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TANK is a negative regulator of Toll-like receptor signaling and is critical for the prevention of autoimmune nephritis.

Kawagoe T, Takeuchi O, Takabatake Y, Kato H, Isaka Y, Tsujimura T, Akira S - Nat. Immunol. (2009)

Bottom Line: Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling.TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages.Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defense, World Premier International Immunology Frontier Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

ABSTRACT
The intensity and duration of immune responses are controlled by many proteins that modulate Toll-like receptor (TLR) signaling. TANK has been linked to positive regulation of the transcription factors IRF3 and NF-kappaB. Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling. TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages. Notably, Tank(-/-) mice spontaneously developed fatal glomerulonephritis owing to deposition of immune complexes. Autoantibody production in Tank(-/-) mice was abrogated by antibiotic treatment or the absence of interleukin 6 (IL-6) or the adaptor MyD88. Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

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TANK negatively regulates the activation of NF-κB and AP-1 as well as gene expression in response to TLR7 stimulation in macrophages(a) Peritoneal macrophages from wild-type (WT) and Tank−/− mice were stimulated with 10 nM R-848 for the indicated periods. Total RNA was extracted and subjected to Northern blot analyses for the expression of Il6, Tnf, Il12b, Ptgs2, Nfkbiz and Nos2. The same membranes were rehybridized with an Actb probe. Data of two independent experiments (lanes marked 1 and 2 represent distinct experiments) are shown. (b,c) Wild-type and Tank−/− macrophages were stimulated with R-848 (10 μM) for the indicated periods. Nuclear extracts were prepared, and the NF-κB (b) and AP-1 (c) DNA-binding activities were determined by EMSA using NF-κB- and AP-1-specific probes. The arrows indicate the induced NF-κB and AP-1 complexes. The results are representative of three independent experiments.
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Figure 4: TANK negatively regulates the activation of NF-κB and AP-1 as well as gene expression in response to TLR7 stimulation in macrophages(a) Peritoneal macrophages from wild-type (WT) and Tank−/− mice were stimulated with 10 nM R-848 for the indicated periods. Total RNA was extracted and subjected to Northern blot analyses for the expression of Il6, Tnf, Il12b, Ptgs2, Nfkbiz and Nos2. The same membranes were rehybridized with an Actb probe. Data of two independent experiments (lanes marked 1 and 2 represent distinct experiments) are shown. (b,c) Wild-type and Tank−/− macrophages were stimulated with R-848 (10 μM) for the indicated periods. Nuclear extracts were prepared, and the NF-κB (b) and AP-1 (c) DNA-binding activities were determined by EMSA using NF-κB- and AP-1-specific probes. The arrows indicate the induced NF-κB and AP-1 complexes. The results are representative of three independent experiments.

Mentions: We examined whether the increased cytokine production in Tank−/− macrophages was evident at the level of transcription. In response to R-848 stimulation, wild-type macrophages showed induction of Il6, Tnf, Il12b, Ptgs2, Nfkbiz and Nos2 gene expression. The expression of these genes was enhanced in Tank−/− macrophages in response to R-848 stimulation (Fig. 4a), indicating that initial TLR-induced gene expression is enhanced in Tank−/− macrophages. Next, we analyzed the activation of the transcription factors NF-κB and AP-1 by electrophoretic mobility shift assays (EMSA). In response to R-848 stimulation, activation of NF-κB and AP-1 was enhanced in Tank−/− macrophages compared to wild-type macrophages (Fig. 4b,c).


TANK is a negative regulator of Toll-like receptor signaling and is critical for the prevention of autoimmune nephritis.

Kawagoe T, Takeuchi O, Takabatake Y, Kato H, Isaka Y, Tsujimura T, Akira S - Nat. Immunol. (2009)

TANK negatively regulates the activation of NF-κB and AP-1 as well as gene expression in response to TLR7 stimulation in macrophages(a) Peritoneal macrophages from wild-type (WT) and Tank−/− mice were stimulated with 10 nM R-848 for the indicated periods. Total RNA was extracted and subjected to Northern blot analyses for the expression of Il6, Tnf, Il12b, Ptgs2, Nfkbiz and Nos2. The same membranes were rehybridized with an Actb probe. Data of two independent experiments (lanes marked 1 and 2 represent distinct experiments) are shown. (b,c) Wild-type and Tank−/− macrophages were stimulated with R-848 (10 μM) for the indicated periods. Nuclear extracts were prepared, and the NF-κB (b) and AP-1 (c) DNA-binding activities were determined by EMSA using NF-κB- and AP-1-specific probes. The arrows indicate the induced NF-κB and AP-1 complexes. The results are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 4: TANK negatively regulates the activation of NF-κB and AP-1 as well as gene expression in response to TLR7 stimulation in macrophages(a) Peritoneal macrophages from wild-type (WT) and Tank−/− mice were stimulated with 10 nM R-848 for the indicated periods. Total RNA was extracted and subjected to Northern blot analyses for the expression of Il6, Tnf, Il12b, Ptgs2, Nfkbiz and Nos2. The same membranes were rehybridized with an Actb probe. Data of two independent experiments (lanes marked 1 and 2 represent distinct experiments) are shown. (b,c) Wild-type and Tank−/− macrophages were stimulated with R-848 (10 μM) for the indicated periods. Nuclear extracts were prepared, and the NF-κB (b) and AP-1 (c) DNA-binding activities were determined by EMSA using NF-κB- and AP-1-specific probes. The arrows indicate the induced NF-κB and AP-1 complexes. The results are representative of three independent experiments.
Mentions: We examined whether the increased cytokine production in Tank−/− macrophages was evident at the level of transcription. In response to R-848 stimulation, wild-type macrophages showed induction of Il6, Tnf, Il12b, Ptgs2, Nfkbiz and Nos2 gene expression. The expression of these genes was enhanced in Tank−/− macrophages in response to R-848 stimulation (Fig. 4a), indicating that initial TLR-induced gene expression is enhanced in Tank−/− macrophages. Next, we analyzed the activation of the transcription factors NF-κB and AP-1 by electrophoretic mobility shift assays (EMSA). In response to R-848 stimulation, activation of NF-κB and AP-1 was enhanced in Tank−/− macrophages compared to wild-type macrophages (Fig. 4b,c).

Bottom Line: Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling.TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages.Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defense, World Premier International Immunology Frontier Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

ABSTRACT
The intensity and duration of immune responses are controlled by many proteins that modulate Toll-like receptor (TLR) signaling. TANK has been linked to positive regulation of the transcription factors IRF3 and NF-kappaB. Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling. TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages. Notably, Tank(-/-) mice spontaneously developed fatal glomerulonephritis owing to deposition of immune complexes. Autoantibody production in Tank(-/-) mice was abrogated by antibiotic treatment or the absence of interleukin 6 (IL-6) or the adaptor MyD88. Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

Show MeSH
Related in: MedlinePlus