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. 2011 Apr 28;472(7344):476-80.
doi: 10.1038/nature09973.

"V体育官网" TLR signalling augments macrophage bactericidal activity through mitochondrial ROS

A Phillip West  1 , Igor E Brodsky, Christoph Rahner, Dong Kyun Woo, Hediye Erdjument-Bromage, Paul Tempst, Matthew C Walsh, Yongwon Choi, Gerald S Shadel, Sankar Ghosh
Affiliations

TLR signalling augments macrophage bactericidal activity through mitochondrial ROS

A Phillip West et al. Nature. .

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 machinery. 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 unclear VSports手机版. 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 assembly. 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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Conflict of interest statement (V体育ios版)

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. TLR1/2/4 signaling induces mROS generation and mitochondrial recruitment to phagosomes
a, RAW cells stimulated as indicated, stained with MitoSOX [mROS], and analyzed by FACS. b, BMM were stimulated as indicated, stained with MitoSOX (top panels) or CM-H2DCFDA [cellular H2O2] (bottom panels), and analyzed by FACS. c, BMM were incubated with uncoated, Pam3CSK4-, or LPS-coated latex beads and mitochondrial networks were immunostained with HSP70 antibodies [Mito]. Confocal Z-stacks were acquired and colocalized beads (red pixels) and mitochondria (green pixels) are displayed in yellow (bottom). Images shown are representative of approximately 100 cells analyzed.
Figure 2
Figure 2. TRAF6 is recruited to mitochondria upon TLR1/2/4, but not TLR3/9, signaling to engage ECSIT on the mitochondrial surface
a-e, RAW cells were stimulated with TLR agonists for the indicated times, cells were fractionated, and extracts were blotted. Purified mitochondrial lysates were immunoprecipitated with ECSIT antibody overnight (d). Heavy chain IgG is indicated by an asterisk (d). Equal amounts of extracts were treated with the indicated amount of proteinase K on ice with or without 0.2% saponin to gently permeabilize mitochondrial membranes (e).
Figure 3
Figure 3. TRAF6-ECSIT signaling regulates generation of mitochondrial and cellular ROS, which requires TRAF6 E3-ubiquitin ligase activity
a-c, WT or ECSIT +/- BMM were transduced with shRNAs and left untreated or stimulated with LPS. Untreated BMM were lysed and extracts blotted for TRAF6 and ECSIT (a). Cells were stained with MitoSOX (b) or CM-H2DCFDA (c) and analyzed by FACS. d-f, WT or TRAF6 null BMM were left untreated or transduced with TRAF6 expressing retroviruses, then stimulated with for the indicated times. Unstimulated BMM were lysed and extracts blotted for TRAF6 expression (d). Cells were stained with MitoSOX (e) and CM-H2DCFDA (f) and analyzed by FACS. Error bars represent s.d. of the mean from triplicate samples.
Figure 4
Figure 4. ECSIT depleted and MCAT transgenic macrophages are less effective at clearing Salmonella
a-c, BMM from WT or ECSIT +/- were transduced with shRNAs and infected with GFP-Salmonella. Cells were fixed and stained with Dapi (a), solubilized in SDS (b), or lysed and plated (c). d-e, WT or MCAT BMM were infected with GFP-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, WT (n=6), MCAT (n=5), and ECSIT +/- (n=5) mice were infected with Salmonella i.p. Five days post infection, spleens (f) and livers (g) were homogenized and CFUs per gram of tissue were determined. Error bars indicate s.e.m., and p values are relative to WT.
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