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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 promotes NF-κB signaling in response to viral and bacterial pathogens(a) NF-κB luciferase reporter induction in response to infection with feline calicivirus (FCV) with feline (fe) serum IgG in wild-type (WT) or Trim21-deficient (K21) MEFs. (b) Respiratory syncytial virus (RSV) with Synagis (Syn) or human (hu) serum IgG in Trim21-deficient MEFs expressing human TRIM21 (K21 T21) or empty vector (K21 EV). (c) Confocal micrographs of HeLa cells 4 h post-infection with Ab-coated GFP-expressing Salmonella enterica serovr Typhimurium (S. Typhimurium). Staining is for anti-LPS Ab and TRIM21. (d) NF-κB luciferase reporter induction in HeLa treated with control (NC) or TRIM21-directed (T21) siRNA 7 h post-challenge with Salmonella in the presence of increasing concentrations of anti-LPS Ab. (e) NF-κB luciferase reporter induction after challenge of wild-type or Trim21-deficient MEFs with Salmonella, LPS or TNF. (f) NF-κB luciferase induction in wild-type or Trim21-deficient MEFs after challenge with Ab-coated wild-type and ΔSifA Salmonella over uncoated bacteria. (g) NF-κB luciferase induction after infection of wild-type MEF or Trim21-deficeint MEFs with a dilution series of ΔSifA Salmonella incubated with PBS or anti-LPS Ab. For panels a, b, d-g, error bars represent SEM from three replicates.
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Figure 6: TRIM21 promotes NF-κB signaling in response to viral and bacterial pathogens(a) NF-κB luciferase reporter induction in response to infection with feline calicivirus (FCV) with feline (fe) serum IgG in wild-type (WT) or Trim21-deficient (K21) MEFs. (b) Respiratory syncytial virus (RSV) with Synagis (Syn) or human (hu) serum IgG in Trim21-deficient MEFs expressing human TRIM21 (K21 T21) or empty vector (K21 EV). (c) Confocal micrographs of HeLa cells 4 h post-infection with Ab-coated GFP-expressing Salmonella enterica serovr Typhimurium (S. Typhimurium). Staining is for anti-LPS Ab and TRIM21. (d) NF-κB luciferase reporter induction in HeLa treated with control (NC) or TRIM21-directed (T21) siRNA 7 h post-challenge with Salmonella in the presence of increasing concentrations of anti-LPS Ab. (e) NF-κB luciferase reporter induction after challenge of wild-type or Trim21-deficient MEFs with Salmonella, LPS or TNF. (f) NF-κB luciferase induction in wild-type or Trim21-deficient MEFs after challenge with Ab-coated wild-type and ΔSifA Salmonella over uncoated bacteria. (g) NF-κB luciferase induction after infection of wild-type MEF or Trim21-deficeint MEFs with a dilution series of ΔSifA Salmonella incubated with PBS or anti-LPS Ab. For panels a, b, d-g, error bars represent SEM from three replicates.

Mentions: Next we investigated whether TRIM21 signaling is pathogen-dependent. We reasoned that a cytoplasmically accessible Ab-bound pathogen should provoke signaling whereas a pathogen that sheds its antibody before penetrating the cytosol should not. We chose two contrasting viruses to test this hypothesis; the non-enveloped feline calicivirus (FCV), which may carry antibodies with it into the cell upon infection and enveloped respiratory syncytial virus (RSV), which fuses at or near the plasma membrane28 and should lose its attached antibodies during membrane fusion. FCV activated a NF-κB luciferase reporter in the presence of pooled feline serum IgG in wild-type but not Trim21-deicient MEFs. In contrast, infection with RSV pre-incubated with antibody resulted in similar NF-κB luciferase reporter activitation in wild-type and Trim21-deficient MEFs (Fig. 6a, b). Similarly, and in contrast to AdV6,10, incubation of RSV with neutralizing antibody resulted in similar levels of neutralization between untreated, TRIM21 shRNA-treated or IFNα-treated HeLa cells (Supplementary Fig. 6).


