Phagocytes from Mice Lacking the Sts Phosphatases Have an Enhanced Antifungal Response to Candida albicans

ABSTRACT Mice lacking expression of the homologous phosphatases Sts-1 and Sts-2 (Sts−/− mice) are resistant to disseminated candidiasis caused by the fungal pathogen Candida albicans. To better understand the immunological mechanisms underlying the enhanced resistance of Sts−/− mice, we examined the kinetics of fungal clearance at early time points. In contrast to the rapid C. albicans growth seen in normal kidneys during the first 24 h postinfection, we observed a reduction in kidney fungal CFU within Sts−/− mice beginning at 12 to 18 h postinfection. This corresponds to the time period when large numbers of innate leukocytes enter the renal environment to counter the infection. Because phagocytes of the innate immune system are important for host protection against pathogenic fungi, we evaluated responses of bone marrow leukocytes. Relative to wild-type cells, Sts−/− marrow monocytes and bone marrow-derived dendritic cells (BMDCs) displayed a heightened ability to inhibit C. albicans growth ex vivo. This correlated with significantly enhanced production of reactive oxygen species (ROS) by Sts−/− BMDCs downstream of Dectin-1, a C-type lectin receptor that plays a critical role in stimulating host responses to fungi. We observed no visible differences in the responses of other antifungal effector pathways, including cytokine production and inflammasome activation, despite enhanced activation of the Syk tyrosine kinase downstream of Dectin-1 in Sts−/− cells. Our results highlight a novel mechanism regulating the immune response to fungal infections. Further understanding of this regulatory pathway could aid the development of therapeutic approaches to enhance protection against invasive candidiasis.

fungal loads in wild-type and Sts Ϫ/Ϫ kidneys at early time points. Figure 1 illustrates that C. albicans within wild-type and Sts Ϫ/Ϫ kidneys proliferates at similar levels in the first 12 h following infection. However, while fungal CFU levels continue to increase after 12 h in wild-type kidneys, they begin to decrease in Sts Ϫ/Ϫ kidneys at between 12 and 18 h (Fig. 1). In this timeframe, similar numbers of leukocytes in wild-type and Sts Ϫ/Ϫ mice have entered the kidneys (16). These data indicate that within 12 to 18 h postinfection, mice lacking the Sts proteins are more effective than wild-type mice at inhibiting fungal growth and eliminating C. albicans cells from the kidney.
Hematopoietic stem cell (HSC)-derived cells contribute to the enhanced resistance of Sts ؊/؊ mice. Having established that enhanced kidney fungal restriction in Sts Ϫ/Ϫ animals becomes evident during the time period when bone marrow leukocyte populations begin to enter the renal compartment, we next determined if Sts Ϫ/Ϫ hematopoietic cells played a role. Transplantation of Sts Ϫ/Ϫ donor marrow into irradiated wild-type or Sts Ϫ/Ϫ recipients enhanced survival of Candida infection relative to the results seen with wild-type donor cells ( Fig. 2A). Additionally, irradiated wild-type and Sts Ϫ/Ϫ mice reconstituted with Sts Ϫ/Ϫ bone marrow displayed a significant reduction in the 24-h fungal burden relative to mice receiving wild-type bone marrow (Fig. 2B). We also noted that Sts Ϫ/Ϫ recipients displayed improved survival relative to wild-type recipients given equivalent amounts of donor marrow ( Fig. 2A), suggesting that a nonhematopoietic component also contributes to the increased survival of Sts Ϫ/Ϫ animals. To address whether phagocytic cells play a critical role in the Sts Ϫ/Ϫ resistance phenotype, we evaluated the 24-h fungal burden in mice that had been administered the phagocyte-depleting agent clodronate 24 h prior to infection (26). As expected, clodronate treatment led to higher fungal burdens. Noticeably, however, it also eliminated the 24-h fungal clearance advantage normally associated with Sts Ϫ/Ϫ animals (Fig. 2C). Together, these data suggest that the hematopoietic cell compartment makes an important contribution to the enhanced resistance of Sts Ϫ/Ϫ mice.
