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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 senses intracellular Ab-bound virus(a) DNA binding ELISA showing NF-κB subunits p65 and p50 binding to consensus oligonucleotides 4 h post challenge of wild-type (WT) MEFs or Trim21-deficient MEFs transduced with empty vector (K21 EV) or expressing human Trim21 (K21 T21) with PBS, anti-adenovirus monoclonal antibody 9C12 (Ab), adenovirus (AdV) or adenovirus-antibody complex (AdV + Ab). (b) Induction of NF-κB luciferase reporter activity in wild-type or Trim21-deficient (K21) MEFs 7 h after challenge with a serial dilution of goat anti-adenovirus, AdV or adenovirus-antibody complex over PBS-treated controls. (c) Induction of NF-κB luciferase reporter activity in wild-type, Trim21-deficient and Trim21-deficient cells expressing human TRIM21. (d) Induction of NF-κB luciferase reporter activity in HeLa cells or HeLa cells depleted of TRIM21 by siRNA. Ab is pooled human serum IgG. (e) Induction of NF-κB luciferase reporter activity after challenge of Trim21-deficient MEFs transduced with empty vector or human TRIM21 with AdV, human serum IgM or AdV + IgM complex. (f) Hot-spot interactions between IgG Fc (cyan) and TRIM21 PRYSPRY domain (yellow) required for complex formation (based on PDB structure 2IWG). (g) Induction of NF-κB luciferase reporter activity in wild-type MEFs challenged with AdV incubated with recombinant 9C12 (r9C12) or point mutant N434D (r9C12 ND). (h) Induction of NF-κB luciferase reporter activity in AdV non-permissive EL4 cells and AdV permissive EL4 CAR 7 h after challenge. For panels a-e, g, h error bars represent SEM from three replicates.
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Figure 1: TRIM21 senses intracellular Ab-bound virus(a) DNA binding ELISA showing NF-κB subunits p65 and p50 binding to consensus oligonucleotides 4 h post challenge of wild-type (WT) MEFs or Trim21-deficient MEFs transduced with empty vector (K21 EV) or expressing human Trim21 (K21 T21) with PBS, anti-adenovirus monoclonal antibody 9C12 (Ab), adenovirus (AdV) or adenovirus-antibody complex (AdV + Ab). (b) Induction of NF-κB luciferase reporter activity in wild-type or Trim21-deficient (K21) MEFs 7 h after challenge with a serial dilution of goat anti-adenovirus, AdV or adenovirus-antibody complex over PBS-treated controls. (c) Induction of NF-κB luciferase reporter activity in wild-type, Trim21-deficient and Trim21-deficient cells expressing human TRIM21. (d) Induction of NF-κB luciferase reporter activity in HeLa cells or HeLa cells depleted of TRIM21 by siRNA. Ab is pooled human serum IgG. (e) Induction of NF-κB luciferase reporter activity after challenge of Trim21-deficient MEFs transduced with empty vector or human TRIM21 with AdV, human serum IgM or AdV + IgM complex. (f) Hot-spot interactions between IgG Fc (cyan) and TRIM21 PRYSPRY domain (yellow) required for complex formation (based on PDB structure 2IWG). (g) Induction of NF-κB luciferase reporter activity in wild-type MEFs challenged with AdV incubated with recombinant 9C12 (r9C12) or point mutant N434D (r9C12 ND). (h) Induction of NF-κB luciferase reporter activity in AdV non-permissive EL4 cells and AdV permissive EL4 CAR 7 h after challenge. For panels a-e, g, h error bars represent SEM from three replicates.

Mentions: To test whether antibody entering the cytoplasm while bound to a pathogen initiates immune signaling, we assayed activated NF-κB subunits p65, p50 and p52 4 h post-challenge with an adenovirus type 5 vector (AdV) in the presence of antibody (Ab) by analyzing binding of the NF-κB subunits to consensus NF-κB DNA oligonucleotides in an ELISA assay. In wild-type mouse embryonic fibroblast (MEF) cells, a substantial increase in activated NF-κB was observed upon infection with adenovirus-antibody complex (AdV + Ab) but not with either component alone (Fig. 1a and Supplementary Fig. 1). The response was dependent upon TRIM21, as activation was not observed in MEFs derived from Trim21-deficient mice. Furthermore, activation in Trim21-deficient MEFs could be restored by ectopic expression of human TRIM21 (Fig. 1a), confirmed by immunoblot analyses (Supplementary Fig. 1). Titration of AdV, Ab or AdV + Ab onto wild-type and Trim21-deficient MEFs revealed that activation of an NF-κB luciferase reporter was dose-dependent and approached saturation at high multiplicity of infection (moi) but was absent at all multiplicities in Trim21-deficient MEFs (Fig. 1b). TRIM21 was not required for constitutive NF-κB signaling in response to other stimuli, as similar activation was observed in wild-type MEFs and Trim21-deficient MEFs transduced with empty vector or human TRIM21 when challenged with lipopolysaccharide (LPS) or tumor necrosis factor (TNF) (Fig. 1c and Supplementary Fig. 1). These results demonstrate that TRIM21 detects intracellular AdV + Ab and activates NF-κB signaling.


