Dcir-deficient (Clec4a2 ^–/–) mice on a C57BL/6J background (13) and wild-type C57BL/6J (WT) mice (CLEA Japan, Tokyo, Japan) were co-housed for at least one week before beginning the study. Male and female mice were used, with no observed sex bias in the results. Mice were maintained under specific-pathogen-free conditions with a gamma-sterilized diet and acidified tap water (0.002 N HCl) ad libitum.

All experiments complied with the “Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the Jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology” (Ministry of Education, Culture, Sports, Science and Technology, Japan, 2006). The Institutional Animal Care and Use Committee from Chiba University approved all protocols (process number: A5-204).

Aspergillus fumigatus MYA4609 (strain CBS 101355 [AF 293], American Type Culture Collection, VA, USA) was maintained in Sabouraud Dextrose Agar (BD, NJ, USA) at 30°C with weekly subcultures. Conidia and germinating hyphae were generated as described by Yoshikawa et al. (6).

To induce pulmonary aspergillosis, sedated mice had their trachea accessed orally using a 20Gx 1 1/4” indwelling needle intravenous cannula (Terumo, Tokyo, Japan). Mice then passively inhaled 1 x 10⁷ conidia cells in 30 μL of phosphate buffer saline (PBS).

To deplete neutrophils, mice were intravenously treated with 200 μg of anti-Ly6G antibody (Selleck Biotechnology, Kanagawa, Japan) or an isotype control (Rat IgG2a, κ, Selleck Biotechnology, Kanagawa, Japan) one day before infection.

To inhibit neutrophil degranulation in vivo, mice received intraperitoneal Nexinhib 20 (Tocris Bioscience, Bristol, United Kingdom) on days 1 and 3 post-infection (30 mg/kg), as previously described (18). At specified post-infection days, the mice were sedated and euthanized; bronchoalveolar lavage fluid (BALF) was collected via tracheal injection of 1 mL ice-cold PBS. Recovered cells underwent flow cytometric analysis (FCM, see section below). Lungs were harvested and processed according to the experiment’s objective.

The Dcir-fused Fc protein chimera was generated as previously described (6). The extracellular domain of Dcir was bound to the Fc portion of human IgG2 in a pIRES bleo3 vector plasmid; a vector lacking the CLR insert served as the control. Binding of Dcir to A. fumigatus isoforms was evaluated using an enzyme-linked immunosorbent assay (ELISA)-like assay, as previously described (6). Optical density (OD) measurements were used to construct binding curves for nonlinear regression analysis.

Neutrophils were isolated from the bone marrows of WT and Clec4a2 ^—/— mice using Percoll gradient centrifugation as previously described (6). Neutrophil purity (> 90%) and Dcir expression were analyzed by FCM.

Neutrophils were plated into 96-well plates containing RPMI-1640 medium (Fujifilm Wako, Osaka, Japan) supplemented with 10% inactivated fetal bovine serum (iFBS; Biosera, Nuaillé, France). Cells were incubated with A. fumigatus isoforms at 37°C and 5% CO₂ for up to 3 h at a ratio of five neutrophils per fungal cell. Cell-free supernatants were collected and stored at –80°C for subsequent cytokine and Lipocalin-2 quantification using a commercial Mouse Lipocalin-2/NGAL DuoSet ELISA kit (R&D systems, MN, USA), or used immediately in other assays.

The remaining neutrophils were lysed with 1% Triton X-100 solution, prepared in-house from polyethylene glycol mono-p-isooctylphenyl ether (Nacalai Tesque, Kyoto, Japan). The number of surviving conidia was determined by seeding on PDA plates for CFU enumeration. Hyphal viability was assessed via the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay.

For degranulation inhibition, upon addition of A. fumigatus to neutrophil cultures, wells were supplemented with 1 μM of Nexinhib 20 (Tocris Bioscience, Bristol, United Kingdom) or DMSO as a vehicle control.

For microscopy, cells were seeded onto glass slides in 24-well plates and incubated with A. fumigatus under the same conditions described above. Slides were stained using the Neat Stain Hematological Stain kit (Polysciences Inc., PA, USA), and images were acquired via optical microscopy.

For assessment of cell viability, the Sytox Green incorporation assay was employed (20). PMA (phorbol 12-myristate 13-acetate; 5 μg/mL) was used as a positive control. Fluorescence in the FITC channel was measured with FCM, and the percentage of positive cells was recorded.

