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TLR signalling augments macrophage bactericidal activity through mitochondrial ROS


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.

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Figure 1: TLR1/2/4 signalling induces mROS generation and mitochondrial recruitment to phagosomes.
Figure 2: TRAF6 is recruited to mitochondria upon TLR1/2/4, but not TLR3/9, signalling to engage ECSIT on the mitochondrial surface.
Figure 3: TRAF6-ECSIT signalling regulates the generation of mitochondrial and cellular ROS, which requires TRAF6 E3-ubiquitin ligase activity.
Figure 4: ECSIT-depleted and MCAT transgenic macrophages are less effective at clearing Salmonella than wild-type macrophages.


  1. Lambeth, J. D. NOX enzymes and the biology of reactive oxygen. Nature Rev. Immunol. 4, 181–189 (2004)

    CAS  Article  Google Scholar 

  2. Arsenijevic, D. et al. 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)

    CAS  Article  Google Scholar 

  3. Rousset, S. et al. The uncoupling protein 2 modulates the cytokine balance in innate immunity. Cytokine 35, 135–142 (2006)

    CAS  Article  Google Scholar 

  4. Sonoda, J. et al. Nuclear receptor ERRα and coactivator PGC-1β are effectors of IFN-γ-induced host defense. Genes Dev. 21, 1909–1920 (2007)

    CAS  Article  Google Scholar 

  5. Vogel, R. O. 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)

    CAS  Article  Google Scholar 

  6. Underhill, D. M. & Ozinsky, A. Phagocytosis of microbes: complexity in action. Annu. Rev. Immunol. 20, 825–852 (2002)

    CAS  Article  Google Scholar 

  7. Koopman, W. et al. Mammalian mitochondrial complex I: Biogenesis, regulation and reactive oxygen species generation. Antioxid. Redox Signal. 10.1089/ars.2009.2743. (2009)

  8. Murphy, M. P. How mitochondria produce reactive oxygen species. Biochem. J. 417, 1–13 (2009)

    CAS  Article  Google Scholar 

  9. Arnoult, D., Carneiro, L., Tattoli, I. & Girardin, S. E. The role of mitochondria in cellular defense against microbial infection. Semin. Immunol. 21, 223–232 (2009)

    CAS  Article  Google Scholar 

  10. Adachi, Y. et al. 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)

    CAS  Article  Google Scholar 

  11. Remer, K. A., Reimer, T., Brcic, M. & Jungi, T. W. Evidence for involvement of peptidoglycan in the triggering of an oxidative burst by Listeria monocytogenes in phagocytes. Clin. Exp. Immunol. 140, 73–80 (2005)

    CAS  Article  Google Scholar 

  12. Werling, D., Hope, J. C., Howard, C. J. & Jungi, T. W. 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)

    CAS  Article  Google Scholar 

  13. West, A. P., Koblansky, A. A. & Ghosh, S. Recognition and signaling by toll-like receptors. Annu. Rev. Cell Dev. Biol. 22, 409–437 (2006)

    CAS  Article  Google Scholar 

  14. Chong, A., Lima, C. A., Allan, D. S., Nasrallah, G. K. & Garduño, R. A. The purified and recombinant Legionella pneumophila chaperonin alters mitochondrial trafficking and microfilament organization. Infect. Immun. 77, 4724–4739 (2009)

    CAS  Article  Google Scholar 

  15. Horwitz, M. A. Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158, 1319–1331 (1983)

    CAS  Article  Google Scholar 

  16. Matsumoto, A., Bessho, H., Uehira, K. & Suda, T. Morphological studies of the association of mitochondria with chlamydial inclusions and the fusion of chlamydial inclusions. J. Electron Microsc. (Tokyo) 40, 356–363 (1991)

    CAS  Google Scholar 

  17. Sinai, A. P., Webster, P. & Joiner, K. A. 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)

    CAS  PubMed  Google Scholar 

  18. Blander, J. M. & Medzhitov, R. Regulation of phagosome maturation by signals from toll-like receptors. Science 304, 1014–1018 (2004)

    ADS  CAS  Article  Google Scholar 

  19. Blander, J. M. & Medzhitov, R. On regulation of phagosome maturation and antigen presentation. Nature Immunol. 7, 1029–1035 (2006)

    CAS  Article  Google Scholar 

  20. Calvo, S. et al. Systematic identification of human mitochondrial disease genes through integrative genomics. Nature Genet. 38, 576–582 (2006)

    CAS  Article  Google Scholar 

  21. Kopp, E. et al. ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1 signal transduction pathway. Genes Dev. 13, 2059–2071 (1999)

    CAS  Article  Google Scholar 

  22. Chen, Z. J. & Sun, L. J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009)

    CAS  Article  Google Scholar 

  23. Bhoj, V. G. & Chen, Z. J. Ubiquitylation in innate and adaptive immunity. Nature 458, 430–437 (2009)

    ADS  CAS  Article  Google Scholar 

  24. Xiao, C. et al. Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis. Genes Dev. 17, 2933–2949 (2003)

    CAS  Article  Google Scholar 

  25. Deng, L. 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)

    CAS  Article  Google Scholar 

  26. Shiloh, M. U. et al. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10, 29–38 (1999)

    CAS  Article  Google Scholar 

  27. Vazquez-Torres, A. & Fang, F. C. Oxygen-dependent anti-Salmonella activity of macrophages. Trends Microbiol. 9, 29–33 (2001)

    CAS  Article  Google Scholar 

  28. Vazquez-Torres, A. & Fang, F. C. Salmonella evasion of the NADPH phagocyte oxidase. Microbes Infect. 3, 1313–1320 (2001)

    CAS  Article  Google Scholar 

  29. Schriner, S. E. et al. Extension of murine life span by overexpression of catalase targeted to mitochondria. Science 308, 1909–1911 (2005)

    ADS  CAS  Article  Google Scholar 

  30. Brodsky, I. E., Ghori, N., Falkow, S. & Monack, D. 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)

    CAS  Article  Google Scholar 

  31. Kang, B. H. et al. Regulation of tumor cell mitochondrial homeostasis by an organelle-specific Hsp90 chaperone network. Cell 131, 257–270 (2007)

    CAS  Article  Google Scholar 

  32. Hoiseth, S. K. & Stocker, B. A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981)

    ADS  CAS  Article  Google Scholar 

  33. Valdivia, R. H. & Falkow, S. Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol. Microbiol. 22, 367–378 (1996)

    CAS  Article  Google Scholar 

  34. Cooper, M. P. et al. Defects in energy homeostasis in Leigh syndrome French Canadian variant through PGC-1α/LRP130 complex. Genes Dev. 20, 2996–3009 (2006)

    CAS  Article  Google Scholar 

  35. Sebastiaan Winkler, G. et al. Isolation and mass spectrometry of transcription factor complexes. Methods 26, 260–269 (2002)

    CAS  Article  Google Scholar 

  36. Walsh, M. C., Kim, G. K., Maurizio, P. L., Molnar, E. E. & Choi, Y. TRAF6 autoubiquitination-independent activation of the NFκB and MAPK pathways in response to IL-1 and RANKL. PLoS ONE 3, e4064 (2008)

    ADS  Article  Google Scholar 

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We would like to thank C. Schindler, B. Reizis and L. Ciaccia for comments on the manuscript. We also thank P. Rabinovitch for MCAT mice, J. Cotney for technical assistance, Z. Zhang for animal maintenance and M. Graham and K. Zichichi for assistance with immuno-electron microscopy. This work was supported by NIH grants to S.G. (R37-AI33443) and G.S.S. (NS-056206).

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Authors and Affiliations



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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Correspondence to Sankar Ghosh.

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The authors declare no competing financial interests.

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West, A., Brodsky, I., Rahner, C. et al. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472, 476–480 (2011).

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