is the chief target organ of C.albicans (Brieland et al., 2001), particularly in neutropenic mice (Fulurija et al., 1996). A log-fold increase in CFU was recovered from kidneys of Mac-1-/- compared to wild-type animals (Fig. 4B) that was associated with increased number of abscesses and organ enlargement (Fig. 4B), coalescing abscesses containing fungal elements and significant neutrophil infiltration. In contrast, abscesses were virtually absent in wild-type animals (Fig. 4C).

Figure 4. Mac-1 is required for resistance to systemic C.albicans infection.

Figure 4

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(A) Mac-1-/- mice and their wild-type cohorts (n=8 in each) were infected intravenously with 5×104 C.albicans and monitored daily for survival. P=0.0011 (log-rank test) (B) Kidneys were harvested at day 4 after infection for gross morphology and visual inspection of abscesses (arrow) (Left panel), and for quantitation of fungal burden in kidney homogenates (Right panel). Each symbol represents data from an individual animal. (C) A representative photomicrograph of periodic acid Schiff (PAS)-stained kidney sections from wild-type and Mac-1-/- mice is shown. Within kidney tissues from Mac-1-/- mice, large collections of neutrophils (abscess formation) surrounded by mixed inflammatory cells are present with abundant fungal forms, which are virtually absent in kidneys from wild-type mice. (D) Kidneys harvested from mice at 16 and 48hrs after infection were homogenized and myeloperoxidase enzyme (MPO) activity was determined and normalized to total protein content. Mean (±s.e.m) of results is given. (E) Confocal intravital microscopy of wild-type and Mac-1-/- mice at the indicated times after injection with live GFP-labeled C.albicans. Mice were also given rhodamine-dextran to delineate blood vessels and anti-Gr-1 to identify neutrophils. Left panels: Representative pictures show neutrophils (blue), blood vessels (red) and C.albicans fungal elements (green). Right panel: Quantitation of percent of neutrophils outside of blood vessels. Data represent mean (±s.e.m.) of 3 independent experiments (F) Analysis of intracellular killing of live unopsonized C.albicans by neutrophils and macrophages. Data represent mean (±s.e.m.) of four experiments. (G) Kidney fungal burden at day 4 in wild-type, CD18-deficient (CD18-/-) and CD18-/- mice with human CD18 restored in neutrophils (hCD18+/mCD18-/-). (H) Renal neutrophil accumulation was evaluated in wild-type and CD18-/- mice at indicated time points after C.albicans infection. Data represent mean (±s.e.m.) of 3 independent experiments **, p<0.01; ***, p<0.001 (unpaired t-test (F); Mann-Whitney test (B,G)). See also Figure S3 and Movie S1.

The increase in susceptibility to infection could not be attributed to a decrease in renal neutrophil accumulation as assessed in kidney homogenates (Fig. 4D). In tissue sections, neutrophil foci were observed in the interstitium of the cortex and scattered cells were observed in the medulla of both wild-type and Mac-1-/- renal samples (Fig. S3A). To rule out trapping of neutrophils within interstitial vessels in Mac-1-/- mice, a more detailed analysis of renal neutrophil accumulation was pursued in real-time in the kidney cortex using laser-scanning confocal intravital microscopy. At 4 and 9 hrs after infection no C.albicans or extravascular neutrophils were detected (Movie S1). At 16hrs after infection, when neutrophil accumulation was measurable in kidney homogenates (Fig. 4D), reproducible C. albicans colonization in the cortex was observed. In this area, greater than >80% of neutrophils in wild-type and Mac-1-/- mice were extravascular (Fig. 4E)(Movie S1). Notably, at sites of colonization and neutrophil infiltration the vasculature was damaged as rhodamine-dextran perfusion (a readout of capillary patency) was diminished in these areas.

