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Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21.

McEwan WA, Tam JC, Watkinson RE, Bidgood SR, Mallery DL, James LC - Nat. Immunol. (2013)

Bottom Line: Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling.Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21.Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.

View Article: PubMed Central - PubMed

Affiliation: Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. wmcewan@mrc-lmb.cam.ac.uk

ABSTRACT
During pathogen infection, antibodies can be carried into the infected cell, where they are detected by the ubiquitously expressed cytosolic antibody receptor TRIM21. Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling. TRIM21 catalyzed the formation of Lys63 (K63)-linked ubiquitin chains and stimulated the transcription factor pathways of NF-κB, AP-1, IRF3, IRF5 and IRF7. Activation resulted in the production of proinflammatory cytokines, modulation of natural killer stress ligands and induction of an antiviral state. Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21. The sensing of antibodies by TRIM21 was stimulated after infection by DNA or RNA nonenveloped viruses or intracellular bacteria. Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.

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TRIM21 signaling is dependent upon TAK1 and stimulates κB, AP-1 and IRF pathways(a) NF-κB luciferase induction in wild-type MEF cells when treated with DMSO or inhibitors 7 h post challenge with AdV, Ab or AdV + Ab. (b) ELISA showing induction of phosphorylated (p-) and total IKKα (Ser176 and Ser180) and p65 (Ser536) 4 hours post-challenge of wild-type (WT) or Trim21-deficient (K21) MEF cells. (c) DNA binding ELISA showing of induction of AP-1 components binding to consensus oligonucleotides 4 h post challenge of Trim21-deficient MEF cells transduced with empty vector (K21 EV) or human TRIM21 (K21 T21). (d) ELISA showing phosphorylated (Ser73) and total c-Jun induction after treatment with indicated stimuli in Trim21-deficient MEF cells or Trim21-deficient MEF cells transduced with human TRIM21. (e) DNA binding ELISA showing induction of IRF3, IRF5, IRF7 and IRF8 binding to DNA response elements 4 h post-infection in wild-type and Trim21-deficient MEF cells. For all panels, error bars represent SEM from three replicates.
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Figure 3: TRIM21 signaling is dependent upon TAK1 and stimulates κB, AP-1 and IRF pathways(a) NF-κB luciferase induction in wild-type MEF cells when treated with DMSO or inhibitors 7 h post challenge with AdV, Ab or AdV + Ab. (b) ELISA showing induction of phosphorylated (p-) and total IKKα (Ser176 and Ser180) and p65 (Ser536) 4 hours post-challenge of wild-type (WT) or Trim21-deficient (K21) MEF cells. (c) DNA binding ELISA showing of induction of AP-1 components binding to consensus oligonucleotides 4 h post challenge of Trim21-deficient MEF cells transduced with empty vector (K21 EV) or human TRIM21 (K21 T21). (d) ELISA showing phosphorylated (Ser73) and total c-Jun induction after treatment with indicated stimuli in Trim21-deficient MEF cells or Trim21-deficient MEF cells transduced with human TRIM21. (e) DNA binding ELISA showing induction of IRF3, IRF5, IRF7 and IRF8 binding to DNA response elements 4 h post-infection in wild-type and Trim21-deficient MEF cells. For all panels, error bars represent SEM from three replicates.

Mentions: K63-linked ubiquitin chains can promote signaling via the kinase complexes TAK1-TAB1-TAB212,15 and IKKα-IKKβ-NEMO16,17. To test whether these complexes are activated by TRIM21, we used inhibitors of TAK1 (5Z-7-oxozeaenol) and IKKα (IKK VII). We also used an inhibitor of the NF-κB pathway at the downstream component, IκB (panepoxydone). NF-κB signaling in response to AdV + Ab was suppressed after treatment with any of the three inhibitors (Fig. 3a), confirming the involvement of both kinase complexes. Next, we analysed the phosphorylation state of individual pathway components. Upon AdV + Ab challenge, we observed an increase in both phosphorylated IKKα and NF-κB p65 in wild-type, but not in Trim21-deficient MEFs (Fig. 3b). Activation of the TAK1 complex can also stimulate the AP-1 transcriptional complex, composed of dimers of Jun and Fos family members. To test whether the AP-1 pathway is also stimulated by TRIM21, we measured Jun phosphorylation and the ability of Fos and Jun to interact with TPA response element (TRE) by DNA binding ELISA. c-Jun was phosphorylated and FosB and Jun bound TRE oligonucleotides only in wild-type, but not Trim21-deficient MEFs stimulated by AdV + Ab complex (Fig. 3c, d). Of interest, c-Fos activation was not TRIM21-dependent and was stimulated by both AdV alone and AdV + Ab, indicative of the activity of other pattern-recognition receptors. TRIM21 has been reported to modulate the activity of IRF transcription factors both positively18,19 and negatively20-22. We tested whether AdV + Ab recognition by TRIM21 was able to stimulate IRFs using a DNA binding assay. We found that IRF3, IRF5 and IRF7, but not IRF8, were specifically activated by AdV + Ab in a TRIM21-dependent manner (Fig 3e). Taken together, our data suggest that recognition of AdV + Ab by TRIM21 stimulates the three principle signaling pathways NF-κB, AP-1 and IRF.


Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21.

