Glutathione Reductase Promotes Fungal Clearance and Suppresses Inflammation during Systemic Candida albicans Infection in Mice

Abstract

Glutathione reductase (Gsr)¹ catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH), which plays an important role in redox regulation. We have previously shown that Gsr facilitates neutrophil bactericidal activities and is pivotal for host defense against bacterial pathogens. However, it is unclear whether Gsr is required for immune defense against fungal pathogens. It is also unclear whether Gsr plays a role in immunological functions outside of neutrophils during immune defense. In this study, we report that Gsr^−/− mice exhibited markedly increased susceptibility to C. albicans challenge. Upon C. albicans infection, Gsr^−/− mice exhibited dramatically increased fungal burden in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration and histological abnormalities in both the kidneys and hearts, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of the glomeruli in the kidneys of Gsr^−/− mice but were not found in wildtype mice. Examination of the neutrophils and macrophages of Gsr^−/− mice revealed several defects. Gsr^−/− neutrophils exhibited compromised phagocytosis, attenuated respiratory burst, and impaired fungicidal activity in vitro. Moreover, upon C. albicans stimulation, Gsr^−/− macrophages produced increased levels of inflammatory cytokines and exhibited elevated p38 and JNK activities, at least in part due to lower mitogen-activated protein kinase phosphatase (Mkp)-1 activity and greater Syk activity. Thus, Gsr-mediated redox regulation is crucial for fungal clearance by neutrophils and the proper control of the inflammatory response by macrophages during host defense against fungal challenge.

Introduction

Candida species are the fourth most common cause of nosocomial infections according to the CDC (1), and it is estimated that approximately 46,000 cases of healthcare-associated invasive candidiasis occur each year in the U.S. (2, 3). Candidemia is one of the most common blood stream infections in the US (4–6). C. albicans accounts for 51% of the nosocomial Candida blood stream infections (4, 7). Thus, there is an urgent need to understand the immune defense against C. albicans. Invasive C. albicans, in both the yeast and hypha forms, trigger a strong immune defense through pattern recognition receptors on innate immune cells such as macrophages and epithelial cells, including TLRs, the C-type lectin receptors (CLRs), and the nucleotide oligomerization domain-like receptors (NLRs) (8). Dectin-1, the most well-studied CLRs in monocytes and macrophages, recognizes C. albicans β-glucans and is involved in both the ligand uptake and phagocytosis (9–11). Engagement of β-glucans with Dectin-1 triggers tyrosine phosphorylation of an immunoreceptor tyrosine activation (ITAM)-like motif, which in turn recruits and activates the Syk tyrosine kinase, leading to activation of both NF-κB and MAPKs through a signaling pathway mediated by Card9 and Raf (9, 12–14). Dectin-2 and Dectin-3 recognize α-mannan-rich structures of C. albicans (9, 15, 16). Unlike Dectin-1, which has an ITAM-like motif, Dectin-2 and -3 do not have an ITAM-like motif (16, 17). Instead, recognition of α-mannans by Dectin-2 and -3 triggers tyrosine phosphorylation of the ITAM motif of FcγR to induce Syk activation and the subsequent NF-κB and MAPK cascades (15, 17). In addition to CLRs, TLRs and NLRs also recognize ligands of C. albicans and elicit multiple signal transduction pathways, ultimately leading to activation of NF-κB and MAPKs (8, 9, 17).

NF-κB regulates the transcription of a myriad of inflammatory cytokine and chemokine genes, including TNF-α, IL-1β, and IL-6, while MAPKs, particularly p38, enhance both the stability and the translation of cytokine and chemokine mRNAs. Mice deficient in TNF-α are highly susceptible to C. albicans infection, highlighting the crucial role of this cytokine in anti-fungal host defense (18). These inflammatory cytokines and chemokines are crucial in the recruitment of neutrophils, the most important leukocytes in the elimination of C. albicans. IL-6 is critical for the development of Th-17 cell-mediated adaptive immunity, which also mobilizes neutrophils against C. albicans (19, 20). Upon encountering C. albicans, neutrophils can engulf the fungal pathogens into the phagosomes through phagocytosis (21). The most important anti-microbial arsenal of neutrophils is the production of microbicidal reactive oxygen species (ROS) through the NADPH oxidase-mediated respiratory burst (21). The phagosomal respiratory burst is initiated by a stimulus-dependent assembly of the NADPH oxidase complex after phagocytosis of microbial pathogens (22, 23). Respiratory burst triggered by microbial particulates starts with recruitment of cytosolic NADPH oxidase subunits, including p47^phox, p40^phox, p67^phox, and Rac2 GTPase to the membrane-associated gp91^phox and p22^phox subunits (23). A number of cell signaling regulators have been shown to mediate NADPH oxidase activation in neutrophils, including Syk, PKC, and ERK and p38 MAPKs (24–27). In the phagosome membrane, NADPH oxidase reduces molecular oxygen to yield O₂^●−, which dismutes to produce H₂O₂. Myeloperoxidase (MPO) released from azurophilic granules catalyzes the conversion of H₂O₂ and Cl⁻ to the highly bactericidal hypochlorous acid in the phagolysosomes (21). The fact that chronic granulomatous disease patients with defects in respiratory burst are susceptible to fungal infections highlights the critical role of respiratory burst in antifungal immune defense (28).

GSH is a major cellular antioxidant, which protects cells from oxidative damage. In the cytosol, GSH reacts with H₂O₂ and is oxidized to GSSG (29, 30). Gsr catalyzes the regeneration of GSH from GSSG (GSSG + NADPH + H⁺ → 2GSH + NADP⁺), utilizing NADPH as an electron donor. Therefore, it is thought that during infections Gsr functions to protect neutrophils from oxidative damage through perpetuating the GSH/GSSG cycle that eliminates harmful H₂O₂ in the cytosol (29). Earlier studies using neutrophils from GSR-deficient patients have demonstrated a critical role of GSR in the neutrophil respiratory burst (31). However, since no overt increase in susceptibility to infections was reported in these GSR-deficient patients (30, 32, 33), the functional importance of GSR in host defense was unclear. Previously, we have studied the effects of Gsr deficiency on host defense against bacterial pathogen using a mouse model of sepsis (34, 35). We have demonstrated that Gsr is crucial for host defense against Escherichia coli, Staphylococcus aureus, and group B Streptococcal infections by facilitating neutrophil respiratory burst and the generation of neutrophil extracellular traps (34, 35). It remains unclear how Gsr deficiency affects host defense against fungal pathogens and whether Gsr functions in phagocytic cells other than neutrophils. In the present studies, we addressed these questions using Gsr-deficient mice and C. albicans fungal pathogen. Our studies demonstrated that Gsr is crucial for host defense against C. albicans, facilitating C. albicans killing by neutrophils and restraining TNF-α production via attenuation of Syk activity and MAPK pathways in macrophages.

