Transgenic mice expressing human IL-37 (hIL-37Tg) have been previously described Nold et al. (2010). Control C57BL/6J (WT) mice were purchased from the Jackson Laboratory and maintained under specific-pathogen free conditions. The mice were fed sterilized laboratory chow (Hope Farms, Woerden, The Netherlands) and water ad libitum. The experiments were approved by the Ethics Committee on Animal Experiments of Radboud University Nijmegen.

Candida albicans ATCC MYA-3573 (UC 820), a strain well described elsewhere (Lehrer and Cline, 1969), was used in all experiments. Candida was grown overnight in Sabouraud broth at 37∘C, cells were harvested by centrifugation, washed twice, and resuspended in culture medium (RPMI-1640 Dutch modification, ICN Biomedicals, Aurora, OH, USA; van der Graaf et al., 2005). To generate pseudohyphae, C. albicans blastoconidia were grown at 37∘C in culture medium, adjusted to pH 6.4 by using hydrochloric acid. Pseudohyphae were killed at 100∘C for 1 h and resuspended in culture medium to a hyphal inoculum size that originated from 10⁶ blastoconidia per ml (referred to as 10⁶ pseudohyphae per ml).

Resident peritoneal macrophages were harvested from groups of five hIL-37Tg and wild-type (WT) mice by injecting 4 ml of sterile phosphate-buffered saline (PBS) intrapertitoneal containing 0.38% sodium citrate (Kullberg et al., 1993). After washing, the cells were resuspended in culture medium in 96-well microtiter plates (Greiner, Alphen, The Netherlands) at 10⁵ cells/well, in a final volume of 200 μl. The cells were stimulated with either control medium or heat killed C. albicans at 1 × 10⁷ microorganisms/ml. After 24 h of incubation at 37∘C, the plates were centrifuged (500 g, 10 min), and the supernatants were removed. The cells were lysed with three freeze-thaw cycles. Samples were stored at -80∘C until cytokine assays were performed.

To assess cytokine production in splenocytes, spleen were gently squeezed into a sterile 200 mm filter chamber. The cells were washed and resuspended in RPMI 1640, counted in a Bürker chamber and adjusted to 5 × 10⁶/ml. 200 μL of the cell suspension was stimulated with 1 × 10⁷ heat-killed C. albicans/ml. Measurement of TNFα and IL-6 concentrations was performed in supernatants collected after 48 h of incubation at 37∘C in 5% CO₂ in 48-well plate.

TNFα was determined by specific radioimmunoassay (detection limit 20 pg/ml), as previously described Netea et al. (1996). IL-6 concentrations were determined by a commercial ELISA (Biosource, Camarillo, CA, USA, detection limit 16 pg/ml) according to the instructions of the manufacturer.

hIL-37Tg mice and WT mice were injected intravenously with C. albicans blastoconidia (1 × 10⁶ CFU/mouse for survival and 5 × 10⁵ CFU/mouse for testing fungal burden) in a 100 μl volume of sterile pyrogen-free PBS. Survival was assessed daily for 2 weeks. Subgroups of five animals were killed on days 3 and 7 of infection. To assess the tissue outgrowth of the microorganisms, the kidneys of the sacrificed animals were removed aseptically, weighed, and homogenized in sterile saline in a tissue grinder. The number of viable Candida cells in the tissues was determined by plating serial dilutions on Sabouraud dextrose agar plates. The CFU were counted after 24 h of incubation at 37∘C, and expressed as log CFU/g tissue.

Sexually mature female C57bl/6 mice (8–12 weeks old, weights 20–25 g) were used. The animals were kept under routine laboratory conditions (21–22∘C, relative humidity 60% and a 12 h light–dark cycle), fed a standard commercial pellet diet (RHM, Hope Farms, The Netherlands), and given acidified tap water ad libitum. The mice were injected intraperitoneal with rat anti-mouse GR1 or isotype control (100 μg in 200 μl PBS; eBioscience) 4 days prior to the experiment to deplete the mice of PMNs. On day zero, the mice were injected intraperitoneal with 1 × 10e5 live C. albicans conidia. After 2 h the mice were anesthetized with isoflurane and blood samples were obtained for measurement of blood cells counts. Subsequently, the mice were sacrificed by cervical dislocation. The number of viable Candida cells in the peritoneal lavage was determined by plating serial dilutions on Sabouraud dextrose agar plates and the CFU were counted after 24 h of incubation at 37∘C and expressed as log CFU.

To assess cytokine production during infection, primed spleen cells from mice on day 7 of infection with 2 × 10⁵ CFU of C. albicans per mouse were stimulated in vitro with heat-killed Candida conidia or pseudohyphae (1 × 10⁶ microorganisms/ml). Spleen cells were washed and resuspended in RPMI1640, counted in a Bürker chamber and the number was adjusted to 5 × 10⁶/ml. 200 μL of the cell suspension was stimulated with 1 × 10⁶ heat killed C. albicans/ml. Measurement of IFNγ, IL-10, and IL-17 concentrations was performed in supernatants collected after 48 h of incubation at 37∘C in 5% CO₂ in 48-well plate.

