Research ArticleINFECTIOUS DISEASES

Mitochondrial cyclophilin D regulates T cell metabolic responses and disease tolerance to tuberculosis

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Science Immunology  11 May 2018:
Vol. 3, Issue 23, eaar4135
DOI: 10.1126/sciimmunol.aar4135
  • Fig. 1 CypD-deficient mice are susceptible to Mtb infection.

    (A) Bacterial burden in the lung, spleen, and liver of WT and Ppif−/− mice (n = 4 to 5 per group) at 7, 14, and 21 days after intravenous infection with 106 H37Rv (two-way ANOVA). (B) Survival of WT and Ppif−/− mice (n = 10 per group) after intravenous infection with 106 H37Rv (log-rank test). (C) Bacterial burden in the lungs after 14, 35, and 90 days of aerosol infection with ~50 H37Rv (n = 5 per group) (two-way ANOVA). (D) Survival of WT and Ppif−/− mice after aersolized infection with ~50 H37Rv (n = 10 per group). *P < 0.05 (log-rank test). (E and F) Representative gross pathology (E) and histology (H&E staining) and inflammatory scores (F) of the lungs of WT and Ppif−/− mice at day 90 after aerosol infection with ~50 H37Rv. Lymphocytes (black arrows) and macrophages (dashed arrows). *P < 0.05 (unpaired t test) (G) Total number of CD4+ (top) and CD8+ T cells (bottom) in the lungs of WT and Ppif−/− mice (n = 5 per group) at day 90 after aerosol infection with ~50 H37Rv. *P < 0.05 (unpaired t test). Data are representative of two independent experiments.

  • Fig. 2 CypD-deficient mice have exacerbated Mtb antigen–specific T cell responses to Mtb infection.

    (A to C) Frequency (A and B) and total number (C) of TB10.4(4–11)-specific CD8+ T cells in the lungs of WT and Ppif−/− mice (n = 5 per group) after 14, 35, and 90 days of aerosol infection with ~50 H37Rv. Phycoerythrin (PE)–conjugated streptavidin was used as a control for the tetramer staining. Numbers above outlined areas (A) indicate the percentage of CD8+ T cells stained with H-2Kb- TB10.4(4–11). *P < 0.05, **P < 0.01, ****P < 0.0001 (two-way ANOVA). (D) Frequency of TNFα- and IFN-γ–producing antigen-specific CD8+ T cells was determined by intracellular staining (ICS) upon restimulation with TB10.4(4–11) peptide at day 35 after aerosol infection with ~50 H37Rv. ****P < 0.0001 (two-way ANOVA). (E) Frequency of T-bet expression in CD8+ T cells was determined by ICS at day 35 after aerosol infection with ~50 H37Rv. *P < 0.05 (unpaired t test). (F to H) Frequency (F to G) and total number (H) of TB10.4(4–11)-specific CD8+ T cells in the lungs of WT and Ppif −/− mice (n = 5 per group) after 10 and 15 days of intravenous infection with H37Rv (~106 CFU). PE-conjugated streptavidin was used as a control for the tetramer staining. Numbers above outlined areas (G) indicate the percentage of CD8+ T cells stained with H-2Kb-TB10.4(4–11). *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-way ANOVA). (I) Schematic of adoptive T cell transfer into Rag1−/− mice followed by aerosol infection with ~50 H37Rv. (J) Bacterial burden in the lungs, spleens, and livers of Rag1-deficient mice reconstituted with either with WT T cells (T cellsWT) or CypD-deficient T cells (T cellsPpif−/−) after 7, 14, and 35 days of infection with H37Rv (n = 5 per group). (K) Frequency of TB10.4(4–11)-specific CD8+ T cells in the lungs of Rag1-deficient mice reconstituted with either with WT T cells (T cellsWT) or CypD-deficient T cells (T cellsPpif−/−) (n = 5 per group) after 7, 14, and 35 days of aerosol infection with H37Rv. **P < 0.01 (unpaired t test). Data in (A) to (H) are representative of three independent experiments. Data in (I) to (K) are representative of two independent experiments.

  • Fig. 3 CypD regulates T cell activation and proliferation.