Materials and Methods

Reagents

Rabbit antibodies against phospho-ERK, phospho-p38, phospho-JNK, phospho-MAPKAPK (MK) 2, phospho-Syk, and total p38 were purchased from Cell Signaling Technology (Danvers, MA). The mouse mAb against total ERK2, APC-labeled anti-mouse CD11b mAb, APC- and PE-Cy5.5-labeled anti-mouse CD45.2 mAbs, the murine MCP-1 and MIP-1α ELISA kits were purchased from BD Biosciences (San Jose, CA). The rabbit polyclonal Abs against gp91^phox, p47^phox, p67^phox, JNK1 (C-17), Mkp-1 (V-15), and Mkp-1 (M-18) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse mAb against phosphotyrosine (4G10) and recombinant active p38 were purchased from Upstate Biotechnology (Lake Placid, NY). The mouse mAb against murine α-tublin and β-actin, luminol, isoluminol, 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), hydrogen peroxide (H₂O₂), and other common chemicals were purchased from Sigma Chemicals (St Louis, MO). The rabbit Ab against the murine Gsr was described previously (36). The rat CD16/CD32 mAb, mouse Syk mAb, Pacific blue- and Brilliant violet 421-labeled anti-mouse Ly6G mAbs, Brilliant violet 785-labeled anti-mouse Ly6C mAb, Pacific blue- and Alexa Fluor 488-labeled anti-mouse F4/80 mAbs were purchased from BioLegend (San Diego, CA). Enzyme-linked immunosorbent assay (ELISA) kits for murine TNF-α and IL-6 were purchased from eBiosciences (San Diego, CA). Murine IL-1β and KC ELISA kits were purchased from BioLegend and R&D Systems (Minneapolis, MN), respectively.

Mice

The Gsr^−/− mice were generated after crossing the Gsr hypomorphic mice (36, 37) with C3H/HeN mice (Harlan Sprague Dawley, Indianapolis, IN) 10 generations (34, 35). WT C3H/HeN and Gsr^−/− mice were kept at 25°C in a specific pathogen-free vivarium with a 12-h alternating light-dark cycle. Both male and female mice between 2 and 6 months of age were used in the study. All animal experiments were approved and conducted according to regulations of the Institutional Animal Care and Use Committee of the Research Institute at Nationwide Children’s Hospital.

C. albicans infection, fungal burden determination, and pathology

C. albicans (SC5414) yeast was grown in YPD medium and C. albicans hyphae were made by additional culture in RPMI 1640 medium overnight (38). Mice were infected intravenously through the tail vein at a dose of 5×10⁵ C. albicans yeast per mouse (39). To assess survival, these mice were monitored over 7 days. To assess blood cytokine levels, tissue and blood fungal burden, and organ pathology, mice were euthanized at specific time-points by ketamine/xylazine overdose to harvest blood and organs. Blood samples were serially diluted in PBS, and 10 μl of the diluted blood was spotted in quadruplicate on YPD plates. To determine fungal burden in organs, organs were excised aseptically, and homogenized in PBS in a Bullet blender at 4°C. The homogenates were serially diluted and spotted in quadruplicate on YPD plates. These plates were cultured at 30°C for 24–48 h to score for colony forming units.

To assess tissue pathology, tissues were fixed in formalin for 24–48 h, washed, and embedded in paraffin to prepare 4-μm sections. Adjacent sections were stained either with H&E or with periodic acid-Schiff (PAS) techniques, as previously described (38, 40).

Kidney MPO activity

Peroxidase activity in kidney homogenates was assessed as previously described (41). Briefly, 10 μl kidney homogenates (20 μg protein) were mixed with 39 μl PBS, 1 μl of 0.03% hydrogen peroxide (H₂O₂), and 50 μl of 200 μM ADHP solution. The assays were performed immediately at 37°C in a 96-well plate, and fluorescence readings were acquired with a SpectraMax® M2 Multimode Microplate Readers (Molecular Device, Sunnyvale, CA), using excitation of 535 nm and emission of 590 nm for a total of 15 kinetic cycles with an interval of 20 s per cycle. Enzyme activities in the wells were defined as the slopes of the fluorescence vs. time curves.

Neutrophil isolation and macrophage derivation

Neutrophils were isolated by negative selection from bone marrow (BM) cells harvested from mouse femurs and tibias using a mouse neutrophil isolation kit (Miltenyl Biotec, Bergisch Gladbach, Germany). Cells were suspended in HBSS containing CaCl₂ and MgCl₂ (Invitrogen, Carlsbad, CA) supplemented with 0.5% BSA (Invitrogen) and 2% FBS (Hyclone, Logan, UT), and counted using a hemocytometer. The purity of neutrophils, evaluated by flow cytometry using Pacific blue-labeled anti-mouse Ly6G, consistently exceeded 90%.

BM cells were cultured on petri dishes in DMEM supplemented with 10% FBS, 25% L929-conditioned medium, and 40 ng/ml of recombinant mouse M-CSF (PeproTech, Rocky Hill, NJ). The cells were refed once with fresh medium after 4 days and cultured for 3 additional days to generate BM-derived macrophages (BMDM).

Leukocyte trafficking to kidneys

To investigate leukocyte trafficking to the kidneys, mice were euthanized by cervical dislocation 12 or 24 h post C. albicans infection. The mice were first perfused through the hearts with 10 ml cold PBS to clear blood from circulation. Kidneys were then excised and enzymatically digested in a solution containing 213 μg/ml Liberase (Roche Diagnostics, Mannheim, Germany), 100 μg/ml DNase I (Roche), and 40 μg/ml collagenase IV (Gibco, Gaithersburg, MD) at 37 °C for 20 min. The cell suspensions were then filtered through a 70-μm strainer, and pelleted by centrifugation, then resuspended in ACK buffer to lyse the red cells. Leukocytes were enriched through centrifugation using Percoll gradient. Leukocyte collected from the 40%–70% Percoll interface were then pelleted and suspended into 500 μl of FACS washing buffer (PBS containing 2.5% FBS). The cells were first blocked with 5 μg anti-CD16/CD32 mAb for 5 min, and then stained with 0.5 μg of APC-labeled anti-mouse CD11b mAb, 2 μg of PE-Cy5.5-labeled anti-mouse CD45.2 mAb, 1 μg of Brilliant violet 421-labeled anti-mouse Ly6G, 1 μg Brilliant violet 785-labeled anti-mouse Ly6C, and 2.5 μg Alexa Fluor 488-labeled anti-mouse F4/80 on ice for 10 min. Subsequently, the cells were washed twice with ice-cold FACS washing buffer prior to flow cytometry on a BD LSR II flow cytometer (BD Biosciences). Cells were gated based on the expression of leukocyte markers. CD45⁺CD11b⁺Ly6G⁺ leukocytes were regarded as neutrophils, and CD45⁺CD11b⁺Ly6C^hi were regarded as monocytes. CD45⁺CD11b⁺F4/80⁺ leukocytes were regarded as macrophages. All flow cytometry data were analyzed using FlowJo software (FlowJo, Ashland, OR).

Neutrophil apoptosis was determined by flow cytometry after staining Percoll gradient-enriched leukocytes with 10 μg/ml anti-CD16/CD32 mAb, 4 μg/ml of APC-labeled anti-mouse CD45.2 mAb, 0.5 μg/ml of Brilliant violet 421-labeled anti-mouse Ly6G, as well as Alexa Flour 488-labeled annexin V and propidium iodide (PI) using an Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit (Invitrogen).

