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Pathogenic fungi regulate immunity by inducing neutrophilic myeloid-derived suppressor cells.

Rieber N, Singh A, Öz H, Carevic M, Bouzani M, Amich J, Ost M, Ye Z, Ballbach M, Schäfer I, Mezger M, Klimosch SN, Weber AN, Handgretinger R, Krappmann S, Liese J, Engeholm M, Schüle R, Salih HR, Marodi L, Speckmann C, Grimbacher B, Ruland J, Brown GD, Beilhack A, Loeffler J, Hartl D - Cell Host Microbe (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics I, University of Tübingen, 72076 Tübingen, Germany. Electronic address: nikolaus.rieber@med.uni-tuebingen.de.

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Fungal MDSC Induction Involves IL-1β, Caspase-8, and ROS(A) Intracellular accumulation and release of IL-1β.Left panel: gating strategy for intracellular cytokine staining. IL-1β was analyzed in CD33+ myeloid cells using intracellular cytokine staining and flow cytometry. Zymosan depleted (20, 100, and 500 μg/ml) and WGP dispersible (20, 100, and 500 μg/ml) were used for 1 hr to stimulate cytokine production.Middle panel: leukocytes isolated from healthy donors (n = 4) were left untreated (empty circles) or were treated for 1 hr with increasing concentrations of zymosan, WGP, A. fumigatus germ tubes, or C. albicans yeasts (each at 2 × 105/ml and 1 × 106/ml). IL-1β synthesis in CD33+ cells was analyzed by intracellular cytokine stainings by flow cytometry. ∗p < 0.05 versus control/untreated conditions.Right panel: co-culture supernatants were collected after incubating isolated PBMCs (5 × 105 cells/ml) with medium only (white bar), A. fumigatus germ tubes (1 × 105 cells/ml), or C. albicans yeasts (1 × 105/ml) for 3 days. IL-1β was quantified by ELISA. ∗p < 0.05 versus medium control conditions.(B) IL-1β signaling is involved in fungal-induced MDSC generation.Left panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with C. albicans yeasts (1 × 105/ml) or recombinant human IL-1β protein (0.01 μg/ml) for 6 days. ∗p < 0.05.Right panel: MDSCs (CD11b+Ly6G+) were quantified in spleens from Il1r−/− and age-matched WT mice 2 days after i.v. infection with 1 × 105 blastospores of C. albicans. ∗p < 0.05.(C) Fungal MDSC generation involves caspase-8. MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with C. albicans yeasts (1 × 105/ml) for 6 days with or without pretreatment (where indicated) with the pan-caspase inhibitor Z-VAD-FMK (10 μM), the caspase-1 inhibitor Z-WEHD-FMK (50 μM), or the caspase-8 inhibitor Z-IETD-FMK (50 μM). IL-1β protein levels were quantified in cell culture supernatants by ELISA (note: two values were below detection limit). Caspase-8 activity was quantified in cell lysates using a luminescent assay. ∗p < 0.05.(D) Fungal MDSC-inducing capacity is ROS dependent. MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with different fungal morphotypes (1 × 105 cells/ml) or zymosan (10 μg/ml) for 6 days. PBMCs were pretreated where indicated with the NADPH oxidase inhibitor DPI (0.1 μM) or the H2O2 converting enzyme catalase (100 U/l). ∗p < 0.05 blocking versus unblocked conditions.(E) Fungal MDSC induction in patients with ROS deficiency.Left panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) from healthy controls (n = 12) or patients with CGD (n = 3) 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) or CGD patients (n = 3) with different fungal morphotypes (1 × 105 cells/ml) for 6 days.In (A)–(E) bars represent means ± SEM.
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fig4: Fungal MDSC Induction Involves IL-1β, Caspase-8, and ROS(A) Intracellular accumulation and release of IL-1β.Left panel: gating strategy for intracellular cytokine staining. IL-1β was analyzed in CD33+ myeloid cells using intracellular cytokine staining and flow cytometry. Zymosan depleted (20, 100, and 500 μg/ml) and WGP dispersible (20, 100, and 500 μg/ml) were used for 1 hr to stimulate cytokine production.Middle panel: leukocytes isolated from healthy donors (n = 4) were left untreated (empty circles) or were treated for 1 hr with increasing concentrations of zymosan, WGP, A. fumigatus germ tubes, or C. albicans yeasts (each at 2 × 105/ml and 1 × 106/ml). IL-1β synthesis in CD33+ cells was analyzed by intracellular cytokine stainings by flow cytometry. ∗p < 0.05 versus control/untreated conditions.Right panel: co-culture supernatants were collected after incubating isolated PBMCs (5 × 105 cells/ml) with medium only (white bar), A. fumigatus germ tubes (1 × 105 cells/ml), or C. albicans yeasts (1 × 105/ml) for 3 days. IL-1β was quantified by ELISA. ∗p < 0.05 versus medium control conditions.(B) IL-1β signaling is involved in fungal-induced MDSC generation.Left panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with C. albicans yeasts (1 × 105/ml) or recombinant human IL-1β protein (0.01 μg/ml) for 6 days. ∗p < 0.05.Right panel: MDSCs (CD11b+Ly6G+) were quantified in spleens from Il1r−/− and age-matched WT mice 2 days after i.v. infection with 1 × 105 blastospores of C. albicans. ∗p < 0.05.(C) Fungal MDSC generation involves caspase-8. MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with C. albicans yeasts (1 × 105/ml) for 6 days with or without pretreatment (where indicated) with the pan-caspase inhibitor Z-VAD-FMK (10 μM), the caspase-1 inhibitor Z-WEHD-FMK (50 μM), or the caspase-8 inhibitor Z-IETD-FMK (50 μM). IL-1β protein levels were quantified in cell culture supernatants by ELISA (note: two values were below detection limit). Caspase-8 activity was quantified in cell lysates using a luminescent assay. ∗p < 0.05.(D) Fungal MDSC-inducing capacity is ROS dependent. MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with different fungal morphotypes (1 × 105 cells/ml) or zymosan (10 μg/ml) for 6 days. PBMCs were pretreated where indicated with the NADPH oxidase inhibitor DPI (0.1 μM) or the H2O2 converting enzyme catalase (100 U/l). ∗p < 0.05 blocking versus unblocked conditions.(E) Fungal MDSC induction in patients with ROS deficiency.Left panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) from healthy controls (n = 12) or patients with CGD (n = 3) 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) or CGD patients (n = 3) with different fungal morphotypes (1 × 105 cells/ml) for 6 days.In (A)–(E) bars represent means ± SEM.

Mentions: C. albicans induces interleukin-1 beta (IL-1β) in vitro (van de Veerdonk et al., 2009) and in vivo (Hise et al., 2009), which is critical for antifungal immunity (Vonk et al., 2006). Recent studies further provided evidence that IL-1β is involved in MDSC homeostasis (Bruchard et al., 2013). We observed an accumulation of intracellular IL-1β protein in CD33+ myeloid cells followed by IL-1β release upon Dectin-1 ligand- and fungal-driven MDSC induction (Figure 4A). IL-1β protein, in turn, was sufficient to drive MDSC generation to a comparable extent as C. albicans did (Figure 4B). Studies in Il1r−/− mice, characterized by an increased susceptibility to C. albicans infection, demonstrated that abrogation of IL-1R signaling decreased MDSC accumulation in vivo (Figures 4B and S4A), and IL-1R antagonism in patients with autoinflammatory diseases decreased MDSCs (Figure S4B). As the inflammasome is the major mechanism driving IL-1β generation in myeloid cells through caspase activities, we blocked caspases chemically. We observed that pan-caspase inhibition largely abolished fungi-induced MDSC generation, which was not recapitulated by caspase-1 inhibition (Figure 4C). We therefore focused on caspase-8, since Dectin-1 activation was shown to trigger IL-1β processing by a caspase-8-dependent mechanism (Ganesan et al., 2014; Gringhuis et al., 2012). Indeed, fungal MDSC induction was paralleled by a substantial increase of caspase-8 activity, and caspase-8 inhibition diminished fungal-induced IL-1β production (Figure 4C) and the potential of fungi to induce MDSCs (Figure 4C). Conversely, supplementing IL-1β partially restored the abrogated MDSC generation upon caspase-8 inhibition (Figure S4C).


Pathogenic fungi regulate immunity by inducing neutrophilic myeloid-derived suppressor cells.

Rieber N, Singh A, Öz H, Carevic M, Bouzani M, Amich J, Ost M, Ye Z, Ballbach M, Schäfer I, Mezger M, Klimosch SN, Weber AN, Handgretinger R, Krappmann S, Liese J, Engeholm M, Schüle R, Salih HR, Marodi L, Speckmann C, Grimbacher B, Ruland J, Brown GD, Beilhack A, Loeffler J, Hartl D - Cell Host Microbe (2015)

Fungal MDSC Induction Involves IL-1β, Caspase-8, and ROS(A) Intracellular accumulation and release of IL-1β.Left panel: gating strategy for intracellular cytokine staining. IL-1β was analyzed in CD33+ myeloid cells using intracellular cytokine staining and flow cytometry. Zymosan depleted (20, 100, and 500 μg/ml) and WGP dispersible (20, 100, and 500 μg/ml) were used for 1 hr to stimulate cytokine production.Middle panel: leukocytes isolated from healthy donors (n = 4) were left untreated (empty circles) or were treated for 1 hr with increasing concentrations of zymosan, WGP, A. fumigatus germ tubes, or C. albicans yeasts (each at 2 × 105/ml and 1 × 106/ml). IL-1β synthesis in CD33+ cells was analyzed by intracellular cytokine stainings by flow cytometry. ∗p < 0.05 versus control/untreated conditions.Right panel: co-culture supernatants were collected after incubating isolated PBMCs (5 × 105 cells/ml) with medium only (white bar), A. fumigatus germ tubes (1 × 105 cells/ml), or C. albicans yeasts (1 × 105/ml) for 3 days. IL-1β was quantified by ELISA. ∗p < 0.05 versus medium control conditions.(B) IL-1β signaling is involved in fungal-induced MDSC generation.Left panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with C. albicans yeasts (1 × 105/ml) or recombinant human IL-1β protein (0.01 μg/ml) for 6 days. ∗p < 0.05.Right panel: MDSCs (CD11b+Ly6G+) were quantified in spleens from Il1r−/− and age-matched WT mice 2 days after i.v. infection with 1 × 105 blastospores of C. albicans. ∗p < 0.05.(C) Fungal MDSC generation involves caspase-8. MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with C. albicans yeasts (1 × 105/ml) for 6 days with or without pretreatment (where indicated) with the pan-caspase inhibitor Z-VAD-FMK (10 μM), the caspase-1 inhibitor Z-WEHD-FMK (50 μM), or the caspase-8 inhibitor Z-IETD-FMK (50 μM). IL-1β protein levels were quantified in cell culture supernatants by ELISA (note: two values were below detection limit). Caspase-8 activity was quantified in cell lysates using a luminescent assay. ∗p < 0.05.(D) Fungal MDSC-inducing capacity is ROS dependent. MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with different fungal morphotypes (1 × 105 cells/ml) or zymosan (10 μg/ml) for 6 days. PBMCs were pretreated where indicated with the NADPH oxidase inhibitor DPI (0.1 μM) or the H2O2 converting enzyme catalase (100 U/l). ∗p < 0.05 blocking versus unblocked conditions.(E) Fungal MDSC induction in patients with ROS deficiency.Left panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) from healthy controls (n = 12) or patients with CGD (n = 3) 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) or CGD patients (n = 3) with different fungal morphotypes (1 × 105 cells/ml) for 6 days.In (A)–(E) bars represent means ± SEM.
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fig4: Fungal MDSC Induction Involves IL-1β, Caspase-8, and ROS(A) Intracellular accumulation and release of IL-1β.Left panel: gating strategy for intracellular cytokine staining. IL-1β was analyzed in CD33+ myeloid cells using intracellular cytokine staining and flow cytometry. Zymosan depleted (20, 100, and 500 μg/ml) and WGP dispersible (20, 100, and 500 μg/ml) were used for 1 hr to stimulate cytokine production.Middle panel: leukocytes isolated from healthy donors (n = 4) were left untreated (empty circles) or were treated for 1 hr with increasing concentrations of zymosan, WGP, A. fumigatus germ tubes, or C. albicans yeasts (each at 2 × 105/ml and 1 × 106/ml). IL-1β synthesis in CD33+ cells was analyzed by intracellular cytokine stainings by flow cytometry. ∗p < 0.05 versus control/untreated conditions.Right panel: co-culture supernatants were collected after incubating isolated PBMCs (5 × 105 cells/ml) with medium only (white bar), A. fumigatus germ tubes (1 × 105 cells/ml), or C. albicans yeasts (1 × 105/ml) for 3 days. IL-1β was quantified by ELISA. ∗p < 0.05 versus medium control conditions.