McEwan WA, Tam JC, Watkinson RE, Bidgood SR, Mallery DL, James LC - Nat. Immunol. (2013)

TRIM21 signaling is dependent upon TAK1 and stimulates κB, AP-1 and IRF pathways(a) NF-κB luciferase induction in wild-type MEF cells when treated with DMSO or inhibitors 7 h post challenge with AdV, Ab or AdV + Ab. (b) ELISA showing induction of phosphorylated (p-) and total IKKα (Ser176 and Ser180) and p65 (Ser536) 4 hours post-challenge of wild-type (WT) or Trim21-deficient (K21) MEF cells. (c) DNA binding ELISA showing of induction of AP-1 components binding to consensus oligonucleotides 4 h post challenge of Trim21-deficient MEF cells transduced with empty vector (K21 EV) or human TRIM21 (K21 T21). (d) ELISA showing phosphorylated (Ser73) and total c-Jun induction after treatment with indicated stimuli in Trim21-deficient MEF cells or Trim21-deficient MEF cells transduced with human TRIM21. (e) DNA binding ELISA showing induction of IRF3, IRF5, IRF7 and IRF8 binding to DNA response elements 4 h post-infection in wild-type and Trim21-deficient MEF cells. For all panels, error bars represent SEM from three replicates.
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Figure 3: TRIM21 signaling is dependent upon TAK1 and stimulates κB, AP-1 and IRF pathways(a) NF-κB luciferase induction in wild-type MEF cells when treated with DMSO or inhibitors 7 h post challenge with AdV, Ab or AdV + Ab. (b) ELISA showing induction of phosphorylated (p-) and total IKKα (Ser176 and Ser180) and p65 (Ser536) 4 hours post-challenge of wild-type (WT) or Trim21-deficient (K21) MEF cells. (c) DNA binding ELISA showing of induction of AP-1 components binding to consensus oligonucleotides 4 h post challenge of Trim21-deficient MEF cells transduced with empty vector (K21 EV) or human TRIM21 (K21 T21). (d) ELISA showing phosphorylated (Ser73) and total c-Jun induction after treatment with indicated stimuli in Trim21-deficient MEF cells or Trim21-deficient MEF cells transduced with human TRIM21. (e) DNA binding ELISA showing induction of IRF3, IRF5, IRF7 and IRF8 binding to DNA response elements 4 h post-infection in wild-type and Trim21-deficient MEF cells. For all panels, error bars represent SEM from three replicates.
Mentions: K63-linked ubiquitin chains can promote signaling via the kinase complexes TAK1-TAB1-TAB212,15 and IKKα-IKKβ-NEMO16,17. To test whether these complexes are activated by TRIM21, we used inhibitors of TAK1 (5Z-7-oxozeaenol) and IKKα (IKK VII). We also used an inhibitor of the NF-κB pathway at the downstream component, IκB (panepoxydone). NF-κB signaling in response to AdV + Ab was suppressed after treatment with any of the three inhibitors (Fig. 3a), confirming the involvement of both kinase complexes. Next, we analysed the phosphorylation state of individual pathway components. Upon AdV + Ab challenge, we observed an increase in both phosphorylated IKKα and NF-κB p65 in wild-type, but not in Trim21-deficient MEFs (Fig. 3b). Activation of the TAK1 complex can also stimulate the AP-1 transcriptional complex, composed of dimers of Jun and Fos family members. To test whether the AP-1 pathway is also stimulated by TRIM21, we measured Jun phosphorylation and the ability of Fos and Jun to interact with TPA response element (TRE) by DNA binding ELISA. c-Jun was phosphorylated and FosB and Jun bound TRE oligonucleotides only in wild-type, but not Trim21-deficient MEFs stimulated by AdV + Ab complex (Fig. 3c, d). Of interest, c-Fos activation was not TRIM21-dependent and was stimulated by both AdV alone and AdV + Ab, indicative of the activity of other pattern-recognition receptors. TRIM21 has been reported to modulate the activity of IRF transcription factors both positively18,19 and negatively20-22. We tested whether AdV + Ab recognition by TRIM21 was able to stimulate IRFs using a DNA binding assay. We found that IRF3, IRF5 and IRF7, but not IRF8, were specifically activated by AdV + Ab in a TRIM21-dependent manner (Fig 3e). Taken together, our data suggest that recognition of AdV + Ab by TRIM21 stimulates the three principle signaling pathways NF-κB, AP-1 and IRF.

Bottom Line: Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling.Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21.Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.

View Article: PubMed Central - PubMed

Affiliation: Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. wmcewan@mrc-lmb.cam.ac.uk

ABSTRACT
During pathogen infection, antibodies can be carried into the infected cell, where they are detected by the ubiquitously expressed cytosolic antibody receptor TRIM21. Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling. TRIM21 catalyzed the formation of Lys63 (K63)-linked ubiquitin chains and stimulated the transcription factor pathways of NF-κB, AP-1, IRF3, IRF5 and IRF7. Activation resulted in the production of proinflammatory cytokines, modulation of natural killer stress ligands and induction of an antiviral state. Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21. The sensing of antibodies by TRIM21 was stimulated after infection by DNA or RNA nonenveloped viruses or intracellular bacteria. Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.

Show MeSH
Related in: MedlinePlus