Candida albicans strain SC5314 (ATCC MYA-2876) and clinical isolates from patients with bloodstream infection (CL 10-1, CA 09-1, CL 15-1, CA 01-1, CL 03-1, and CA 03-1, National Taiwan University Hospital) were used in this study. SC5314, clinical isolates, and GFP-expressing strain OG1 (25) were cultured at 30°C on yeast–peptone–dextrose (YPD) agar (DIFCO) plate. Mice were injected intravenously with Candida yeasts prepared in RPMI 1640 medium. Candida yeasts were opsonized in phenol red free HBSS containing 10% fresh mouse or human serum at room temperature for 30 min.

Bone marrow cells were harvested from the femurs and suspended in dPBS buffer before overlaid on discontinuous percoll gradients (55, 62, and 81% in the order from top to bottom) (GE Healthcare). After centrifugation at 1,400 × g for 30 min, cells at the interface between 62 and 81% gradients were harvested and washed. Flow cytometric analysis showed that 92% of cells were CD11b⁺Ly6G⁺. For adoptive transfer, 5 × 10⁶ neutrophils isolated from gal3^+/+, gal3^−/−, and littermate mice were injected into recipient mice through the tail vein. Recipient mice were given Candida intravenously 1 h later.

Fresh peripheral venous bloods were collected from healthy volunteers with no history of candidiasis in BD Vacutainer^® Plus Plastic K2EDTA Tubes or serum separator tubes for different purposes. Neutrophils were isolated as described previously (26). Briefly, bloods were diluted in Mg²⁺- and Ca²⁺-free DPBS and overlaid on Ficoll-Paque solution (Ficoll-Paque™ PLUS, GE Healthcare). After centrifugation at 600 × g for 15 min, the pellet containing neutrophils and red blood cells (RBCs) was subject to dextran sedimentation (3%) to separate RBCs from neutrophils. Residual RBCs were lysed by lysis buffer. Cell viability was determined by trypan blue exclusion.

Neutrophils from human or mice were stimulated with opsonized Candida for 5 and 15 min. Cells were stained with rat anti-gal3 and fluorochrome-conjugated goat anti-rat antibodies. After fixation in 4% PFA and permeabilization in saponin (Sigma)-containing perm/wash buffer, cells were stained with Alexa 647-rat anti-gal3 antibody. Samples were acquired by BD FACSCanto II and analyzed by BD FACSDiva software.

Kidneys were cut into small pieces and treated with 0.2 mg/ml Liberase TM (Roche) at 37°C for 30 min. Pellets were resuspended in 45% percoll and overlaid on 81% percoll. Cells at the interface (45/81%) were collected after centrifugation. Cells stained positive for CD45 were taken as infiltrating cells. After exclusion of Ly6G⁺ neutrophils, cells were divided into three distinct populations according to the expression of MHC II and F4/80 (MHC II⁺F4/80⁺ as macrophages, MHC II⁻F4/80⁺ as monocytes, and MHC II⁺F4/80⁻ as dendritic cells). Detailed description is given in Methods in Supplementary Material.

Bone marrow neutrophils were incubated in phenol red free HBSS containing 10 µM CM-H₂DCFDA (Life Technologies) for 30 min. After replenishment with fresh HBSS or with HBSS containing kinase inhibitors, cells were stimulated with opsonized Candida yeasts (MOI = 0.5). Oxidative DCF was analyzed by flow cytometry. To assess ROS production by renal infiltrating neutrophils isolated from infected mice, neutrophils collected from percoll gradients were stained with CM-H₂DCFDA and subject to flow cytometric analysis.

Human neutrophils were pretreated with mock (0.5% DMSO) or 250 μM of TD139 (3,3′-deoxy-3,3-bis(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside) (27) for 4 h before addition of CM-H₂DCFDA. Cells were then stimulated by opsonized Candida for 20 min before subject to flow cytometric analysis.

The assay was adopted from Methods in Molecular Biology (28). Briefly, Candida yeasts were grown in Sabouraud-dextrose broth (pH = 5.6) in 37°C overnight. Bone marrow neutrophils and CD45⁺Ly6G⁺ renal infiltrating cells were subject to killing assay in 96-well plate with 5 × 10⁵ cells and 1 × 10⁴ opsonized Candida yeasts. Wells containing opsonized Candida yeasts without neutrophils was used as control. Twenty minutes was allowed for phagocytosis after which supernatants in control wells and experimental groups were plated on YPD plate. The number of ingested yeasts (N_0h) = the number of yeasts in control well − the number of uningested yeasts. The rest of the wells were incubated for another 1.5 h and the cells were lysed to release intracellular yeasts. The lysates were plated. % Killing = (N_0h − N_1.5h)/N_0h × 100%, where N = the number of intracellular Candida.