Enhanced candidacidal activity of Sts ؊/؊ leukocytes ex vivo. The regulatory role of the Sts proteins in innate leukocyte populations has not been explored. Both Sts-1 and Sts-2 are expressed by marrow cells at levels comparable to those within peripheral blood leukocytes and splenic cells (see Fig. S1A in the supplemental material). Therefore, we utilized an in vitro cell/fungal coculture assay to examine directly the interaction of bone marrow cells with fungal cells ex vivo (27). Marrow cells isolated from uninfected wild-type and Sts Ϫ/Ϫ mice were placed in culture and incubated with C. albicans cph1⌬ efg1⌬ cells. The cph1⌬ efg1⌬ mutant was used because it fails to undergo hyphal growth (28), thereby facilitating accurate quantification of fungal growth. After 24 h, we recovered equivalent CFU levels in cocultures containing untreated wild-type or Sts Ϫ/Ϫ marrow cells (Fig. 3A). In the presence of the immune cell activator phorbol myristate acetate (PMA), fewer fungal CFU were obtained (Fig. 3A). Significantly, PMA-treated Sts Ϫ/Ϫ marrow cells were more efficient at inhibiting fungal growth than treated wild-type marrow cells (Fig. 3A). This suggests that the antifungal   Marrow cell types associated with the initial innate immune response to systemic C. albicans infection include neutrophils and monocytes (29)(30)(31). Sts-1 and Sts-2 were found to be highly expressed in both cell populations ( Fig. S1B and C [for cell purity data, see Fig. S2]). In order to examine the role of Sts in the antifungal responses of bone marrow monocytes and neutrophils, cells were purified from preparations of murine marrow. Sts Ϫ/Ϫ marrow monocytes exhibited 10-fold-greater inhibition of fungal growth than the corresponding wild-type cultures (Fig. 3B). As in the case of total bone marrow cell coculture, the increased fungal-growth-inhibitory properties displayed by Sts Ϫ/Ϫ monocytes was evident only when cells were pretreated with PMA. In contrast to marrow monocytes, PMA-treated neutrophils lacking Sts expression were less efficient at inhibiting fungal growth than wild-type neutrophils, although both were better at inhibiting fungal growth than untreated cells (Fig. 3C). We also examined the different phagocyte populations that can be obtained by culturing bone marrow ex vivo in the presence of different cytokines. These include bone marrow-derived dendritic cells (BMDCs) (32), bone marrow-derived macrophages (BMDMs) (33), and bone marrow-derived monocytes (BMD monocytes) (34) (see Fig. S1D to F). Similarly to bone marrow cells and marrow monocytes, Sts Ϫ/Ϫ BMDCs exhibited a significantly greater ability to restrict C. albicans growth ex vivo than wild-type BMDCs (Fig. 3D). In contrast, no differences were observed in the growth-inhibitory properties of wild-type and Sts Ϫ/Ϫ BMDMs and BMD monocytes ( Fig. 3E and F). Cumulatively, our results suggest that the Sts proteins play a negative role in regulating antifungal properties of select phagocyte populations.
Increased Candida-induced ROS production in cells lacking Sts expression. Because Sts Ϫ/Ϫ BMDCs displayed enhanced fungal growth suppression ex vivo (Fig. 3D), we next investigated their antifungal effector functions. Members of the C-type lectin receptor (CLR) superfamily, including Dectin-1, are among the surface receptors engaged by fungal cells (35,36). We stimulated cells with zymosan, a crude preparation of yeast cell wall extract that engages antifungal TLRs and CLRs, and observed a significantly greater zymosan-induced reactive oxygen species (ROS) response in Sts Ϫ/Ϫ BMDC cultures than in wild-type BMDCs. In particular, both the rate of ROS production and the peak ROS signal were significantly enhanced in cells lacking Sts (Fig. 4A, left). We then evaluated the ROS response of BMDCs treated with C. albicans cells. After addition of either live or heat-killed (HK) C. albicans to BMDC cultures, production of ROS became evident, although the onset of the ROS response was delayed relative to cells treated with zymosan ( Fig. 4A, middle and right panels). Similarly to the response seen following zymosan treatment, ROS production by Sts Ϫ/Ϫ BMDCs following challenge with fungal cells was significantly augmented relative to the ROS response of wild-type BMDCs. In contrast to BMDCs, wild-type and Sts Ϫ/Ϫ neutrophils (Fig. 4B) and BMD monocytes (Fig. S3A) did not display any differences in fungus-induced ROS production, while BMDMs of both genotypes did not generate a fungus-induced ROS response (Fig. S3B). Stimulation of BMDCs with heat-killed C. albicans cph1⌬ efg1⌬ cells also produced a heightened ROS response in Sts Ϫ/Ϫ cells relative to wild-type cells (Fig. S4). Together, our data indicate that the Sts proteins negatively regulate the activation of fungus-induced ROS production in BMDCs.
