Calcineurin regulates innate antifungal immunity in neutrophils

Abstract

Patients taking immunosuppressive drugs, like cyclosporine A (CsA), that inhibit calcineurin are highly susceptible to disseminated fungal infections, although it is unclear how these drugs suppress resistance to these opportunistic pathogens. We show that in a mouse model of disseminated Candida albicans infection, CsA-induced susceptibility to fungal infection maps to the innate immune system. To further define the cell types targeted by CsA, we generated mice with a conditional deletion of calcineurin B (CnB) in neutrophils. These mice displayed markedly decreased resistance to infection with C. albicans, and both CnB-deficient and CsA-treated neutrophils showed a defect in the ex vivo killing of C. albicans. In response to the fungal-derived pathogen-associated molecular pattern zymosan, neutrophils lacking CnB displayed impaired up-regulation of genes (IL-10, Cox2, Egr1, and Egr2) regulated by nuclear factor of activated T cells, the best characterized CnB substrate. This activity was Myd88 independent and was reproduced by stimulation with the beta(1,3) glucan curdlan, indicating that dectin-1, rather than toll-like receptors, is the upstream activator of calcineurin. Our results suggest that disseminated fungal infections seen in CsA-treated patients are not just a general consequence of systemic suppression of adaptive immunity but are, rather, a result of the specific blockade of evolutionarily conserved innate pathways for fungal resistance.

Figures

Figure 1.

CsA suppresses innate resistance to C. albicans infection. (A) Kaplan-Meier survival curve showing the survival of C. albicans–infected mice. Rag2−/− or WT control mice were infected with 105C. albicans yeasts by i.v. injection and then treated daily with 200 mg/kg CsA or vehicle control (n = 11–12 mice per group). Mice were then monitored daily for survival. P < 0.0001 by log-rank test comparing CsA-treated to vehicle-treated groups, with no significant difference between either Rag2−/− or WT groups. Results are representative of three independent experiments. (B) Mice were infected as in A and histological analysis of the kidneys was performed 4 d after infection. C. albicans were visualized by PAS stain (purple, arrow). Histology is representative of four per group. Results are representative of three independent experiments. Bars: (left) 500 µm; (right) 100 µm. (C) CsA- or vehicle-treated Rag2−/− or WT mice were infected with C. albicans as in A. Homogenates of the kidney were made 4 d after infection and C. albicans quantitated by serial dilution and colony counting. Each dot represents the mean of two measurements taken from a single mouse. P < 0.0001 by comparing either CsA-treated group to the corresponding vehicle-treated group by an unpaired Student’s t test. Results are representative of two independent experiments. Horizontal bars show the mean of the group. Error bars show SD.

Figure 2.

CnBLysM mice display increased susceptibility to fungal infection. (A) Kaplan-Meier survival curve showing the survival of C. albicans–infected mice. CnBfl/fl or CnBLysM mice were infected with 105C. albicans yeasts by i.v. injection (n = 10–11 mice per group). Mice were then monitored daily for survival. P < 0.0001 by the log-rank test. Results are representative of three independent experiments. (B) CnBLysM or control CnBfl/fl mice were infected with C. albicans as in A. Homogenates of the kidney were made 4 d after infection and C. albicans quantitated by serial dilution and colony counting. Each dot represents the mean of two measurements taken from a single mouse. P < 0.0001 by an unpaired Student’s t test. Results are representative of three independent experiments. Horizontal bars show the mean of the group. Error bars show SD. (C) Histological analysis of the kidneys of C. albicans–infected mice 4 d after challenge. C. albicans were visualized by PAS stain (purple color). Histology is representative of four mice per group. Results are representative of three independent experiments. Bars, 100 µm.

Figure 3.

Zymosan activates the calcineurin–NFAT pathway. (A) Primary neutrophils were isolated and treated with CsA or vehicle for 15 min before mixing 1:1 with C. albicans yeasts, strain SC 5314. At the indicated time points, aliquots were taken and CFUs of C. albicans determined by serial dilutions on YPD agar. Relative killing was determined by normalization to C. albicans added to medium without neutrophils. Results are representative of three independent experiments. (B) Expression of NFATc1, NFATc2, and NFATc3 was determined in neutrophils and macrophages as indicated by real-time PCR. Results are representative of three independent experiments. (C) Primary neutrophils stimulated with 100 µg/ml zymosan and blotted for expression of NFATc1. Results are representative of three independent assays. (D) Primary neutrophils were stimulated with zymosan for 30 min, and nuclear and cytoplasmic fractions were blotted for NFATc1. Results are representative of three independent experiments. (E) Primary neutrophils were stimulated with the indicated concentrations of zymosan for 12 h, and the supernatant levels of IL-10 and IL-6 were quantified by ELISA. Results are representative of three independent experiments. (F) Primary neutrophils were stimulated with zymosan for 6 h and the expression of Cox2 was determined by real-time PCR. Results are representative of three independent experiments. (G) NFATc1 NFATc3Mx neutrophils and neutrophils from double heterozygous NFATc1Mx/+ NFATc3Mx/+ mice were isolated and stimulated with 100 µg/ml zymosan. 12 h later, supernatants were harvested and IL-10 levels determined by ELISA. Results are representative of three independent experiments. Error bars show SD.

Figure 4.

Dectin-1, and not TLRs, are upstream of CnB activation. (A) Primary neutrophils from the indicated genotypes were pretreated with CsA for 15 min and stimulated with 100 µg/ml curdlan. IL-10 secretion was determined after 12 h by ELISA. Results are representative of three independent experiments. (B) Primary neutrophils from the indicated genotypes were pretreated with CsA for 15 min and stimulated with 100 µg/ml curdlan. IL-10 secretion was determined after 12 h by ELISA. Results are representative of two independent experiments. Error bars show SD.