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Delinking CARD9 and IL-17: CARD9 Protects against Candida tropicalis Infection through a TNF-α-Dependent, IL-17-Independent Mechanism.

Whibley N, Jaycox JR, Reid D, Garg AV, Taylor JA, Clancy CJ, Nguyen MH, Biswas PS, McGeachy MJ, Brown GD, Gaffen SL - J. Immunol. (2015)

Bottom Line: Consistently, WT mice depleted of TNF-α were more susceptible to C. tropicalis, and CARD9-deficient neutrophils and monocytes failed to produce TNF-α following stimulation with C. tropicalis Ags.However, TNF-α treatment of neutrophils in vitro enhanced their ability to kill C. tropicalis.Moreover, CARD9-dependent production of TNF-α enhances the candidacidal capacity of neutrophils, limiting fungal disease during disseminated C. tropicalis infection.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261;

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TNF-α responses are impaired in CARD9−/− mice and are required for protection against C. tropicalis infection. (A) WT or CARD9−/− neutrophils or monocytes were isolated from naive bone marrow and treated or not with HK C.t for 24 h in vitro. TNF-α in supernatants was measured by ELISA. Data are representative of two or three experiments (n = 2/group). Bar graphs show mean ± SEM. ****p < 0.0001, unpaired Student t test. (B–E) Mice were infected with 1 × 104 CFU/g C. tropicalis yeast cells, and blood was harvested on the indicated days. Cells were cultured or not with HK C.t for 3 h, and TNF-α produced by CD45+CD11b+Ly6G+ neutrophils (B and C) and CD45+CD11b+Ly6G−Ly6C+ monocytes (D and E) was measured by flow cytometry. Data are pooled from (C) or are representative of (E) two experiments (each data point represents an individual mouse). Cells were gated through leukocyte, single cell, and live cell gates. *p < 0.05, **p < 0.01, Mann-Whitney U test. (F) Kidneys and spleens were harvested on the indicated days, and gene expression was assessed by quantitative PCR. Data are representative of two experiments (n = 4–10 mice). Bar graphs show mean ± SEM. *p < 0.05, ****p < 0.0001, Kruskal–Wallis and post hoc Dunn multiple-comparisons tests. (G) WT mice were treated with TNF-α inhibitor (etanercept) every 2 d starting on day −1 (“continuous”) or on day 2 or 5 (“delayed”). Data are pooled from two or three experiments (WT, n = 11; WT + TNF-α inhibitor continuous, n = 13; WT + TNF-α inhibitor delayed, n = 10). (H) CARD9−/− mice were treated or not with etanercept every 2 d starting on day −1 postinfection. Data are pooled from two experiments (CARD9−/−n = 7, CARD9−/− + TNF-α inhibitor, n = 8). For (G) and (H), *p < 0.05, ****p < 0.0001, log-rank (Mantel–Cox) test. ns, not significant.
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fig04: TNF-α responses are impaired in CARD9−/− mice and are required for protection against C. tropicalis infection. (A) WT or CARD9−/− neutrophils or monocytes were isolated from naive bone marrow and treated or not with HK C.t for 24 h in vitro. TNF-α in supernatants was measured by ELISA. Data are representative of two or three experiments (n = 2/group). Bar graphs show mean ± SEM. ****p < 0.0001, unpaired Student t test. (B–E) Mice were infected with 1 × 104 CFU/g C. tropicalis yeast cells, and blood was harvested on the indicated days. Cells were cultured or not with HK C.t for 3 h, and TNF-α produced by CD45+CD11b+Ly6G+ neutrophils (B and C) and CD45+CD11b+Ly6G−Ly6C+ monocytes (D and E) was measured by flow cytometry. Data are pooled from (C) or are representative of (E) two experiments (each data point represents an individual mouse). Cells were gated through leukocyte, single cell, and live cell gates. *p < 0.05, **p < 0.01, Mann-Whitney U test. (F) Kidneys and spleens were harvested on the indicated days, and gene expression was assessed by quantitative PCR. Data are representative of two experiments (n = 4–10 mice). Bar graphs show mean ± SEM. *p < 0.05, ****p < 0.0001, Kruskal–Wallis and post hoc Dunn multiple-comparisons tests. (G) WT mice were treated with TNF-α inhibitor (etanercept) every 2 d starting on day −1 (“continuous”) or on day 2 or 5 (“delayed”). Data are pooled from two or three experiments (WT, n = 11; WT + TNF-α inhibitor continuous, n = 13; WT + TNF-α inhibitor delayed, n = 10). (H) CARD9−/− mice were treated or not with etanercept every 2 d starting on day −1 postinfection. Data are pooled from two experiments (CARD9−/−n = 7, CARD9−/− + TNF-α inhibitor, n = 8). For (G) and (H), *p < 0.05, ****p < 0.0001, log-rank (Mantel–Cox) test. ns, not significant.