Respiratory burst assays

Neutrophil and macrophage respiratory burst ex vivo was assessed by bioluminescence in a 96-well format. Briefly, purified neutrophils were pre-incubated in 180 μl of HBSS containing 0.5% BSA, 2% FBS, 100 μM luminol or isoluminol, 1.6 unit of horse radish peroxidase (EMD Biosciences, San Diego, CA) at room temperature for 10 min. Heat-killed, mouse serum-opsonized yeast (in 20 μl) were added into the wells to achieve a MOI of 10. PMA (dissolved in 20 μl reaction buffer) was directly added to the wells to achieve a concentration of 0.5 μM. Luminescence in each well was measured using a Synergy 2 luminometer (BioTek, Winooski, VT) for 1 s every 2 min consecutively for a given period. Cumulative respiratory burst activity was calculated as the integral of luminescence over a given time period. Respiratory burst in macrophages were measured similarly, with the following minor changes. BMDM were seeded at a density of 1×10⁵ cells per well in 200 μl of complete medium on the day prior to the respiratory burst experiment. Before the respiratory burst experiment, 180 μl medium was removed from the wells, and replaced with HBSS containing 0.5% BSA, 2% FBS, 100 μM luminol or isoluminol, 1.6 unit of horse radish peroxidase. To assess intracellular oxidant production, 10 units of superoxide dismutase (Sigma Chemicals) and 400 units of catalase (Worthington Biochemical, Lakewood, NJ) were added in the luminol reactions, to eliminate the extracellular superoxide and hydrogen peroxide.

To assess oxidative burst activity in vivo, we carried out luminol bioluminescence imaging essentially as described by Gross et al. (42). Following C. albicans infection, each mouse was given one dose of luminol (200 μg/g b.w.) i.p. at a given time, and immediately placed in an IVIS Spectrum imaging system (PerkinElmer, Waltham, MA) to collect luminescent images (exposure time, 5 min; FOV, D; binning, 8).

Phagocytosis

Heat-killed C. albicans were labeled using a pHrodo™ Red Microscale Labeling Kit (Thermo Fisher Scientific, Waltham, MA), according to manufacturer’s recommendation. Yeast cells were then washed three times with PBS and opsonized with 10% mouse serum at 37°C for 60 min. Neutrophils suspended in HBSS containing CaCl₂, MgCl₂, 0.5% BSA, and 2% FBS were incubated with pHrodo red-labeled, heat-killed, serum-opsonized yeast at a MOI of 3 or 10, either at 37°C or at 0°C for 30 min in a volume of 100 μl. Then, 1 μg of rat anti-mouse CD16/CD32 mAb and 0.5 μg of rat Pacific blue-conjugated anti-mouse Ly6G mAb were added into the neutrophil/yeast mixture to stain neutrophils on ice for 20 min. Subsequently, the cells centrifuged at 300×g for 5 min at 4°C, washed three time with 1 ml of ice-cold FACS washing buffer (PBS containing 0.5% BSA and 0.1% NaN₃), and finally suspended in ice-cold FACS washing buffer for flow cytometry.

To evaluate phagocytosis of C. albicans by macrophages, BMDM were plated onto 35-mm petri dishes in 2 ml DMEM containing 10% FBS at a density of 10⁶ cells per plate. On the next day, pHrodo red-labeled heat-killed C. albicans were added into the medium at a MOI of 10. Then the plates were centrifuged at 300×g for 2 min at 37°C to sediment the yeast. After 30 min incubation at 37°C in 5% CO₂, the plates were washed twice with ice-cold PBS. Then 500 μl of PBS containing 0.5% BSA, 0.5 μg of Pacific blue-labeled anti-mouse F4/80 mAb, and 0.5 μg of anti-mouse CD16/CD32 mAb were added into the plates, and the plates were incubated on ice for 10 min. Subsequently, the plates were washed twice with ice-cold FACS washing buffer, and cells were collected by scrapping for flow cytometry. Pacific blue-positive cells (Ly6G⁺ neutrophils or F4/80⁺ macrophages) that also displayed red fluorescence were regarded as neutrophils that engulfed C. albicans. All flow cytometry data were analyzed using FlowJo software (FlowJo, Ashland, OR).

C. albicans killing by neutrophils

Neutrophils isolated from BM were mixed with C. albicans yeast opsonized for 60 min with 10% normal mouse serum at a multiplicity of 10:1. Then the neutrophils/C. albicans mixtures were mixed with 10 volumes of sterile H₂O, serially diluted, and plated onto YPD agar plates. The plates were incubated at 37°C for approximately 24 h. Viable C. albicans cells were scored by counting the colonies formed on the YPD plates. The percent killing was calculated using the formula: % killing=(colony number of control - colony number of neutrophil-treated)/colony number of control×100.

Neutrophil subcellular fractionation

Neutrophils purified from mouse BM were stimulated with 0.5 μM PMA for 15 min or left unstimulated. Neutrophils were lysed, and the lysates were fractionated into membrane and cytosolic fractions by ultracentrifugation, as described (43).

Macrophage response to C. albicans stimulation

BMDM (8–10×10⁶ cells per plate) were seeded in 10 ml of DMEM (Invitrogen) containing 10% FBS and penicillin/streptomycin. To stimulate BMDM with C. albicans, heat-killed C. albicans were added into macrophage culture, and mixed thoroughly with medium. The plates were then centrifuged at 300×g for 2 min to sediment C. albicans. To harvest the cells, BMDM were washed twice with ice-cold PBS, and then lysed in 500 μl of lysis buffer, as previously described (44, 45). Cell lysates were centrifuged at 20,800×g for 10 min, and the supernatants were used for Western blot analysis.

To assess cytokine production, BMDM (5×10⁵ cells per well) were seeded in 1 ml of complete medium on 12-well plates. On the next day, heat-killed C. albicans opsonized with mouse serum were added into the medium and mixed thoroughly. The plates were then centrifuged at 300×g for 2 min to sediment the yeast to the bottom of the wells. The cells were incubated at 37°C in humidified air containing 5% CO₂ for 24 or 48 h to harvest medium for ELISA.

TNF-α mRNA stability

BMDM were first stimulated with heat-killed C. albicans for 5.5 h, and then treated with either DMSO or SB203580 (final concentration, 10 μM) for an additional 30 min. Actinomycin D was added into the culture medium. Total RNA were harvested from the cells using Trizol (Invitrogen) after 0, 30, 60, and 90 min. TNF-α mRNA levels were quantified by qRT-PCR to assess TNF-α mRNA decay, as previously described (46, 47).

ELISA and Western blot analysis

ELISA and Western blot analyses were performed as previously described (40, 44, 48).

Mkp-1 activity assay

WT and Gsr^−/− BMDM were stimulated with heat-killed C. albicans at an MOI of 5 for 4 h or left unstimulated. Soluble lysates (~900 μg protein) were precleaned with protein A-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ) at 4˚C for 30 min on a rotator, and then incubated with two rabbit polyclonal Abs against Mkp-1 (4 μg each, Mkp-1 (V15) and Mkp-1 (M18)) and protein A-Sepharose beads (25 μl) overnight at 4˚C on a rotator. Subsequently, the beads were washed twice with 1 ml lysis buffer and twice with lysis buffer supplemented with 150 mM NaCl. Recombinant active p38 (60 ng in 60 μl reaction buffer containing 50 mM Tris-HCl, pH7.4, 2 mM DTT, and 5 mM MgCl₂) was then incubated with the immunoprecipitates at 30˚C. A fraction (10 ng) of phospho-p38 was taken out at 0, 10, 30, and 90 min and mixed immediately with NuPAGE sample buffer containing 62.5 mM orthovanadate. The levels of phospho-p38 were assayed by Western blot analysis.

Statistics

The mortality data were compared using Kaplan-Meier’s Log-Rank test. The values of various biomarkers were compared over time and between conditions using one-way ANOVA. Differences were identified using a modified t-test. p values of less than 0.05 were considered significant.

