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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 independent of TLR, FcR, ADIN and PAMPs(a) NF-κB luciferase reporter induction in Trim21-deficient MEFs expressing empty vector (K21 EV) or human TRIM21 (K21 T21) with control or inhibitor peptides of TLR signaling pathway components MyD88 and TRIF. (b) NF-κB luciferase reporter induction in MEFs after treatment with DMSO or an inhibitor of FcR kinase Syk. (c) NF-κB luciferase reporter induction in wild-type (WT) or Trim21-deficient MEFs after control (NC), MDA-5-directed or RIG-I-directed siRNA treatment by antibody-coated AdV or FCV. (d) Immunoblot for MDA-5, RIG-I and β–actin from wild-type or Trim21-deficient MEFs after control, MDA-5-directed or RIG-I-directed siRNA treatment. (e) Fold neutralization of adenovirus, measured as relative infectivity of PBS-treated AdV over 9C12-treated AdV on wild-type and Trim21-deficient MEFs following treatment with proteasome inhibitor epoxomicin or DMSO. (f) DNA binding ELISA showing induction of IRF7 activation over PBS-treated controls in DMSO or epoxomicin treated Trim21-deficient MEFs expressing empty vector or human TRIM21 4 h after challenge with AdV + Ab. (g) NF-κB luciferase reporter induction in Trim21-deficient MEFs expressing empty vector or human TRIM21 following transfection of biotin beads, anti-biotin Ab or complexed beads and Ab. Error bars in panels a-c, e, g represent SEM of three replicates.
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Figure 7: TRIM21 signaling is independent of TLR, FcR, ADIN and PAMPs(a) NF-κB luciferase reporter induction in Trim21-deficient MEFs expressing empty vector (K21 EV) or human TRIM21 (K21 T21) with control or inhibitor peptides of TLR signaling pathway components MyD88 and TRIF. (b) NF-κB luciferase reporter induction in MEFs after treatment with DMSO or an inhibitor of FcR kinase Syk. (c) NF-κB luciferase reporter induction in wild-type (WT) or Trim21-deficient MEFs after control (NC), MDA-5-directed or RIG-I-directed siRNA treatment by antibody-coated AdV or FCV. (d) Immunoblot for MDA-5, RIG-I and β–actin from wild-type or Trim21-deficient MEFs after control, MDA-5-directed or RIG-I-directed siRNA treatment. (e) Fold neutralization of adenovirus, measured as relative infectivity of PBS-treated AdV over 9C12-treated AdV on wild-type and Trim21-deficient MEFs following treatment with proteasome inhibitor epoxomicin or DMSO. (f) DNA binding ELISA showing induction of IRF7 activation over PBS-treated controls in DMSO or epoxomicin treated Trim21-deficient MEFs expressing empty vector or human TRIM21 4 h after challenge with AdV + Ab. (g) NF-κB luciferase reporter induction in Trim21-deficient MEFs expressing empty vector or human TRIM21 following transfection of biotin beads, anti-biotin Ab or complexed beads and Ab. Error bars in panels a-c, e, g represent SEM of three replicates.

Mentions: To exclude the possibility that signaling is due to recognition of AdV + Ab immune complexes by FcR or innate sensors other than TRIM21, we performed infection experiments in the presence of specific inhibitors or knockdown of these pathways. Addition of MyD88 and TRIF inhibitors efficiently blocked signaling in response to LPS (Supplementary Fig. 7), but had no effect on AdV + Ab stimulation of NF-κB (Fig. 7a), confirming that signaling is TLR-independent. Inhibition of cytosolic tyrosine kinase Syk prevented signaling in response to cross-linked surface bound antibody (Supplementary Fig. 7) but did not prevent signaling in response to AdV + IgG or AdV + IgM (Fig. 7b), confirming that TRIM21 signaling is FcR-independent. Similarly, knockdown of intracellular nucleic acid receptors, MDA-5 and RIG-I failed to impair signaling in response to antibody-bound AdV or FCV (Fig. 7c,d).