Mac-1-/- neutrophils failed to kill unopsonized C.albicans ex vivo (Fig. 4F). This was specific for neutrophils as macrophages lacking this integrin exhibited normal intracellular killing (Fig. 4F). Thus Mac-1 is dispensable for renal neutrophil accumulation following fungal infection but is essential for anti-fungal cytotoxic functions of tissue-recruited neutrophils. Next, we compared CD18-deficient mice with the same expressing human CD18 selectively in neutrophils. The latter reinstates expression of CD18 integrins including Mac-1 on neutrophils to 50% of wild-type levels and markedly restores zymosan phagocytosis (Fig. S3B-S3E). As observed with Mac-1-/- mice, the kidney fungal burden was much higher in CD18-null mice compared to wild-type counterparts (Fig. 4G) that was not a consequence of defective renal neutrophil recruitment (Fig. 4H). Importantly, restoration of CD18 in neutrophils resulted in a significant reduction in fungal burden compared to CD18-/- mice (Fig. 4G). These data reveal the importance of neutrophil CD18 in fungal resistance at a step downstream of neutrophil recruitment.

Protection against C.albicans requires Vav proteins

The importance of Vav proteins in C.albicans clearance was evaluated. Inoculation with 5×104 colony forming units (CFU) resulted in significant mortality in Vav1,3-/- mice compared to wild-type counterparts (Fig. 5A) as did 1×105 CFU (Fig. S4). Results in Vav1,3-/- mice phenocopied Mac-1-/- mice including an increase in kidney fungal burden (Fig. 5B), the presence of renal abscesses containing fungal elements (Fig. 5C) and robust neutrophil accumulation at all time points tested (Fig. 5D and Fig 5E). Ex vivo, Vav1,3-/- neutrophils failed to kill C. albicans (Fig. 5F) while macrophages deficient in Vav1,3 retained this function (Fig. 5F). The importance of Vav specifically in hematopoietic cells in antifungal resistance in vivo was tested by generating mice chimeric for Vav1,3 expression by bone marrow transplantation. Lack of Vav in circulating hematopoietic cells led to increased kidney fungal burden that was comparable to mice lacking Vav in all cells while a deficiency in Vav only in resident tissues were relatively resistant to infection (Fig. 5G).

Figure 5. Vav1,3 proteins are required for resistance to C.albicans infection.

Figure 5

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(A) Survival curves of wild-type and Vav1,3-deficient mice infected intravenously with 5×104 CFU of C.albicans (n=8 in each). P=0.0009 (log-rank test) (B) Images of kidneys harvested from wild-type and Vav1,3-/- mice 4 days after infection (Left panel) and quantification of fungal burden in kidney homogenates (Right panel). (C) A representative photomicrograph of PAS-stained renal sections from wild-type and Vav1,3-/- mice is shown. (D) Evaluation of tissue neutrophil accumulation in kidney at the indicated days after infection. Mean (±s.e.m) of results is given. (E) Laser-scanning confocal intravital microscopy of wild-type and Vav-1,3-/- mice as described in Fig. 4E. Data represent mean (±s.e.m.) of 3 independent experiments (F) Analysis of intracellular killing of live unopsonized C.albicans by neutrophils and macrophages. Data represent mean (±s.e.m.) of 3 independent experiments. (G) Wild-type (WT) and Vav 1,3 deficient (V1,3-/-) recipient mice were reconstituted with bone marrow from wild-type or Vav1,3-/- donors. At day 4 after C.albicans innoculation, the CFU in kidney homogenates was determined. **, p<0.01; ***, p<0.001 (unpaired t-test (F); Mann-Whitney test (B,G)). See also Figure S4.

