Calcineurin regulates innate antifungal immunity in neutrophils

To determine whether CsA promotes fungal infection through effects on the innate or the adaptive immune system, Rag2^−/− mice, which lack an adaptive immune system, and WT mice were used in a model of disseminated fungal infection. Because C. albicans is the most common fungal pathogen in humans, we used a model of disseminated C. albicans infection in mice (Diekema et al., 2002; Schelenz, 2008). Mice of both genotypes were infected with 10⁵ C. albicans yeasts by i.v. injection and then treated daily with 200 mg/kg CsA or vehicle. Both CsA-treated Rag2^−/− and CsA-treated WT mice showed a median survival of 4 d after challenge with the yeasts (Fig. 1 A). In contrast, the majority of both Rag2^−/− and WT mice treated with vehicle survived the 14-d experiment. Treatment with CsA in the absence of infection did not alter mouse survival (Fig. S1 A). Similar results were also obtained using a lower (40 mg/kg) dose of CsA (Fig. S1 B). To confirm that the CsA-treated mice died as a result of failure to control C. albicans infection, histological analysis of the kidneys and quantitative assessments of renal C. albicans burdens 4 d after infection were performed (Fig. 1, B and C). C. albicans was stained using the periodic acid-Schiff (PAS) stain. The kidneys of both Rag2^−/− and WT mice treated with CsA showed fulminant infection with germinating hyphal C. albicans forms with no obvious difference in disease severity. In contrast, C. albicans levels were substantially lower in the kidneys of vehicle-treated mice. Collectively, these data indicate that CsA acts on the innate immune system to promote susceptibility to infection with C. albicans.

To both further define the cell type responsible for suppression of antifungal immunity by CsA and to establish that the suppression is the result of a calcineurin-dependent effect of CsA, we generated mice with a conditional deletion of a CnB floxed allele by cre recombinase expressed under the lysozyme M promoter (hereafter, CnB^LysM mice; Clausen et al., 1999; Neilson et al., 2004). To determine the efficiency of cre-mediated recombination in different myeloid cell lineages, we measured CnB mRNA levels in neutrophils sorted from the BM and spleen as a CD11b^high Gr-1^high population (Fig. S1 C). Analysis of CnB expression by real-time PCR showed efficient deletion (∼90%) of CnB in neutrophils at both sites. Macrophages were also sorted from the spleen as CD11b^int F4/80⁺ cells. In contrast to the efficient deletion seen in neutrophils, however, splenic macrophages showed no appreciable deletion of CnB. Peritoneal macrophages showed only modest deletion (∼50%). Hence, we attribute the effects described in the next paragraph primarily to the function of CnB in neutrophils, although we cannot rule out an additional contribution from CnB in certain macrophage subsets.

CnB^lysM mice and littermate controls were infected with 10⁵ C. albicans, as in the previous section, and monitored for survival (Fig. 2 A). CnB^lysM mice succumbed to C. albicans infection ∼7 d after challenge. As in the experiment presented in Fig. 1, the majority of control mice survived the 14-d experiment. CFU counts measured from kidney tissue 4 d after infection (Fig. 2 B) revealed a significantly higher renal burden of C. albicans in CnB^lysM mice compared with controls, indicating death from a failure to control infection. Likewise, histological analysis of the kidneys revealed extensive PAS-positive lesions in CnB^lysM mice containing germinating C. albicans hyphae and sheets of tissue-infiltrating neutrophils. In contrast, inflammatory infiltrates were much less frequent in control mice, and, when found, appeared to be nearly devoid of PAS-positive material, indicating effective fungal clearance (Fig. 2 C). Thus, similar to CsA-treated Rag2^−/− mice, CnB^lysM mice display a dramatically increased susceptibility to systemic fungal infection.