In addition to ROS production, fungus-stimulated phagocytes produce diverse proinflammatory cytokines, including tumor necrosis factor alpha (TNF-␣), interleukin-6 (IL-6), and IL-1␤ (37). In the case of the latter, activation of the NLRP3 inflammasome downstream of the fungal receptor Dectin-1 results in upregulation of IL-1␤ secretion (38). In contrast to the differential ROS responses observed between wild-type and Sts Ϫ/Ϫ BMDCs, we detected no differences in the production of TNF-␣, IL-6, or IL-1␤ by cells lacking Sts expression relative to wild-type cells ( Fig. 4C to E). Additionally, wild-type and Sts Ϫ/Ϫ cells upregulated Nos2 expression to similar extents following stimulation, resulting in identical levels of NO production ( Fig. 4F and G). These results suggest that the Sts proteins regulate a subset of phagocyte antifungal effector functions that includes the production of reactive oxygen species.
Sts regulates ROS production downstream of fungal receptor Dectin-1. Dectin-1 is a CLR that is stimulated by C. albicans and mediates activation of numerous downstream pathways (39,40). Among the ligands that stimulate Dectin-1 are polymeric forms of ␤-glucan that are components of fungal cell walls (10). To investigate the involvement of Dectin-1, we treated BMDCs with purified particulate ␤-1,3 glucan polymers and found that Sts Ϫ/Ϫ BMDCs demonstrated significantly increased ROS production relative to wild-type cells (Fig. 5A). Unlike polymeric ␤-glucan, soluble monomeric ␤-glucan acts in a competitive inhibitory fashion and blocks access to the Dectin-1 ligand-binding surface (41). After addition of soluble ␤-glucan to BMDC cocultures, the enhanced ROS response of Sts Ϫ/Ϫ cells following zymosan or HK C. albicans stimulation was inhibited (Fig. 5B). The elevated ROS response of Sts Ϫ/Ϫ BMDCs was also abrogated following addition of a blocking anti-Dectin-1 antibody to cocultures but not following addition of a control antibody (Fig. 5C). Importantly, we noted no difference in levels of surface expression of Dectin-1 on wild-type and Sts Ϫ/Ϫ BMDCs (Fig. 5D). Similarly, no differences in the stimulation-dependent downregulation of Dectin-1 from the cell surface were observed (Fig. 5E). These results suggest that the  Sts regulates levels of Syk activation induced by C. albicans. Among the first known biochemical events following Dectin-1 stimulation are upregulation of Src family member kinase activity and phosphorylation of Shp2 phosphatase (42,43). Spleen tyrosine kinase (Syk), a nonreceptor protein kinase that is highly expressed in a variety of phagocytes, is then activated (44). We examined activation levels of Src family kinases in stimulated wild-type and Sts Ϫ/Ϫ cells using a phosphospecific antibody. No differences were observed in the levels of Src kinase activation following stimulation of BMDCs with zymosan or wild-type C. albicans. (Fig. 6A). Further, no differences between stimulated wild-type and Sts Ϫ/Ϫ cells in Shp2 phosphorylation levels were noted (Fig. 6B). In contrast, stimulation of wild-type and Sts Ϫ/Ϫ cells with either zymosan or C. albicans resulted in enhanced Syk tyrosine phosphorylation in Sts Ϫ/Ϫ cells ( Fig. 6C; quantified in Fig. S5A). These observations suggest that the Sts phosphatases regulate Dectin-1 signaling at the level of Syk phosphorylation.
Engagement of Dectin-1 by fungus-associated ligands leads to activation of a number of intracellular signaling pathways. To investigate pathways downstream of Syk in Sts Ϫ/Ϫ phagocytes, we examined activation of PLC␥2, a downstream substrate of Syk (40). PLC␥2 was found to be hyperphosphorylated in stimulated Sts Ϫ/Ϫ BMDCs relative to wild-type cells (Fig. 6D; quantified in Fig. S5B). We also examined the activation of extracellular signal-regulated kinase-1/2 (ERK1/2) and phosphatidylinositol  3-kinase (PI3K), signaling molecules that play critical roles in transcriptional activation downstream of C-type lectin receptors. No differences were observed in the kinetics or extent of ERK1/2 activation (Fig. 6E), or in PI3K activation, as indicated by the kinetics and extent of Akt phosphorylation (Fig. 6F). Finally, levels of activation of p65 NF-B in wild-type and Sts Ϫ/Ϫ cells following stimulation with either zymosan or C. albicans were identical (Fig. 6G). These results suggest that the Sts proteins could regulate a specific Syk-PLC␥2-ROS signaling axis downstream of Dectin-1 that is independent of the pathways regulating Dectin-1/Syk-induced cytokine gene expression.