DISCUSSION

An absence of Dectin-1 or its effector Card9 increases incidences of mucocutaneous candidiasis in patients (Ferwerda et al., 2009; Glocker et al., 2009). Moreover, Leukocyte adhesion deficiency I (LADI) patients that lack expression of Mac-1 and other members of the CD18 family have recurrent localized and disseminated Candidal and Aspergillus infection (Lekstrom-Himes and Gallin, 2000) that correlates with a failure of their neutrophils to respond to zymosan (Ross et al., 1987). Our results demonstrate that Dectin-1 activation of Mac-1 on recruited neutrophils is essential for neutrophil phagocytic functions associated with fungal clearance and likely serves as an early amplification loop to localize and contain fungal pathogens (Figure 6). The lack of invasive candidiasis in patients lacking Dectin-1 and Card9 may be because in the absence of Dectin-1, Mac-1 activation is superseded by other pattern recognition receptors in an evolving infection. In support of this, Mac-1 activation by Mg2+ restored phagocytosis in Dectin-1-/- neutrophils. Moreover, Dectin-1 deficient human neutrophils were reported to exhibit normal yeast phagocytosis in the presence of serum and under adherent conditions (Ferwerda et al., 2009), both of which have the ability to activate Mac-1 independently of Dectin-1. In addition to serving as a pattern recognition receptor, Mac-1 binds complement iC3b-opsonized targets. Under opsonizing conditions as well, Mac-1 and Vav proteins were required for zymosan uptake. The Dectin-1 to Mac-1 pathway described herein maybe specific for the yeast forms of C.albicans prevalent in human invasive candidiasis (Larone, 2002) as the molecular mechanisms involved in clearance of hyphae and conidia fundamentally differ. For example, although yeast forms engage Dectin-1, hyphae forms shield β-glucans by mannans thus preventing activation of Dectin-1 signaling pathways (Netea et al., 2008). The molecular mechanisms of clearance may also diverge as hyphae trigger neutrophil spreading and extracellular release of ROS while conidia fungal forms promote phagocytosis induced ROS (Boyle et al., 2011). In showing the specificity of the Dectin-1-Vav-Mac-1 axis for neutrophil but not macrophage mediated anti-fungal activities our results suggest that this mechanism evolved to localize cytotoxic responses of circulating neutrophils to infected tissues. There are potentially broad implications of this paradigm in neutrophil mediated host defense.

Figure 6. Model of neutrophil mediated anti-fungal cytotoxicity.

Figure 6

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C.albicans blastoconidia (yeast) engage macrophages, which generate cytokines that activate the endothelium to recruit circulating neutrophils. Engagement of Dectin-1 on transmigrated neutrophils leads to phosphorylation (-P) of its ITAM by Src kinases, and recruitment and phosphorylation of Syk kinase leading to Vav/PLCγ activation and Ca2+ release. These steps result in Mac-1 activation and it's binding to C.albicans. Ligand-bound Mac-1 recruits p-Vav and contributes to the Dectin-1 initiated signaling complexes leading to activation of downstream targets including Pyk-2, PAK and ERK. These events together promote cytoskeletal rearrangement required for phagocytosis and activation of the NADPH oxidase.

Before the identification of Dectin-1, Mac-1 was considered the major fungal recognition receptor on phagocytes in vitro (Ross, 2000). This was reiterated in a recent study that reported the dominance of Mac-1 in fungal responses in human neutrophils; however, Dectin-1 function was only partially blocked (60-70%) using anti-hDectin-1 antibody (van Bruggen et al., 2009). In light of our results, it is plausible that zymosan engagement of the remaining Dectin-1 is likely sufficient, and required to activate Mac-1. Integrin activation is associated with alterations in its conformation leading to changes in monomeric affinity, which is mediated by Rap GTPase dependent signaling. It also correlates with integrin clustering that alters the number of bonds that it can form, which is regulated by the cytoskeleton (Hynes, 2002). Although Dectin-1 induced Mac-1 activation was Vav1,3 dependent, Rap1 GTPase activation was not sufficient for integrin activation in the absence of Vav. Thus we postulate that Vav is not required for changes in the affinity of Mac-1 following zymosan stimulation but may be responsible for PLCγ induced Ca2+ flux and cytoskeletal changes required for integrin clustering. Indeed, inhibition of PLCγ or Ca2+ abolished zymosan induced Mac-1 activation.