When neutrophils encounter a pathogen, they attempt to limit infection by both directly killing the offending agent and producing molecules that promote an effective inflammatory response, including cytokines and prostanoids. To explain the observation that mice deficient in calcineurin activity in innate immune cells succumb rapidly to C. albicans, the response of calcineurin-deficient neutrophils to this pathogen was further explored in vitro. First, we tested the role of calcineurin in fungicidal responses. Both CnB-deficient and CsA-treated neutrophils were defective in the ability to kill C. albicans within 30 min (Fig. 3 A). Similar results were obtained with two C. albicans strains, SC5314 (Fig. 3 A) and NCCLS 11 (Fig. S1 E). CsA was not seen to directly affect C. albicans survival in the absence of neutrophils (Fig. S1 D). Likewise, C. albicans that were pretreated with CsA or vehicle, washed thoroughly, and then added to neutrophils were found to induce equivalent IL-10 production, making it unlikely that possible direct effects of CsA on yeast metabolism complicate the interpretation of these results (Fig. S1 D). Despite this difference in killing, classical effector pathways mediating the fungicidal activity of neutrophils, including degranulation, as monitored by myeloperoxidase release, and the production of nitric oxide, were unaltered by treatment with CsA (Fig. S2, A–C; Aratani et al., 1999). Likewise, CsA treatment or CnB deficiency did not affect reactive oxygen species (ROS) production or phagocytosis in response to zymosan or C. albicans (Fig. S4, E–G). Collectively, these data suggest that calcineurin regulates the ability of neutrophils to kill C. albicans through a novel anti-microbial pathway.

Next, we examined the transcriptional effector mechanisms downstream of CnB in neutrophils leading to the control of inflammatory responses to fungal pathogens. Because NFAT transcription factors are the best characterized targets of calcineurin, we examined the expression of NFATc1, NFATc2, and NFATc3 in neutrophils and macrophages. Although macrophages expressed NFATc1, NFATc2, and NFATc3, neutrophils only expressed NFATc1 and NFATc3 (Fig. 3 B). We then examined if pathogen-associated molecule patterns (PAMPs) from the fungal cell wall can activate the calcineurin–NFAT pathway. Neutrophils were purified from the BM of WT mice and stimulated with zymosan, an extract of yeast cell walls rich in β(1,3)-glucans. A substantial increase in NFATc1 protein was seen after 6 h of stimulation (Fig. 3 C). To directly demonstrate that zymosan is able to activate NFATs, neutrophils were stimulated with zymosan and the activation status of NFATc1 was determined by monitoring nuclear translocation (Fig. 3 D). Nuclear translocation of NFATc1 occurred after 30 min, demonstrating that zymosan activates NFATs in a manner consistent with previous reports in other myeloid cell types (Goodridge et al., 2007).

To elucidate the importance of the calcineurin–NFAT pathway on the antifungal transcriptional responses in neutrophils, WT, CnB-deficient, and CsA-treated neutrophils were stimulated with zymosan. Both CnB-deficient and CsA-treated neutrophils were defective in IL-10 production as measured by ELISA in supernatants 12 h after stimulation (Fig. 3 E). Previously, the induction of IL-10 after zymosan stimulation has been noted to be dectin-1 dependent (Saijo et al., 2007). Additional analysis by real-time PCR of neutrophils 6 h after zymosan stimulation showed that other known NFAT target genes, including Cox2, Egr1, and Egr2, were all induced by zymosan in WT cells and that this response was significantly blunted in CnB-deficient and CsA-treated neutrophils (Fig. 3 F and Fig. S2 D; Goodridge et al., 2007; Lazarevic et al., 2009). In contrast, the production of IL-6 was not altered in CnB-deficient or CsA-treated neutrophils (Fig. S2 E).

Notably, although these in vitro studies focus on the functions of CnB in neutrophils, CnB can function similarly in other myeloid lineages. Splenic macrophages isolated from CnB^LysM mice displayed improved deletion (∼70%) of CnB after 5 d of ex vivo culture in the presence of 20 ng/ml M-CSF (Fig. S3 A). CsA-treated or CnB-deficient macrophages displayed a defect in IL-10 secretion in response to zymosan similar to that seen in neutrophils (Fig. S3 B). Similarly, treatment with CsA blocked IL-10 and IL-12 secretion by primary splenic dendritic cells (Fig. S3C, D). Because neutrophils were not observed to produce detectable IL-12 in response to zymosan, the effects of CnB/CsA on antifungal responses are likely to be both broad, in that they affect many cell lineages, and specific with respect to their effects in any given lineage.

Because NFATc1 and NFATc3 are the major NFAT family members expressed in neutrophils, we tested whether they were responsible for the production of IL-10 downstream of CnB. NFATc1 and NFATc3 floxed alleles were deleted using the inducible Mx-cre/poly I:C system. The resulting NFATc1 NFATc3 doubly deficient neutrophils (NFATc1^Mx NFATc3^Mx) showed defective IL-10 production when stimulated with zymosan, which is similar to the defect seen in CnB-deficient neutrophils (Fig. 3 G). Mice with a deletion of only NFATc1 or NFATc3 alone showed little to no defect in IL-10 production (Fig. S4 A). Given that NFATc1 and NFATc3 mediate the production of IL-10 downstream of calcineurin, we examined if they might also be involved in promoting candidal killing. Interestingly, in contrast to neutrophils deficient in CnB (Fig. 3 A), NFATc1^Mx NFATc3^Mx neutrophils showed no defect in killing C. albicans in vitro (Fig. S4 B). Moreover, NFATc1^Mx NFATc3^Mx mice showed no significant alteration in survival in a model of disseminated Candidemia (Fig. S4 C).