DISCUSSION
Combination therapy that pairs the use of traditional antibiotics with agents to enhance beneficial host immune responses is considered an important therapeutic goal for the treatment of intractable infections and of those for which antibiotic resistance is a looming concern (11, 12). Interestingly, genetic inactivation of the Sts proteins dramatically improves host survival following lethal doses of intravenous C. albicans, suggesting that they are possible targets to enhance host antifungal immunity. Importantly, the resistance of Sts Ϫ/Ϫ mice is accompanied by rapid fungal clearance within the kidney, sharply decreased levels of inflammatory molecules, and an absence of inflammatory lesions. While our previous analysis revealed key differences in the immunological responses of wild-type versus Sts Ϫ/Ϫ mice (16), it did not provide mechanistic insights into how Sts inactivation alters immune responses and increases protection from systemic infection. In this study, therefore, we sought to identify underlying cellular and molecular components of the enhanced antifungal immune response displayed by Sts Ϫ/Ϫ mice.
Increased resistance to systemic C. albicans is mediated by Sts ؊/؊ leukocytes. Comparative analyses of fungal clearance within infected mice provided an important clue into underlying mechanisms. Critically, C. albicans grew similarly in wild-type and Sts Ϫ/Ϫ kidneys during the first 12 h postinfection. As kidney-resident phagocytes represent the primary innate immune cell population within uninfected kidneys (45), this observation suggests that the initial responses of resident phagocytes do not account for the differential abilities of wild-type and Sts Ϫ/Ϫ mice to control the infection. However, levels of fungal CFU within infected kidneys begin to differ between 12 and 18 h postinfection (Fig. 1), with Sts Ϫ/Ϫ CFU beginning to decline at a time when large numbers of leukocytes enter the renal compartment to counteract the infection (23,31). This suggests that a key contribution is made by Sts Ϫ/Ϫ bone marrow-derived leukocytes, a hypothesis that is supported by our radiation chimera studies in which animals reconstituted with Sts Ϫ/Ϫ marrow had improved fungal clearance and enhanced survival relative to animals receiving wild-type marrow (Fig. 2). Ex vivo coculture analysis also demonstrated increased fungal growth suppression associated with Sts Ϫ/Ϫ phagocytes (Fig. 3). Together, these observations suggest that the Sts phosphatases negatively regulate intrinsic antifungal responses within key leukocyte populations.
Increased ROS production downstream of Dectin-1-Syk signaling in Sts ؊/؊ cells. After stimulating BMDCs with fungal ligands, we observed significantly heightened ROS production in Sts Ϫ/Ϫ cells but no differences in other antifungal responses such as cytokine production or generation of nitric oxide (Fig. 4). ROS production is known to be induced following engagement of the CLR Dectin-1 (46), and we confirmed the involvement of Dectin-1 using both a competitive inhibitor and blocking antibodies. While the signaling pathway from Dectin-1 to initiation of the ROS response has not been fully elucidated, one established component is the Syk kinase (38,44,47). Using a phosphospecific antibody that recognizes the tyrosine phosphorylated activation loop of Syk, we observed hyperphosphorylation of Syk in Sts Ϫ/Ϫ BMDCs following stimulation with zymosan or infection with C. albicans (Fig. 6). Under these conditions, there was no evidence of increased activation of upstream components such as Src kinases or Shp2. Therefore, our data suggest that Sts regulates signaling events downstream of Dectin-1 at the level of Syk phosphorylation and activation, perhaps by direct dephosphorylation of Syk. The idea of a role for Sts in regulating Syk activity in BMDCs is supported by studies in other cell types that demonstrated that Syk is a Sts target (19,21,22).