Both Dectin-1 and Mac-1-deficient mice exhibit decreases in survival following C.albicans infection. In Dectin-1-/- animals, this has been attributed to a decrease in neutrophil recruitment albeit the analysis was done following acute infection in the peritoneum (Taylor et al., 2007) and not in the infected kidney, the primary target organ of infection and predictor of mortality in invasive candidiasis (Brieland et al., 2001; Odds, 1988). A deficiency in Mac-1, all CD18 integrins or Vav 1,3 didn't impact renal neutrophil influx. Abundant extravasated neutrophils in proximity to C.albicans in the renal interstitium of both Mac-1-/- and Vav1,3-/- mice. Thus Mac-1 and indeed CD18 integrins are not required for renal neutrophil recruitment suggesting that recruitment to the specialized vasculature of this organ has distinct requirements for leukocyte adhesion receptors (Mayadas et al., 1999). The lack of effect of Mac-1 deficiency on neutrophil recruitment contrasts with the conclusion in the C.albicans hyphae model. However, in this case as well, recruitment was examined in the peritoneum after local introduction of hyphae (Soloviev et al., 2011). Despite normal neutrophil recruitment in Mac-1-/- and Vav1,3-/- mice, a significant increase in fungal colonization in the kidney that correlated with defects in C.albicans intracellular killing was observed. Together our results suggest a model wherein Dectin-1 induced Mac-1 activation via Vav proteins in renal-accumulated neutrophils promotes the phagocytic clearance of C.albicans (Figure 6). Contrary to our study, a previous study showed a small reduction in fungal burden in the kidney in Mac-1-/- mice following C.albicans blastoconidia infection although survival was not specifically measured (Romani et al., 2004). The analysis on a mixed background mouse strain and use of a different fungal strain as that used in the current study may account for such a disparity (Dostert and Tschopp, 2007).

Vav activated by Dectin-1 is pivotal in relaying signals from fungal components. Syk and src kinases are activated by zymosan and serve as docking sites for Vav proteins, which in other cell types, are key hubs in signaling to downstream effectors that link to cytoskeletal rearrangements. Vav-Tyr174 phosphorylation may facilitate stepwise relief of its autoinhibition leading not only to GEF activity but also allowing it to interact with other molecules to form a functional signaling complex (Bustelo, 2000). Vav phosphorylation was observed in Mac-1-/- neutrophils yet the phosphorylation of molecules anticipated to be downstream of Vav such as PLCγ or PAK was suppressed. One explanation is that signaling downstream of Mac-1 involves a signaling pathway other than the Vav-PLCγ axis. However, it is also likely that in addition to phosphorylation, Vav's localization into a microsignalosomes (Weber et al., 2008) that may be supported by integrin containing lipid rich domains is required for the recruitment of signaling molecules such as PLCγ (Nakayama et al., 2008; Rodriguez et al., 2003) and PAK (Krautkramer et al., 2004). Our work suggests that Vav controls Ca2+ release. Reconstitution of Vav-null T cells with Vav1 lacking its GEF domain rescues several T-cell receptor responses including Ca2+ mobilization (Miletic et al., 2009). By extension, both Vav's GEF and adaptor functions may play essential roles in Dectin-1-dependent signaling. The overlap of signaling pathways impaired in Mac-1 and Vav1,3-/- neutrophils and the absolute requirement for Vav proteins in zymosan induced ROS, a Dectin-1 dependent process, indicate that in addition to integrin activation, Vav1,3 are recruited into a complex that is required for signaling downstream of Dectin-1 and activated Mac-1 (Figure 6).

Mac-1 and Vav proteins were not required for phagocytosis or C.albicans killing in macrophages even though Dectin-1 contributes to phagocytosis in this cell type (Taylor et al., 2007). The differential requirement for Mac-1 may reflect a need for tighter regulation of cytotoxic functions of circulating cells such as neutrophils versus macrophages (Hynes, 2002). Dectin-1 alone may serve as a phagocytic receptor in macrophages as its stable expression in nonprofessional phagocytes can result in zymosan uptake (Herre et al., 2004). Alternatively, Dectin-2 (Saijo and Iwakura, 2011) present on macrophages but not on neutrophils may replace Mac-1's role. The dispensable role of Vav proteins in macrophages may be attributed to differential signaling requirements in these cells versus neutrophils, which is consistent with Dectin-1 signaling being cell context dependent (Goodridge et al., 2009; Herre et al., 2004).