Phagocytosis and killing of C. albicans blastoconidia were assessed as described elsewhere (Vonk et al., 2006). Exudate peritoneal phagocytes from groups of five hIL-37Tg mice and WT mice were elicited by an i.p. injection of 10% proteose peptone. After 72 h, cells were collected in separate sterile tubes by washing the peritoneal cavity with 4 ml of ice-cold PBS that contained 50 U/ml heparin. Phagocytes were centrifuged (for 10 min at 2250 g), counted in a Bürker chamber, and resuspended in culture medium. 5 × 10⁵ cells were dispensed into the wells of a 96-well flat bottom plate (Costar), allowed to adhere for 2 h, and washed to remove non-adherent cells. Subsequently, the cells were incubated with 1 × 10⁴ CFU C. albicans, which were opsonized for 45 min at 24∘C in modified Eagle’s medium (MEM; Gibco Life Technologies) containing 2.5% fresh mouse serum (effector:target ratio, 40:1). After 15 min, supernatants were aspirated, and monolayers were gently washed with MEM to remove non-ingested microorganisms. The supernatant and well washings that contained the non-ingested Candida blastoconidia were plated in serial dilutions on Sabouraud agar plates. The percentage of phagocytosed microorganisms was defined as [1–(number of uningested CFU/CFU at the start of incubation)] × 100. Killing of C. albicans by phagocytes was assessed in the same monolayers. After removal of the non-phagocytized Candida blastoconidia, 200μl of culture medium, consisting of Sabouraud in MEM (50% vol/vol), was added to the monolayers. After 3 h of incubation at 37∘C in air and 5% CO₂, the wells were scraped gently with a plastic paddle and washed with 200μl distilled H₂O to achieve lysis of phagocytes in three cycles, and 10-fold dilutions of each sample were spread on Sabouraud agar plates and incubated at 37∘C for 24 h. The percentage of yeast killed by the phagocytes was determined as follows: [1–(CFU after incubation/number of phagocytized CFU)] (Nold et al., 2010) × 100. Phagocyte-free incubations of blastoconidia were included as a control for yeast viability. In earlier experiments we have shown that 90–97% of attached/internalized C. albicans are intracellular (Vonk et al., 2006).

The differences between groups were analyzed by the Mann–Whitney U-test. The level of significance between groups was set at p < 0.05. The data are presented as cumulative results of all experiments performed.

To investigate the role of IL-37 in the host defense against invasive C. albicans infection, hIL-37Tg mice, and WT mice were infected with C. albicans blastospores. The survival during disseminated candidiasis was 20% for WT-type mice after 2 weeks, whereas all mice had died by day 10 in the hIL-37Tg group (Figure 1A). The fungal burden in the kidneys, the target organ of disseminated candidiasis in mice (Spellberg et al., 2005), was not significantly different at day 3 (mean ± SD; WT: 3.6 ± 0.83, IL-37Tg 3.8 ± 0.44 log CFU/g kidney), and 1-log higher in the hIL-37Tg mice than in WT mice on day 7 of infection (p < 0.05; Figure 1B). Histology revealed an accumulation of Candida in the pyelum of the kidneys of hIL-37Tg mice which was not observed in the WT mice (Figure 1C).

In order to understand the enhanced lethality and greater fungal burden of the hIL-37Tg mice, we investigated the dynamics of the influx and function of neutrophils. Peritoneal cells were harvested and counted 4 h after intraperitoneal injection of heat-killed Candida. A significant lower influx of neutrophils was apparent 4 h after challenge with heat killed Candida in the hIL37Tg mice compared to WT mice (Figure 2A). Phagocytosis of C. albicans by hIL-37Tg cells was similar to that by cells of WT mice (37 vs. 39% phagocytized in 15 min; p > 0.05). hIL-37Tg cells killed 92% of phagocytized Candida blastoconidia after 3 h, which was not different from the killing activity of cells from WT cells (88%, p > 0.05, Figure 2B).

To assess whether the decrease of neutrophils within the first hours has a significant biological effect, we investigated the difference of fungal burden between neutrophil depleted mice and non-neutrophil depleted mice in a short in vivo Candida infection model. The lack of neutrophils at the site of infection resulted in a log increase in fungal growth within 2 h compared to mice in which neutrophil influx was normal (Figure 2C). These differences support the importance of early influx of neutrophils in restricting Candida growth in vivo.

To investigate the inhibitory effect of IL-37 at the level of cytokine production, resident peritoneal macrophages, or naïve splenocytes of hIL-37Tg and WT mice were exposed to heat-killed C. albicans blastoconidia and pseudohyphae in vitro. Cytokine production by unstimulated macrophages of each mouse strain was below the detection limit for TNFα and IL-6. However, after stimulation with C. albicans pseudohyphae, the production of TNFα was significantly lower in macrophages from the hIL-37Tg mice compared to cells from WT mice. There were no differences in the production of IL-6 in the same cultures (Figure 3).

Proinflammatory T helper responses such as Th1 and Th17 responses play an important role in fungal host defense (Gow et al., 2012). The Th17 response during disseminated fungal infection can be both protective and detrimental (Huang et al., 2004; Zelante et al., 2007; Lin et al., 2009). To study the impact of IL-37 on proinflammatory adaptive immune responses, we investigated the Th1 and Th17 characteristic cytokines IFNγ and IL-17. Splenocytes isolated from hIL-37Tg mice with a higher fungal burden at day 7 produced significant more IL-17 in response to C. albicans pseudohyphae (Figure 4). IFNγ production in response to C. albicans pseudohyphae was undetectable in WT and hIL-37Tg splenocytes at day 7 (Figure 4). Since IL-37 mainly has anti-inflammatory effects (Nold et al., 2010; Boraschi et al., 2011), we investigated levels of IL-10 in cells from IL-37Tg There was no significant difference in IL-10 production in splenocytes from hIL-37Tg compared to WT cells in response to C. albicans, although hIL-37Tg mice showed a trend toward an increased IL-10 production (Figure 4).