The use of human leukocytes from healthy donors with written informed consent complies with the Declaration of Helsinki (Ethical committee Erlangen approval no. 4055 and no. 111_12 B). PMBCs were obtained from leukoreduction system chambers by density centrifugation (35). Monocytes were positively selected from PBMC using α-CD14 microbeads (Miltenyi Biotec), purity was ≥90%. For culture, RPMI1640 was supplemented with 10% (v/v) fetal calf serum (FCS, Biochrom) and 100 U/ml penicillin and 100 μg/ml streptomycin (cRPMI). A total of 50 U/ml GM-CSF (Genzyme) or 50 U/ml M-CSF (Peprotech) were added for differentiation of macrophages. 50 U/ml GM-CSF and 250 U/ml IL-4 (Peprotech) were added for differentiation of DC. Cells were cultured at a density of 0.8 × 10⁶ cells/ml (GM-CSF macrophages, DC) or 1.6 × 10⁶ cells/ml (M-CSF macrophages) for 6–7 days without change of media at 37°C with 5% CO₂ humidified air.

C57BL/6, Clec4e^−/−, Balb/c, and Stat6^−/− mice were bred at the Präklinische Experimentelle Tierzentrum of the Medical Faculty of the Friedrich-Alexander-University Erlangen-Nürnberg. Mice were housed and humanely sacrificed according to regional government regulations. Bone marrow cells from femurs and tibiae were differentiated to macrophages by culture in complete Dulbecco’s Modified Eagle Medium containing 10% FCS, antibiotics, and 50 μM β-mercaptoethanol (cDMEM) plus 10% L929-cell conditioned medium as a source of M-CSF, as previously described (36). On day 7, adherent macrophages were harvested by Accutase (Sigma) treatment, washed and counted. For generation of BMDC, bone marrow cells were cultured in cDMEM containing 10% conditioned medium of X63 cells producing GM-CSF for 7–8 days before harvesting. Peritoneal exudate cells (PEC) were obtained on day 4 after injection of mice with 2 ml of 4% thioglycollate by flushing the peritoneal cavity with 10 ml of ice-cold PBS.

Human antigen-presenting cells were stimulated by adding M-CSF (Peprotech, 50 U/ml), GM-CSF (Genzyme, 50 U/ml), or IL-4 (Peprotech, 250 U/ml unless otherwise stated) to the cell culture medium before plating of cells into 48 well cell-culture plates in a concentration of 0.85 × 10⁶/ml. Mouse antigen-presenting cells were stimulated with 10 ng/ml IL-4 (Peprotech), 10 ng/ml LPS (E. coli serotype O55:B5, Sigma) by adding the substance to the cell culture medium before plating of the cells into 24 well cell-culture plates in a concentration of 0.5 × 10⁶/ml. TDB (Polar Avanti, 5 μg/ml) was used plate-bound as previously described (4, 37), coating with isopropanol only was used as a negative control.

RNA was isolated with Trifast (Peqlab), cDNA was transcribed using a cDNA synthesis kit (Applied Biosystems). Expression levels of the housekeeping genes hypoxanthine-guanin-phosphoribosyltransferase (Hprt, mouse) or cyclophilin A (PPIA, human), as of the genes of interest, were analyzed using primer/probe combinations selected from the Roche Universal Probe Library (Roche). For human MINCLE, the primer/probe combination was selected using the software PrimerExpress (Applied Biosystems). All primers and probes used have been described (28) and were purchased from Metabion. ΔCT values were calculated as ΔCT = CT_{(housekeeping gene)} − CT_{(gene of interest)} (such that higher values indicate higher relative expression), and ΔΔCT values referred to the calibrator as indicated.

Cytokine concentration of murine G-CSF and TNF-α was analyzed by sandwich ELISA (DuoSet ELISA, R&D Systems) using cell-free cell culture supernatants of cells stimulated as indicated.

The 2 × 10⁵ BMM of C57BL/6 and Mincle−/− mice were stimulated with LPS (10 ng/ml) or TDB (5 μg/ml) in the presence or absence of IL-4 (10 ng/ml), as well as with IL-4 alone, for 16 h in F-bottom 96 well cell-culture plates. Cell surface expression of Mincle and Mcl was analyzed by flow cytometry. Staining was performed by using anti-Mincle (clone 4A9, MBL) and anti-Mcl (clone 3A4) (38) as primary antibodies, and anti-rat IgG1 conjugated to APC as secondary antibody (eBioscience) in a final concentration of 1 μg/ml for anti-Mincle and anti-rat IgG-APC or 2.27 μg/ml for anti-Mcl. Fc receptors were blocked by adding anti-mouse CD16/32 (clone 93, eBioscience) in a final concentration of 2.5 μg/ml before staining. Cells were stained with primary antibodies for 20 min at 4°C, washed, and then stained with secondary antibody for 20 min at 4°C. Flow cytometry data were acquired on a FACSCanto II instrument (BD); analysis was carried out using FlowJo (version 10).

Statistical analysis was performed using Prism5 (GraphPad Software, v5.01). Student’s t-test was applied as indicated for non-paired and paired testing between two groups. *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05.