In view of the findings that both Dectin-1 and Mac-1 have been implicated in fungal responses our data provide a mechanistic explanation for how these two receptors coordinate fungal-induced neutrophil cytotoxic responses. Importantly, we demonstrate that Dectin-1 activation of Mac-1 via Vav proteins and PLCγ/Ca2+ represents a pathway for integrin activation relevant to host defense. The in vivo findings allow us to clearly establish a role for Mac-1 and Vav 1,3 to cytotoxic responses of recruited neutrophils thus offering Vav as an attractive therapeutic target for enhancing tissue localized immunity to fungal and potentially other microbial infections.

EXPERIMENTAL PROCEDURES

Mice

Vav1,3-deficient (Vav1,3–/–) (Fujikawa et al., 2003) were backcrossed to C57BL/6J for 6 generations and maintained with similarly derived wild-type (WT) counterparts. Mac-1-deficient (Mac-1-/-)/B6F9 mice (Hirahashi et al., 2006), MyD88/TRIF (MyD88–/–TRIF–/–) deficient mice (Yamamoto et al., 2003) and Dectin-1 deficient (Dectin-1–/–) mice backcrossed to C57Bl/6 (Saijo et al., 2007) were as described and C57BL/6J wild-type mice served as their controls. All mice were bred and maintained in virus- and antibody-free facilities. Dectin-1–/– mice (Taylor et al., 2007) backcrossed to 129sv/ev were maintained as described (Parti et al., 2010) and control 129sv/ev mice for these animals were purchased from Taconic. All presented data were derived from neutrophils from Dectin-1–/– mice described in Taylor et al., 2007 (Taylor et al., 2007). The data were confirmed in neutrophils harvested from Dectin-1–/– mice described in Saijo et al, 2007 (Saijo et al., 2007) and neutrophils only from these animals were used in the zymosan binding assay. All the experiments were approved by the Harvard Medical School Animal Care and Use Committee.

Reagents and Antibodies

Curdlan, zymosan, laminarin, PMA, luminol, cytochalasin D were obtained from Sigma-Aldrich. The following Abs were used for western blot analysis: rabbit anti-p-Syk, rabbit anti-p-Src, rabbit anti-p-Akt, rabbit anti-p-PAK1/2, rabbit anti-p-Pyk2, rabbit anti-p-p40(phox) and mouse anti-p-p44/42 MAPK (Cell Signaling Technology); rabbit anti-Rap1, rabbit anti-p-Vav (Santa Cruz Biotechnology) and β-actin (Sigma). Rat anti-Dectin-1 (2A11; AbD serotec) and rat IgG control (BD Biosciences) were used in functional blocking assays.

Respiratory burst, phagocytosis and iC3b-SRBC rosetting

Mature bone marrow derived neutrophils were isolated as described (Hirahashi et al., 2006). Peritoneal macrophages were collected by lavage from mice injected intraperitoneally 4 days before with 3% thioglycollate. Peripheral blood human neutrophils from healthy donors were isolated using density gradients. Real-time ROS generation was monitored by luminol-enhanced chemiluminescence. Neutrophils (3 × 106 cells) were transferred to PBS plus Ca2+/Mg2+ containing 50μM luminol. Curdlan or zymosan was added at 100μg/ml. Dectin-1 antibody 2A11 or IgG control was added at 10μg/ml. Chemiluminescence was measured using a AutoLumat LB-953 tube luminometer (Berthold). For phagocytosis, FITC-zymosan was incubated with cells at a 10:1 ratio at 37°C for 30min. A fluorescence microscope was used to assess the percentage of cells with ≥1 internalized FITC-zymosan.

Zymosan binding assay was adopted from (Philip Taylor et al 2004). After 20min on ice, neutrophils and FITC-zymosan at 10:1 with 0.5% BSA were centrifuged at 350g and left for 1h on ice before resuspension and FACs analysis.

C3-RBC rosetting: Sheep red blood cells (RBC) (Lampire) were incubated with anti-sheep RBC IgM (Cedarlane) followed by incubation without or with C5 deficient serum (Sigma). Neutrophils were treated with PMA or zymosan for 15 min at 37°C. Cells were washed, C3-RBCs were added, the samples were spun down at 800rpm and incubated at 37°C for 15 min. After washing, the percentage of neutrophils with ≥2 rosetted RBCs were counted.

