Pathogenic fungi regulate immunity by inducing neutrophilic myeloid-derived suppressor cells.
Mechanistically, pathogenic fungi induce neutrophilic MDSCs through the pattern recognition receptor Dectin-1 and its downstream adaptor protein CARD9.Fungal MDSC induction is further dependent on pathways downstream of Dectin-1 signaling, notably reactive oxygen species (ROS) generation as well as caspase-8 activity and interleukin-1 (IL-1) production.These studies define an innate immune mechanism by which pathogenic fungi regulate host defense.
Affiliation: Department of Pediatrics I, University of Tübingen, 72076 Tübingen, Germany. Electronic address: firstname.lastname@example.org.
- Aspergillus fumigatus/immunology*
- Candida albicans/immunology*
- Host-Pathogen Interactions*
- Immune Tolerance*
- CARD Signaling Adaptor Proteins/metabolism
- Cells, Cultured
- Disease Models, Animal
- Lectins, C-Type/metabolism
- Signal Transduction
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fig3: Fungi Induce MDSCs through a Dectin-1-, Syk-, and CARD9-Mediated Mechanism(A) Fungal factors mediating MDSC induction are heat resistant. MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with medium only (negative control), untreated, or heat-denatured (95°C, 30 min) supernatants (SNT) of A. fumigatus germ tubes (4%) for 6 days. The x-fold induction of MDSCs compared to control conditions is depicted. ∗p < 0.05 versus control conditions.(B) Dectin-1 and Syk are involved in fungal MDSC induction. MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with A. fumigatus germ tubes (1 × 105/ml), hyphae (1 × 105/ml), and C. albicans yeasts (1 × 105/ml) for 6 days. Where indicated, PBMCs were pretreated for 60 min with anti-Dectin-1 blocking antibody (15 μg/ml), soluble WGP (1 mg/ml), and a Syk inhibitor (100 nM). ∗p < 0.05 blocking versus unblocked conditions.(C) Dectin-1/CARD9 ligands mimic fungal MDSC induction. MDSCs were generated in vitro by incubating isolated PBMCs with the Dectin-1/CARD9 ligands zymosan depleted (10 μg/ml), dispersible WGP (20 μg/ml), or curdlan (10 μg/ml). p < 0.05 versus control conditions.(D) Dectin-1/CARD9 ligands induce functional MDSCs. The suppressive effects of CD33+-MACS-isolated MDSCs were analyzed on CD4+ and CD8+ T cell proliferation (CFSE polyclonal proliferation assay). MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with zymosan depleted (10 μg/ml) or dispersible WGP (20 μg/ml). MDSC, T cell ratio was 1:6.(E) Fungal MDSC induction in patients with genetic Dectin-1 or CARD9 deficiency.Left panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) from healthy controls (n = 12), an individual with Dectin-1 deficiency, or patients with CARD9 deficiency (n = 2) with the Dectin-1/CARD9 ligands zymosan depleted (10 μg/ml) or dispersible WGP (20 μg/ml).Right panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) from healthy controls (n = 12), an individual with genetically proven Dectin-1 deficiency, or patients with CARD9 deficiency (n = 2) with different fungal morphotypes (1 × 105 cells/ml) for 6 days.(F) CARD9 is involved in fungi-induced MDSC recruitment in vivo. Card9−/− mice and age-matched wild-type mice were challenged intranasally with 1 × 106A. fumigatus conidia for 3 days. On the fourth day, a BAL was performed, and CD11b+Ly6G+ MDSCs were quantified by flow cytometry. In (B), (C), and (E) bars represent means ± SEM.
The potency of A. fumigatus to induce MDSCs was most pronounced for germ tubes and hyphae, morphotypes characteristic for invasive fungal infections (Figure 1A) (Aimanianda et al., 2009; Hohl et al., 2005; Moyes et al., 2010). The MDSC-inducing fungal factor was present in conditioned supernatants and was heat resistant (Figure 3A), pointing to β-glucans as the bioactive component. We therefore focused on Dectin-1 as β-glucan receptor and key fungal sensing system in myeloid cells. Fungi-induced MDSCs expressed Dectin-1, and blocking Dectin-1 prior to fungal exposure diminished the MDSC-inducing effect, while blocking of TLR 4 had no effect (Figures 3B and S3). Furthermore, Dectin-1 receptor activation mimicked the generation of neutrophilic MDSCs phenotypically and functionally (Figures 3C and 3D). Dectin-1 receptor signaling was confirmed by blocking of the spleen tyrosine kinase Syk, which acts downstream of Dectin-1 (Figure 3B). We further used cells from human genetic Dectin-1 deficiency and used Dectin-1 knockout mice for fungal infection models. The potential of fungi or fungal patterns to induce neutrophilic MDSCs was diminished in human and, albeit to a lesser extent, murine Dectin-1 deficiency (Figures 3E and S1D). We analyzed the role of caspase recruitment domain 9 (CARD9), a downstream adaptor protein and key transducer of Dectin-1 signaling, in fungi-mediated MDSC generation in patients with genetic CARD9 deficiency and Card9 knockout mice. These approaches demonstrated that CARD9 signaling was involved in fungal MDSC induction in the human and the murine system (Figures 3E and 3F).