To examine if NFATs contribute to cytokine responses to zymosan in other myeloid cells, the expression of NFATc1-c3 was considered in primary splenic dendritic cells. NFATc1-c3 transcripts were present and the expression of NFATc1 and NFATc2 was induced in response to zymosan stimulation. On this basis, we generated dendritic cells deficient for NFATc1 and NFATc2 by breeding NFATc1 floxed allele NFATc2^−/− mice to the CD11c-cre deleter strain to generate NFATc1^CD11c NFATc2^−/− mice. Splenic dendritic cells from these mice displayed defects in IL-12 and IL-10 production similar those seen with CsA treatment (Fig. S3 D). Dendritic cells lacking only NFATc1 or NFATc2 alone displayed little to no defect in IL-10 secretion (Fig. S3 F). Thus, the specific combination of NFATs responsible for cytokine production in response to zymosan varies from tissue to tissue.

Thus, CnB mediates two distinct pathways in the response to C. albicans. One, which is likely to be NFAT independent, is responsible for the immediate killing of C. albicans, and the other is mediated by NFATc1 and NFATc3 and is involved in transcriptional responses to this pathogen.

Zymosan has been shown to activate both the dectin-1 receptor through β(1,3)-glucans and TLRs, particularly TLR2 (Dillon et al., 2006). Previous studies indicate that activation of dectin-1, but not TLRs, promotes the sustained elevations in intracellular calcium that are necessary to activate calcineurin (Xu et al., 2009). To directly test whether the activation of dectin-1, and not TLRs, is upstream of the effects of CsA, WT or CnB-deficient neutrophils were stimulated with curdlan, which contains dectin-1–stimulatory β(1,3)-glucans but lacks TLR-stimulatory motifs (Gringhuis et al., 2009). Similar to the results obtained with zymosan, both CsA-treated and CnB-deficient neutrophils showed a defect in IL-10 production after stimulation with curdlan (Fig. 4 A). As expected, CsA did not alter the production of IL-10, TNF, or IL-6 in neutrophils stimulated with LPS, a pure TLR4 agonist (Fig. S4 D). To confirm that contaminants with TLR-stimulating capacity in the curdlan preparation were not confounding the interpretation of this experiment, neutrophils were isolated from MyD88^−/− mice (Fig. 4 B). In the absence of MyD88, all TLR signaling, except for the activation of the TRIF pathway by TLR4 and TLR3, which are not targeted by fungal pathogens, is defective (Kawai et al., 1999; Hoebe et al., 2003; Yamamoto et al., 2003). WT and MyD88^−/− neutrophils were equally sensitive to the ability of CsA to block IL-10 production downstream of curdlan (Fig. 4 B). These results make it unlikely that TLR-stimulatory contaminants confound the interpretation of curdlan as a selective activator of dectin-1 and not TLRs.

In summary, CsA acts on the innate immune system to suppress resistance in a model of disseminated fungal infections. This activity maps to the function of CnB in neutrophils, where it contributes to both immediate killing of C. albicans yeasts and transcriptional responses downstream of dectin-1.

Although calcineurin inhibitors are some of the most potent immunosuppressants available, significant toxicity limits their clinical use (Farkas et al., 2009). Understanding the biology of these adverse effects is critical for the rational design of immunosuppressants with greater clinical utility. In particular, patients taking calcineurin inhibitors are susceptible to infection with a range of opportunistic fungal pathogens, the most common of which is C. albicans (Diekema et al., 2002; Schelenz, 2008). In this paper, we demonstrate that CsA promotes fungal infection through suppression of innate immune antifungal responses, a function which is separable from its action to prevent T cell–mediated allograft rejection. Furthermore, these actions likely reflect inhibition of responses to fungal PAMPs through the dectin-1 receptor, although recent work has identified several related receptors, such as dectin-2, Mincle, and CLEC-2, that use similar signaling pathways and may likewise function upstream of CnB (Matsumoto et al., 1999; Ariizumi et al., 2000a; Wells et al., 2008; Kerrigan et al., 2009; Robinson et al., 2009; Yamasaki et al., 2009). Thus, the disseminated fungal infections observed in CsA-treated patients are not a consequence of broad inhibition of T cell responses but rather reflect specific blockade of evolutionarily conserved innate pathways for sensing fungal pathogens. Our results also help explain the clinical observation that disseminated fungal infections occur more frequently in patients with neutropenia than those with congenital defects in lymphocyte development (De Rosa et al., 2009). Moreover, this study highlights a broader role for calcineurin and NFATs in immune responses beyond antigen receptor signaling because this pathway regulates responses in the myeloid compartment.