Five million neutrophils were stimulated with opsonized Candida. Cells were lysed in 500 µl of non-denaturing lysis buffer. Cell lysates were mixed with 1.5 µg of mouse anti-gal3 antibody (B2C10) or 1 µg of anti-Syk polyclonal antibody at 4°C overnight. The mixture was mixed with 50 µl of agarose beads slurry under rotary agitation at 4°C for 4 h. Lysate beads mixture was washed with IP washing buffer. Beads were then mixed with 25 µl of sample buffer and boiled to elute immunoprecipitates.

Kidney and brain were homogenized in a tissue grinder with 1 ml of RPMI 1640 medium. One to ten serial dilutions were made and 0.1 ml was plated on YPD agar. Colonies were counted after incubation at 30°C for 2 days.

Three million human neutrophils were premixed with Nucleofector^® Solution (Lonza) plus supplement in total volume of 100 µl followed by addition of 2 µM of scrambled siRNA or siRNA targeting gal3 (Santa Cruz Biotechnology). Cells were transfected by using Nucleofector^® Program Y-001 in the nucleofector II device and incubated in RPMI complete medium for 7 h before further experiment. Knockdown efficiency was about 47%. Cell viability was determined by trypan blue exclusion.

Mann–Whitney test (non-parametric analysis) was used to compare the difference between two groups. Mortality after Candida infection was analyzed by log-rank and Fisher’s exact test. Statistical significance was defined as p < 0.05.

Human study was approved by the Institutional Review Board of the National Taiwan University Hospital (Permit Number: 201103121IRB, 201401074INA). All written informed consents were received from participants prior to inclusion in this study. Mouse study was carried out in strict accordance with the recommendations in the Guidebook for the Care and Use of Laboratory Animals, The Third Edition, 2007, published by The Chinese-Taipei Society of Laboratory Animal Sciences. The experimental protocol was approved by the Committee on the Ethics of Animal Experiments of the National Taiwan University College of Medicine (Permit Number: 20120031, 20130238, and 20140304).

Previous studies revealed that recombinant gal3 positively regulates neutrophil functions when added exogenously, here we focused our study on the role of cell intrinsic gal3 in neutrophils. Fluorescence microscopic (Figure 1A) and flow cytometric analyses (Figures 1B,C) showed that above 80% of unstimulated neutrophils expressed low levels of gal3 in the cytosol and stimulation with opsonized Candida increased intracellular gal3 expression as early as 5 min after stimulation (Figure 1B). While dendritic cells and monocytes/macrophages constitutively express gal3 on the surface (29, 30), gal3 was not detected on the surface of unstimulated neutrophils (Figure 1C). Stimulation by Candida induced gal3 redistribution to the cell surface in about 20% of neutrophils (Figure 1C). It was noted that intracellular gal3 was diffusely distributed (Figure 1D) and 70.0 ± 8.8% and 85.9 ± 4.6% of the intracellular gal3 at 5 and 15 min time points, respectively, did not colocalize with opsonized Candida (Figures 1D,E). By tracking the movement of cytosolic gal3 in neutrophils upon encountering GFP-expressing Candida, we found that gal3 moved toward engulfed Candida (Figure S1 in Supplementary Material). At the point when gal3 was closest to Candida (7.56 min), it still did not associate with the organism (Figure S1 and Video 1 in Supplementary Material). These results together indicate that stimulation by opsonized Candida upregulates gal3 expression in neutrophils and that intracellular gal3 does not bind to the engulfed organism.

