We selected adult APECED patients presenting with CMC and autoimmunity, as confirmed by genetic analysis of the AIRE gene; two of these patients have a Pro326Leu substitution, one has two deleted residues (exon 5 Ser187 and exon 9 Gln358) and another stop codon in exon 5. Blood samples were collected from patients and healthy donors and were then processed and shipped according to the protocols approved by the Institutional Ethics Committee, the Ministry of Health of Brazil, and the Helsinki Convention.

The following reagents were used: puromycin, piceatannol, and polybrene (Sigma-Aldrich); RPMI 1640 culture medium and Dulbecco’s PBS (Invitrogen); anti-Dectin-1, anti-Dectin-2 (H-46), anti-AIRE (H-300 and C-2), anti-Syk (4D10), anti-CARD9 (C-17) (Santa Cruz Biotechnology); anti-Dectin-1 (AF1756), anti-Dectin-2 (AF3114) (R&D Systems); and human IgG (Abcam 90285). Lentiviral particles specific for AIRE knockdown and control shRNA Lentiviral particles (shRNA; sc-37669-V and sc-108080) (Santa Cruz Biotechnology) were used in the transduction of THP-1 cell.

The myelomonocytic THP-1 cell line was cultured in RPMI 1640 supplemented with 10% inactivated fetal bovine serum (iFBS), 2 mM l-glutamine, 10 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 2 mM sodium pyruvate, and 100 µM MEM non-essential amino acids, in a humid 5% CO₂ atmosphere at 37°C. 2.5 × 10⁶ THP-1 cells were differentiated to macrophage-like THP-1 cells using 100 U/mL IFN-γ and 1000 U/mL TNF-α for 24–48 h in 6-well plates, following overnight resting in RPMI media. This time point was selected based upon detection of AIRE protein by western blotting (data not shown). Only adherent cells were used in the experiments. Peripheral blood mononuclear cells (PBMCs) from healthy donors and AIRE patients were purified using a Ficoll Paque gradient (GE Healthcare Life Sciences). Human monocytes were isolated by adherence and differentiated into macrophages by incubation with 5 ng/mL GM-CSF (R&D Systems) for 6 days. The C. albicans strain used was ATCC SC5314 (kindly provided by Dr. Joachim Morschhauser from Institut Fur Molekulare Infektionsbiologie, Wurzburg, Germany) (31), and it was cultured in Sabouraud medium with 10% iFBS for 3 h to induce hyphal differentiation.

2 × 10⁴ THP-1 cells were cultured in supplemented RPMI medium in 96-well plates for 24 h. Then, 8 mg/mL polybrene was added prior to cell transduction with lentiviral particles at a multiplicity of infection (MOI) of 1:10. Cells were incubated for 5 h, and then the culture medium was replaced with fresh medium. After 48 h culture stabilization, fresh medium containing 1.5 mg/mL puromycin was added for 72 h to select transduced cells. The puromycin concentration was then increased to 3 mg/mL, and the cells were incubated for an additional 72 h. AIRE expression in the transduced clones was monitored by western blotting, flow cytometry, and RT-PCR, using anti-AIRE (H-300 and C-2), donkey anti-rabbit Alexa Fluor 647 (a31573) (Molecular Probes), and human AIRE primers (sc-37669-PR). For overexpression of AIRE, 3 × 10⁶ HEK293T cells were transfected with pLenti-AIRE (NM_000383) Human Tagged ORF, mGFP tagged (RC213497L2, OriGene) using FuGENE 6 Transfection Reagent (Promega) for overexpression of AIRE.

