Open Access

TSC1 Suppresses Macrophage Necroptosis for the Control of Infection by Fungal Pathogen Candida albicans

Tiantian Li, Yadong Xie, Lei Shi, Yumeng Sun, Jing Wen, Zihou Deng, Haibing Zhang, Huabin Li, Jinbo Yang and Hui Xiao
ImmunoHorizons February 1, 2021, 5 (2) 90-101; DOI: https://doi.org/10.4049/immunohorizons.2000093

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Macrophage death triggered by C. albicans.

    (A and B) RAW 264.7 cells and BMDMs were either untreated or pretreated with Ac-YVAD-CMK (YVAD, 50 μM), z-DEVD-FMK (DEVD, 30 μM), ferrostatin-1 (Fer-1, 5 μM), GSK’872 (5 μM), or necrostatin-1 (Nec-1, 30 μM) for 1 h, then infected with live C. albicans (MOI: 1) for various times, then cell death was measured by PI-staining or LDH release (n = 3). (C and D) WT, Mlkl−/−, and Ripk3−/− BMDMs were uninfected (UI) or infected with live C. albicans (MOI: 1) for 4 h. PI (5 μg/ml) was added 10 min prior to harvest. Images were collected under a fluorescence microscope and percentage of PI+ macrophages (PI+ macrophage numbers per field/total macrophage numbers per field) were calculated (UI, n = 3; C. albicans, n = 15). White arrows indicate PI-positive BMDMs. Scale bar, 50 μm. (E) WT, Mlkl−/− and Ripk3−/− BMDMs were uninfected (UI) or infected with live C. albicans (MOI: 1) for 4 and 8 h with or without z-VAD (40 μM), then cell cytotoxicity was measured by LDH release (n = 3). The data are representative of three independent experiments and shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA.

  • FIGURE 2.
    FIGURE 2.

    Tsc1−/− BMDMs are highly susceptible to C. albicans–induced necroptosis.

    (A) Immunoblots of protein lysates from WT and Tsc1M/N−/− BMDMs infected with live C. albicans (MOI: 1) for various times with indicated Abs. (B) WT and Tsc1M/N−/− BMDMs were uninfected (UI) or infected with live C. albicans (MOI: 1) for 8 h with or without z-VAD (40 μM). Cell death was measured by LDH release (n = 3). (C–E) WT and Tsc1M/N−/− BMDMs (C and D) or WT, Tsc1M/N−/−, and Tsc1M/N−/−Mlkl−/− BMDMs (E and F) were uninfected (UI) or infected with live C. albicans (MOI: 1) for 4 h. PI (5 μg/ml) was added 10 min prior to harvest. Images were collected under a fluorescence microscope, and percentage of PI+ macrophages (PI+ macrophage numbers per field/total macrophage numbers per field) were calculated (UI, n = 3; C. albicans, n = 15). White arrows indicate PI-positive BMDMs. Scale bar, 50 μm. The data are representative of three independent experiments and shown as mean ± SEM. **p < 0.01, ***p < 0.001, by unpaired Student t test (B and D) or one-way ANOVA (F).

  • FIGURE 3.
    FIGURE 3.

    Dectin-1 and TLR4 elicits hyper necroptosis in Tsc1−/− BMDMs.

    (A) WT and Mlkl−/− BMDMs were untreated (NT) or treated with HKCA-Y (MOI: 2), ZymD (100 μg/ml), mannan (M, 100 μg/ml), and zymosan (ZymA, 100 μg/ml) with or without z-VAD (Z, 40 μM) overnight, then cell cytotoxicity was measured by LDH release (n = 3). (B) WT and Mlkl−/− BMDMs were untreated (NT) or treated with LPS (L, 100 ng/ml), poly(I:C) (pIC, 10 μg/ml), and Pam3CSK4 (Pam3, 100 ng/ml) with or without z-VAD (Z, 40 μM) overnight, then cell cytotoxicity was measured by LDH release (n = 3). (C) WT and Tsc1M/N−/− BMDMs were untreated (NT) or treated with HKCA-Y (MOI: 2), ZymD (100 μg/ml), mannan (M, 100 μg/ml), and zymosan (ZymA, 100 μg/ml) as well as LPS (L, 100 ng/ml), poly(I:C) (pIC, 10 μg/ml), Pam3CSK4 (Pam3, 100 ng/ml), and Sel1 (100 ng/ml) with or without z-VAD (Z, 40 μM) and necrostatin-1 (Nec-1, 30 μM) overnight; then cell cytotoxicity was measured by LDH release (n = 3). (D) WT, Tsc1M/N−/−, and Tsc1M/N−/−Mlkl−/− BMDMs were untreated (NT) or treated with ZymD (100 μg/ml), mannan (M, 100 μg/ml), LPS (L, 100 ng/ml), and poly(I:C) (pIC, 10 μg/ml) with or without z-VAD (Z, 40 μM) overnight, then cell cytotoxicity was measured by LDH release (n = 3). The data are representative of three independent experiments and shown as mean ± SEM. **p < 0.01, ***p < 0.001, by unpaired Student t test (A–C) or one-way ANOVA (D).

