A Human Dectin-2 Deficiency Associated With Invasive Aspergillosis

Abstract Immunocompromised patients are highly susceptible to invasive aspergillosis. Herein, we identified a homozygous deletion mutation (507 del C) resulting in a frameshift (N170I) and early stop codon in the fungal binding Dectin-2 receptor, in an immunocompromised patient. The mutated form of Dectin-2 was weakly expressed, did not form clusters at/near the cell surface and was functionally defective. Peripheral blood mononuclear cells from this patient were unable to mount a cytokine (tumor necrosis factor, interleukin 6) response to Aspergillus fumigatus, and this first identified Dectin-2–deficient patient died of complications of invasive aspergillosis.

Immunocompromised patients are highly susceptible to invasive aspergillosis. Herein, we identified a homozygous deletion mutation (507 del C) resulting in a frameshift (N170I) and early stop codon in the fungal binding Dectin-2 receptor, in an immunocompromised patient. The mutated form of Dectin-2 was weakly expressed, did not form clusters at/near the cell surface and was functionally defective. Peripheral blood mononuclear cells from this patient were unable to mount a cytokine (tumor necrosis factor, interleukin 6) response to Aspergillus fumigatus, and this first identified Dectin-2-deficient patient died of complications of invasive aspergillosis.
Invasive fungal infections including invasive aspergillosis (IA) represent a severe disease burden in immunocompromised patients such as acute myeloid leukemia patients and allogeneic hematopoietic stem cell transplant (HSCT) recipients. Absence of a robust antifungal immune response permits fungal colonization, invasive growth, and disease. IA mortality is unacceptably high (30%-80%) in allogeneic HSCT recipients [1,2]. Therefore, patients are empirically treated with antifungal therapies.

Ethics Statement
The study was approved by the National Institute for Social Care and Health Research Ethics Committee (reference number 14/ WA/1119). Written informed consent was obtained from patients in the study. Animal work was performed according to institutional and United Kingdom (UK) Home Office guidelines. This study was performed in accordance with the Project License. Procedures were approved by the Cardiff University Animal Welfare and Ethical Review Body and UK Home Office. The animal care and use protocol adhered to the Animals (Scientific Procedures) Act 1986.

Genetic Analysis
RNA was extracted from patient blood samples using PAXgene Blood RNA kit (Qiagen), and complementary DNA (cDNA) was generated using a Reverse Transcription Kit (Thermo Fisher Scientific). Dectin-2 DNA was amplified and sequenced from patient cDNA by polymerase chain reaction (PCR) using primers (Supplementary Table 1).

Structural Analysis
The structure of wild-type (WT) Dectin-2, Protein Data Bank accession code 5VYB, was used as the starting model. The Coot program was used to implement mutations and model readjustment. REFMAC5 (CCP4) was used to regularize model geometry.

Determination of Dectin-2 Expression
Forty-eight hours after transfection of HEK293T cells and 72 hours after infection of D1D2 DKO BMDMs, RNA was extracted using TRIZOL (Thermo Fisher Scientific) and purified using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using the TaqMan Reverse Transcription Kit (Invitrogen). CLEC6A/ Dectin-2 mRNA was quantified by quantitative PCR using ABI Taqman Primer/Probe Sets (Thermo Fisher Scientific) and normalized against HPRT1. Dectin-2 protein expression was measured by intracellular flow cytometry staining with anti-FLAG (L5 BioLegend) or by surface flow cytometry staining with anti-Dectin-2 (545943 R&D).

Fungal Cultures
Aspergillus fumigatus 13073 (American Type Culture Collection [ATCC]) was cultured on potato dextrose agar for 7 days at 37°C. Conidia were harvested and passed through a 40-μM filter to remove hyphal fragments. Resting conidia were washed and resuspended in DPBS [7]. Candida albicans SC5314 (ATCC) was cultured on YPD agar plates overnight at 30°C, then cultured in YPD broth for 16 hours at 30°C with shaking, washed with DPBS, and resuspended in DPBS [5].
Data were analyzed using GraphPad Prism. Data are presented as mean ± standard error of the mean. One-way analysis of variance (ANOVA) followed by Tukey posttest or 2-way ANOVA followed by Bonferroni posttest was used for statistical analysis for multiple groups. Nonnormally distributed data were transformed by Y = sqrt (Y + 0.5) and ANOVA. P values < .05 were considered statistically significant.

CLEC6A (Dectin-2) Mutation
The patient possessed a homozygous base pair deletion (507delC) in exon 6 of Dectin-2 (CLEC6A) (Supplementary Figure 1A), which causes a frame shift (N170I) and premature termination of Dectin-2 ( Figure 1A). Loss of Cys176 removes a disulphide bridge, while loss of Asp191 removes a Ca 2+ and Na + stabilizing bridge ( Figure 1B) and loss of the final β-strand would leave a large hole at the core of the protein ( Figure 1C), resulting in failure of the protein to fold. Furthermore, mutant Dectin-2 could not bind its ligand (Supplementary Figure 1B  and 1C). Therefore, this Dectin-2 mutation likely has serious functional and clinical consequences.
Based on computational modeling, we hypothesized that mutant Dectin-2 would not produce a stable protein product. HEK293T cells expressing mutant Dectin-2 displayed increased RNA levels but minimal protein levels compared to WT Dectin-2 (Supplementary Figure 1D, 1E and 1F). Similarly, Dectin-1-Dectin-2 DKO BMDMs expressing mutant Dectin-2 displayed normal RNA levels ( Figure 1D) but minimal protein levels compared to BMDMs expressing WT Dectin-2 ( Figure  1E and 1F). Furthermore, while WT Dectin-2 clustered at/near the cell surface, mutant Dectin-2 was expressed at low levels throughout the cytosol and did not form clusters ( Figure 1G and Supplementary Figure 1G). Together, these data indicate that Dectin-2 N170I does not form a stable protein product, is minimally expressed, and is therefore functionally defective.