Mentions: TNF-α is produced rapidly upon disseminated C. albicans infection and is required for host defense (47, 48). Monocytes are prominent producers of TNF-α, although this cytokine can potentially be produced by many cell types, including neutrophils (49). Given the rapid mortality of infected CARD9−/− mice and the requirement for neutrophils and monocytes in this setting, we hypothesized that TNF-α production by these cell types is important for host defense against C. tropicalis. To test this hypothesis, we first measured TNF-α production by WT and CARD9−/− bone marrow neutrophils and monocytes that were stimulated with HK C.t in vitro. Strikingly, CARD9−/− neutrophils and monocytes were completely defective in TNF-α production following HK C.t stimulation (Fig. 4A). To determine whether CARD9−/− neutrophils and monocytes were impaired in TNF-α production during C. tropicalis infection, we assessed TNF-α levels in the blood of WT and CARD9−/− mice by flow cytometry on days 2 and 5. In line with in vitro data, both neutrophils and monocytes isolated from the blood of CARD9−/− mice on days 2 and 5 were severely impaired in TNF-α production in response to HK C.t compared with WT mice (Fig. 4B–E). Thus, CARD9 mediates the production of TNF-α by neutrophils and monocytes during disseminated C. tropicalis infection.


Delinking CARD9 and IL-17: CARD9 Protects against Candida tropicalis Infection through a TNF-α-Dependent, IL-17-Independent Mechanism.

Whibley N, Jaycox JR, Reid D, Garg AV, Taylor JA, Clancy CJ, Nguyen MH, Biswas PS, McGeachy MJ, Brown GD, Gaffen SL - J. Immunol. (2015)