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 promotes NF-κB signaling in response to viral and bacterial pathogens(a) NF-κB luciferase reporter induction in response to infection with feline calicivirus (FCV) with feline (fe) serum IgG in wild-type (WT) or Trim21-deficient (K21) MEFs. (b) Respiratory syncytial virus (RSV) with Synagis (Syn) or human (hu) serum IgG in Trim21-deficient MEFs expressing human TRIM21 (K21 T21) or empty vector (K21 EV). (c) Confocal micrographs of HeLa cells 4 h post-infection with Ab-coated GFP-expressing Salmonella enterica serovr Typhimurium (S. Typhimurium). Staining is for anti-LPS Ab and TRIM21. (d) NF-κB luciferase reporter induction in HeLa treated with control (NC) or TRIM21-directed (T21) siRNA 7 h post-challenge with Salmonella in the presence of increasing concentrations of anti-LPS Ab. (e) NF-κB luciferase reporter induction after challenge of wild-type or Trim21-deficient MEFs with Salmonella, LPS or TNF. (f) NF-κB luciferase induction in wild-type or Trim21-deficient MEFs after challenge with Ab-coated wild-type and ΔSifA Salmonella over uncoated bacteria. (g) NF-κB luciferase induction after infection of wild-type MEF or Trim21-deficeint MEFs with a dilution series of ΔSifA Salmonella incubated with PBS or anti-LPS Ab. For panels a, b, d-g, error bars represent SEM from three replicates.
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Figure 6: TRIM21 promotes NF-κB signaling in response to viral and bacterial pathogens(a) NF-κB luciferase reporter induction in response to infection with feline calicivirus (FCV) with feline (fe) serum IgG in wild-type (WT) or Trim21-deficient (K21) MEFs. (b) Respiratory syncytial virus (RSV) with Synagis (Syn) or human (hu) serum IgG in Trim21-deficient MEFs expressing human TRIM21 (K21 T21) or empty vector (K21 EV). (c) Confocal micrographs of HeLa cells 4 h post-infection with Ab-coated GFP-expressing Salmonella enterica serovr Typhimurium (S. Typhimurium). Staining is for anti-LPS Ab and TRIM21. (d) NF-κB luciferase reporter induction in HeLa treated with control (NC) or TRIM21-directed (T21) siRNA 7 h post-challenge with Salmonella in the presence of increasing concentrations of anti-LPS Ab. (e) NF-κB luciferase reporter induction after challenge of wild-type or Trim21-deficient MEFs with Salmonella, LPS or TNF. (f) NF-κB luciferase induction in wild-type or Trim21-deficient MEFs after challenge with Ab-coated wild-type and ΔSifA Salmonella over uncoated bacteria. (g) NF-κB luciferase induction after infection of wild-type MEF or Trim21-deficeint MEFs with a dilution series of ΔSifA Salmonella incubated with PBS or anti-LPS Ab. For panels a, b, d-g, error bars represent SEM from three replicates.
Mentions: Next we investigated whether TRIM21 signaling is pathogen-dependent. We reasoned that a cytoplasmically accessible Ab-bound pathogen should provoke signaling whereas a pathogen that sheds its antibody before penetrating the cytosol should not. We chose two contrasting viruses to test this hypothesis; the non-enveloped feline calicivirus (FCV), which may carry antibodies with it into the cell upon infection and enveloped respiratory syncytial virus (RSV), which fuses at or near the plasma membrane28 and should lose its attached antibodies during membrane fusion. FCV activated a NF-κB luciferase reporter in the presence of pooled feline serum IgG in wild-type but not Trim21-deicient MEFs. In contrast, infection with RSV pre-incubated with antibody resulted in similar NF-κB luciferase reporter activitation in wild-type and Trim21-deficient MEFs (Fig. 6a, b). Similarly, and in contrast to AdV6,10, incubation of RSV with neutralizing antibody resulted in similar levels of neutralization between untreated, TRIM21 shRNA-treated or IFNα-treated HeLa cells (Supplementary Fig. 6).

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