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

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.

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.

Figure 2. TRIM21 RING domain synthesizes K63-linked ubiquitin chains

(a-c) Immunoblots of in vitro ubiquitination reactions with K63-specific E2 conjugation enzymes UBC13 and UEV1A with (a) titration of TRIM21, (b) ubiquitin or mutants K63R or K63 only and (c) full-length TRIM21 or RING (ΔRING) and PRYSPRY (ΔSPRY) domain deletions. The target of each immunoblot is indicated below each panel, TRIM21 (T21), Ubiquitin (Ub). (d) Induction of NF-κB luciferase reporter in wild-type (WT) or Trim21-deficient (K21) MEF cells transduced with indicated TRIM21 constructs (EV, empty vector) upon infection with AdV + Ab. (e) Induction of NF-κB luciferase reporter upon challenge with Ab, AdV or AdV + Ab in wild-type MEF treated with negative control siRNA (NC si) or UBC13-directed siRNA. (f) Induction of NF-κB luciferase reporter upon challenge with Ab, AdV or AdV + Ab in HeLa cells treated with control or one of two UBC13-directed siRNA treatments. For panels d-f, error bars represent SEM from three replicates.

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.

Figure 4. TRIM21 signaling initiates production of proinflammatory cytokines

(a) Quantitative RT-PCR showing induction of cytokine transcripts after challenge with human serum IgG (Ab), AdV, AdV + Ab or poly I:C in wild-type (WT) or Trim21-deficient (K21) MEFs. Data are represented as fold change above PBS-treated control at 7 h post-infection. (b) ELISAs showing concentration of cytokine protein in wild-type and Trim21-deficient MEF supernatant 72 h post-challenge with indicated treatment. (c) ELISAs showing concentration of cytokines produced by Trim21-deficient expressing empty vector (K21 EV) or human TRIM21 (K21 T21) 72 h post-challenge. (d) ELISA showing concentration of IL-6 of wild-type MEF cell supernatant incubated with DMSO or panepoxydone (PPD) 72 h after challenge with indicated treatment. (e) ELISA showing concentration of IL-6 in supernatant of Trim21-deficient MEF expressing the indicated constructs or transduced with empty vector 72 h post-challenge. (f) Quantitative RT-PCR showing induction of IFN-β transcripts in wild-type and Trim21-deficient MEFs by AdV + Ab over AdV only. For all panels error bars represent SEM from three replicates.

Figure 5. Detection of intracellular antibody-bound pathogens promotes an antiviral state

(a) Fluorescence-activated cell sorting (FACS) data showing percent of wild-type (WT) or Trim21-deficient (K21) MEF cells positive for surface expression of NKG2D ligands RAE-1γ and H60 and NKG2A ligand Qa-1. (b) FACS data showing change in fluorescence intensity after staining for surface MHC class Ia allele H2-D^b expression over PBS-treated control in wild-type and Trim21-deficient MEFs. (c, d) Sindbis GFP virus infection of wild-type MEF ‘reporter’ cells which were incubated with fresh media (DMEM), media supplemented with mouse IFNα or supernatant (sup) derived from wild-type or Trim21-deficient MEFs challenged with AdV, Ab or AdV + Ab. (c) Fluorescence microscope images showing wild-type MEF reporter cell monolayers 12 h after Sindbis GFP infection. (d) Percent GFP positive wild-type reporter MEF cells relative to DMEM-treated control. (E) Fold neutralization of adenovirus by monoclonal antibody 9C12 relative to PBS-treated adenovirus after treatment of wild-type and Trim21-deficient reporter cells with Fresh media (DMEM), media supplemented with IFNα or supernatant transferred from wild-type or Trim21-deficient MEFs that were either PBS-treated or challenged with AdV + Ab. (f) Propidium iodide staining showing proportion of cells in G₁, S or G₂ 24 h after challenge with AdV or AdV complexed with recombinant 9C12 (r9C12) or 9C12 bearing a point mutation N434D (r9C12 ND). For panels a, b, d-f, error bars represent SEM from three replicates.

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.

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.

Figure 8. TRIM21 mediates an inflammatory response in primary human and mouse cells

(a) Immunoblot for TRIM21 and β-actin in normal human lung fibroblasts (NHLF) treated with control (NC) or TRIM21-directed (T21) siRNA. NF-κB luciferase reporter induction in control or TRIM21-directed siRNA (si) treated NHLF after treatment with (b) LPS or (c) AdV complexed with goat polyclonal antibody (pAb) or human serum IgG (huIgG) or RSV complexed with Synagis or huIgG. Data are represented as fold increase in NF-κB induction of virus-antibody complex over PBS-treated virus. For both adenovirus antibodies, P < 0.01, n=6, unpaired t-test (*); for both RSV antibodies, the difference was not significant (N.S.). (d) NHLF NF-κB luciferase induction after treatment with the indicated inibitors. Untreated (UT), panepoxydone (PPD), 5Z-7-oxozeaenol (5Z7O), IKK inhibitor (IKK VII), Syk inhibitor (Syk I). For panels b-d error bars represent SEM of six replicates. (e) ELISA showing IL-6 concentration in supernatant of NHLFs treated with DMSO or inhibitors PPD or 5Z7O. The concentration of IL-6 produced was significantly reduced after TRIM21 knockdown P < 0.001, n=3, unpaired t-test (**). (f) DNA binding ELISA showing binding of NF-κB subunits p65 and p50 4 h post-infection in bone marrow derived macrophages (BMDM) collected from C57BL/6 WT or Trim21^−/− mice aged 8-10 weeks. (g) ELISA showing cytokine concentration in supernatant from BMDM 72 h post-challenge. Error bars in panels e-g represent SEM of three replicates.