(B) IL-1β signaling is involved in fungal-induced MDSC generation.Left panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with C. albicans yeasts (1 × 105/ml) or recombinant human IL-1β protein (0.01 μg/ml) for 6 days. ∗p < 0.05.Right panel: MDSCs (CD11b+Ly6G+) were quantified in spleens from Il1r−/− and age-matched WT mice 2 days after i.v. infection with 1 × 105 blastospores of C. albicans. ∗p < 0.05.(C) Fungal MDSC generation involves caspase-8. MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with C. albicans yeasts (1 × 105/ml) for 6 days with or without pretreatment (where indicated) with the pan-caspase inhibitor Z-VAD-FMK (10 μM), the caspase-1 inhibitor Z-WEHD-FMK (50 μM), or the caspase-8 inhibitor Z-IETD-FMK (50 μM). IL-1β protein levels were quantified in cell culture supernatants by ELISA (note: two values were below detection limit). Caspase-8 activity was quantified in cell lysates using a luminescent assay. ∗p < 0.05.(D) Fungal MDSC-inducing capacity is ROS dependent. MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) with different fungal morphotypes (1 × 105 cells/ml) or zymosan (10 μg/ml) for 6 days. PBMCs were pretreated where indicated with the NADPH oxidase inhibitor DPI (0.1 μM) or the H2O2 converting enzyme catalase (100 U/l). ∗p < 0.05 blocking versus unblocked conditions.(E) Fungal MDSC induction in patients with ROS deficiency.Left panel: MDSCs were generated in vitro by incubating isolated PBMCs (5 × 105 cells/ml) from healthy controls (n = 12) or patients with CGD (n = 3) 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) or CGD patients (n = 3) with different fungal morphotypes (1 × 105 cells/ml) for 6 days.In (A)–(E) bars represent means ± SEM.
Mentions: C. albicans induces interleukin-1 beta (IL-1β) in vitro (van de Veerdonk et al., 2009) and in vivo (Hise et al., 2009), which is critical for antifungal immunity (Vonk et al., 2006). Recent studies further provided evidence that IL-1β is involved in MDSC homeostasis (Bruchard et al., 2013). We observed an accumulation of intracellular IL-1β protein in CD33+ myeloid cells followed by IL-1β release upon Dectin-1 ligand- and fungal-driven MDSC induction (Figure 4A). IL-1β protein, in turn, was sufficient to drive MDSC generation to a comparable extent as C. albicans did (Figure 4B). Studies in Il1r−/− mice, characterized by an increased susceptibility to C. albicans infection, demonstrated that abrogation of IL-1R signaling decreased MDSC accumulation in vivo (Figures 4B and S4A), and IL-1R antagonism in patients with autoinflammatory diseases decreased MDSCs (Figure S4B). As the inflammasome is the major mechanism driving IL-1β generation in myeloid cells through caspase activities, we blocked caspases chemically. We observed that pan-caspase inhibition largely abolished fungi-induced MDSC generation, which was not recapitulated by caspase-1 inhibition (Figure 4C). We therefore focused on caspase-8, since Dectin-1 activation was shown to trigger IL-1β processing by a caspase-8-dependent mechanism (Ganesan et al., 2014; Gringhuis et al., 2012). Indeed, fungal MDSC induction was paralleled by a substantial increase of caspase-8 activity, and caspase-8 inhibition diminished fungal-induced IL-1β production (Figure 4C) and the potential of fungi to induce MDSCs (Figure 4C). Conversely, supplementing IL-1β partially restored the abrogated MDSC generation upon caspase-8 inhibition (Figure S4C).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics I, University of Tübingen, 72076 Tübingen, Germany. Electronic address: nikolaus.rieber@med.uni-tuebingen.de.

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Related in: MedlinePlus