Intracellular ROS levels were measured using a commercial DCFDA/H2DCFDA-Cellular ROS Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Fluorescence was detected via FCM (FITC channel).

For extracellular ROS measurement, a cytochrome C reduction assay was performed to measure superoxide (O₂ ^-) in the supernatants as previously described (21). Neutrophils were plated in 96-well plates with 10% iFBS in HBSS and incubated with A. fumigatus or 100 nM PMA. The cell-free supernatants were combined with 1 mg/mL cytochrome C, with or without 100 U/mL superoxide dismutase (SOD) (stock solutions prepared in HBSS; Nacalai Tesque, Kyoto, Japan). Absorbance was recorded at 550, 540, and 560 nm. The OD at 550 nm was adjusted by the average OD between 540 and 560 nm (ODcorr) for calculations. The O₂ ^-concentration was calculated using the formula:

where the constant 21.1 is the extinction coefficient for reduced cytochrome C; results are expressed as mmol/μL.

Gelatinase activity in neutrophil cultures was assessed via zymography. Cell-free supernatants from A. fumigatus-stimulated neutrophil cultures were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using an in-house 6% polyacrylamide gel with 1 mg/mL gelatin under non-reducing and non-denaturing conditions. Gels were renatured (Zymogram Renaturing Buffer, Novex, CA, USA), developed overnight at 37°C (Zymogram Developing Buffer, Novex), and stained with Bio-Safe Coomassie G-250 stain (Bio-Rad, USA). Digested regions appeared as clear bands on a blue background.

For MMP9 detection, supernatants were run on SDS-PAGE (non-reducing, non-denaturing), transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA), and analyzed by western blot. For PLCγ2/pPLCγ2 detection, neutrophil lysates underwent SDS-PAGE (reducing, denaturing) before transfer to PVDF membranes. Primary antibodies included: Rb mAb to MMP9 (Abcam, MA, USA), PLCgamma2 Rabbit Ab, phospho-PLCγ2 (Tyr1217) Ab (Cell Signaling, MA, USA), and anti-β-actin pAb (MBL, Tokyo, Japan). HRP donkey anti-rabbit IgG (Biolegend, CA, USA) served as the secondary antibody. Zymography and western blot images were quantified using ImageJ (version 1.53 for Mac OS X, NIH, USA).

Intracellular Ca²⁺ levels were estimated by FCM using the fluorescent probe 4-Fluo AM (Dojindo, Tokyo, Japan). After stimulation with A. fumigatus, mean fluorescence intensity (MFI) values were recorded using FCM (Fluo-4AM) in the FITC channel.

For cell phenotyping, cells were stained for FCM analysis as previously described. Reagents and antibodies are listed in Supplementary Table 1 . Data were acquired with a FACS Verse flow cytometer (8-color, BD, NJ, USA) and analyzed using FlowJo (v.10.7.1 for Mac OS X, BD, NJ, USA). Gating strategies are shown in Supplementary Figure 3 .

Cytokines were measured with the BD Cytometric Bead Array assay per the manufacturer’s instructions. Data were acquired on a FACS Verse and analyzed using FCAP Array software (v.3.0.1; BD). Detection limits were as follows: IL-1β,  1.9 pg/mL; IL-6, 1.4 pg/mL; TNF, 2.8 pg/mL; IL-10, 9.6 pg/mL; MCP-1, 2.7 pg/mL; KC, 0.1 pg/mL; CCL3/MIP-1α, 2.3 pg/mL; and CCL4/MIP-1β, 0.6 pg/mL.

Statistical analyses were conducted using GraphPad Prism (v. 10 for OSX, GraphPad Inc., CA, USA). Data were screened for outliers using the ROUT method, and group comparisons were made using non-parametric tests, regardless of normality results. The statistical test employed for each analysis, sample size, and number of replicates are disclosed in the figure legends. A p-value < 0.05 was considered statistically significant.

In a previously described model of acute disseminated aspergillosis, immunocompetent WT mice were shown to develop transient, self-limiting disease, while dectin-1 knockout mice readily succumbed to the infection (6), facilitating the rapid screening of susceptibility factors.

We employed this model to initially assess the potential contribution of Dcir to antifungal defense ( Supplementary Figure 1 ). Clec4a2 ^—/— mice experienced no mortality, less weight loss ( Supplementary Figure 1A ) (area under the curve [AUC]: mean 47.22 vs. 17.51, p = 0.0279), and lower spleen A. fumigatus burden than WT mice ( Supplementary Figure 1B ; 5 days post infection [dpi] mean 106360 vs. 45259, p = 0.0377), with only IL-1β levels markedly reduced among cytokines ( Supplementary Figure 1C ). These results suggest that Dcir restricts pathogen clearance rather than promoting anti-fungal responses.