    (A) Rag-deficient mice were used as recipients for splenic T cells (CD3+) purified from naïve Ppif−/− or WT mice and labeled with CFSE. (B) CD8+ T cell proliferation (B) and the percentage of undivided CD8+ T cells (C) after 3 days of adoptive transfer. ****P < 0.0001 (unpaired t test). (C) Proliferation of CFSE-labeled CD8+ T cells purified from WT (gray) and Ppif−/− (black line) after 3 days of stimulation with plate-bound anti-CD3 and anti-CD28. (D) CD8+ T cells were stimulated for 48 hours with anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) and analyzed for TNFα and IFN-γ production by ICS. **P < 0.01, ****P < 0.0001 (unpaired t test). (E to F) Frequency of CD8+ T cells expressing CD69 and CD25 after 24 hours of stimulation with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml). MFI of CD69 and CD25 (F). ****P < 0.0001 (unpaired t test). (G) Frequency of proliferation of CFSE-labeled CD8+ T cells purified from WT OT-I or CypD-deficient OT-I after 3 days of coculture with BMDC loaded with OVA protein. Frequency of cells that undergo two or more cycles of proliferation (right). **P < 0.01 (unpaired t test). (H) Frequency of pulmonary Ki67+ CD8+ T cells at days 14 and 28 after aerosol infection with ~50 H37Rv. *P < 0.05 (unpaired t test). (I) MFI of Ki67, Bcl-2, and activated caspase 3 from TB10.4(4–11)-specific CD8+ T cell from lungs of WT and Ppif−/− mice at day 14 after infection with virulent with ~50 H37Rv via aerosol. *P < 0.05 (unpaired t test). (J to K) Frequency of pulmonary active caspase3+ CD8+ T cells (J) and 7AAD+ CD8+ T cells (K) at days 14 and 28 after aerosol infection with ~50 H37Rv. Data are representative of three independent experiments.

  • Fig. 4 CypD regulates T cell metabolism and proliferation via mROS.

    (A and B) ECAR (A) and lactate (B) measurements of Ppif−/− and WT CD8+T cells activated for 48 hours with anti-CD3 (5 μm/ml) and anti-CD28 (2 μg/ml). **P < 0.01, ****P < 0.0001 (unpaired t test). (C) The oxygen consumption measured on the bioflux analyzer as a function of time (OCR). Mitochondrial function was analyzed through the addition of mitochondrial inhibitors in the following order: oligomycin A (O) (1.5 μM), FCCP (F) (2 μM), and antimycin A (A) (2 μM). (D) The energetic status of CD8+-activated T cells from WT (black square) and Ppif−/− (open circle) mice was assessed 48 hours after activation. The bioenergetics flux was represented as the change in ECAR on the x axis in milli-pH unit/minute (mpH/min) versus the OCR on the y axis. Each symbol represents one mouse per genotype, with n = 3. (E and F) Metabolite consumption on a per-cell basis between WT and CypD-deficient CD8+ T effector cells was assessed. Glucose (glc) consumption (E) and glutamine consumption (F) in the media was measured after 48 hours of in vitro activation. ***P < 0.001 (unpaired t test). (G) WT and CypD-deficient CD8+T cells were activated for 48 hours, and their percent of double-positive IFN-γ+/TNFα+ production was assessed by ICS and flow cytometry. Before restimulation for ICS, CD8+ T cells were either pretreated with 2 mM 2-DG, Na+ oxamate, or vehicle. ****P < 0.0001 (unpaired t test). (H) Nonmitochondrial OCR measured from bioenergetics flux analysis between WT and CypD-deficient CD8+ T effector cells. *P < 0.05 (unpaired t test). (I) mROS production measured as the MFI of MitoSOX dye in CD8+ T effector cells generated from WT and Ppif−/− mice. **P < 0.01 (unpaired t test). (J to M) ECAR (J), OCR (K), CFSE proliferation assay (L), and quantification (M) of WT (top) and CypD-deficient (bottom) CD8+ T cells in the presence of vehicle (gray) or 2.5 μM of CoQ (black line) stimulated for 3 days with plate-bound α-CD3 (1 μg/ml) and α-CD28 (0.5 μg/ml). **P < 0.01, ****P < 0.0001 (two-way ANOVA). Data are representative of at least three independent experiments.