Further evidence for heightened Syk kinase activity in Sts Ϫ/Ϫ BMDCs lies in the observation that PLC␥2, a putative Syk substrate, displays increased phosphorylation following stimulation of the Dectin-1 pathway. Interestingly, we did not observe any differences in the levels of activation of the mitogen-activated protein kinase (MAPK) and PI3K pathways, two signaling pathways also thought to be downstream of Syk (48). We also observed no differences between wild-type and Sts Ϫ/Ϫ cells in production of TNF-␣, IL-6, and IL-1␤, which are three cytokines that have been shown to lie downstream of the location of Syk activation (47). Together, these data highlight complexity in the regulation of Syk signaling that heretofore has not been described. In particular, the distinct effects on downstream effector pathways in Sts Ϫ/Ϫ cells suggest that Syk-mediated activation of downstream pathway components occurs in a differentially regulated manner. Whether this involves interaction of activated Syk with multiple distinct regulatory factors or differential subcellular localization of activated Syk is currently unclear. How Sts deficiency and increased Syk activation together lead selectively to increased ROS production is currently being investigated.
Cell-specific regulation of antifungal responses by Sts. Interestingly, our ex vivo analysis data suggest that Sts regulates leukocytes in a cell-specific manner. In particular, while Sts Ϫ/Ϫ BMDCs displayed increased candidacidal activity, BM-derived macrophages and BM-derived monocytes were unaffected by Sts inactivation. Furthermore, although bone marrow monocytes and neutrophils both expressed high levels of the Sts proteins, only Sts Ϫ/Ϫ monocytes displayed enhanced C. albicans growth-suppressive properties. The underlying basis for a cell-specific role for Sts in regulating antifungal responses is currently unclear, but it could indicate important differences in the manner in which different innate immune cells respond to fungal pathogens. These observations are consistent with a previous report from a study demonstrating that different bone marrow-derived cell lineages exhibit differential responses to fungal ligands (49). It will be interesting to determine how the cell specificity observed ex vivo influences the in vivo immune response to fungal infection.
Increasing resistance to C. albicans infection. Similarly to the Sts proteins, two other gene products (Jnk1 and Cbl-b) have recently been shown to negatively regulate phagocyte signaling pathways such that the corresponding gene deletions result in mice that have increased resistance to C. albicans bloodstream infection (12,(50)(51)(52)(53). Jnk1, a member of the MAPK family of enzymes, negatively regulates activation of the transcription factor NFATc1. NFATc1 induces expression of the CLR CD23, and CD23 expression is upregulated in Jnk1 Ϫ/Ϫ mice phagocytes, resulting in elevated levels of inducible nitric oxide synthase (iNOS) (Nos2) expression. Therefore, the protection of Jnk1 Ϫ/Ϫ mice from systemic candidiasis appears to stem from increased fungusinduced NO production (50). The ubiquitin ligase Cbl-b mediates Dectin-1 internalization and degradation. In its absence, Dectin-1 surface expression is stabilized, resulting in Syk hyperactivation and enhanced phagocyte antifungal responses (51)(52)(53). Interestingly, while Cbl-b Ϫ/Ϫ BMDCs demonstrate increases in both ROS and cytokine production levels following infection with C. albicans, Sts Ϫ/Ϫ cells display an augmented ROS response without concomitant increases in cytokine production. Nonetheless, an intriguing property common to Jnk1 Ϫ/Ϫ , Cbl-b Ϫ/Ϫ , and Sts Ϫ/Ϫ mutant mice is increased activation of phagocyte signaling pathways downstream of fungal CLRs, with consequent increases in antifungal effector activities. These observations offer insights into developing novel immune-enhancing therapeutics that could be paired with traditional antifungal antibiotics to ameliorate the destructive effects of systemic C. albicans infection.

MATERIALS AND METHODS
Mouse strains and cells. The generation of C57/B6 mice containing the Sts mutations has been previously described (17,54,55). Mice were housed in the Stony Brook University Animal Facility in BMDCs were differentiated as previously described (32). Briefly, cells were cultured in RPMI medium containing 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 10 U/ml penicillin/streptomycin (Pen/Strep), 55 M ␤-mercaptoethanol (BME), and 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF). On days 3, 6, and 8, cells were provided fresh growth media. All BMDC experiments utilized nonadherent cells grown for 9 to 10 days in culture. BMDMs were cultured in DMEM containing 30% L929 cell supernatant (33), 20% FBS, and 1 mM sodium pyruvate for 4 days, after which cells were harvested, counted, and utilized as described. During the derivation of BMDMs, day 4 nonadherent cells were harvested as BM-derived monocytes (34).