Despite the defect in the ex vivo killing of C. albicans by CsA-treated or CnB-deficient neutrophils, these mutant neutrophils did not display significant defects in any of several effector processes, such as phagocytosis, ROS production, myeloperoxidase degranulation, or NO production, suggesting that CnB instigates an as yet uncharacterized candidacidal effector mechanism. The disassociation between transcriptional responses to yeast/zymosan and immediate C. albicans killing seen in NFATc1 NFATc3-deficient neutrophils suggests that CnB mediates two pathways involved in the response to fungal pathogens.

In investigating the transcriptional pathways downstream of CnB, the production of IL-10 in particular was dependent on CnB and NFATc1/c3. Interestingly, although IL-10 is most often cited as an antiinflammatory cytokine and, indeed, directly suppresses the phagocytic and candidacidal activities of neutrophils in vitro, the role of IL-10 in C. albicans resistance in vivo is less clear. Recent studies have correlated high IL-10 production with reduced renal C. albicans burden and tissue damage in i.v. models of C. albicans infection (Roilides et al., 2000; Tavares et al., 2000; MacCallum et al., 2009). Further work is needed to define the balance between pro- and antiinflammatory cytokines that provides for efficient protection from invasive fungal infection. Moreover, it is possible that both the nature and relative importance of the transcriptional response may vary depending on the route and kinetics of fungal exposure.

In summary, the results presented in this paper implicate the calcineurin signaling axis as critical for fungal responses in myeloid cells and specifically implicate the innate immune system as the target of CsA most relevant to the pathogenesis of disseminated fungal infections. In vitro, CnB contributes both to the immediate killing of C. albicans yeasts and to cytokine responses, particularly IL-10 production, to fungal PAMPs such as zymosan and curdlan. We propose that the combined defect in candidacidal activity and IL-10 production by calcineurin inhibition leads to both increased pathogen burden and a robust unchecked inflammatory response that promotes excessive tissue damage and lethality. These results refocus inquiry into the mechanism of disseminated fungal infections in CsA-treated patients away from the effects of CsA to suppress adaptive immune responses and toward a specific examination of dectin-1–dependent responses in myeloid cells.

The generation of NFATc1 floxed allele mice was previously described (Aliprantis et al., 2008; Horsley et al., 2008). The lysozyme M–cre strain was purchased from The Jackson Laboratory. The CD11c-cre strain was a gift from B. Reizis (Columbia University, New York, NY). CalcineurinB and Nfatc3 floxed allele mice were a gift from G. Crabtree (Stanford University, Palo Alto, CA; Neilson et al., 2004; Canté-Barrett et al., 2007). Rag2^−/− mice were maintained on the BALB/c background. CnB^LysM mice were maintained on a mixed C57BL/6 × 129 background. For NFATc1 NFATc3^Mx mice, Mx-cre was induced by three injections every 2 d of 500 µg poly I:C (Kühn et al., 1995). All mice were housed at the Harvard School of Public Health, and experimental protocols were approved by the Harvard Institutional Animal Care and Use Committee.

Neutrophils were isolated by flushing femoral and tibial BM and lysing red blood cells in ACK buffer. The remaining cells were then layered over a three-part percoll density gradient (55, 65, and 75% isotonic percoll/PBS) and centrifuged at 500 g for 30 min at room temperature. The interface between the 65 and 75% layers was collected as the neutrophil-enriched fraction. This fraction was determined to be ∼95% Gr-1^hi CD11b^hi B220⁻ CD4⁻ CD8⁻ neutrophils by FACS analysis. Microscopic analysis of nuclear morphology confirmed the FACS determination of purity. For stimulation and in vitro killing assays, neutrophils were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, glutamine, penicillin, streptomycin, and 2-mercaptoethanol. Neutrophils were stimulated with 100 µg/ml zymosan (from Saccharomyces cerevisiae; Sigma-Aldrich) or 100 µg/ml curdlan (from Alcaligenes faecalis; Sigma-Aldrich) as indicated. Where CsA was used, cells were pretreated for 15 min with CsA before stimulation. For in vitro studies, CsA was dissolved in ethanol and diluted 1,000× to obtain the indicated concentration. For in vivo studies CsA was prepared as an emulsion in 15% (volume/volume) ethanol/castor oil.