Functional Consequences of CLEC6A Mutation
To determine the functional consequences of Dectin-2 N170I, we tested whether the patients in our study were able to mount an effective immune response against A. fumigatus. Most WT PBMCs produced LPS-and A. fumigatus-induced cytokines. However, mutant Dectin-2 PBMCs only produced LPS-induced cytokines and not A. fumigatus-induced cytokines (Figure 2A). To confirm a role for Dectin-2 in A. fumigatus-induced cytokine production, we utilized Dectin-2 KO cells. Dectin-2 KO BMDCs displayed reduced A. fumigatus-induced ( Figure 2B) and C. albicans-induced cytokine production compared to WT controls, whereas they displayed normal LPS-induced cytokine production ( Figure 2C). However, Dectin-2 did not contribute to fungal killing (Supplementary Figure 2A). We next investigated whether Dectin-2 mediated binding to A. fumigatus. Dectin-1-Dectin-2 DKO BMDMs expressing mutant Dectin-2 displayed a modest reduction in binding A. fumigatus compared to DKO BMDMs expressing WT Dectin-2 (Supplementary Figure 2B and 2C). Aspergillus fumigatus-induced trained immunity could be significantly reduced by blocking Dectin-2 in human monocytes (Supplementary Figure  2D). Together, these data indicate that Dectin-2 N170I has detrimental consequences for antifungal immunity. In agreement with this, the patient was diagnosed with probable IA and lung abnormalities consistent with fungal infection, which progressively worsened until death (Supplementary Table 2).

DISCUSSION
Herein, we characterized a novel Dectin-2 N170I mutation identified in an HSCT recipient who died of complications of IA. This mutation results in truncation of Dectin-2 and radically alters the receptor's tertiary structure, leading to significantly reduced expression. Dectin-2 is important for fungal binding, trained immunity, and fungal-induced cytokine production.
Multiple polymorphisms in CLRs and their signaling component CARD9 increase susceptibility to fungal infection [9,11], some even without immunosuppression [8]. Two patients with reduced CARD9 protein expression developed IA [12], and HSCT recipients with the Dectin-1 Y238X SNP, which results in a truncated CLR, displayed increased susceptibility to IA [9]. Here, we demonstrate that the Dectin-2 N170I mutation also results in a truncated CLR. The Dectin-2 mutation was identified in the recipient prior to SCT; however, up to 70% of tissue resident cells remain from host origin and may persist for up to 1 year [13][14][15]. The patient displayed signs of A. fumigatus infection <1 year post-HSCT, when host cells expressing mutant Dectin-2 were likely present in the lung. While the patient died of complications of IA, additional patients would be required to confirm a direct link between Dectin-2 N170I and IA.
Structural modeling of Dectin-2 N170I predicted incorrect folding of the protein. Consistent with this, we showed reduced expression of Dectin-2 N170I at the cell surface despite the presence of RNA and low level of dispersed intracellular protein. These  results suggest that Dectin-2 N170I forms an unstable structure, is poorly transported to the cell membrane, and is minimally expressed, similar to Dectin-1 Y238X [8,9]. Furthermore, Dectin-2 predominantly recognizes mannose, and hence Aspergillus, through its EPN motif, a structure lost in Dectin-2 N170I [4]. Dectin-2 generates robust cytokine and chemokine responses against A. fumigatus [4], and mice deficient in Dectin-2 are susceptible to C. albicans infection [5]. Here, we found a significant role for Dectin-2 mediating A. fumigatus-and C. albicans-induced tumor necrosis factor (TNF) and interleukin 6 (IL-6) secretion. Similarly, PBMCs from CARD9-deficient patients display impaired fungal-induced cytokine production [16], and mice with TNF blockade or IL-6 deficiency are highly susceptible to IA [17,18]. Dectin-2 has previously been shown to bind to A. fumigatus hyphae [4], and we observed a modest reduction in binding of mutant Dectin-2 to A. fumigatus compared to WT Dectin-2. Importantly, we observed that Dectin-2 mediated A. fumigatus-induced trained immunity, further supporting the importance of Dectin-2 during IA.
Our research is the first to functionally characterize a Dectin-2 mutation associated with decreased antifungal responses. Furthermore, the Dectin-2 mutant patient developed and died of complications of IA. The Dectin-2 N170I mutation renders the CLR functionally null, and loss of Dectin-2 results in defective antifungal responses. Identifying mutations that increase a patient's fungal susceptibility may permit a personalized approach and enable targeted prophylaxis of patients at high risk of fungal disease [11].

Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes