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TLR signalling augments macrophage bactericidal activity through mitochondrial ROS

Abstract

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.

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Acknowledgements

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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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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Correspondence to Sankar Ghosh.

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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). https://doi.org/10.1038/nature09973

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