Results

Gsr^−/− mice exhibit defective immune defense but elevated inflammatory response after C. albicans infection

To understand the tissue expression profile of Gsr, we isolated liver, lung, skeletal muscle, spleen, heart, kidney, brain, thymus, and eyes from WT and Gsr^−/− mice. These organs were homogenized and tissue proteins were extracted. BM cells were flushed out from femurs and tibias and BM proteins were extracted. Western blot analyses were performed using a rabbit polyclonal Ab generated in house (SFig. 1). A clear band of ~53 kDa was detected in the liver, lung, spleen, kidney, brain, thymus, BM, and eyes of WT mice, but not in these tissues of Gsr^−/− mice. A WT-specific band was not detected in skeletal muscle and heart tissues, suggesting that Gsr expression in muscle tissues was substantially lower than in other tissues. The relatively high expression of Gsr in BM, spleen, and thymus suggests that Gsr may play an important function in immune defense against pathogens.

To assess the role of Gsr in immune defense against fungal pathogens, we infected WT and Gsr^−/− mice intravenously with live SC5414 C. albicans, and monitored the survival of these mice over 7 days (Fig. 1A). WT mice exhibited substantial resilience against C. albicans, with the first fatality occurring 4 days after C. albicans infection and 70% of the mice surviving after 7 days. In contrast to WT mice, Gsr^−/− mice were considerably more sensitive to C. albicans infection. Substantial mortality was already observed one day after infection, and continued to increase rapidly. After 7 days, only 30% of the mice survived. The differences in survival rates (p<0.05) clearly illustrate the critical role of Gsr in host defense against fungal infections. In addition, the high mortality observed in Gsr^−/− mice correlated with greater fungal burdens in the kidneys at 24 and 48 h after infection than in WT mice (Fig. 1B). In keeping with this observation, severe inflammatory lesions associated with heavy C. albicans hyphae accumulation were observed in the kidneys of C. albicans-infected Gsr^−/− mice but not in the kidneys of infected WT mice (Fig. 1C), as viewed by H&E and PAS staining, respectively. The fungal lesion in the kidneys of C. albicans-infected Gsr^−/− mice contained almost entirely C. albicans hyphae (Fig. 1D). No C. albicans was detected in the blood or the spleens in either WT or Gsr^−/− mice 24 or 48 h post inoculation (data not shown). Furthermore, inflammatory lesions were observed in the hearts of C. albicans-infected Gsr^−/− mice but not in the hearts of WT mice (SFig. 2). Although inflammatory lesions with abundant neutrophils were frequently seen in the hearts of C. albicans-infected Gsr^−/− mice, fungi were not detected in the hearts (data not shown). The presence of a larger number of neutrophils in the kidneys of C. albicans-infected Gsr^−/− mice was also consistent with the greater in vitro MPO activity in the kidney homogenates of infected Gsr^−/− mice than in those of the infected WT mice (Fig. 1E).

Figure 1. Survival curve and kidney sections of WT and Gsr^−/− mice after C. albicans challenge.

Mice were infected i.v. with 5×10⁵ CFU of C. albicans (SC5314). A. Survival curve of WT and Gsr^−/− mice. *, p<0.05 (Kaplan-Meier’s Log-Rank test). n_WT=10; n_Gsr^−/−=17. B. Kidney fungal burden in WT and Gsr^−/− mice. WT and Gsr^−/− mice were infected i.v. with 5×10⁵ CFU of C. albicans and sacrificed 24 or 48 h later to harvest kidneys for the assessment of fungal burdens by culture. *, p<0.05 (t-test). Graph on the right depicts the dramatic contrast between the decline and increase in fungal burden in WT and Gsr^−/− mice, respectively. C. H&E and PAS stained kidney sections. Kidney tissues were harvested 48 h post infection. Adjacent sections were stained with H&E and PAS. D. A high resolution image of fungal lesion in the kidney section of a C. albicans-infected Gsr^−/− mouse after PAS staining. E. MPO activity in kidney homogenates. MPO activity was assayed using ADHP and H₂O₂ as substrates through measuring the kinetics of fluorescence (ex 535 nm/em 590 nm).

Leukocyte recruitment plays a crucial role in immune response, and defects in the trafficking of leukocytes, particularly phagocytes, could compromise immune defense and lead to organ dysfunction of the host (8, 49, 50). To examine whether Gsr^−/− mice have defects in leukocyte trafficking during C. albicans infection, we assessed the cell numbers of neutrophils, macrophages, monocytes in the kidneys shortly (12 or 24 h) after C. albicans infection (Fig. 2A). Leukocytes were enriched by Percoll gradient centrifugation from enzymatically digested kidneys, and the numbers of neutrophils, macrophages, and monocytes were determined by flow cytometry. At 12 h post C. albocans infection, the number of neutrophils in the kidneys of Gsr^+/+ and Gsr^−/− mice were similar. By 24 h, substantially more neutrophils were seen in the kidneys of Gsr^−/− mice than in those of Gsr^+/+ mice. In fact, fewer neutrophils were seen in the kidneys of Gsr^+/+ mice at 24 h than at 12 h. While both macrophages and monocytes were seen in Gsr^−/− mice, their numbers did not differ significantly between Gsr^+/+ and Gsr^−/− mice at either 12 or 24 h time-point. Since neutrophils are the most potent leukocytes mediating C. albicans killing (8, 51), we assessed whether there were differences in the viabilities of the neutrophils in the kidneys between Gsr^+/+ and Gsr^−/− mice (Fig. 2B). Necrotic and apoptotic neutrophils were assessed based on the staining by PI and annexin V. No differences were seen in the portions of necrotic, early or later apoptotic neutrophils between C. albicans-infected Gsr^+/+ and Gsr^−/− mice. Thus, despite increased numbers of neutrophils in the kidneys of Gsr^−/− mice, counter-intuitively these mice exhibited substantially elevated fungal burden and increased mortality, strongly supporting a defective fungicidal response in Gsr^−/− mice.

Figure 2.

Leukocyte numbers and neutrophil viability in the kidneys of C. albicans-infected Gsr^+/+ and Gsr^−/− mice. Mice were infected i.v. with 5×10⁵ CFU of C. albicans (SC5314) and then sacrificed at the indicated time to isolate kidneys. Leukocytes were enriched through Percoll gradient centrifugation, counted, and stained with different leukocyte markers for flow cytometry. Cells were first gated on forward scatter (FSC) and side scatter (SSC) to exclude cell debris, and viable cells were then gated for CD45⁺CD11b⁺Ly6G⁺F4/80⁻ neutrophils, CD45⁺CD11b⁺Ly6C^hi monocytes, and CD45⁺CD11b⁺F4/80⁺ macrophages. A. Numbers of neutrophils, macrophages, and monocytes in the kidneys at 12 and 24 h post C. albicans infection. Values represent means±SE (n=4–5). *, p<0.05 (Two-way ANOVA). B. Representative annexin V-Alexa 488 vs PI contour plots. Leukocytes were enriched from enzymatically digested kidneys from Gsr^+/+ or Gsr^−/− mice 24 h post C. albicans infection. These cells were then stained with CD45, Ly6G, annexin V, and PI, and subsequently assessed by flow cytometry. Neutrophils were gated as CD45+Ly6G+ cells, and further divided as necrotic (PI⁺), early apoptotic (annexin V⁺), late apoptotic (annexin V⁺ PI⁺), and live cells (annexin V⁻PI⁻). C. Percentage of live, necrotic, early apoptotic, late apoptotic, and total dead cells. Values represent means±SE (n=4–5).