3 × 10⁶ macrophages-like THP-1 cells stimulated with hyphae for 10, 20, or 30 min were submitted to RNA extraction using Trizol reagent (Life Technologies). Preparation of complementary DNA (cDNA) was performed using 2 μg of RNA, 0.8 µL of reverse transcriptase M-MLV, 4 μL of 5× Reaction Buffer M-MLV, 2 μL of 10 mM dNTPs, 32 U/μL of RiboLock™ Ribonuclease Inhibitor (Fermentas), and 320 ng of oligo-dT primer (Integrated DNA Technologies). This mixture was incubated at 42°C for 60 min and then at 70°C for 10 min. cDNA was stored at −20°C. After preparation of cDNA, quantification of gene expression by real-time PCR was performed. Amplification conditions were standardized for each transcript. A comparative relationship between reaction cycles (CT) was used to determine gene expression relative to PPIA control (housekeeping gene). The detection of the gene of interest was performed using Quantstudio apparatus (Applied Biosystems). Commercially available probes (Life Technologies) were used for PPIA (Hs_99999904). The following sequences of primers, detected using SybrGreen^® (Life Technologies) were also used for this study: KiCqStart Primers Human H_Clec7a (Dectin-1) (NM_022570), H_Syk (NM_001135052), H-Card9 (NM_052813), H_Clec6 (Dectin-2) (NM_001007033), and H_AIRE (NM_000383) (Sigma Aldrich). The quantification method was 2 − ΔΔCt (32) using unstimulated THP-1 samples as a normalizer.

Wild-type and AIRE-knockdown macrophage-like THP-1 cells (3 × 10⁶ cells) were stimulated with Candida hyphae. Lysates were obtained using Pierce RIPA buffer. The protein concentrations were determined using a Pierce BCA Protein Assay Kit (Life Technologies), and equivalent amounts of protein were subjected to SDS-PAGE. The membrane was incubated with primary antibody for 1 h, followed by incubation with an IRDye 800CW, IRDye 680RD (LI-COR), or horseradish peroxidase (Sigma Aldrich) secondary antibody. Bands were visualized with an Odyssey^® CLx Infrared Imaging System (LI-COR) or ImageQuant LAS 500 (GE Healthcare Life Sciences). 18 × 10⁶ macrophage-like THP-1 cells, control, or stimulated with hyphae was lysed with immunoprecipitation buffer (500 mM Tris pH7.4; 500 mM NaCl; 500 mM EDTA pH8.0; 200 mM EGTA; 1% TRITON X-100). Total protein was quantified using Bradford reagent (BioRad) before immunoprecipitation with anti-AIRE (C-2), anti-Dectin-2 (H-46) (Santa Cruz Biotechnology), and control IgG. Macrophage-like THP-1 cells (2.5 × 10⁶ cells) were stimulated with Candida hyphae for 10, 20, or 30 min. The cytoplasmic fraction was separated using Buffer 1 (10 mM HEPES pH7.9, 50 mM NaCl, 0.5 M sucrose, 0.1 mM EDTA, and 0.5% TRITON X-100). The nuclear pellet was washed using 1 mL Wash Buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF), and nuclear fraction extraction was performed using Buffer 2 (20 mM Tris-HCl, 1.5 mM MgCl₂, 400 mM NaCl, 0,2 mM EDTA, 1 mM PMSF, and 15% Glycerol). Western blotting was performed using anti-AIRE (H-300), anti-AIRE (C-2), anti-CARD9, anti-Syk (4D10), anti-Dectin-1 (Santa Cruz Biotechnology), and anti-Dectin-2 (AF3114) (R&D Systems). Anti-β-Actin (I-19), anti-GAPDH (FL-335) (Santa Cruz Biotechnology), and anti-Lamin B1 (Abcam16048) were used as loading controls.

After macrophage differentiation, cells were trypsinized and adjusted to 2 × 10⁵ cells/mL. Cells were then incubated for 45 min at 37°C on poly-l-lysine-treated slides to promote cellular adhesion. Macrophage-like THP-1 cells were subsequently stimulated with C. albicans hyphae for different times. Fc receptors were blocked using human IgG to prevent non-specific binding, followed by staining for anti-Dectin-1 (AF1756), anti-Dectin-2 (AF3114) (R&D Systems), or anti-Dectin-2 (H-46) (Santa Cruz Biotechnology). Cells were then fixed with 100 µL CytoFix/CytoPerm (BD Bioscience), according to the manufacturer’s recommendations, and stained for anti-AIRE (H-300), anti-Syk (4D10), or anti-CARD9 (C-17) (Santa Cruz Biotechnology) on ice to reduce non-specific binding. The cells were washed with wash buffer (BD Bioscience) and stained with fluorescence secondary antibody (Molecular Probes). Slides were mounted with Gel Mount (Vectashield) and sealed with nail polish prior to image acquisition using a Zeiss confocal microscopy equipped with Yokogawa CSU-10 spinning disk and Hamamatsu ORCA-ER camera. Confocal micrographs were analyzed by Volocity software (PerkinElmer) to obtain area and mean fluorescence intensity (MFI). 30 cells from 3 independent experiments were measured. All antibodies were validated with a species-specific IgG negative control at the same concentration (Figure S1A in Supplementary Material). To validate the specificity of the AIRE antibody used for confocal microscopy, AIRE-mGFP was overexpressed in HEK293T cells as described earlier. Cells were fixed using CytoFix/CytoPerm (BD Biosciences) or FoxP3/Transcription Factor Fix/Perm solution (Thermo Fisher Scientific) at room temperature and gently washed with Perm wash (BD Biosciences) or Perm buffer (Thermo Fisher Scientific). Staining for GFP was performed with anti-GFP (BioLegend) directly conjugated to AlexaFluor 488 (BioLegend). Staining for AIRE was performed with anti-AIRE (Santa Cruz Biotechnology) followed by goat anti-rabbit IgG conjugated to AlexaFluor 568. Coverslips were mounted using using VectaShield with DAPI (Vector Laboratories). Images were acquired in Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems) using LASAFx software (Figure S1B in Supplementary Material).

Wild-type and AIRE-knockdown macrophage-like THP-1 cells were treated with 10 µg/mL piceatannol (selective Syk inhibitor) or vehicle control and stimulated with hyphae or yeast for 20 or 30 min. To evaluate lysosome production, cells were incubated with LysoTracker Red for 30 min and washed and stimulated with C. albicans. Next, the cells were fixed with 4% paraformaldehyde in PBS for 10 min and washed and resuspended in 80 µL PBS. Samples were analyzed by Imagestream X Mark II Imaging Flow Cytometer (Amnis). Flow cytometry image data analysis was performed on LysoTracker/Candida-GFP double positive cells, following by the analysis of the internalization function using IDEAS software (Amnis). The internalization gate was generated using THP-1 macrophages stimulated at 4°C. Human macrophages were stimulated with yeast (MOI 1:5) and hyphae (MOI 1:2) for 30 min on coverslips for phagocytosis evaluations using cells from healthy donors and patients. After being washed, the cells were stained with panoptic dye (Laborclin), and C. albicans were counted under a light microscope. As control, human macrophages or macrophage-like THP-1 cells were stimulated with E. coli (MOI 1:5) for 30 min, incubated with gentamicin for 45 min, washed and cultured in Luria Bertani agar. The number of colony-forming units (CFU) was counted after overnight incubation at 37°C.

Macrophage-like THP-1 cells were stimulated with hyphae for 6 h. Supernatants were collected and assessed for IL-1β and TNF-α using BioLegend’s LEGENDplex™ kits. Human macrophages were stimulated with hyphae or 100 ng/mL lipopolysaccharide (LPS; Sigma Aldrich), and cytokine secretion was evaluated using IL-6 (eBioscience) and TNF-α (BD Biosciences) ELISA kits. A standard curve was generated using purified protein to obtain a R² value of 0.99.

To evaluate the FS formation, macrophage-like THP-1 cells were stimulated with C. albicans hyphae for 10, 20, or 30 min (Figure 1A). Dectin-1 and Dectin-2 expression increased at the membrane following contact with hyphae compared to unstimulated macrophage-like THP-1 cells (Figures 1B,C). Analysis of macrophage in contact with hyphae demonstrated higher percentages of Dectin-1 and Dectin-2 at the FS after 20 min stimulation compared to levels in the cells as a whole (Figures 1D,E). In addition, expression of Dectin-2 was higher than Dectin-1 at the FS. This was expected since the cell wall of hyphae binds more strongly to Dectin-2 than to Dectin-1. Moreover, the amount of Dectin-1 did increase after 30 min (Figures 1F,G) as did the co-localization of Dectin-1 and Dectin-2 at the FS (Figures 1H,I).

To determine any potential role of Dectin-2 in the recruitment of Dectin-1 to the nascent FS, we first blocked Dectin receptors in macrophage-like THP-1 cells using either an anti-Dectin-1 or anti-Dectin-2 antibody or both for 30 min prior to hyphal stimulation (Figure S2 in Supplementary Material). Blocking Dectin-1 increased the accumulation of Dectin-2 at the FS compared with control IgG-treated macrophages. Since these receptors likely share a Syk-dependent signaling pathway, blocking Dectin-1 may increase signaling promoting Dectin-2 activation. After blocking Dectin-2, however, there was no significant change in Dectin-1 accumulation at the FS (Figures S2A,C in Supplementary Material). To verify that Dectin-1 recruitment was initiated by its own ligation and signaling, we blocked both Dectin receptors before hyphal stimulation. This blockage resulted in decreased accumulation of both receptors at the FS (Figures S2B,D in Supplementary Material). These findings suggest that Dectin-1 recruitment is mainly attributed to its own ligation and activation.

First, we investigated whether FS activation is modulating the transcription of Dectin-1, Dectin-2, Syk, Card9, and AIRE. We observed that only Dectin-2 and AIRE mRNA significantly increased the transcription after hyphae stimulation (Figure S3B in Supplementary Material). Since Dectin-2 localizes to the FS and we observed increased mRNA expression levels of Dectin-2 and AIRE, we next evaluated whether AIRE might be involved in the Dectin-2 signaling pathway. Macrophage-like THP-1 cells were stimulated with hyphae to evaluate AIRE and Syk recruitment to the FS. This stimulation led to considerable co-localization of Dectin-2, Syk, and AIRE (Figure 2A) as well as increased AIRE and Syk expression at the FS (Figures 2B,C). Interestingly, the cell surface expression of AIRE, as well as the accumulation of Dectin-1 and Dectin-2 receptors at the FS, reached peaks at 20 min (Figures 1D,E and 2B). However, the percentage of AIRE accumulated at the FS remained relatively constant across all time points (Figures 2B–D). Thus, AIRE accumulation did not change because its expression was increased throughout the cell and not only at the FS. Although Syk is important for Dectin receptor activation, Syk expression remained consistent at all time points (Figures 2C–E). An increase in AIRE and Syk co-localization, however, was detected after 20 min of hyphal stimulation (Figures 2F,G). Moreover, we separated cytoplasmic and nuclear fractions to confirm AIRE expression in macrophage-like THP-1 cells stimulated with hyphae. AIRE was identified in two isoforms, with higher expression at 20 min in the cytoplasmic fraction while it was increased after hyphae stimulation at the nuclear fraction (Figure 2H). To determine whether Dectin-2, Syk, and AIRE actually physically interact, AIRE or Dectin-2 was immunoprecipitated from hyphae-stimulated macrophage-like THP-1 cells. AIRE was identified in molecular complexes with Dectin-2 and Syk, in macrophage-like THP-1 cells that had been activated with C. albicans hyphae (Figure 2I). Moreover, AIRE was increased compared to unstimulated cells. Thus, Dectin-2 localizes to the FS as part of a molecular complex with both Syk and AIRE.

