The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan

Abstract

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

Keywords: Aspergillus fumigatus; fungi; host-pathogen interactions; immune mechanisms; innate immunity; mycology.

FIG 1

A. fumigatus and galactomannan activate Dectin-2-expressing CLR reporter cells. Reporter cells were stimulated for 18 h using either live or heat-killed germlings of A. fumigatus Af293 at a multiplicity of infection (MOI) of 20:1 (A) or unmodified FLPs, β-1,3 glucan (BG) FLPs, S. cerevisiae mannan FLPs, and A. fumigatus galactomannan (GALM) FLPs at an effector to target ratio of 30:1 (B). LacZ activity was measured in total cell lysates using CPRG as a substrate. Error bars represent the standard error of the mean (SEM) of duplicate wells, and each experiment was repeated three times.

FIG 2

Recombinant Dectin-2 Fc protein binds galactomannan FLPs and A. fumigatus. (A) Unmodified FLPs, β-1,3 glucan FLPs, and galactomannan FLPs were incubated with soluble recombinant murine Dectin-2-human IgG1 Fc protein conjugate, control IgG1-Fc protein, or in buffer only, incubated with anti-human IgG1 conjugated to Alexa Fluor 488 and analyzed by flow cytometry. Dectin-2-Fc bound to galactomannan FLPs but not to either unmodified or β-1,3 glucan FLPs. (B) FLPs were incubated for 2 h in binding buffer alone or containing Dectin-2-Fc protein or Dectin-2-Fc preincubated for 30 min with a Dectin-2-neutralizing antibody. Dectin-2-neutralizing antibody disrupted the association of Dectin-2-Fc with galactomannan FLPs. (C) A. fumigatus CEA10 swollen conidia and early germlings were incubated with Dectin-2-Fc protein or control protein, IgG1-Fc, followed by incubation with anti-human IgG Fc conjugated to Alexa Fluor 647 and analyzed by confocal microscopy. Dectin-2-Fc protein, but not control IgG1-Fc, bound intensely to the early hyphal structure and to swollen conidia.

FIG 3

Galactomannan FLPs activate Dectin-2-dependent signaling in macrophages. (A) Immunoblot of immortalized wild-type C57BL/6 macrophages or C57BL/6 macrophages expressing Dectin-2 stimulated with live C. albicans (SC5314), unmodified FLPs (UM FLP), galactomannan FLPs (GALM FLP), β-1,3 glucan FLPs (BG FLP), or mannan FLPs at an MOI of 10:1 for 1 h at 37°C and probed for both total Syk and phosphorylated (active) Syk. Overexpression of Dectin-2 increased phosphorylation of Syk in response to C. albicans, galactomannan and mannan FLPs, but not to unmodified or β-1,3 glucan FLPs. (B) Heatmap displaying the genes differentially expressed in Dectin-2^OE macrophages stimulated with GALM-FLP compared with unstimulated. The heatmap depicts the log₂ fold change in Dectin-2^OE cells and wild-type cells demonstrating a higher degree of change for most genes in the Dectin-2^OE cells compared to those of the WT. (C) Immortalized wild-type C57BL/6 macrophages or C57BL/6 macrophages expressing Dectin-2 were stimulated overnight with unmodified (UM), galactomannan (GALM), or mannan (MAN) FLPs at a target to effector ratio of 10:1. Prior to FLP stimulation, macrophages were pretreated for 30 min with either a Dectin-2-neutralizing antibody or an isotype control antibody as indicated. Supernatants were analyzed for TNF-α using ELISA. Both galactomannan and mannan FLPs induced robust TNF-α production by macrophages expressing Dectin-2, which was blocked by Dectin-2-neutralizing antibody but not the isotype control. The significance indicated is in relation to the wild-type cell stimulated with the same FLPs. (D) BMDMs from wild-type C57BL/6 and Dectin-2^−/− mice were stimulated with LPS (500 ng/mL), and unmodified (UM) or galactomannan (GALM) FLPs at an effector to target ration of 20:1 for 18 h. Supernatants were analyzed for TNF-α using ELISA. Dectin-2^−/− BMDM produced less TNF-α when stimulated with galactomannan FLP compared with wild-type BMDM. Error bars represent standard error of three biological replicates, statistics were calculated with two-way analysis of variance (ANOVA) using PRISM 9 software (not significant, ns; ***, P < 0.001; ****, P < 0.0001).

FIG 4

Deficiency of Dectin-2 leads to increased immune cell recruitment and decreased pulmonary TNF-α levels but is dispensable for survival. (A to E) Immunocompetent wild-type and Dectin-2^−/− mice were infected via oropharyngeal inhalation of 4 × 10⁷ conidia of A. fumigatus CEA10. Lung tissue was harvested 48 h postinfection and digested to obtain a single cell suspension that was then analyzed by flow cytometry. Dectin-2-infected mice had (A) increased infiltration of live CD45⁺ cells compared with that of the wild type. There was a significant increase in the total number of (B) neutrophils (7-AAD^lo, CD45⁺, CD90.2⁻, CD19⁻, SiglecF⁻, Ly6G⁺) in Dectin-2^−/− mice compared with wild type. However, there was no difference in the percentage of CD45⁺ cells that were neutrophils (C). The data shown are representative data from a single experiment. Each point represents an individual animal and horizontal lines represent the median. One-way ANOVA was performed using PRISM 9 software to determine statistical significance with Šídák’s multiple comparison test (not significant, ns; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (D) Immunocompetent wild-type and Dectin-2^−/− mice were infected with 4 × 10⁷ CEA10. After 48 h, lungs were extracted and fungal burden was quantified using CFU. There was no difference in fungal burden between wild-type and Dectin-2^−/− mice. Each point represents an individual animal, and horizontal lines represent the median fungal burden in the lungs. Data shown are from a single experiment consisting of 5 infected and 1 PBS control per mouse strain. Each experiment was repeated at least twice, and concordant results were obtained in each experiment. (E) Immunocompetent wild-type and Dectin-2^−/− mice were infected with 4 × 10⁷ CEA10 or PBS, and after 48 h, lungs were harvested and homogenized for cytokine analysis. Infected Dectin-2^−/− mice had significantly less TNF-α compared with wild-type mice, and there was no difference in TNF-α between PBS-stimulated mice. Each point represents an individual animal, and boxes represent the mean with error bars representing the standard deviation. Two-way ANOVA was performed using PRISM 9 software to determine statistical significance with Šídák’s multiple comparison test (not significant, ns; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (F) Wild-type and Dectin-2^−/− mice were immunosuppressed with corticosteroids and infected intranasally with 2.5 × 10⁴ conidia of A. fumigatus strain CEA10. There was no significant difference in survival between wild-type and Dectin-2^−/− mice as determined based upon Kaplan-Meyer and Cox analysis using PRISM 9 software.