Next, in a standard pulmonary aspergillosis model that more closely mimics natural infection, WT and Clec4a2 ^—/— mice were intratracheally infected with A. fumigatus ( Figure 1 ). Although WT and Clec4a2 ^—/— mice both cleared the infection efficiently, the knockout group showed a lower fungal burden at mid-infection (5 dpi; Figure 1A ; mean 68267 vs. 38604, p = 0.0315). This was also observed in lung sections harvested at the same time point and stained with Grocott (silver-based) stain (black structures against the green-stained lung tissue) in Clec4a2 ^—/— mice ( Figure 1B ). However, pulmonary cytokine and chemokine levels were similar between groups ( Figure 1C ), suggesting equivalent inflammatory responses in WT and Clec4a2 ^—/— mice. Overall, these results suggest that Dcir deficiency enhances fungal elimination.

Neutrophils are crucial for Aspergillus clearance (22) and neutrophil recruitment is altered in Dcir-deficient mice in experimental models of chemical hepatitis (23) and DSS-induced colitis (24). Thus, we investigated phagocyte recruitment by analyzing the phenotypes of innate immune cells within the lungs and BALF harvested 5 dpi.

Neutrophils constituted the predominant cell population in the lung and BALF infiltrates of infected mice ( Figure 2A ), with no significant differences between WT and Clec4a2 ^—/— mice ( Supplementary Figure 2 ). While Clec4a2 ^—/— mice exhibited a statistically significant reduction in alveolar macrophages (Mϕ), inflammatory Mϕ, and dendritic cells counts ( Supplementary Figure 2 ), the frequencies of these subsets remained low, making up less than 10% of the total infiltrate. Thus, the response of Clec4a2 ^—/— mice was not related to improved phagocyte recruitment.

Considering that neutropenia is a critical risk factor for aspergillosis (25, 26) and that neutrophils dominate the cellular infiltrates, we determined whether neutrophil-depleted Clec4a2 ^—/— mice retained improved infection clearance compared to the WT group. Neutrophils were depleted using an anti-Ly6G antibody before infection ( Figure 2B ) and fungal burden was subsequently analyzed. Neutrophil depletion abolished the protective effect conferred by Dcir deficiency ( Figure 2C ; isotype: mean 23695 vs. 9850, p<0.0001; anti-Ly6G: mean 18574 vs. 22933, p = 0.1222). Additionally, Dcir was significantly expressed by neutrophils ( Figure 2D ), confirming previous findings (27, 28). These results suggest that Dcir directly regulates neutrophil effector functions against A. fumigatus.

To further investigate this mechanism, in vitro assays were conducted wherein bone marrow-derived neutrophils were incubated with different A. fumigatus isoforms to assess their phagocytic killing function. Clec4a2 ^—/— neutrophils exhibited enhanced hyphal killing ( Figure 2E ; mean 60.88 vs. 78.27, p = 0.0391). However, the absence of Dcir did not uniformly enhance conidial killing. As A. fumigatus spores evade dectin-1 recognition due to hydrophobin A masking binding sites on the conidial surface (29), we explored whether a similar mechanism might affect Dcir sensing. Using a chimeric Dcir soluble protein comprising the receptor’s extracellular domain fused to a human IgG Fc fragment, we evaluated binding to A. fumigatus isoforms. Unexpectedly, Dcir bound conidia and hyphae ( Figure 3 ), suggesting that evasion analogous to dectin-1 is not operative. These data support the hypothesis that, while Dcir binds both forms, it specifically modulates neutrophil effector functions against hyphae rather than conidia.

Therefore, neutrophils from Clec4a2 ^—/— mice eliminated fungi more effectively, likely explaining their enhanced clearance capability, despite unchanged tissue inflammation or cell recruitment.

Next, the effector mechanisms through which Dcir regulates fungicidal activity against A. fumigatus were investigated. Neutrophils eliminate pathogens via phagocytosis, programmed cell death (NETosis), oxidative stress, and degranulation (30). Given the size disparity between neutrophils and A. fumigatus hyphae, phagocytosis was ruled out and we focused on the other possibilities.