To evaluate the role of AIRE in macrophages with regard to the FS, AIRE was knocked down in THP-1 cells, and the FS formation compared to that in control cells. There was no overall change in the total expression levels of Dectin-1, Dectin-2, Syk, and CARD9 in the AIRE-knockdown cells (Figures S4A–E in Supplementary Material). Given that these were maintained at physiological levels, we next examined whether AIRE is involved in FS formation. Wild-type and AIRE-knockdown macrophage-like THP-1 cells were stimulated with hyphae for 20 or 30 min, and then Dectin-1 and Dectin-2 recruitment to the FS was analyzed (Figure 3). We observed decreased accumulation of both receptors at the FS in 20 and 30 min in AIRE knockdown macrophages (Figures 3A–G). To evaluate if this has any functional significance, we asked whether protein signaling was affected in AIRE knockdown cells. Importantly, decreased Syk amount was observed at the FS at both time points in AIRE-knockdown macrophages and decreased CARD9 recruitment was identified at 30 min (Figures 4A–G). These results suggest that the delayed FS formation is attributed to decreases in Dectin-1 and Dectin-2 recruitment and protein signaling.

Based on the observed delay in FS formation in AIRE-knockdown macrophage-like THP-1 cells, we determined if the phagocytosis of C. albicans might be affected. Notably, decreased yeast and hyphal conjugation as well as internalization was observed in AIRE-knockdown macrophage-like THP-1 cells when compared to controls in 20 and 30 min (Figures 5A–C). However, E. coli phagocytosis was not affected in AIRE-knockdown macrophage-like THP-1 cells (Figure S5 in Supplementary Material). To determine whether this effect of AIRE was upstream of Syk as previously proposed, yeast and hyphal phagocytosis were measured in AIRE-knockdown macrophage-like THP-1 cells treated with piceatannol, a Syk inhibitor. In this setting, the reduced levels were not further altered by Syk inhibition in AIRE-knockdown macrophage-like THP-1 cells, only in control cells (Figures S6A,B in Supplementary Material), showing that AIRE is important for yeast and hyphal phagocytosis and that it requires a Syk-dependent pathway in macrophage-like THP-1 cells.

Since Dectin-1 is also recruited to the FS after 30 min of hyphal stimulation (Figure 1F) and it is important for lysosomal maturation (33), we determined if lysosomal production was affected by absence of AIRE. Wild-type and AIRE-knockdown macrophage-like THP-1 cells were stimulated with yeast or hyphae for 20 or 30 min. No difference was observed between the wild-type and AIRE-knockdown macrophage-like THP-1 cells stimulated with hyphae. However, simulation of macrophages with yeast resulted in decreased lysosome production in the AIRE-knockdown macrophage-like THP-1 cells at 30 min compared with that in the wild-type macrophage-like THP-1 cells (Figures 5D,E). This suggests that AIRE can be involved in lysosomal production by Dectin-1 in macrophages stimulated with yeast.

Cytokines are pivotal to both innate and adaptive immunity, and their production increases following Dectin ligation. We evaluated IL-1β and TNF-α secretion in wild-type and AIRE-knockdown macrophage-like THP-1 cells stimulated with hyphae. As might be predicted from the interrupted Dectin signaling complex in the absence of normal AIRE function, AIRE-knockdown macrophage-like THP-1 cells secreted lower levels of IL-1β and TNF-α after stimulation with hyphae for 6 h (Figure 5F). This demonstrated that the physiologic inflammatory response after hyphal stimulation requires AIRE in macrophage-like THP-1 cells.

To determine if those mechanisms are relevant to APECED patients, we evaluated human monocyte-derived macrophages from APECED patients with AIRE mutations as well as from healthy donors. Decreased Dectin-2 expression throughout the entire cell surface of APECED patient macrophages was detected after 30 min of stimulation with C. albicans hyphae compared to those from healthy donors (Figure 6). In addition, the APECED macrophages exhibited low phagocytic activity following stimulation with yeast or hyphae for 30 min compared to that in the healthy donor cells (Figure 7A). Macrophages from APECED patients also showed reduced IL-6 and TNF-α secretion after 24 h of hyphal stimulation compared with those from healthy donors (Figure 7B). Importantly, they were not globally impaired as there was no detectable difference in cytokine secretion from those macrophages after LPS stimulation (Figure S7 in Supplementary Material). Thus, the CMC observed in AIRE-deficient patients may be associated with the delay in FS formation and associated defective fungal-specific macrophage signal generation, phagocytosis, and cytokine production.