TLR signalling augments macrophage bactericidal activity through mitochondrial ROS

Journal name:
Nature
Volume:
472,
Pages:
476–480
Date published:
DOI:
doi:10.1038/nature09973
Received
Accepted
Published online

Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase machinery1. However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to mouse macrophage bactericidal activity, although the mechanisms linking innate immune signalling to mitochondria for mROS generation remain unclear2, 3, 4. Here we demonstrate that engagement of a subset of Toll-like receptors (TLR1, TLR2 and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of a TLR signalling adaptor, tumour necrosis factor receptor-associated factor 6 (TRAF6), to mitochondria, where it engages the protein ECSIT (evolutionarily conserved signalling intermediate in Toll pathways), which is implicated in mitochondrial respiratory chain assembly5. Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT- and TRAF6-depleted macrophages have decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS levels by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These results reveal a novel pathway linking innate immune signalling to mitochondria, implicate mROS as an important component of antibacterial responses and further establish mitochondria as hubs for innate immune signalling.

At a glance

Figures

  1. TLR1/2/4 signalling induces mROS generation and mitochondrial recruitment to phagosomes.
    Figure 1: TLR1/2/4 signalling induces mROS generation and mitochondrial recruitment to phagosomes.

    a, RAW cells stimulated as indicated, stained with MitoSOX (mROS) and analysed by fluorescence-activated cell sorting (FACS). Pam3, Pam3CSK4; PIC, poly(I:C); Rot, rotenone; AA, antimycin A. b, BMDMs stimulated as indicated, stained with MitoSOX (top panels) or CM-H2DCFDA (cellular H2O2, bottom panels) and analysed by FACS. c, BMDMs were incubated with uncoated, Pam3CSK4-coated or LPS-coated latex beads, mitochondrial networks were immunostained with HSP70 antibodies (Mito) and confocal Z-stacks were acquired. Co-localized beads (red pixels) and mitochondria (green pixels) are displayed in yellow (bottom). Images shown are representative of approximately 100 cells analysed at ×63 original magnification.

  2. TRAF6 is recruited to mitochondria upon TLR1/2/4, but not TLR3/9, signalling to engage ECSIT on the mitochondrial surface.
    Figure 2: TRAF6 is recruited to mitochondria upon TLR1/2/4, but not TLR3/9, signalling to engage ECSIT on the mitochondrial surface.

    ae, RAW cells were stimulated with TLR agonists for the indicated times, cells were fractionated and extracts were western-blotted. cyt., cytoplasmic localization; p.m./endos., plasma membrane/endosomes localization; mito., mitochondrial localization. d, Purified mitochondrial lysates were immunoprecipitated with ECSIT antibody overnight. e, Equal amounts of extracts were treated with the indicated amount of proteinase K (1×, 33ngμl−1; 0.1×, 3.3ngμl−1) on ice with or without 0.2% saponin to permeabilize mitochondrial membranes.

  3. TRAF6-ECSIT signalling regulates the generation of mitochondrial and cellular ROS, which requires TRAF6 E3-ubiquitin ligase activity.
    Figure 3: TRAF6-ECSIT signalling regulates the generation of mitochondrial and cellular ROS, which requires TRAF6 E3-ubiquitin ligase activity.

    ac, Wild-type (WT) or Ecsit+/−(+/−) BMDMs were transduced with short hairpin RNAs (sh), then either left untreated or stimulated with LPS. a, Untreated BMDMs were lysed and extracts blotted for TRAF6 and ECSIT. b, c, Cells were stained with MitoSOX (b) or CM-H2DCFDA (c) and analysed by FACS. df, wild-type or TRAF6-null BMDMs were left alone or transduced with TRAF6-expressing retroviruses, then either left unstimulated or stimulated for the indicated times with LPS or LTA. d, Unstimulated BMDMs were lysed and extracts blotted for TRAF6 expression. e, f, Cells were stained with MitoSOX (e) or CM-H2DCFDA (f) and analysed by FACS. All error bars represent s.d. of the mean from triplicate samples.

  4. ECSIT-depleted and MCAT transgenic macrophages are less effective at clearing Salmonella than wild-type macrophages.
    Figure 4: ECSIT-depleted and MCAT transgenic macrophages are less effective at clearing Salmonella than wild-type macrophages.

    ac, BMDMs from wild-type or Ecsit+/−mice were transduced with shRNAs and infected with GFP-expressing Salmonella (GFP Sal). Cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI) (a), solubilized in SDS (b) or lysed and plated (c). d, e, wild-type or MCAT BMDMs were infected with GFP-expressing Salmonella. Cells were solubilized in SDS (d) or fixed and DAPI-stained (e). Triplicate wells were pooled and blotted (b, d), and error bars (c) represent s.d. from triplicate samples. f, g, wild-type (n = 6), MCAT (n = 5) and Ecsit+/− (n = 5) mice were infected with Salmonella intraperitoneally . Five days after infection, spleens (f) and livers (g) were homogenized and colony-forming units (c.f.u.) per gram of tissue were determined. Error bars indicate s.e.m. and P values are relative to wild-type.

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Author information

Affiliations

  1. Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA

    • A. Phillip West &
    • Igor E. Brodsky
  2. Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA

    • Christoph Rahner
  3. Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA

    • Dong Kyun Woo &
    • Gerald S. Shadel
  4. Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA

    • Hediye Erdjument-Bromage &
    • Paul Tempst
  5. Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA

    • Matthew C. Walsh &
    • Yongwon Choi
  6. Department of Microbiology and Immunology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA

    • Sankar Ghosh
  7. Present address: Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA.

    • Igor E. Brodsky

Contributions

A.P.W. designed and performed experiments and wrote the paper; I.E.B. generated GFP-expressing Salmonella, helped to design and perform bacterial challenge experiments and edited the paper; C.R. assisted with immuno-electron microscopy; D.K.W. provided MCAT tissues for generating BMDMs; H.E.B. and P.T. performed mass spectrometry analysis; M.C.W. and Y.C. provided reagents and technical advice for experiments involving Traf6-knockout cells; G.S.S. designed experiments and edited the paper; S.G. designed experiments and wrote the paper.

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The authors declare no competing financial interests.

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