To unravel the role of upregulated gal3 in neutrophils, gal3^+/+ and gal3^−/− neutrophils were allowed to ingest opsonized Candida, and the subsequent cellular response was assessed. We found that gal3^−/− neutrophils produced higher levels of ROS than gal3^+/+ cells (Figure 2A). While gal3^−/− and gal3^+/+ cells were comparable in their abilities to engulf Candida (Figure 2B), gal3^−/− neutrophils killed ingested Candida more efficiently than gal3^+/+ cells (Figure 2C). Moreover, suppressing ROS completely abolished killing of ingested Candida yeasts by both gal3^+/+ and gal3^−/− neutrophils (Figure 2D). These results indicate that both gal3^+/+ and gal3^−/− neutrophils kill ingested Candida through a ROS-dependent mechanism and that gal3 suppresses neutrophil fungicidal activity through inhibiting ROS production. Interestingly, CR3-deficient (Itgam^−/−) neutrophils were significantly less efficient in producing ROS (Figure 3A) and killing of opsonized Candida (Figure 3B) than WT cells, but such functions were not affected in dectin-1-deficient (clec7a^−/−) neutrophils (Figures S2A,B in Supplementary Material). Immunofluorescence staining revealed that CR3 congregated around but did not colocalize with engulfed Candida (Figure 3C). Thus, it appears that ROS production as a result of CR3 engagement is responsible for neutrophil fungicidal activity. In addition, gal3 deficiency promoted the abilities of dectin-1-deficient neutrophils to produce ROS (Figure S2C in Supplementary Material) and kill Candida (Figure S2D in Supplementary Material), whereas the presence or absence of gal3 did not make any difference in ROS production (Figure 3D) and the ability to kill Candida by Itgam^−/− cells (Figure 3E). It reveals that the suppressive effect of gal3 on neutrophil anti-Candida function is through CR3. It is worth noting that gal3 expression increased by twofold in Itgam^−/− neutrophils after stimulation by opsonized Candida compared to that without stimulation (Figure 3F), revealing that gal3 upregulation is independent of CR3. Moreover, we found that majority (>72%) of the intracellular gal3 in the cell population we tracked did not colocalize with CR3 (Figures 3G,H). These results together suggest that although neutrophil production of ROS is through CR3 engagement with Candida, gal3 does not directly interact with CR3.

It is reported that CR3 engagement activates neutrophil killing of Candida through Syk signaling (31). Our data showed that the levels of pSyk in CR3-deficient neutrophils were completely abolished after stimulation (Figure 4A) while that in dectin-1-deficient cells were comparable to WT cells (Figure S2E in Supplementary Material). Treatment with Syk inhibitor Bay 61-3606 dose-dependently reduced neutrophil ROS production (Figure 4B). Importantly, the levels of pSyk and p-p40^phox were significantly higher in gal3^−/− than in gal3^+/+ neutrophils after stimulation (Figure 4C). Reciprocal co-immunoprecipitation (Figure 4D) and immunofluorescence staining (Figure 4E) revealed that gal3 directly interacted with Syk after stimulation. Therefore, ROS production by neutrophils in response to opsonized Candida is Syk-dependent and gal3, by modulating Syk activation, regulates CR3 downstream signaling pathway(s) leading to ROS production.

Previous studies revealed the role of gal3 in cell recruitment in vivo (32, 33). To address whether gal3 also regulates cell infiltration in systemic candidiasis, we analyzed renal infiltrating neutrophils (CD45⁺Ly6G⁺), macrophages (CD45⁺Ly6G⁻MHCII⁺F4/80⁺), monocytes (CD45⁺Ly6G⁻MHCII⁻F4/80⁺), and dendritic cell (CD45⁺Ly6G⁻MHCII⁺F4/80⁻) populations (34). The results showed that gal3 deficiency did not affect the recruitment of neither neutrophils nor macrophages (Figures 5A–C). Gal3 deficiency, however, reduced monocyte (Figure 5D; Figure S4A in Supplementary Material) but enhanced dendritic cell (Figure 5E; Figure S4A in Supplementary Material) infiltration. In addition, the levels of chemokines were not different between gal3^−/− and gal3^+/+ mice after infection (Figure S3 in Supplementary Material). Interestingly, we found that renal infiltrating macrophages in gal3^−/− mice expressed higher levels of MHC II (Figure 5C), iNOS, and lower CD206 than in gal3^+/+ mice (Figure S4B in Supplementary Material).

Peripheral blood neutrophils from infected gal3^−/− were compared to those from gal3^+/+ mice for the levels of activation marker CD11b (35). Results in Figure 6A showed that CD11b on neutrophils from gal3^−/− mice were significantly higher than cells from gal3^+/+ mice after infection (Figure 6A). Moreover, renal infiltrating neutrophils of infected gal3^−/− mice produced significantly higher amount of ROS (Figure 6B) and their ability to kill Candida (Figure 6C) was higher than cells of gal3^+/+ mice. These results demonstrated that gal3 negatively regulates neutrophil functions not only in vitro but also in vivo.