Rap1 activation and calcium flux assay

GTP-loaded Rap1 in neutrophils treated with zymosan was determined in pull-down assays as previously described (Ghandour et al., 2007) with additional 5mM Diisopropylfluorophosphate in the lysis buffer.

For Ca2+ flux, 2×107 neutrophils in PBS plus Ca2+/Mg2+ were incubated with 2uM Indo-1 AM and 0.04% F-127 (Invitrogen) at 37°C for 45 min. 2×106 cells were washed and stimulated with zymosan. The Ca2+ flux was monitored on a LSRII flow cytometer in real time for 8min. Data were analyzed with Flowjo software.

In vitro C.albicans killing assay

This assay was based on a published protocol (Johnnidis et al., 2008). 5×105 C. albicans (CA) were incubated with or without 5×105 phagocytes in 96-well plates (Falcon) in RPMI media. Surviving CA were incubated with Alamar blue (Invitrogen) at 1:10 dilution in PBS and fluorescence was measured using a SpectraMax M2 plate reader (Molecular Devices). Killing was calculated as [1- (Number of CA incubated with phagocytes)/(Number of CA incubated without phagocytes)].

In vivo model of systemic candidiasis

C.albicans (SC5314; American Type Culture Collection) were cultured and mice were infected with blastoconidia (yeast forms) essentially as described (Hirai et al., 2006; Taylor et al., 2007). 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 four days after infection. The left kidneys were photographed and homogenized for enumeration of fungal burden. The right kidneys were fixed for histological analysis. For intravital microscopy, mice were given 1×105 live GFP-labeled C. albicans (Cormack et al., 1997).

For tissue neutrophil accumulation an MPO assay was performed as previously described (Huang et al., 2004). MPO activity in supernatants of homogenized kidney tissue was measured with TMB substrate kit (Pierce). MPO protein quantity was calculated based on a recombinant mouse Myeloperoxidase standard (R&D systems). Total protein content was measured with BCA Protein Assay Kit (Pierce) and MPO quantity was normalized to protein content.

In vivo confocal microscopic imaging of neutrophils in the kidney cortex

Mice were anesthetized by intraperitoneal injection of ketamine-xylazine and intravital microscopy of the kidney was performed essentially as described (Fan et al., 2010). Briefly, mice were placed on a heated plate of a motorized x-y-z translational stage. The anti-Gr-1 antibody conjugated with Alexa 647 was injected intravenously for neutrophil staining. After surgical exteriorization of the left kidney, 70 kDa TRITC-dextran (50μl of 5% w/v) was injected intravenously to stain blood vessels. A coverslip was applied to the top of the exposed kidney and the tissue was imaged with a laser-scanning confocal microscope with a 40X 0.6 N.A. objective. Images were acquired within 1 hour after the kidney exteriorization.

Statistical analysis

ANOVA with Bonferroni post-tests was used when making multiple statistical comparisons on a single data set. Two-tailed unpaired t-test was used for analysis of two groups. For the analysis of nonparametrically distributed data, the two-tailed Mann-Whitney test was used. Survival data were analyzed with the log-rank test. Results were considered statistically significant with P values of less than 0.05.

Supplementary Material

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Highlights.

The β-glucan receptor Dectin-1 activates the integrin Mac-1 on neutrophils via Vav1,3

Dectin-1 and Mac-1 antifungal responses depend on a Vav-dependent signaling pathway

Mac-1 and Vav1,3 are essential for resistance to C.albicans blastoconidia infection

Mac-1 and Vav are required for C.albicans clearance but not renal neutrophil influx

ACKNOWLEDGEMENTS

We are grateful to Dr. Gordon D. Brown (Univ. of Aberdeen, Aberdeen, UK) for giving us permission to use materials from Dectin-1-/- mice, Dr. Yoichiro Iwakura (Instit. of Medical Science, Univ of Tokyo, Japan) for providing Dectin-1-/- mice and Dr. Brendan Cormack (John Hopkins, Baltimore, MD) for GFP-C.albicans. This work was supported by the National Institutes of Health R01 AR050800 and HL065095 (T.M.) and K01 AR054984 (X.C.), and the American Lung Association (A.U).

Footnotes

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COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

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