Since inflammatory cytokines, particularly TNF-α, play a crucial role in the orchestration of the anti-fungal immune defense (52–54), we assessed the production of inflammatory cytokines in WT and Gsr^−/− mice. Blood was harvested at different times following infection, and cytokine levels in the blood were measured by ELISA (Fig. 3). The levels of TNF-α, IL-6, MCP-1, and MIP-1α were comparable in WT and Gsr^−/− mice at 6 h post-infection. Clear differences in the blood levels of IL-6 and MCP-1 between WT and Gsr^−/− mice were seen at 24 h post-infection. By 48 h, the levels of all five serum cytokines (TNF-α, IL-6, MCP-1, MIP-1α, and KC) were substantially higher in Gsr^−/− mice than in WT mice. These results clearly indicate that Gsr^−/− mice can mount an inflammatory response as the result of C. albicans infection.

Figure 3. Gsr deletion results in exaggerated inflammatory response following C. albicans infection.

WT and Gsr^−/− mice were infected i.v. with 5×10⁵ CFU of C. albicans. Mice were sacrificed 6, 24, or 48 hours later to harvest blood for assessment of blood cytokines by ELISA. Values are means ± SE. *, p<0.05 (t-test, n=4).

Compromised fungicidal activity of Gsr-deficient neutrophils

Respiratory burst in phagocytes play a pivotal role in the containment and killing of fungal pathogens (55, 56). We assess the respiratory burst activity in vivo following C. albicans infection, by luminol bioluminescence (42). In un-infected (control) mice, respiratory burst activity was undetectable in either Gsr^+/+ or Gsr^−/− mice (Fig. 4, Upper panel). Respiratory burst activity was elevated in the abdominal region of Gsr^+/+ mice 12 h after C. albicans infection (Fig. 4, Mid panel) and plummeted to undetectable levels by 24 h (Fig. 4, Lower panel). No respiratory activity was detected at either 12 or 24 h in C. albicans-infected Gsr^−/− mice (Fig 4).

Figure 4. Gsr deficiency leads to diminished respiratory burst activity in C. albicans-infected mice.

The respiratory burst burst activity in vivo in C. albicans-challenged mice (5×10⁵ CFU i.v. per mouse) was detected by luminol bioluminescence. Uninfected (control) or C. albicans-infected mice (12 or 24 h post challenge) were administered luminol (200 μg/g b.w.) to collect luminescent images (exposure time, 5 min; FOV, D; binning, 8). Images were from a representative experiment.

To understand the basis underlying the immunological defect of Gsr^−/− mice against C. albicans, we assessed the phagocytic activities of neutrophils purified from WT and Gsr^−/− mice. Phagocytosis of C. albicans by Ly6G⁺ neutrophils was assessed by flow cytometry (Fig. 5A). The percentage of WT Ly6G⁺ neutrophils that engulfed C. albicans was 99.2%, while the percentage of Gsr^−/− neutrophils that engulfed C. albicans was 95.5%. Although the percentage of neutrophils that engulfed C. albicans did not markedly differ between WT and Gsr^−/− groups, WT neutrophils engulfed substantially more C. albicans than did Gsr^−/− neutrophils on a per cell basis, indicated by greater mean fluorescent intensity (12,036 vs 5,204 arbitrary units/cell for WT and Gsr^−/− cells, respectively). The results suggest that Gsr is required for effective phagocytosis of C. albicans in neutrophils.

Figure 5. Gsr deletion results in compromised yeast phagocytosis by neutrophils.

A. Phagocytosis of C. albicans by WT and Gsr^−/− neutrophils. Purified BM neutrophils were incubated with pHrodo red-tagged C. albicans (MOI=10) at 0°C or 37°C for 30 min, and then labeled with Ly6G–Pacific Blue on ice for 20 min. Cells were subsequently washed twice with cold FACS buffer and analyzed by flow cytometry. Ly6G⁺ neutrophils cells were analyzed for the presence of pHrodo red-tagged C. albicans. Solid lines depict Gsr^+/+ and dashed lines depict Gsr^−/− neutrophils. B. Fungal killing assays. Serum-opsonized C. albicans yeast were incubated with either purified BM neutrophils or medium for 60 min at an MOI of 10:1, and then the neutrophils were lyzed by hypo-osmolarity. The mixtures were then serially diluted, and plated onto YPD agar plates to culture at 37°C for approximately 24 h. Viable C. albicans cells were scored by counting the colonies formed on the YPD plates. The percentage of killing was calculated using the medium-treated C. albicans as references. Values are means ± SE. *, p<0.05 (t-test, n=3).

To assess whether Gsr^−/− neutrophils exhibit a decline in fungicidal activity, in vitro fungicidal assays were performed. Live C. albicans yeast were incubated with WT and Gsr^−/− neutrophils at a MOI of 10 for 60 min, and then neutrophils were lysed by hypo-osmolarity. The surviving C. albicans yeast were cultured on YPD plates and quantified. Compared with WT neutrophils that killed 33% of C. albicans yeast, Gsr^−/− neutrophils killed only 17% of C. albicans yeast, clearly indicating a defect in fungicidal activity (Fig. 5B).

Since the NADPH oxidase-mediated respiratory burst is a major microbicidal mechanism of neutrophils (21, 56) and a diminished respiratory burst activity was seen in C. albicans-infected Gsr^−/− mice in vivo (Fig. 6), we assessed respiratory burst activity in WT and Gsr^−/− neutrophils. Respiratory burst was measured by chemiluminescence in vitro. The kinetics of total ROS production in WT and Gsr^−/− neutrophils assessed using luminol after stimulation with C. albicans are depicted in Figure 6A (Upper graph). Intracellular ROS was measured in the presence of SOD and catalase in order to eliminate extracellular ROS (Fig. 6A, Middle graph). Extracellular ROS was detected using isoluminol (Fig. 6A, Lower graph), as the cell membrane is impermeable to isoluminol. In WT neutrophils, both extracellular and intracellular production of ROS increased with C. albicans stimulation and reached peak levels at about 5–6 h, followed by a decline. ROS production was also enhanced by C. albicans stimulation in Gsr^−/− neutrophils, but did not reach the levels of WT neutrophils. The cumulative production of ROS both intracellularly and extracellularly in Gsr^−/− neutrophils following C. albicans stimulation were significantly lower than those in WT neutrophils (Fig. 6B). Similarly, lower ROS production was also observed in Gsr^−/− neutrophils following PMA stimulation (Fig. 6B).

Figure 6. Gsr deletion impairs respiratory burst of neutrophils.

Purified BM neutrophils were stimulated with heat-killed C. albicans (MOI=10) or PMA (0.5 μM). Respiratory burst was measured using luminol or isoluminol in the presence or absence of SOD and catalase in 96-well plates in a Biotek Synergy 2 luminometer. A. Top: kinetics of total respiratory burst activity. Middle: kinetics of intracellular respiratory burst activity. Bottom: kinetics of extracellular burst activity. Values represent the means. Differences between Gsr^+/+ and Gsr^−/− are significant (p<0.05, two-way ANOVA, n=4). B. Cumulative respiratory burst activity over 8 h. Values are means±SE. *, p<0.05 (t-test, n=4). C. PMA-stimulated PMN respiratory burst of Gsr^+/+ (Left) and Gsr^−/− (Right) with the presence (▲ or △) and absence (● or ) of vitamin E (0.2 mM), measured using luminol.