TNF-α responses are impaired in CARD9−/− mice and are required for protection against C. tropicalis infection. (A) WT or CARD9−/− neutrophils or monocytes were isolated from naive bone marrow and treated or not with HK C.t for 24 h in vitro. TNF-α in supernatants was measured by ELISA. Data are representative of two or three experiments (n = 2/group). Bar graphs show mean ± SEM. ****p < 0.0001, unpaired Student t test. (B–E) Mice were infected with 1 × 104 CFU/g C. tropicalis yeast cells, and blood was harvested on the indicated days. Cells were cultured or not with HK C.t for 3 h, and TNF-α produced by CD45+CD11b+Ly6G+ neutrophils (B and C) and CD45+CD11b+Ly6G−Ly6C+ monocytes (D and E) was measured by flow cytometry. Data are pooled from (C) or are representative of (E) two experiments (each data point represents an individual mouse). Cells were gated through leukocyte, single cell, and live cell gates. *p < 0.05, **p < 0.01, Mann-Whitney U test. (F) Kidneys and spleens were harvested on the indicated days, and gene expression was assessed by quantitative PCR. Data are representative of two experiments (n = 4–10 mice). Bar graphs show mean ± SEM. *p < 0.05, ****p < 0.0001, Kruskal–Wallis and post hoc Dunn multiple-comparisons tests. (G) WT mice were treated with TNF-α inhibitor (etanercept) every 2 d starting on day −1 (“continuous”) or on day 2 or 5 (“delayed”). Data are pooled from two or three experiments (WT, n = 11; WT + TNF-α inhibitor continuous, n = 13; WT + TNF-α inhibitor delayed, n = 10). (H) CARD9−/− mice were treated or not with etanercept every 2 d starting on day −1 postinfection. Data are pooled from two experiments (CARD9−/−n = 7, CARD9−/− + TNF-α inhibitor, n = 8). For (G) and (H), *p < 0.05, ****p < 0.0001, log-rank (Mantel–Cox) test. ns, not significant.
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fig04: TNF-α responses are impaired in CARD9−/− mice and are required for protection against C. tropicalis infection. (A) WT or CARD9−/− neutrophils or monocytes were isolated from naive bone marrow and treated or not with HK C.t for 24 h in vitro. TNF-α in supernatants was measured by ELISA. Data are representative of two or three experiments (n = 2/group). Bar graphs show mean ± SEM. ****p < 0.0001, unpaired Student t test. (B–E) Mice were infected with 1 × 104 CFU/g C. tropicalis yeast cells, and blood was harvested on the indicated days. Cells were cultured or not with HK C.t for 3 h, and TNF-α produced by CD45+CD11b+Ly6G+ neutrophils (B and C) and CD45+CD11b+Ly6G−Ly6C+ monocytes (D and E) was measured by flow cytometry. Data are pooled from (C) or are representative of (E) two experiments (each data point represents an individual mouse). Cells were gated through leukocyte, single cell, and live cell gates. *p < 0.05, **p < 0.01, Mann-Whitney U test. (F) Kidneys and spleens were harvested on the indicated days, and gene expression was assessed by quantitative PCR. Data are representative of two experiments (n = 4–10 mice). Bar graphs show mean ± SEM. *p < 0.05, ****p < 0.0001, Kruskal–Wallis and post hoc Dunn multiple-comparisons tests. (G) WT mice were treated with TNF-α inhibitor (etanercept) every 2 d starting on day −1 (“continuous”) or on day 2 or 5 (“delayed”). Data are pooled from two or three experiments (WT, n = 11; WT + TNF-α inhibitor continuous, n = 13; WT + TNF-α inhibitor delayed, n = 10). (H) CARD9−/− mice were treated or not with etanercept every 2 d starting on day −1 postinfection. Data are pooled from two experiments (CARD9−/−n = 7, CARD9−/− + TNF-α inhibitor, n = 8). For (G) and (H), *p < 0.05, ****p < 0.0001, log-rank (Mantel–Cox) test. ns, not significant.
Mentions: TNF-α is produced rapidly upon disseminated C. albicans infection and is required for host defense (47, 48). Monocytes are prominent producers of TNF-α, although this cytokine can potentially be produced by many cell types, including neutrophils (49). Given the rapid mortality of infected CARD9−/− mice and the requirement for neutrophils and monocytes in this setting, we hypothesized that TNF-α production by these cell types is important for host defense against C. tropicalis. To test this hypothesis, we first measured TNF-α production by WT and CARD9−/− bone marrow neutrophils and monocytes that were stimulated with HK C.t in vitro. Strikingly, CARD9−/− neutrophils and monocytes were completely defective in TNF-α production following HK C.t stimulation (Fig. 4A). To determine whether CARD9−/− neutrophils and monocytes were impaired in TNF-α production during C. tropicalis infection, we assessed TNF-α levels in the blood of WT and CARD9−/− mice by flow cytometry on days 2 and 5. In line with in vitro data, both neutrophils and monocytes isolated from the blood of CARD9−/− mice on days 2 and 5 were severely impaired in TNF-α production in response to HK C.t compared with WT mice (Fig. 4B–E). Thus, CARD9 mediates the production of TNF-α by neutrophils and monocytes during disseminated C. tropicalis infection.

Bottom Line: Consistently, WT mice depleted of TNF-α were more susceptible to C. tropicalis, and CARD9-deficient neutrophils and monocytes failed to produce TNF-α following stimulation with C. tropicalis Ags.However, TNF-α treatment of neutrophils in vitro enhanced their ability to kill C. tropicalis.Moreover, CARD9-dependent production of TNF-α enhances the candidacidal capacity of neutrophils, limiting fungal disease during disseminated C. tropicalis infection.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261;

Show MeSH
Related in: MedlinePlus