Consistent with higher neutrophil fungicidal functions, gal3^−/− mice had better survival (Figure 7A) and lower fungal burdens in kidneys and brain (Figure 7B) than gal3^+/+ mice when systemically infected with Candida. While gal3^+/+ mice sustained multiple abscesses in the kidney with their kidney weights being about 2.3-fold of that of uninfected mice, gal3^−/− mice exhibited only minimal gross renal pathology and relatively less change in kidney weights (Figure 7C). The creatinine and blood urea nitrogen levels were also significantly lower in gal3^−/− mice than in gal3^+/+ mice (Figure 7D). Both gal3^+/+ and gal3^−/− mice exhibited chronic-active nephritis and pyelonephritis with inflammatory infiltrates on day 2 after infection. Starting on day 3, nephritis became severe in gal3^+/+ mice, but remained minimal/slight in gal3^−/− mice (Figure 7E), indicating that severe inflammation and tissue damage occurred to gal3^+/+ but not to gal3^−/− mice. Additionally, while hyphal/pseudohyphal forms of Candida were found in both infected gal3^+/+ and gal3^−/− mice on 18 h after infection, these forms increased and persisted in gal3^+/+ mice, but became almost completely absent in gal3^−/− mice by 3 days after infection (Figure S5 in Supplementary Material). These results together show that gal3 deficiency ameliorates systemic candidiasis by reducing fungal burdens, renal pathology, and mortality.

Adoptive transfer experiments were performed to investigate whether gal3 regulates neutrophil functions in vivo is a cell intrinsic event. We found that adoptively transferred gal3^−/− neutrophils cleared Candida infection more efficiently than gal3^+/+ neutrophils whether the recipient mice were gal3^+/+ (Figure 8A) or gal3^−/− (Figure 8B). Transfer of neutrophils from littermate donors also demonstrated the suppressive effect of gal3 on neutrophils in clearing Candida (Figure 8C). When neutrophil cytosolic factor 1-deficient (ncf-1^−/−) mice were used as recipients, transfer of gal3^−/− neutrophils also resulted in significantly less fungal burdens than transfer of gal3^+/+ cells (Figure 8D). The results confirm that it is ROS produced by transferred neutrophils but not that by cells in the recipient mice that mediates host defense against Candida. Taken together, these results indicate that cell intrinsic gal3 regulates neutrophil anti-Candida function.

Human neutrophils were like mouse neutrophils (Figure 1A) constitutively expressed gal3 in the cytosol but not on the surface (Figure 9A). Surface gal3 became detectable as early as 5 min after encountering opsonized Candida (Figure 9A). Interestingly, silencing Lgals3 in primary human neutrophils by siRNA significantly enhanced ROS production (Figure 9B). Treatment with gal3 inhibitor TD139, a derivative of thiodigalactoside, augmented human neutrophil ROS production (Figure 9C) and reduced fungal burden in mice with systemic candidiasis (Figure 9D). These results demonstrate that gal3 is a negative regulator of human neutrophil ROS response and point to the possibility of employing TD139 as a treatment strategy to restore neutrophil functions against Candida infection.

We tested C. albicans isolates from six patients with bloodstream candidiasis and showed that all six clinical isolates induced WT neutrophil ROS production (Figure 10A). Although the levels of ROS induced by different isolates varied, gal3 deficiency enhanced its production (Figure 10A). Isolates CL 15-1, CL 03-1, and CA 03-1, like SC5314, were more susceptible to killing by gal3^−/− neutrophils than by gal3^+/+ neutrophils (Figure 10B), while isolates CL 10-1, CA 09-1, and CA 01-1 were highly and equally susceptible (>90%) to killing by both gal3^−/− and gal3^+/+ neutrophils (Figure 10B). Consistent with the observation about the role of gal3 in systemic SC5314 infection, gal3^−/− mice were more efficient than gal3^+/+ mice in clearing CA 03-1 infection (Figure 10C) and had better survival (Figure 10D). Thus, gal3 negatively regulates neutrophil ROS production in response to not only SC5314 but also other clinical isolates and the negative effect of gal3 on neutrophil killing is more pronounced on otherwise “hard-to-kill” isolates.