Previously, it has been shown that vitamin E pre-treatment of neutrophils isolated from GSR-deficient human subjects rescued their pentose phosphatase shunt activity (33), a metabolic reaction responsible for the generation of NADPH, a substrate for neutrophil respiratory burst. We assessed whether vitamin E alleviates the respiratory burst defects of Gsr^−/− neutrophils (Fig. 6C). Neutrophils were preincubated with vitamin E for 30 min prior to the initiation of respiratory burst by PMA. While vitamin E enhanced the respiratory burst by sustaining the maximal respiratory burst activity in WT neutrophils, vitamin E did not markedly change the kinetics of PMA-triggered respiratory burst in Gsr^−/− neutrophils (Fig. 6C).

To understand the mechanism underlying the respiratory burst defect in Gsr^−/− neutrophils, we studied the membrane assembly of NADPH oxidase subunits and signal transduction pathways known to regulate NADPH oxidase activation. WT and Gsr^−/− neutrophils purified from BM were stimulated with or without PMA, lyzed, and fractionated by centrifugation into cytosolic and membrane fractions. Different NADPH subunits were detected by Western blot analysis (Fig. 7A). Consistent with previous reports that gp91^phox localizes in the membrane as a glycosylated protein (57, 58), gp91^phox was detected in the membrane fraction in both WT and Gsr^−/− neutrophils. In response to PMA stimulation, the abundance of membrane-associated gp91^phox actually decreased in both groups. In contrast, both p67^phox and p47^phox subunits were predominantly localized in the cytosolic fractions. However, in response to PMA stimulation, a small fraction of both p47^phox and p67^phox were translocated into the membrane fractions. No differences were found between WT and Gsr^−/− neutrophils in the localization of these subunits, suggesting that Gsr deficiency does not affect the assembly of the NADPH oxidase complexes.

Figure 7. Assembly of NADPH oxidase complexes, ERK and p38 activation, and tyrosine phosphorylation after stimulation in wildtype and Gsr^−/− neutrophils.

A. Subcellular localization of different NADPH oxidase subunits in WT and Gsr^−/− neutrophils before and after PMA stimulation. Purified WT and Gsr^−/− neutrophils were stimulated with 0.5 μM PMA for 15 min or left unstimulated. Cells were lysed and fractionated into cytosolic and membrane fractions by ultracentrifugation. The subcellular fractions were subjected to Western blot analyses, using Abs against gp91^phox, p47^phox, and p67^phox subunits of the NADPH oxidase. B. ERK and p38 activities in control and PMA-stimulated neutrophils. Purified WT and Gsr^−/− neutrophils were stimulated with 0.5 μM PMA for 15 min or left unstimulated, and lysed. Cell lysates were subjected to Western blot analysis using Abs against phospho-ERK, phospho-MEK, phospho-p38, and Gsr. Blots were stripped and probed with a mAb against β-actin to verify comparable protein loading. C. Differential tyrosine phosphorylation and ERK activation in WT and Gsr^−/− neutrophils following PMA and C. albicans stimulation. Purified BM neutrophils were stimulated with 0.5 μM PMA for 30 min, or with heat-killed C. albicans yeast (MOI=5) for 60 min, or left unstimulated. Neutrophils were lysed and cell lysates were subject to Western blot analysis using an Ab against phospho-ERK or phosphotyrosine (4G10). A blot was stripped and reprobed with a mAb against β-actin. The positions of molecular mass markers were shown on the right side of the blot. Asterisks indicate several tyrosine phosphorylated proteins in C. albicans-stimulated neutrophils.

A number of protein kinases, including ERK and p38 MAPKs as well as Syk tyrosine kinase have been shown to regulate neutrophil respiratory burst (22, 24, 59). To assess whether alterations in these kinases could contribute to the defective respiratory burst of Gsr^−/− neutrophils, we assayed the activities of ERK and p38 and examined global tyrosine phosphorylation in stimulated neutrophils (Figs. 7B & C). Interestingly, PMA-induced ERK activity was substantially stronger in Gsr^−/− neutrophils than in similarly treated WT neutrophils, although PMA-stimulated p38 activity was similar in the two groups (Fig. 7B). C. albicans-induced ERK activity was also slightly stronger in Gsr^−/− neutrophils than in WT neutrophils (Fig. 7C, Upper panel). Furthermore, PMA-induced tyrosine phosphorylation was dramatically stronger in Gsr^−/− neutrophils than in WT neutrophils (Fig. 7C, Lower panel). These tyrosine-phosphorylated proteins triggered by PMA covered a wide range of molecular masses, from ~20 –200 kDa. C. albicans also triggered the tyrosine phosphorylation of several proteins in the 30–65 kDa range in Gsr^−/− neutrophils (marked with asterisks in Fig. 7C), but not in WT neutrophils.

Enhanced inflammatory activity of Gsr^−/− macrophages

To understand whether macrophages in Gsr^−/− mice contribute to the abnormality in the immune defense against C. albicans, BMDM were stimulated with different doses of heat-killed C. albicans. C. albicans were heat-killed to avoid the complication of varying stimuli due to potential differences in the killing of C. albicans between WT and Gsr^−/− macrophages. The medium was harvested after 24 or 48 h, and assayed for cytokine production. TNF-α production was dramatically increased following C. albicans stimulation in both groups. Significantly more TNF-α was produced by Gsr^−/− macrophages than by WT macrophages (Fig. 8A). More IL-6 and IL-1β were also produced by Gsr^−/− macrophages than by WT cells following C. albicans stimulation. The enhanced cytokine production in Gsr^−/− macrophages was not caused by differences in phagocytic activity between WT and Gsr^−/− macrophages, since the phagocytic activity of the two groups were similar (Fig. 8B). These results clearly indicate that Gsr^−/− macrophages are functionally competent in the detection of C. albicans.

Figure 8. Gsr deletion in macrophages results in exaggerated cytokine production following C. albicans stimulation with little effect on macrophage phagocytic activity.

A. C. albicans-induced cytokine production by WT and Gsr^−/− macrophages. WT and Gsr^−/− BMDM were stimulated with heat-killed C. albicans (MOI=30, 100) for 24 or 48 h, and cytokines in the medium were quantified by ELISA. *, p<0.05 (t-test, n=4). B. Phagocytosis of C. albicans by WT and Gsr^−/− BMDM. WT and Gsr^−/− BMDM were incubated with pHrodo red-tagged C. albicans (MOI= 3 or 10) at 37°C for 30 min, and then labeled with Pacific blue-labelled anti-mouse F4/80 on ice for 10 min. Cells were subsequently washed twice with cold FACS buffer and analyzed by flow cytometry. F4/80⁺ BMDM were analyzed for the presence of pHrodo red-tagged C. albicans. Solid lines depict Gsr^+/+ and dashed lines depict Gsr^−/− cells.

Since MAPKs, particularly ERK, have been shown to regulate the production of inflammatory cytokines following C. albicans stimulation (60–62), we assessed whether Gsr deficiency alters MAPK activities (Fig. 9A). Upon stimulation with heat-killed C. albicans, ERK activity was rapidly increased in both WT and Gsr^−/− macrophages, and reached peak levels at ~15 min. Although the peak levels of ERK activity, indicated by the density of the phospho-ERK bands, were not different between WT and Gsr^−/− macrophages, ERK activity was slightly higher at the later time points (2 and 4 h) in Gsr^−/− macrophages. C. albicans stimulation also triggered a rapid increase in the activities of JNK and p38 within 15 min in both WT and Gsr^−/− macrophages. However, JNK activity rebounded by 4 h in Gsr^−/− macrophages but not in WT macrophages. In WT macrophages, p38 activity declined after the peak and then modestly rebounded between 2 and 4 h. In contrast, p38 activity slightly decreased at 45 min and then increased dramatically between 2 to 4 h in Gsr^−/− macrophages. Substantially elevated MK2 activity (63), which is regulated by p38 (64), was seen in C. albicans-stimulated Gsr^−/− macrophages but not in WT macrophages.