Neutrophils accumulated around the hyphal structures ( Figure 4A ); however, significant cell death was not detected following A. fumigatus stimulation within our experimental time frame, as assessed by SYTOX probe incorporation ( Figure 4B ). Consequently, the increased killing of A. fumigatus-hyphae promoted by Clec4a2 ^—/— cells ( Figure 2B ) appears to occur independently of neutrophil death.

The oxidative stress induced in A. fumigatus-stimulated neutrophils was assessed in sequence. Tokieda et al. (24) reported that Clec4a2 ^—/— neutrophils exhibit impaired intracellular ROS production when exposed to lipopolysaccharide. In alignment with these findings, lower ROS production was observed in response to A. fumigatus and the positive control TBHP, in Dcir-deficient cells ( Figure 4C ). Although the underlying molecular mechanisms responsible for this intrinsic defect remain to be elucidated, its impact might be minimal in terms of fungal killing, given that even the inactivation of internalized conidia can also be achieved via non-oxidative mechanisms (31).

Subsequently, extracellular ROS production was also evaluated, which originates from a distinct source compared to intracellular ROS (32). Measurement of O₂ ^- in culture supernatants using the cytochrome C assay revealed no significant difference between WT and Clec4a2 ^—/— cells, despite the pronounced oxidative burst induced by A. fumigatus ( Figure 4D ). These findings suggest that oxidative stress does not account for the increased fungicidal activity observed in the knockout cells.

Interestingly, the supernatants from A. fumigatus-activated neutrophil cultures exhibited gelatinase activity, which was higher in Dcir-deficient cells ( Figure 4E ; mean 1.153 vs. 1.990, p = 0.0492). However, other secreted proteins, as cytokines, were not equally enhanced in the knockout cells ( Figure 4F ), suggesting that enzyme release is being independently favored. Neutrophil gelatinases are stored in granules and can be exocytosed sequentially (33), hinting that Clec4a2 ^—/— neutrophils may exert increased degranulatory responses.

To determine whether Clec4a2 ^—/— neutrophils exhibit enhanced degranulation following A. fumigatus challenge, the levels of two proxy markers—metalloproteinase [MMP]-9 and Lipocalin-2/NGAL (34)—were quantified, confirming an increase in both markers in the supernatants of knockout cells ( Figure 5A , mean 35.71 vs. 46.91, p = 0.0465; Figure 5B , mean 1585 vs. 1775, p = 0.0043). Notably, elevated NGAL concentrations were also detected in the lungs of infected Clec4a2 ^—/— mice, supporting the in vivo relevance of the findings ( Figure 5C ; lung: mean 2090 vs. 2416, p = 0.0207).

Degranulation initiation is linked to increased cytoplasmic Ca²⁺, mobilized from intracellular stores (33). Using the fluorescent probe 4-Fluo AM, Ca²⁺ levels in Clec4a2 ^—/— cells were monitored, revealing a more pronounced increase upon A. fumigatus stimulation compared to controls ( Figure 5D ; mean 1.048 vs. 1.223, p = 0.0499).

Various receptors can drive Ca²⁺ mobilization via their specific stimulatory pathways; however, a common signaling hub upstream of Ca²⁺ mobilization is phospholipase PLCγ2 phosphorylation/activation (35). Clec4a2 ^—/— neutrophils displayed enhanced PLCγ2 phosphorylation after A. fumigatus stimulation ( Figure 5E ; pPLCγ2: mean 0.1837 vs. 0.3543, p = 0.0305). These data suggest that Dcir negatively regulates the signaling cascade leading to neutrophil degranulation.

To assess the importance of degranulation in A. fumigatus killing, this pathway was pharmacologically inhibited using Nexinhib 20—a neutrophil-specific drug (18). Inhibition of this pathway abolished the enhanced antifungal effect observed in Dcir-deficient neutrophils ( Figure 5F ; vehicle: mean 34.83 vs. 56.43, p = 0.0408; Nex20: mean 28.00 vs. 40.00, p = 0.4349). These findings were validated in vivo, wherein Nexinhib 20 was administered during infection. Despite the lack of changes in pulmonary neutrophil influx ( Figure 5G ), Nexinhib 20 treatment neutralized the advantage of Dcir knockout ( Figure 5H ; sham: mean 48782 vs. 24681, p = 0.0491; Nex20: mean 47576 vs. 54130, p = 0.5804), consistent with the effects observed following antibody-mediated neutrophil depletion ( Figure 2 ).

Collectively, these results demonstrate that Dcir modulates the neutrophil-driven elimination of A. fumigatus by dampening the degranulation process.