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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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Fungi Induce Functional MDSCs In Vitro and In Vivo(A) Fungal morphotypes differentially induce MDSCs.Left panel: MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with medium only (negative control), or different morphotypes of A. fumigatus (conidia, 5 × 105/ml; germ tubes, 1 × 105/ml; hyphae, 1 × 105/ml) or C. albicans (yeasts, 1 × 105/ml; hyphae, 1 × 105/ml). The x-fold induction of MDSCs compared to control conditions is depicted. ∗p < 0.05.Right panel: representative histograms of fungi-induced MDSCs (CD11b+CD33+CD14−CD16+CXCR4+).(B) Fungi-induced MDSCs suppress T cells. The suppressive effects of CD33+-MACS-isolated MDSCs were analyzed on CD4+ and CD8+ T cell proliferation. MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with A. fumigatus germ tubes (1 × 105/ml) or C. albicans yeasts (1 × 105/ml) for 6 days. Different MDSC-to-T cell ratios were assessed (1:2, 1:4, 1:6, 1:8, and 1:16). The lower bar graphs represent the proliferation index compared to control conditions as means ± SEM.(C) MDSCs in patients with fungal infections.Left panel: MDSCs were characterized as CD14− cells expressing CD33, CD66b, CD16, CD11b, and CXCR4 in the PBMC fraction. The gray line shows unstained controls. MDSCs were quantified in peripheral blood from healthy controls, immunosuppressed patients without fungal infections (disease controls, n = 5), or immunosuppressed patients with invasive fungal infections (invasive A. fumigatus infections, n = 9, and invasive C. albicans infections, n = 6). ∗p < 0.05.Right panel: representative CFSE stainings, showing the effect of MDSCs isolated (MACS) from patients with invasive A. fumigatus infections (left) or invasive C. albicans infections (right) on CD4+ and CD8+ T cell proliferation.(D) Fungi induce MDSCs in mice in vivo.Upper left panel: C57/BL6 (n = 3 mice per treatment group) or BALB/c (n = 4 mice per treatment group) wild-type mice were not infected (white bars) or challenged intranasally with 1 × 104 (light gray bar) or 1 × 106 (dark gray bar) A. fumigatus conidia for 3 days. On the fourth day, a bronchoalveolar lavage (BAL) was performed, and CD11b+Ly6G+ MDSCs were quantified by FACS. The x-fold induction of CD11b+Ly6G+ MDSCs in the BAL compared to control non-infected conditions is depicted. ∗p < 0.05.Upper right panel: C57BL/6 mice were not infected (white bars) or injected via the lateral tail vein with 2.5 × 105 (light gray bar) or 5 × 105 (dark gray bar) blastospores of C. albicans. On the fifth day, mice were sacrificed, and CD11b+Ly6G+ MDSCs in the spleen were quantified by FACS. The x-fold induction of CD11b+Ly6G+ MDSCs in the spleen compared to control non-infected conditions is depicted. n = 5 mice per treatment group. ∗p < 0.05.Lower panel: bone marrow-isolated murine CD11b+Ly6G+ MDSCs were co-cultured for 3 days with T cells (CD4+ splenocytes) at a 1:2 (MDSCs:T cell) ratio. T cell proliferation was analyzed using the CFSE assay with and without MDSCs.(E) Adoptive transfer of MDSCs modulates survival in fungal infection. For adoptive transfer experiments, CD11b+Ly6G+ MDSCs were isolated from the bone marrow of BALB/c mice by MACS and checked for T cell suppression. In (A)–(D) bars represent means ± SEM.Upper panel: adoptive MDSC transfer was performed by intravenous (i.v.) injection of 5 × 106 MDSCs per animal. Seven mice received MDSCs, while seven mice served as non-MDSC control animals. A total of 2 hr after the MDSC transfer, mice were i.v. injected with 1 × 105 blastospores of C. albicans. Mice were weighed daily and monitored for survival and signs of morbidity.Lower panel: for invasive pulmonary A. fumigatus infection survival studies, mice were immunosuppressed by treatment with cyclophosphamide, and MDSC transfer was performed by i.v. injection of 4 × 106 MDSCs per animal. Five mice received MDSCs, while five mice served as non-MDSC control animals. After the MDSC transfer, mice were challenged intranasally with 2 × 105A. fumigatus conidia and were monitored for survival.
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fig1: Fungi Induce Functional MDSCs In Vitro and In Vivo(A) Fungal morphotypes differentially induce MDSCs.Left panel: MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with medium only (negative control), or different morphotypes of A. fumigatus (conidia, 5 × 105/ml; germ tubes, 1 × 105/ml; hyphae, 1 × 105/ml) or C. albicans (yeasts, 1 × 105/ml; hyphae, 1 × 105/ml). The x-fold induction of MDSCs compared to control conditions is depicted. ∗p < 0.05.Right panel: representative histograms of fungi-induced MDSCs (CD11b+CD33+CD14−CD16+CXCR4+).(B) Fungi-induced MDSCs suppress T cells. The suppressive effects of CD33+-MACS-isolated MDSCs were analyzed on CD4+ and CD8+ T cell proliferation. MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with A. fumigatus germ tubes (1 × 105/ml) or C. albicans yeasts (1 × 105/ml) for 6 days. Different MDSC-to-T cell ratios were assessed (1:2, 1:4, 1:6, 1:8, and 1:16). The lower bar graphs represent the proliferation index compared to control conditions as means ± SEM.(C) MDSCs in patients with fungal infections.Left panel: MDSCs were characterized as CD14− cells expressing CD33, CD66b, CD16, CD11b, and CXCR4 in the PBMC fraction. The gray line shows unstained controls. MDSCs were quantified in peripheral blood from healthy controls, immunosuppressed patients without fungal infections (disease controls, n = 5), or immunosuppressed patients with invasive fungal infections (invasive A. fumigatus infections, n = 9, and invasive C. albicans infections, n = 6). ∗p < 0.05.Right panel: representative CFSE stainings, showing the effect of MDSCs isolated (MACS) from patients with invasive A. fumigatus infections (left) or invasive C. albicans infections (right) on CD4+ and CD8+ T cell proliferation.(D) Fungi induce MDSCs in mice in vivo.Upper left panel: C57/BL6 (n = 3 mice per treatment group) or BALB/c (n = 4 mice per treatment group) wild-type mice were not infected (white bars) or challenged intranasally with 1 × 104 (light gray bar) or 1 × 106 (dark gray bar) A. fumigatus conidia for 3 days. On the fourth day, a bronchoalveolar lavage (BAL) was performed, and CD11b+Ly6G+ MDSCs were quantified by FACS. The x-fold induction of CD11b+Ly6G+ MDSCs in the BAL compared to control non-infected conditions is depicted. ∗p < 0.05.Upper right panel: C57BL/6 mice were not infected (white bars) or injected via the lateral tail vein with 2.5 × 105 (light gray bar) or 5 × 105 (dark gray bar) blastospores of C. albicans. On the fifth day, mice were sacrificed, and CD11b+Ly6G+ MDSCs in the spleen were quantified by FACS. The x-fold induction of CD11b+Ly6G+ MDSCs in the spleen compared to control non-infected conditions is depicted. n = 5 mice per treatment group. ∗p < 0.05.Lower panel: bone marrow-isolated murine CD11b+Ly6G+ MDSCs were co-cultured for 3 days with T cells (CD4+ splenocytes) at a 1:2 (MDSCs:T cell) ratio. T cell proliferation was analyzed using the CFSE assay with and without MDSCs.(E) Adoptive transfer of MDSCs modulates survival in fungal infection. For adoptive transfer experiments, CD11b+Ly6G+ MDSCs were isolated from the bone marrow of BALB/c mice by MACS and checked for T cell suppression. In (A)–(D) bars represent means ± SEM.Upper panel: adoptive MDSC transfer was performed by intravenous (i.v.) injection of 5 × 106 MDSCs per animal. Seven mice received MDSCs, while seven mice served as non-MDSC control animals. A total of 2 hr after the MDSC transfer, mice were i.v. injected with 1 × 105 blastospores of C. albicans. Mice were weighed daily and monitored for survival and signs of morbidity.Lower panel: for invasive pulmonary A. fumigatus infection survival studies, mice were immunosuppressed by treatment with cyclophosphamide, and MDSC transfer was performed by i.v. injection of 4 × 106 MDSCs per animal. Five mice received MDSCs, while five mice served as non-MDSC control animals. After the MDSC transfer, mice were challenged intranasally with 2 × 105A. fumigatus conidia and were monitored for survival.

Mentions: We analyzed the effect of the human-pathogenic fungi A. fumigatus and C. albicans on human immune cells and noticed the appearance of a cell population that was different from monocytes (CD14−), and expressed the myeloid markers CD33+, CD11b+, CD16+, and CXCR4 (Figures 1A and S1A). Fungi-induced myeloid cells strongly suppressed both CD4+ and CD8+ T cell proliferation in a dose-dependent manner (Figure 1B), which defines MDSCs. Fungi-induced MDSCs also suppressed innate natural killer (NK) cell responses, without affecting cell survival (Figure S2). In contrast to growth factor-induced MDSCs, fungi-induced MDSCs dampened Th2 responses, which play essential roles in fungal asthma (Kreindler et al., 2010) (Figure S1B). We quantified MDSCs in patients with invasive fungal infections and challenged mice with A. fumigatus or C. albicans. MDSCs accumulated in both A. fumigatus- and C. albicans-infected patients compared to healthy and disease control patients without fungal infections (Figure 1C). Murine studies further showed that systemic or pulmonary fungal challenge with C. albicans (invasive disseminated candidiasis) or A. fumigatus (pulmonary aspergillosis), as the clinically relevant routes of infection, dose-dependently triggered the recruitment of MDSCs in both immunocompetent and immunosuppressed conditions, with a stronger MDSC induction seen in immunocompetent animals (Figures 1D and S1C). MDSCs expressed neutrophilic markers in both man and mice, resembling the neutrophilic subtype of MDSCs (Rieber et al., 2013), while monocytic MDSC subsets were not induced (Figure S1D). Fungi-induced MDSCs functionally suppressed T cell proliferation (Figure 1C), while autologous conventional neutrophils failed to do (Figure S1E).


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)

Fungi Induce Functional MDSCs In Vitro and In Vivo(A) Fungal morphotypes differentially induce MDSCs.Left panel: MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with medium only (negative control), or different morphotypes of A. fumigatus (conidia, 5 × 105/ml; germ tubes, 1 × 105/ml; hyphae, 1 × 105/ml) or C. albicans (yeasts, 1 × 105/ml; hyphae, 1 × 105/ml). The x-fold induction of MDSCs compared to control conditions is depicted. ∗p < 0.05.Right panel: representative histograms of fungi-induced MDSCs (CD11b+CD33+CD14−CD16+CXCR4+).(B) Fungi-induced MDSCs suppress T cells. The suppressive effects of CD33+-MACS-isolated MDSCs were analyzed on CD4+ and CD8+ T cell proliferation. MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with A. fumigatus germ tubes (1 × 105/ml) or C. albicans yeasts (1 × 105/ml) for 6 days. Different MDSC-to-T cell ratios were assessed (1:2, 1:4, 1:6, 1:8, and 1:16). The lower bar graphs represent the proliferation index compared to control conditions as means ± SEM.(C) MDSCs in patients with fungal infections.Left panel: MDSCs were characterized as CD14− cells expressing CD33, CD66b, CD16, CD11b, and CXCR4 in the PBMC fraction. The gray line shows unstained controls. MDSCs were quantified in peripheral blood from healthy controls, immunosuppressed patients without fungal infections (disease controls, n = 5), or immunosuppressed patients with invasive fungal infections (invasive A. fumigatus infections, n = 9, and invasive C. albicans infections, n = 6). ∗p < 0.05.Right panel: representative CFSE stainings, showing the effect of MDSCs isolated (MACS) from patients with invasive A. fumigatus infections (left) or invasive C. albicans infections (right) on CD4+ and CD8+ T cell proliferation.