  • Fig. 5 CypD-deficient T cells are pathological.

    (A) Schematic diagram for generation of mixed BM chimeric mice. Twelve weeks after reconstitution, mice (n = 4 to 5 per group) were infected via aerosol route with ~50 H37Rv. (B) Immunoblot of CypD expression in purified T cells from T cellWT or T cellPpif−/− chimeric mice after 12 weeks of reconstitution. (C to D) Frequency of TB10.4(4–11)-specific CD8+ T cell in the lung of T cellWT or T cellPpif−/− chimeric mice after 35 days of H35Rv infection. PE-conjugated streptavidin was used as a control for the tetramer staining. Numbers above outlined areas indicate the percentage of CD8+ T cells stained with H-2Kb–TB10.4(4–11). *P < 0.05 (unpaired t test). (E and F) Frequency of TNFα- and IFN-γ–producing antigen-specific CD8+ T cells was determined by ICS upon restimulation with TB10.4(4–11) peptide at day 35 after aerosol infection with H37Rv. ***P < 0.001 (unpaired t test). (G) Survival of WT and Ppif−/− mice (n = 9 to 10 mice per group) temporarily depleted of T cells with anti-CD8 (150 μg per mouse) and anti-CD4 (150 μg per mouse) or IgG-control antibody at days 7 and 14 after intravenous infection with H37Rv (~5 × 105 CFU). Survival was monitored by log-rank test. (H) Schematic of adoptive T cell transfer from WT or Ppif−/− into Rag1-deficient mice followed by aerosol infection with H37Rv. (I) Survival of Rag-deficient mice (n = 7 per group) after adoptive transfer of T cells from Ppif−/−, WT mice, or equal number of Ppif−/− T cells and WT T cells (1:1) following aerosol infection with ~50 H37Rv. Survival was monitored by log-rank test. *P < 0.05, **P < 0.01, ***P < 0.001. (J) Representative lung histology (H&E staining) of Rag-deficient mice that received T cells from Ppif−/− mice before death after aerosol infection with ~50 H37Rv. Dense polymorphous inflammatory composed of macrophages (green arrows), lymphocytes (dashed arrows), and neutrophils (wide arrows). Data are representative of two independent experiments.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/23/eaar4135/DC1

    Material and Methods

    Fig. S1. CyD-deficient macrophages are more resistance to necrosis after infection with virulent strain of Mtb.

    Fig. S2. CypD regulates T cell responses independent of sex during Mtb infection.

    Fig. S3. CypD-deficient mice presents enhanced T cell response after infection with Mtb.

    Fig. S4. CypD minimally affects innate immune cells and B cell responses after Mtb infection.

    Fig. S5. CypD regulates T cells proliferation and activation.

    Fig. S6. CypD regulates T cell metabolism.

    Table S1. Raw data sets.

  • Supplementary Materials

    Supplementary Material for:

    Mitochondrial cyclophilin D regulates T cell metabolic responses and disease tolerance to tuberculosis

    Fanny Tzelepis, Julianna Blagih, Nargis Khan, Joshua Gillard, Laura Mendonca, Dominic G. Roy, Eric H. Ma, Philippe Joubert, Russell G. Jones, Maziar Divangahi*

    *Corresponding author. Email: maziar.divangahi{at}mcgill.ca

    Published 11 May 2018, Sci. Immunol. 3, eaar413 (2017)
    DOI: 10.1126/sciimmunol.aar4135

    This PDF file includes:

    • Material and Methods
    • Fig. S1. CyD-deficient macrophages are more resistance to necrosis after infection with virulent strain of Mtb.
    • Fig. S2. CypD regulates T cell responses independent of sex during Mtb infection.
    • Fig. S3. CypD-deficient mice presents enhanced T cell response after infection with Mtb.
    • Fig. S4. CypD minimally affects innate immune cells and B cell responses after Mtb infection.
    • Fig. S5. CypD regulates T cells proliferation and activation.
    • Fig. S6. CypD regulates T cell metabolism.

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    Other Supplementary Material for this manuscript includes the following:

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