Figure 9. C. albicans-stimulated Gsr^−/− BMDM exhibit augmented p38, JNK, and Syk activities, attenuated Mkp-1 activity, and enhanced TNF-α mRNA stability.

A. The kinetics of ERK, JNK, and p38 activation following C. albicans stimulation. WT and Gsr^−/− BMDM were stimulated with heat-killed C. albicans (MOI=5) for the indicated lengths of time, and harvested. Cell lysates were subjected to Western blot analysis using Abs against total or phospho-specific protein kinases. The blot was stripped and blotted with a rabbit Ab against Gsr. B. Dephosphorylation of phospho-p38 by Mkp-1 immunoprecipitated from WT and Gsr^−/− BMDM. BMDM (10⁷ cells) were either left untreated (Upper panel) or stimulated with heat-killed C. albicans for 4 h (Lower panel). Cells were lysed and Mkp-1 was immunoprecipitated using protein A-Sepharose beads and polyclonal Abs against Mkp-1. The immunoprecipitates were then incubated with recombinant phospho-p38 for different times. The reaction was stopped and the mixtures were resolved using 10% NuPAGE gels. The same amount of phospho-p38 was run on the right side as a positive control. Western blot analysis was performed using a phospho-p38-specific Ab. Ca, C. albicans. C. p38-dependent increase in TNF-α mRNA stability in C. albicans-stimulated Gsr^−/− BMDM. BMDM were stimulated with heat-killed C. albicans (MOI=5) for 5.5 h, and then treated with DMSO or 10 μ SB203580 for 30 min. Subsequently, cells were treated with actinomycin D (5 μg/ml) for different times to isolate total RNA. TNF-α mRNA levels were assessed by qRT-PCR and normalized to 18s. TNF-α mRNA levels at time 0 were set as 100%. Results from a representative experiment are shown. D. Augmented Syk activity and global tyrosine phosphorylation in Gsr^−/− BMDM following hypha stimulation. WT and Gsr^−/− BMDM were stimulated with heat-killed C. albicans hyphae (MOI=5) for the indicated lengths of time, and harvested. Cell lysates were subjected to Western blot analysis using a polyclonal Ab against phospho-Syk (Upper panel), a mouse mAb against total Syk (Middle panel), and the 4G10 mAb against phosphotyrosine (Lower panel). The positions of the molecular mass standards were marked on the left of the blots.

In macrophages, the activity of JNK and p38 are negatively regulated by Mkp-1, a dual specificity protein phosphatase (40, 44, 48, 65), and attenuated Mkp-1 activity could enhance both JNK and p38 activities. To measure the activity of Mkp-1, we immunoprecipitated Mkp-1 from unstimulated and C. albicans-stimulated macrophages, and assessed the enzymatic activity of Mkp-1 in vitro using recombinant active p38 as a substrate (Fig. 9B). Incubation of active p38 with immunoprecipitated Mkp-1 resulted in a time-dependent decline in p38 phosphorylation, detected by Western blotting (Fig. 9B). Compared to Mkp-1 immunoprecipitated from WT macrophages, the activity of Mkp-1 immunoprecipitated from Gsr^−/− macrophages was lower than that of WT macrophages, indicated by the greater phospho-p38 levels at the corresponding time-points.

Since p38 has been shown to regulate the stability of the mRNAs containing AU-rich elements in their 3’ untranslated regions of a variety of cytokines, including TNF-α (66), we assessed the stability of TNF-α mRNA in C. albicans-stimulated Gsr^+/+ and Gsr^−/− macrophages (Fig. 9C). While the half-life of TNF-α mRNA in C. albicans-treated Gsr^+/+ macrophages was 24 min, the half-life was increased to 49 min in similarly treated Gsr^−/− macrophages (Fig. 9C). The p38 inhibitor, SB203580, almost completely abrogated the increase in the stability of TNF-α mRNA in Gsr^−/− macrophages.

Syk plays a critical role in the orchestration of the signal transduction pathways in response to C. albicans stimulation (11–13). Syk activation is associated with autophosphorylation at the Tyr525/526 site (67). We assessed whether C. albicans-stimulated Syk phosphorylation at the Tyr525/526 site in Gsr^−/− macrophages is different from that in WT cells (Fig. 9D). C. albicans hypha stimulation only slightly increased Syk phosphorylation at the Tyr525/526 site in WT macrophages. In contrast, Syk phosphorylation at the Tyr525/526 site was markedly increased in Gsr^−/− macrophages after exposure to C. albicans hyphae, although the total Syk levels were similar to those in WT macrophages. Syk phosphorylation at Tyr525/526 was detectable within 15 min following C. albicans stimulation and lasted for at least 4 h. The levels of tyrosine phosphorylation in multiple proteins were dramatically increased in C. albicans hypha- stimulated Gsr^−/− macrophages but not in WT macrophages. Taken together, these results strongly support an unexpected role of Gsr in the restraint of the inflammatory response of macrophages following C. albicans infection through the negative regulation of the Syk tyrosine kinase cascade.

Discussion

Gsr^−/− mice were found to be highly susceptible to C. albicans. In the absence of Gsr, C. albicans infection caused a markedly elevated fungal burden (Fig. 1B), blood cytokine storm (Fig. 3), and substantially increased mortality (Fig. 1A). Since large numbers of inflammatory leukocytes, including neutrophils, were found in the proximity of the fungal lesions in the kidneys of Gsr^−/− mice (Figs. 1C & D), Gsr deficiency did not appear to hinder the leukocyte trafficking to the kidneys. The higher kidney fungal burdens in Gsr^−/− mice (Figs. 1B & C) in spite of the greater numbers of neutrophils in the kidneys (Figs. 1C, 1E, and 2A) clearly indicate a profound defect in the fungicidal mechanisms in these mice. In support of this notion, we observed a marked decline in the phagocytosis activity and a significant impairment in fungicidal activity in Gsr^−/− neutrophils (Fig. 5). The decreased respiratory burst activity of Gsr^−/− neutrophils (Fig. 6) provides a mechanistic explanation for the markedly diminished respiratory burst activity in vivo (Fig. 4) and compromised immune defense against C. albicans in Gsr^−/− mice. Gsr^−/− neutrophils appeared to undergo normal NAPDH oxidase assembly on the membrane and was associated with profound enhancements of overall tyrosine phosphorylation and ERK activation (Fig. 7). We found that Gsr also plays a significant role in macrophages. In response to heat-killed C. albicans, Gsr^−/− macrophages exhibited elevated Syk, p38 and JNK activities (Fig. 9), and produced increased inflammatory cytokines TNF-α, IL-1β, and IL-6 (Fig. 8A), although Gsr deficiency in macrophages had little impact on C. albicans uptake (Fig. 8B). Our studies demonstrate that although Gsr^−/− mice are competent in detecting C. albicans pathogens and mobilizing inflammatory leukocytes to the infection site, their neutrophils are impaired in capturing and killing of these pathogens. Our studies revealed critical functions of Gsr in both neutrophils and macrophages, two key cell types of the innate immune defense.