In our recent characterization of CLR expression on primary human antigen-presenting cells, we observed higher expression of MINCLE mRNA in macrophages generated with M-CSF or GM-CSF compared to DC differentiated in the presence of GM-CSF plus IL-4 (28). This effect was confirmed in independent experiments using cells from unrelated healthy donors, showing a strong reduction in the ΔCT values from around −4.05 (M-CSF) and −5.0 (GM-CSF) to −10.4 (GM-CSF + IL-4) (Figure 1A). Quantitative RT-PCR analysis of mRNA expression for a range of CLR showed that the reduced expression in DC compared to GM-CSF-derived macrophages was selectively observed for MINCLE, MCL, DECTIN2, and CLEC12A whereas expression levels of the other tested CLR were not significantly different (Figure 1B). Since the presence of IL-4 in the cell culture media was the main difference in the differentiation protocols for GM-CSF macrophages and DC, we tested whether IL-4 alone would be sufficient to downregulate MINCLE expression in monocyte cultures (Figure 1C). MINCLE expression was highest in freshly isolated CD14⁺ monocytes and declined over time during culture in media alone. However, addition of IL-4 caused a stronger reduction, and this effect was detectable at all time points tested (6–144 h). The presence of GM-CSF kept MINCLE expression at a high level, but additional IL-4 induced strong downregulation, which was again detectable already after 6h, but increased over time (Figure 1C). The inhibitory effect of IL-4 on MINCLE expression by monocytes was dose dependent and already detected with 0.25 U/ml IL-4 but more pronounced at concentrations above 2.5 U/ml (Figure 1D). Having shown that IL-4 inhibits MINCLE expression in monocytes early during differentiation cultures, we next asked whether the same effect was seen when adding IL-4 to macrophages or DC after differentiation. While the expression of MINCLE in monocyte-derived DC cultured in media only was relatively variable and the reduction observed for the average values in DC treated with IL-4 was not significant, both types of macrophages responded with significant downregulation of MINCLE when treated with IL-4 for 48 h (Figure 1E).

We next asked whether the regulatory action of IL-4 is conserved between species and tested the expression of mouse macrophages and DC treated for 48 h with IL-4 (Figure 2). IL-4 was used at the saturating concentration of 10 ng/ml (39). Mincle mRNA levels in M-CSF-driven BMM under basal conditions were lower than those in d4 thioglycollate-elicited peritoneal exsudate cells (PEC) or GM-CSF-driven BMDC, but IL-4 decreased Mincle expression in all three cell types significantly (Figure 2A). Mcl expression was comparably high in untreated BMM, PEC, and BMDC and strongly inhibited by IL-4 (Figure 2B). Dectin-2 mRNA was highest in BMDC and significantly downregulated by IL-4 in BMM and BMDC (Figure 2C). In contrast, IL-4 did not inhibit Dectin-1 expression in mouse APC, but in fact increased Dectin-1 mRNA in BMM, confirming a previous report (34) (Figure 2D). Together, the pattern of IL-4 effects on expression of the CLR family members examined was very similar between mouse and human, with significant downregulation of Mincle, Mcl, and Dectin-2, whereas Dectin-1 was not affected or even upregulated by IL-4.

Expression of Mincle is strongly inducible by pattern recognition receptor signaling, such as TLR or CLR (4, 25, 40). Therefore, we examined the effect of IL-4 on Mincle mRNA expression in BMM treated with the TLR4 ligand LPS or the Mincle ligand TDB (Figure 3). Stimulation with LPS or TDB for 48 h significantly induced Mincle expression, as expected; the concomitant exposure to IL-4 inhibited Mincle upregulation by TDB, but not by LPS (Figure 3A). Priming of BMM with LPS or LPS + IL-4 overnight, followed by removal of LPS and washing before addition of IL-4, also abrogated the inhibitory effect of IL-4 on Mincle expression (Figure 3B). Thus, the inhibitory effect of IL-4 on Mincle expression could be overcome by stimulation of macrophages with the TLR4 ligand LPS.

We next employed flow cytometry to determine the cell surface levels of Mincle and Mcl in resting and stimulated macrophages (Figures 4A,B). Confirming previous reports (40, 41), only weak staining with the Mincle- and Mcl-specific monoclonal antibodies (4A9 and 3A4) was detected in resting macrophages. In fact, using Mincle-deficient macrophages as staining controls, only a minimal increase in the fluorescence signal for Mincle was observed. Overnight stimulation with TDB caused upregulation of Mincle and Mcl on a significant fraction of the macrophage population. However, this increase was almost completely prevented by co-treatment with IL-4. In contrast, the LPS-induced surface staining for Mincle and Mcl was found on the entire macrophage population and was only weakly reduced in the presence of IL-4. Together, the changes in cell surface protein levels and mRNA expression of Mincle and Mcl showed a close correlation.

We reasoned that inhibition of Mincle expression in macrophages treated with IL-4 may functionally impair the response to the Mincle ligand TDB. To test this hypothesis, we determined the levels of the cytokines G-CSF and TNF, which are robustly secreted by macrophages stimulated with TDB or with LPS although the levels varied considerably between independent experiments (Figures 5A,B). Macrophages treated with IL-4 or media alone did not produce significant amounts of G-CSF or TNF (data not shown). Concomitant treatment with IL-4 strongly and consistently inhibited the production of both cytokines in response to TDB (Figures 5A,B). In contrast, LPS-induced G-CSF was inhibited less strongly by IL-4 (Figure 5A), and the LPS-induced release of TNF was not affected by IL-4 (Figure 5B).

IL-4 triggers Jak-Stat signaling, with Stat6 being the pivotal transcription factor for IL-4-induced gene expression (42). We investigated whether the inhibitory effect of IL-4 was also dependent on this transcription factor by using Stat6^−/− BMM in comparison to the respective Balb/c wild type control BMM (Figure 6). The Stat6 genotype had no effect on the basal (data not shown) or TDB-induced Mincle expression (Figure 6A). However, IL-4 inhibited the basal (data not shown) and TDB-induced Mincle expression in Balb/c BMM, but not in Stat6^−/− BMM (Figure 6A). Similarly, the TDB-inducible expression of G-CSF at the mRNA and protein level was strongly inhibited in Balb/c BMM, but not in Stat6^−/− BMM, where in fact a moderate increase in G-CSF protein secretion production was evident (Figures 6B,C).