(D) Fungi induce MDSCs in mice in vivo.Upper left panel: C57/BL6 (n = 3 mice per treatment group) or BALB/c (n = 4 mice per treatment group) wild-type mice were not infected (white bars) or challenged intranasally with 1 × 104 (light gray bar) or 1 × 106 (dark gray bar) A. fumigatus conidia for 3 days. On the fourth day, a bronchoalveolar lavage (BAL) was performed, and CD11b+Ly6G+ MDSCs were quantified by FACS. The x-fold induction of CD11b+Ly6G+ MDSCs in the BAL compared to control non-infected conditions is depicted. ∗p < 0.05.Upper right panel: C57BL/6 mice were not infected (white bars) or injected via the lateral tail vein with 2.5 × 105 (light gray bar) or 5 × 105 (dark gray bar) blastospores of C. albicans. On the fifth day, mice were sacrificed, and CD11b+Ly6G+ MDSCs in the spleen were quantified by FACS. The x-fold induction of CD11b+Ly6G+ MDSCs in the spleen compared to control non-infected conditions is depicted. n = 5 mice per treatment group. ∗p < 0.05.Lower panel: bone marrow-isolated murine CD11b+Ly6G+ MDSCs were co-cultured for 3 days with T cells (CD4+ splenocytes) at a 1:2 (MDSCs:T cell) ratio. T cell proliferation was analyzed using the CFSE assay with and without MDSCs.(E) Adoptive transfer of MDSCs modulates survival in fungal infection. For adoptive transfer experiments, CD11b+Ly6G+ MDSCs were isolated from the bone marrow of BALB/c mice by MACS and checked for T cell suppression. In (A)–(D) bars represent means ± SEM.Upper panel: adoptive MDSC transfer was performed by intravenous (i.v.) injection of 5 × 106 MDSCs per animal. Seven mice received MDSCs, while seven mice served as non-MDSC control animals. A total of 2 hr after the MDSC transfer, mice were i.v. injected with 1 × 105 blastospores of C. albicans. Mice were weighed daily and monitored for survival and signs of morbidity.Lower panel: for invasive pulmonary A. fumigatus infection survival studies, mice were immunosuppressed by treatment with cyclophosphamide, and MDSC transfer was performed by i.v. injection of 4 × 106 MDSCs per animal. Five mice received MDSCs, while five mice served as non-MDSC control animals. After the MDSC transfer, mice were challenged intranasally with 2 × 105A. fumigatus conidia and were monitored for survival.
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fig1: Fungi Induce Functional MDSCs In Vitro and In Vivo(A) Fungal morphotypes differentially induce MDSCs.Left panel: MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with medium only (negative control), or different morphotypes of A. fumigatus (conidia, 5 × 105/ml; germ tubes, 1 × 105/ml; hyphae, 1 × 105/ml) or C. albicans (yeasts, 1 × 105/ml; hyphae, 1 × 105/ml). The x-fold induction of MDSCs compared to control conditions is depicted. ∗p < 0.05.Right panel: representative histograms of fungi-induced MDSCs (CD11b+CD33+CD14−CD16+CXCR4+).(B) Fungi-induced MDSCs suppress T cells. The suppressive effects of CD33+-MACS-isolated MDSCs were analyzed on CD4+ and CD8+ T cell proliferation. MDSCs were generated by incubating PBMCs (5 × 105/ml) from healthy donors with A. fumigatus germ tubes (1 × 105/ml) or C. albicans yeasts (1 × 105/ml) for 6 days. Different MDSC-to-T cell ratios were assessed (1:2, 1:4, 1:6, 1:8, and 1:16). The lower bar graphs represent the proliferation index compared to control conditions as means ± SEM.(C) MDSCs in patients with fungal infections.Left panel: MDSCs were characterized as CD14− cells expressing CD33, CD66b, CD16, CD11b, and CXCR4 in the PBMC fraction. The gray line shows unstained controls. MDSCs were quantified in peripheral blood from healthy controls, immunosuppressed patients without fungal infections (disease controls, n = 5), or immunosuppressed patients with invasive fungal infections (invasive A. fumigatus infections, n = 9, and invasive C. albicans infections, n = 6). ∗p < 0.05.Right panel: representative CFSE stainings, showing the effect of MDSCs isolated (MACS) from patients with invasive A. fumigatus infections (left) or invasive C. albicans infections (right) on CD4+ and CD8+ T cell proliferation.