C. albicans SC5314 was grown for 16 h in YPD broth and washed three times with PBS. Yeasts were then counted on a hemocytometer and suspended at a concentration of 10⁶/ml, and 100 µl was injected into 8-wk-old mice of the indicated genotypes via i.v. tail vein injection. C. albicans was observed microscopically before injection and found to be >99% yeast/hyphal forms. Mice were monitored daily for 14 d. For determination of candidal burdens, mice were euthanized 4 d after injection by CO₂ narcosis, and kidneys were harvested and homogenized by mechanical disruption in PBS. Serial dilutions of this homogenate were plated on YPD agar and colonies enumerated to determine quantitative fungal CFU counts. For histological analysis of the kidney, animals were euthanized, and kidneys were harvested and fixed in 4% paraformaldehyde. They were then dehydrated through an ethanol/xylene series and embedded in paraffin. Sections were cut and stained with the PAS stain or hematoxylin and eosin.

For mRNA expression studies, RNA was isolated using trizol (Invitrogen). cDNA was synthesized using the iScript kit (Bio-Rad Laboratories), and real-time PCR was performed using SYBR green mix (Applied Biosystems). Primers for NFATc1, NFATc2, NFATc3, EGR1, and EGR2 were used as previously described (Lazarevic et al., 2009).

Nuclear/cytoplasmic fractionation was performed using the NE-PER kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The subsequent fractions were run on SDS-PAGE and blotted using anti-NFATc1 (clone 7A6; BD), Sp1 (PEP2; Santa Cruz Biotechnology, Inc.), and β-actin (13E5; Cell Signaling Technology).

C. albicans strain NCCLS 11 was obtained from American Type Culture Collection. Primary neutrophils were mixed 1:1 with C. albicans yeasts with gentle agitation at 37°C. At the indicated times, 10-µl aliquots were taken, neutrophils lysed by resuspending in 1 ml of water, and C. albicans CFU determined by serial dilution and colony counting on YPD agar.

Primary neutrophils were cultured at a density of 2 × 10⁵ cells/well in a 96-well plate. At the indicated times after stimulation with 100 µg/ml zymosan or a 1:1 addition of live Candia yeasts, aliquots were taken and mixed 1:1 with a solution of 2 mM luminol, and chemiluminescence was measured on a luminometer (2010; Monolight).

Dendritic cells were purified from the single cell suspensions of splenocytes using positive selection with anti-CD11c magnetic beads (Miltenyi Biotec), according to the manufacturer’s instructions. The resulting cells were confirmed to be >85% CD11c^hi dendritic cells by FACS analysis.

For macrophage cultures, total splenocytes were cultured in 24-well tissue culture plates in the presence of 20 ng/ml M-CSF for 5 d. Nonadherent cells were removed by daily washing. The resulting cells were confirmed to by >95% CD11b⁺ macrophages by FACS.

All statistical tests were calculated using Prism. For survival curves, error bars reflect standard error. All other values graphed are mean ± SD.

Fig. S1. provides controls for in vivo and in vitro studies using C. albicans. Fig. S2 shows that IL-6 production, ROS production, and neutrophil degranulation do not require CnB. Fig. S3 shows the contribution of CnB/NFATs to cytokine secretion in dendritic cells and macrophages. Fig. S4 shows no appreciable difference in susceptibility to fungal infections in NFATc1^Mx NFATc3^Mx mice, that CsA does not alter cytokine secretion after LPS stimulation, and unaltered phagocytosis after treatment of neutrophils with CsA.

The authors thank Gerald Crabtree and Boris Reizis for generously sharing the CnB floxed allele and CD11c-cre lines, Antony Segal, for helpful discussion, Kelly Tsang, Arnold Park, Zachary Swartz, and Shivesh Punit for technical assistance, Dorothy Zhang for assistance with histology, and Marc Wein, Vania Lazarevic, and Fabio Martinon for their thoughtful review of the manuscript.

This work was supported by National Institutes of Health grants AI43953 and AI31541 (L.H. Glimcher), Burroughs Wellcome Fund CAMS award (A. Aliprantis), and K08AR054859 (A. Aliprantis).

L.H. Glimcher declares that she is a Member of the Board of Directors of and holds equity in Bristol-Myers Squibb. The authors have no other conflicting financial interests.