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
- NOX enzymes and the biology of reactive oxygen. Nature Rev. Immunol. 4, 181–189 (2004)
- Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nature Genet. 26, 435–439 (2000) et al.
- The uncoupling protein 2 modulates the cytokine balance in innate immunity. Cytokine 35, 135–142 (2006) et al.
- Nuclear receptor ERRα and coactivator PGC-1β are effectors of IFN-γ-induced host defense. Genes Dev. 21, 1909–1920 (2007) et al.
- Cytosolic signaling protein Ecsit also localizes to mitochondria where it interacts with chaperone NDUFAF1 and functions in complex I assembly. Genes Dev. 21, 615–624 (2007) et al.
- Phagocytosis of microbes: complexity in action. Annu. Rev. Immunol. 20, 825–852 (2002) &
- Mammalian mitochondrial complex I: Biogenesis, regulation and reactive oxygen species generation. Antioxid. Redox Signal. doi:10.1089/ars.2009.2743. (2009) et al.
- How mitochondria produce reactive oxygen species. Biochem. J. 417, 1–13 (2009)
- The role of mitochondria in cellular defense against microbial infection. Semin. Immunol. 21, 223–232 (2009) , , &
- IFN-γ primes RAW264 macrophages and human monocytes for enhanced oxidant production in response to CpG DNA via metabolic signaling: roles of TLR9 and myeloperoxidase trafficking. J. Immunol. 176, 5033–5040 (2006) et al.
- Evidence for involvement of peptidoglycan in the triggering of an oxidative burst by Listeria monocytogenes in phagocytes. Clin. Exp. Immunol. 140, 73–80 (2005) , , &
- Differential production of cytokines, reactive oxygen and nitrogen by bovine macrophages and dendritic cells stimulated with Toll-like receptor agonists. Immunology 111, 41–52 (2004) , , &
- Recognition and signaling by toll-like receptors. Annu. Rev. Cell Dev. Biol. 22, 409–437 (2006) , &
- The purified and recombinant Legionella pneumophila chaperonin alters mitochondrial trafficking and microfilament organization. Infect. Immun. 77, 4724–4739 (2009) , , , &
- Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158, 1319–1331 (1983)
- Morphological studies of the association of mitochondria with chlamydial inclusions and the fusion of chlamydial inclusions. J. Electron Microsc. (Tokyo) 40, 356–363 (1991) , , &
- Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J. Cell Sci. 110, 2117–2128 (1997) , &
- Regulation of phagosome maturation by signals from toll-like receptors. Science 304, 1014–1018 (2004) &
- On regulation of phagosome maturation and antigen presentation. Nature Immunol. 7, 1029–1035 (2006) &
- Systematic identification of human mitochondrial disease genes through integrative genomics. Nature Genet. 38, 576–582 (2006) et al.
- ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1 signal transduction pathway. Genes Dev. 13, 2059–2071 (1999) et al.
- Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009) &
- Ubiquitylation in innate and adaptive immunity. Nature 458, 430–437 (2009) &
- Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis. Genes Dev. 17, 2933–2949 (2003) et al.
- Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000) et al.
- Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10, 29–38 (1999) et al.
- Oxygen-dependent anti-Salmonella activity of macrophages. Trends Microbiol. 9, 29–33 (2001) &
- Salmonella evasion of the NADPH phagocyte oxidase. Microbes Infect. 3, 1313–1320 (2001) &
- Extension of murine life span by overexpression of catalase targeted to mitochondria. Science 308, 1909–1911 (2005) et al.
- Mig-14 is an inner membrane-associated protein that promotes Salmonella typhimurium resistance to CRAMP, survival within activated macrophages and persistent infection. Mol. Microbiol. 55, 954–972 (2005) , , &
- Regulation of tumor cell mitochondrial homeostasis by an organelle-specific Hsp90 chaperone network. Cell 131, 257–270 (2007) et al.
- Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981) &
- Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol. Microbiol. 22, 367–378 (1996) &
- Defects in energy homeostasis in Leigh syndrome French Canadian variant through PGC-1α/LRP130 complex. Genes Dev. 20, 2996–3009 (2006) et al.
- Isolation and mass spectrometry of transcription factor complexes. Methods 26, 260–269 (2002) et al.
- TRAF6 autoubiquitination-independent activation of the NFκB and MAPK pathways in response to IL-1 and RANKL. PLoS ONE 3, e4064 (2008) , , , &
- Supplementary Information (1.6M)
This file contains Supplementary Figures 1-15 with legends and Supplementary Table 1.