(D) Fungi induce MDSCs in mice in vivo.Upper left panel: C57/BL6 (n = 3 mice per treatment group) or BALB/c (n = 4 mice per treatment group) wild-type mice were not infected (white bars) or challenged intranasally with 1 × 104 (light gray bar) or 1 × 106 (dark gray bar) A. fumigatus conidia for 3 days. On the fourth day, a bronchoalveolar lavage (BAL) was performed, and CD11b+Ly6G+ MDSCs were quantified by FACS. The x-fold induction of CD11b+Ly6G+ MDSCs in the BAL compared to control non-infected conditions is depicted. ∗p < 0.05.Upper right panel: C57BL/6 mice were not infected (white bars) or injected via the lateral tail vein with 2.5 × 105 (light gray bar) or 5 × 105 (dark gray bar) blastospores of C. albicans. On the fifth day, mice were sacrificed, and CD11b+Ly6G+ MDSCs in the spleen were quantified by FACS. The x-fold induction of CD11b+Ly6G+ MDSCs in the spleen compared to control non-infected conditions is depicted. n = 5 mice per treatment group. ∗p < 0.05.Lower panel: bone marrow-isolated murine CD11b+Ly6G+ MDSCs were co-cultured for 3 days with T cells (CD4+ splenocytes) at a 1:2 (MDSCs:T cell) ratio. T cell proliferation was analyzed using the CFSE assay with and without MDSCs.(E) Adoptive transfer of MDSCs modulates survival in fungal infection. For adoptive transfer experiments, CD11b+Ly6G+ MDSCs were isolated from the bone marrow of BALB/c mice by MACS and checked for T cell suppression. In (A)–(D) bars represent means ± SEM.Upper panel: adoptive MDSC transfer was performed by intravenous (i.v.) injection of 5 × 106 MDSCs per animal. Seven mice received MDSCs, while seven mice served as non-MDSC control animals. A total of 2 hr after the MDSC transfer, mice were i.v. injected with 1 × 105 blastospores of C. albicans. Mice were weighed daily and monitored for survival and signs of morbidity.Lower panel: for invasive pulmonary A. fumigatus infection survival studies, mice were immunosuppressed by treatment with cyclophosphamide, and MDSC transfer was performed by i.v. injection of 4 × 106 MDSCs per animal. Five mice received MDSCs, while five mice served as non-MDSC control animals. After the MDSC transfer, mice were challenged intranasally with 2 × 105A. fumigatus conidia and were monitored for survival.
Mentions: We analyzed the effect of the human-pathogenic fungi A. fumigatus and C. albicans on human immune cells and noticed the appearance of a cell population that was different from monocytes (CD14−), and expressed the myeloid markers CD33+, CD11b+, CD16+, and CXCR4 (Figures 1A and S1A). Fungi-induced myeloid cells strongly suppressed both CD4+ and CD8+ T cell proliferation in a dose-dependent manner (Figure 1B), which defines MDSCs. Fungi-induced MDSCs also suppressed innate natural killer (NK) cell responses, without affecting cell survival (Figure S2). In contrast to growth factor-induced MDSCs, fungi-induced MDSCs dampened Th2 responses, which play essential roles in fungal asthma (Kreindler et al., 2010) (Figure S1B). We quantified MDSCs in patients with invasive fungal infections and challenged mice with A. fumigatus or C. albicans. MDSCs accumulated in both A. fumigatus- and C. albicans-infected patients compared to healthy and disease control patients without fungal infections (Figure 1C). Murine studies further showed that systemic or pulmonary fungal challenge with C. albicans (invasive disseminated candidiasis) or A. fumigatus (pulmonary aspergillosis), as the clinically relevant routes of infection, dose-dependently triggered the recruitment of MDSCs in both immunocompetent and immunosuppressed conditions, with a stronger MDSC induction seen in immunocompetent animals (Figures 1D and S1C). MDSCs expressed neutrophilic markers in both man and mice, resembling the neutrophilic subtype of MDSCs (Rieber et al., 2013), while monocytic MDSC subsets were not induced (Figure S1D). Fungi-induced MDSCs functionally suppressed T cell proliferation (Figure 1C), while autologous conventional neutrophils failed to do (Figure S1E).

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.

Show MeSH
Related in: MedlinePlus