For neutrophil purification, bone marrow cells were suspended in 4 ml of phosphate-buffered saline (PBS), placed on 3 ml Lymphoprep reagent (Axis-Shield, Oslo, Norway) (1.077 g/ml), and spun at 2,000 rpm. Alternatively, a murine neutrophil enrichment kit (Miltenyi) was utilized to obtain marrow neutrophils. Bone marrow monocytes were obtained with a murine monocyte isolation kit (Miltenyi) or an EasySep mouse monocyte kit (Stem Cell Technologies), according to the instructions of the manufacturers.
Mouse infections. Candida albicans infections were carried out as previously described (16). Cells were harvested, washed twice in PBS, and counted, and cell counts were confirmed by plating dilutions onto yeast extract-peptone-dextrose (YPD) plates. Female mice were inoculated with 2.5 ϫ 10 5 CFU via the lateral tail vein and monitored for 28 days. For clodronate depletion experiments, mice were administered by intravenous (i.v.) injection 1 mg clodronate/liposome formulation or control liposomes (Encapsula Nano Sciences, Brentwood, TN) 24 h prior to inoculation with C. albicans (1 ϫ 10 5 CFU) (26). To obtain kidney CFU, kidneys were excised at the indicated times postinfection, placed in 5 ml PBS, and homogenized. The number of CFU per gram of tissue was determined by plating homogenate serial dilutions onto YPD medium plates and incubating at 30°C.
Derivation and use of radiation chimeras. Female mice (8 to 10 weeks of age) were dosed with 1,100 rads from a gamma cell irradiator (GammaCell 40; AEC Ltd.) and administered 8 ϫ 10 6 bone marrow cells via tail vein injection within 1 h. Chimeric mice were housed for 12 weeks and then utilized for survival or organ CFU assays. Graphing and statistical analysis of survival after infection were carried out using a log rank test (Mantel-Haenszel test) with SigmaPlot software (SigmaPlot Systat Software, Inc., San Jose, CA).
Ex vivo C. albicans coculture assay. Nonfilamentous C. albicans mutant cph1Δ efg1Δ cells (28) were grown overnight, reinoculated into fresh medium, and grown to an optical density at 600 nm (OD 660 ) of 0.7 to 0.9. Cells were washed twice in PBS and coincubated with cells obtained from male or female mice in RPMI media at a multiplicity of infection (MOI) of 0.0375 to 0.125, with or without 10 ng/ml PMA, for 24 h in a 96-well plate. Wells were washed once with water and collected in 1 ml of deionized water to lyse nonfungal cells. Fungal CFU were obtained by plating serial dilutions onto YPD plates.
Pathway analysis. Cells obtained from male or female mice were stimulated, washed, and lysed in buffer containing 0.05 M Tris, 0.15 M sodium chloride, 5 M EDTA, 0.2 mM pervanadate, 0.5 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 1ϫ Roche protease inhibitors. Lysates were clarified by centrifugation, subjected to SDS-PAGE, and transferred to nitrocellulose (Whatman). Membranes were probed with specific antibody and the appropriate secondary antibody and were developed with an Odyssey CLx imaging system (Li-COR). Immunoprecipitations were conducted by rotating lysates with specific antibody for 2 h at 4°C, followed by 1 h at 4°C with protein A Sepharose beads (Sigma). Beads were washed three times in lysis buffer, and proteins were eluted with 2ϫ Laemmli sample buffer and separated by SDS-PAGE. Dectin-1 downregulation was evaluated using a FACScan cytometer (Cytek Biosciences).
Measurement of ROS and NO production. Levels of reactive oxygen species were measured as previously described (56). Briefly, BMDCs (1 ϫ 10 5 cells/well) or neutrophils (4 ϫ 10 5 cells/well) were plated in triplicate wells of a 96-well plate. Stimuli were prepared in RPMI media containing 600 M luminol and 16 units of HRP/ml. Reagent medium (100 l) was added to 100 l of preplated cells, and luminescence was measured at regular intervals on a Filtermax F5 96-well plate reader using Softmax Pro software (Molecular Devices, Sunnyvale, CA). Griess reagent was used to evaluate levels of NO 2 production in cell culture supernatants per the instructions of the manufacturer (Promega).
Cytokine measurements. Cells were placed in a 6-well tissue culture plate and stimulated with zymosan (100 g/ml) or heat-killed SC5314 (MOI of 2). The supernatant was collected and frozen at Ϫ80°C until measured. IL-6, TNF-␣ (BioLegend), and IL-1␤ (Thermo Fisher Invitrogen) levels were measured by enzyme-linked immunosorbent assay (ELISA) according to provided instructions.