The function of Gsr in neutrophils

While exactly how Gsr facilitates neutrophil-mediated C. albicans killing is not completely understood, few possible mechanisms could be considered. First, it is possible that Gsr protects the fungicidal machinery of neutrophils by maintaining the level of GSH. Upon encountering fungi or yeast, neutrophils produce large amounts of ROS in the phagolysosomes, which are crucial for the killing of the pathogens (21, 68). However, phagolysosomal ROS, H₂O₂ in particular, could also leak into the cytosol, leading to harmful oxidation of cellular components. In the cytosol, GSH can detoxify H₂O₂ and becomes GSSG. Because Gsr catalyzes the regeneration of GSH from GSSG, it is conceivable that WT neutrophils would be able to maintain a significant level of GSH in the cytosol to detoxify cytosolic ROS and thereby protect the cells. In contrast, GSH cannot be regenerated in Gsr^−/− neutrophils and upon depletion of GSH, ROS leaked out from the phagolysosomes will remain unopposed in reacting with the cellular organelles and enzymes, including those responsible for the generation of NADPH from glucose. The notable increase in global protein tyrosine phosphorylation in Gsr^−/− neutrophils following PMA stimulation is also consistent with the exposure to elevated oxidants; H₂O₂ is known to inhibit tyrosine phosphatases, thereby leading to global protein tyrosine phosphorylation (69–71). Such oxidative damage could impair the phagocytosis of C. albicans, halt the pentose monophosphate pathway, and terminate NADPH oxidase-mediated respiratory burst. It is worth noting that the cessation of the respiratory burst in Gsr^−/− neutrophils following PMA stimulation was not caused by death of these cells, as the vast majority of the neutrophils were not stained by PI (data not shown).

A second but related possibility is that the compromised respiratory burst in Gsr^−/− neutrophils may be the result of a dramatic signaling disarray. In Gsr^−/− neutrophils, PMA stimulation resulted in a profound increase in both global protein tyrosine phosphorylation and ERK activation (Fig. 7C). We speculate that the substantially elevated global tyrosine phosphorylation may only represent the ‘tip of the iceberg’ in a dramatic signaling disarray, as other biological processes could also be affected but not easily detectable. Nonetheless, it is conceivable that the aberrant signaling disarray ultimately leads to the termination of the intricately organized respiratory burst process. Neutrophil signaling disarray or enzymatic inhibition in Gsr^−/− neutrophils during the phagocytosis may also explain why fewer yeast particles are engulfed and killed by Gsr^−/− neutrophils than by WT cells (Fig. 5), since respiratory burst occurs simultaneously as cells engulf yeast particles (72). While ERK has been shown to regulate the respiratory burst through phosphorylation of p47^phox (22, 24, 59), the elevated ERK activity in Gsr^−/− neutrophils (Figs. 7B & C) cannot explain the decline in respiratory burst activity in these cells. Since ERK activation in response to PMA is mediated by PKCs, a kinase family critical for neutrophil respiratory burst (22), based on the greater ERK activity we reason that a defect in PKC activation in Gsr^−/− neutrophils is unlikely. A third possibility is that Gsr may facilitate the assembly or modification of the NADPH oxidase subunits, although we think that this is unlikely. In PMA-stimulated neutrophils, both p67^phox and p47^phox subunits of the NADPH oxidase complex were recruited to the membrane and the membrane-associated gp91^phox subunit underwent similar changes in both WT and Gsr^−/− cells after PMA stimulation (Fig. 7A). These results combined with the normal initial respiratory burst in the first few minutes following PMA stimulation in Gsr^−/− neutrophils (33, 34) strongly suggest that NADPH oxidase assembly, at least in the early phase, is not affected by Gsr deficiency.

The function of Gsr in macrophages

Another interesting finding of this study is that Gsr^−/− mice and macrophages produce higher amounts of cytokines and chemokines than do their WT counterparts (Figs. 3 & 8). While increased fungal burden may contribute to the elevated cytokines and chemokines in C. albicans-challenged Gsr^−/− mice, Gsr^−/− macrophages also appeared to exhibit enhanced intrinsic inflammatory responses following fungal stimulation. This is reflected in the elevated cytokine production in vitro following stimulation with heat-killed C. albicans (Fig. 8) and greater levels of p38 and JNK activities (Fig. 9A). Given that C. albicans used in these experiments were heat-killed and both WT and Gsr^−/− macrophages engulfed similar amounts of C. albicans (Fig. 8B), the increased cytokine production and elevated p38 and Syk activities strongly suggest a greater sensitivity of Gsr^−/− cells to C. albicans stimulation. We think that two mechanisms could contribute to the enhanced cytokine production in Gsr^−/− macrophages. First, the increased Syk activity and enhanced global tyrosine phosphorylation may enhance the inflammatory response to yeast through augmenting the ERK pathway (Fig. 9A, D). Second, p38 may contribute to the inflammatory response by enhancing the stabilities and the translation of the AU-rich element-containing cytokine mRNAs. TNF-α mRNA was substantially more stable in Gsr^−/− macrophages than in the wildtype macrophages, and TNF-α mRNA stabilization caused by Gsr deficiency was almost completely abolished by the p38 inhibitor (Fig. 9C), highlighting the critical role of enhanced p38 activation in the augmented inflammatory response. Enhanced mRNA stability may also contribute to the increased production of other cytokines, such as IL-6 and IL-1β (Fig. 8).

While it is not entirely unclear how Gsr deficiency causes enhanced Syk and MAPK activation, based on the biochemical function of Gsr we can make some speculation. Many protein kinases, particularly tyrosine kinases and MAPKs whose activation requires tyrosine phosphorylation, are negatively regulated by protein tyrosine phosphatases (73). Protein tyrosine phosphatases rely on a cysteine residue in the catalytic site, (H/V)CX₅R(S/T), for tyrosine dephosphorylation. The MAPK phosphatases have a similar catalytic site (VHCX5R(S/T)) (74, 75), with cysteine being essential for MAPK dephosphorylation. If the thiol residue in their catalytic sites is oxidized by ROS, these phosphatases become functionally inactive. GSH may play a crucial role in the protection of these cysteine residues in tyrosine phosphatases and dual specificity phosphatases including Mkp-1. In the absence of Gsr, GSSG cannot be regenerated into GSH. Once GSH is depleted by cytosolic ROS as the result of respiratory burst, cytosolic proteins such as protein tyrosine phosphatases and Mkps will become vulnerable to oxidation, leading to exuberant tyrosine kinase and MAPK activation. This model is consistent with the observation that Mkp-1 activity in Gsr^−/− macrophages is lower than that of wildtype macrophages (Fig. 9B). This model also explains why sustained Syk, JNK, and p38 activation and global tyrosine phosphorylation only occurs hour(s) after C. albicans stimulation, because GSH depletion only occurs after a period of respiratory burst. The respiratory burst in neutrophils induced by PMA is substantially more robust than that induced by yeast in neutrophils (Fig. 6B) or macrophages (76). Therefore, it is not surprising that dramatically greater global tyrosine phosphorylation is seen in PMA-stimulated Gsr^−/− neutrophils than in yeast- or PMA-treated macrophages (Fig. 7C & 9C). The difference in the amounts of ROS produced by neutrophils and macrophages may also explain why Gsr deficiency preferentially compromises the phagocytosis of neutrophils (Fig. 5A) but not macrophages (Fig. 8B). Because neutrophils produce far more ROS than the macrophages (76), Gsr deficiency is expected to result in greater oxidative damage and have a larger impact on phagocytosis in neutrophils than in macrophages.

Key Points.

• Gsr^−/− mice exhibited markedly increased susceptibility to C. albicans challenge.

• Gsr^−/− neutrophils display defects in fungicidal activity.

• Gsr^−/− macrophages exhibit an enhanced inflammatory response.