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 senses intracellular Ab-bound virus(a) DNA binding ELISA showing NF-κB subunits p65 and p50 binding to consensus oligonucleotides 4 h post challenge of wild-type (WT) MEFs or Trim21-deficient MEFs transduced with empty vector (K21 EV) or expressing human Trim21 (K21 T21) with PBS, anti-adenovirus monoclonal antibody 9C12 (Ab), adenovirus (AdV) or adenovirus-antibody complex (AdV + Ab). (b) Induction of NF-κB luciferase reporter activity in wild-type or Trim21-deficient (K21) MEFs 7 h after challenge with a serial dilution of goat anti-adenovirus, AdV or adenovirus-antibody complex over PBS-treated controls. (c) Induction of NF-κB luciferase reporter activity in wild-type, Trim21-deficient and Trim21-deficient cells expressing human TRIM21. (d) Induction of NF-κB luciferase reporter activity in HeLa cells or HeLa cells depleted of TRIM21 by siRNA. Ab is pooled human serum IgG. (e) Induction of NF-κB luciferase reporter activity after challenge of Trim21-deficient MEFs transduced with empty vector or human TRIM21 with AdV, human serum IgM or AdV + IgM complex. (f) Hot-spot interactions between IgG Fc (cyan) and TRIM21 PRYSPRY domain (yellow) required for complex formation (based on PDB structure 2IWG). (g) Induction of NF-κB luciferase reporter activity in wild-type MEFs challenged with AdV incubated with recombinant 9C12 (r9C12) or point mutant N434D (r9C12 ND). (h) Induction of NF-κB luciferase reporter activity in AdV non-permissive EL4 cells and AdV permissive EL4 CAR 7 h after challenge. For panels a-e, g, h error bars represent SEM from three replicates.
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Figure 1: TRIM21 senses intracellular Ab-bound virus(a) DNA binding ELISA showing NF-κB subunits p65 and p50 binding to consensus oligonucleotides 4 h post challenge of wild-type (WT) MEFs or Trim21-deficient MEFs transduced with empty vector (K21 EV) or expressing human Trim21 (K21 T21) with PBS, anti-adenovirus monoclonal antibody 9C12 (Ab), adenovirus (AdV) or adenovirus-antibody complex (AdV + Ab). (b) Induction of NF-κB luciferase reporter activity in wild-type or Trim21-deficient (K21) MEFs 7 h after challenge with a serial dilution of goat anti-adenovirus, AdV or adenovirus-antibody complex over PBS-treated controls. (c) Induction of NF-κB luciferase reporter activity in wild-type, Trim21-deficient and Trim21-deficient cells expressing human TRIM21. (d) Induction of NF-κB luciferase reporter activity in HeLa cells or HeLa cells depleted of TRIM21 by siRNA. Ab is pooled human serum IgG. (e) Induction of NF-κB luciferase reporter activity after challenge of Trim21-deficient MEFs transduced with empty vector or human TRIM21 with AdV, human serum IgM or AdV + IgM complex. (f) Hot-spot interactions between IgG Fc (cyan) and TRIM21 PRYSPRY domain (yellow) required for complex formation (based on PDB structure 2IWG). (g) Induction of NF-κB luciferase reporter activity in wild-type MEFs challenged with AdV incubated with recombinant 9C12 (r9C12) or point mutant N434D (r9C12 ND). (h) Induction of NF-κB luciferase reporter activity in AdV non-permissive EL4 cells and AdV permissive EL4 CAR 7 h after challenge. For panels a-e, g, h error bars represent SEM from three replicates.
Mentions: To test whether antibody entering the cytoplasm while bound to a pathogen initiates immune signaling, we assayed activated NF-κB subunits p65, p50 and p52 4 h post-challenge with an adenovirus type 5 vector (AdV) in the presence of antibody (Ab) by analyzing binding of the NF-κB subunits to consensus NF-κB DNA oligonucleotides in an ELISA assay. In wild-type mouse embryonic fibroblast (MEF) cells, a substantial increase in activated NF-κB was observed upon infection with adenovirus-antibody complex (AdV + Ab) but not with either component alone (Fig. 1a and Supplementary Fig. 1). The response was dependent upon TRIM21, as activation was not observed in MEFs derived from Trim21-deficient mice. Furthermore, activation in Trim21-deficient MEFs could be restored by ectopic expression of human TRIM21 (Fig. 1a), confirmed by immunoblot analyses (Supplementary Fig. 1). Titration of AdV, Ab or AdV + Ab onto wild-type and Trim21-deficient MEFs revealed that activation of an NF-κB luciferase reporter was dose-dependent and approached saturation at high multiplicity of infection (moi) but was absent at all multiplicities in Trim21-deficient MEFs (Fig. 1b). TRIM21 was not required for constitutive NF-κB signaling in response to other stimuli, as similar activation was observed in wild-type MEFs and Trim21-deficient MEFs transduced with empty vector or human TRIM21 when challenged with lipopolysaccharide (LPS) or tumor necrosis factor (TNF) (Fig. 1c and Supplementary Fig. 1). These results demonstrate that TRIM21 detects intracellular AdV + Ab and activates NF-κB signaling.

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