  • FIGURE 4.
    FIGURE 4.

    mTORC1 activation is crucial in regulating macrophage necroptosis.

    (A) WT and Tsc1M/N−/− BMDMs were pretreated with or without 100 nM rapamycin (Rapa) for 1 h, then stimulated with ZymD (100 μg/ml) or LPS (L, 100 ng/ml) plus z-VAD (Z, 40 μM) or z-VAD plus necrostatin-1 (Nec-1, 30 μM) overnight; then cell cytotoxicity was measured by LDH release (n = 3). (B and C) WT, Tsc1M/N−/−, and Tsc1/mTorM/N−/− BMDMs; (B) or WT and RptorM/N−/− BMDMs; WT and RictorM/N−/− BMDMs (C) were untreated (NT) or treated with ZymD (100 μg/ml), mannan (M, 100 μg/ml), LPS (L, 100 ng/ml), and poly(I:C) (pIC, 10 μg/ml) with or without z-VAD (Z, 40 μM) overnight; then cell cytotoxicity was measured by LDH release (n = 3). The data are representative of three independent experiments and shown as mean ± SEM. **p < 0.01, ***p < 0.001, by unpaired Student t test (C) or one-way ANOVA (A and B).

  • FIGURE 5.
    FIGURE 5.

    TSC1 regulates the activation of necroptotic signaling.

    (A–D) Immunoblots of protein lysates from WT and Tsc1M/N−/− BMDMs treated with live C. albicans (MOI: 1), ZymD (100 μg/ml), and Sel1 (100 ng/ml) with or without z-VAD (40 μM) for various times. p-RIPK1 (S166), p-RIPK3 (S232), and p-MLKL (S345) were used. The data are representative of three independent experiments.

  • FIGURE 6.
    FIGURE 6.

    Tsc1M/N−/− mice are more vulnerable to C. albicans infection.

    (A and B) Six- to eight-week-old WT and Tsc1M/N−/− littermates (n = 11) were infected with C. albicans (4 × 104 fungal cells per mouse) by i.v. injection; weight loss (A) and survival (B) were documented daily. (C) Six- to eight-week-old WT and Mlkl−/− littermates (n = 11–12) were infected with C. albicans (4 × 104 fungal cells per mouse) by i.v. injection, survival was documented daily. (D) Quantification of C. albicans in the kidney, liver, and spleen of WT and Tsc1M/N−/− mice (n = 6) 3 d postinfection with C. albicans as in (A), analyzed by serial dilution of homogenized tissues and presented as CFU per gram of tissue. (E and F) The percentages (E) and numbers (F) of resident macrophages (CD11b+F4/80hi), monocyte-derived macrophages (CD11b+F4/80lo), and neutrophils of the kidney in WT and Tsc1M/N−/− mice either uninfected (UI) or 3 d postinfection with C. albicans were analyzed by flow cytometry (n = 6–7), cells were pregated as CD45+. The data are representative of three independent experiments and shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, analyzed by unpaired Student t test (A, D, and F) or by log-rank test (B and C).

  • FIGURE 7.
    FIGURE 7.

    C. albicans infection induces necroptotic cell death of macrophages and neutrophils.

    (A–C) Immunofluorescent staining on kidney sections from WT and Tsc1M/N−/− (n = 2–4) mice either uninfected (UI) or 3 d postinfection with C. albicans (4 × 104 fungal cells per mouse). Representative images were captured, and double-positive cells were counted (UI, n = 4; C. albicans, n = 8). White arrows indicate double-positive cells. Scale bar, 50 μm. (D) Six- to eight-week-old WT, Tsc1M/N−/−, and Tsc1M/N−/−Mlkl−/− mice (n = 12) were infected with C. albicans (5 × 104 fungal cells per mouse) by i.v. injection, and survival was monitored daily. The data are representative of three independent experiments and shown as mean ± SEM. *p < 0.05, **p < 0.01, analyzed by unpaired Student t test (A–C) or by log-rank test (D).

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