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 independent of TLR, FcR, ADIN and PAMPs(a) NF-κB luciferase reporter induction in Trim21-deficient MEFs expressing empty vector (K21 EV) or human TRIM21 (K21 T21) with control or inhibitor peptides of TLR signaling pathway components MyD88 and TRIF. (b) NF-κB luciferase reporter induction in MEFs after treatment with DMSO or an inhibitor of FcR kinase Syk. (c) NF-κB luciferase reporter induction in wild-type (WT) or Trim21-deficient MEFs after control (NC), MDA-5-directed or RIG-I-directed siRNA treatment by antibody-coated AdV or FCV. (d) Immunoblot for MDA-5, RIG-I and β–actin from wild-type or Trim21-deficient MEFs after control, MDA-5-directed or RIG-I-directed siRNA treatment. (e) Fold neutralization of adenovirus, measured as relative infectivity of PBS-treated AdV over 9C12-treated AdV on wild-type and Trim21-deficient MEFs following treatment with proteasome inhibitor epoxomicin or DMSO. (f) DNA binding ELISA showing induction of IRF7 activation over PBS-treated controls in DMSO or epoxomicin treated Trim21-deficient MEFs expressing empty vector or human TRIM21 4 h after challenge with AdV + Ab. (g) NF-κB luciferase reporter induction in Trim21-deficient MEFs expressing empty vector or human TRIM21 following transfection of biotin beads, anti-biotin Ab or complexed beads and Ab. Error bars in panels a-c, e, g represent SEM of three replicates.
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Figure 7: TRIM21 signaling is independent of TLR, FcR, ADIN and PAMPs(a) NF-κB luciferase reporter induction in Trim21-deficient MEFs expressing empty vector (K21 EV) or human TRIM21 (K21 T21) with control or inhibitor peptides of TLR signaling pathway components MyD88 and TRIF. (b) NF-κB luciferase reporter induction in MEFs after treatment with DMSO or an inhibitor of FcR kinase Syk. (c) NF-κB luciferase reporter induction in wild-type (WT) or Trim21-deficient MEFs after control (NC), MDA-5-directed or RIG-I-directed siRNA treatment by antibody-coated AdV or FCV. (d) Immunoblot for MDA-5, RIG-I and β–actin from wild-type or Trim21-deficient MEFs after control, MDA-5-directed or RIG-I-directed siRNA treatment. (e) Fold neutralization of adenovirus, measured as relative infectivity of PBS-treated AdV over 9C12-treated AdV on wild-type and Trim21-deficient MEFs following treatment with proteasome inhibitor epoxomicin or DMSO. (f) DNA binding ELISA showing induction of IRF7 activation over PBS-treated controls in DMSO or epoxomicin treated Trim21-deficient MEFs expressing empty vector or human TRIM21 4 h after challenge with AdV + Ab. (g) NF-κB luciferase reporter induction in Trim21-deficient MEFs expressing empty vector or human TRIM21 following transfection of biotin beads, anti-biotin Ab or complexed beads and Ab. Error bars in panels a-c, e, g represent SEM of three replicates.
Mentions: To exclude the possibility that signaling is due to recognition of AdV + Ab immune complexes by FcR or innate sensors other than TRIM21, we performed infection experiments in the presence of specific inhibitors or knockdown of these pathways. Addition of MyD88 and TRIF inhibitors efficiently blocked signaling in response to LPS (Supplementary Fig. 7), but had no effect on AdV + Ab stimulation of NF-κB (Fig. 7a), confirming that signaling is TLR-independent. Inhibition of cytosolic tyrosine kinase Syk prevented signaling in response to cross-linked surface bound antibody (Supplementary Fig. 7) but did not prevent signaling in response to AdV + IgG or AdV + IgM (Fig. 7b), confirming that TRIM21 signaling is FcR-independent. Similarly, knockdown of intracellular nucleic acid receptors, MDA-5 and RIG-I failed to impair signaling in response to antibody-bound AdV or FCV (Fig. 7c,d).

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