TRIM21enhances IL-17A signaling and drives autoimmune myocarditis by promoting TRAF3 lysosomaldegradation in cardiac fibroblasts

Figures

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  Fig. 1.. TRIM21 is significantly upregulated in hearts of human DCM and EAM mice. (A) TRIM21 mRNA levels in human cardiac tissue were analyzed in three datasets, including nonfailing donors (NFD; n = 166) and DCM patients (n = 166) from GSE141910, donors with normal left ventricular ejection fraction (N-LVEF; n = 8) and DCM patients (n = 47) from GSE120895, and healthy controls (HC; n = 6) and chronic Chagasic cardiomyopathy (CCC; n = 8) from GSE191081. (B) Trim21 expression in cardiac tissues from EAM mice at the indicated time points (Day 0: n = 8; Day 14: n = 6; Day 21: n = 7; Day 35: n = 8) was detected by qPCR. (C) TRIM21 protein levels in cardiac tissues from EAM mice at the indicated time points detected by western blot (n = 3). (D) Representative images of H&E staining with inflammation scoring (Top), Sirius Red staining with fibrosis score (Middle), and TRIM21 IHC with positive area score (Bottom) in EAM hearts at the indicated time points (n = 6 per time point). Error bars represent group means ± SD (A) or group median (B and D Right). Statistical analysis: Data in (A) were analyzed by unpaired two-tailed Student’s t test. Data in (B and D Right) were analyzed by one-way ANOVA…

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  Fig. 2.. TRIM21 deficiency protects against EAM in mice. (A–D) Six-week-old WT and Trim21 KO mice were immunized with MyHCα614-629 peptide emulsified in CFA to induce EAM and were evaluated by day 21 (n = 6 per group). (A) Representative echocardiography images with EF and FS. (B) Serum CK-MB activity. (C) Representative gross cardiac morphology, H&E staining with inflammation scores, and Sirius Red staining with fibrosis scores. (D) qPCR of Tnfα, Il6, Cxcl1, Cxcl5, and Cxcl10 in heart tissues and Gapdh normalization. (E) Heart-infiltrating immune cell populations (n = 6 per group). Estimated number and percentages of CD45+ cells in live single cells. Percentages of CD45+CD11b+Ly6G+ neutrophils and CD45+CD11b+Ly6C+ Mo/Mφ in CD45+ cells. Percentage of CD45+CD11b+Ly6Chigh Mo/Mφ in CD45+ CD11b+Ly6C+ Mo/Mφ cells. Error bars represent group means ± SD, and each data point represents an individual mouse. Statistical analysis: Data in (A Right, B and C Right and D) were analyzed by two-way ANOVA with Sidak multiple comparisons test. Data in (E) were analyzed using unpaired two-tailed Student’s t test. Significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

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  Fig. 3.. TRIM21 expression in nonhematopoietic cells is required for EAM progression. (A–D) Irradiated WT or Trim21 KO mice were transplanted with WT or Trim21 KO bone marrow to generate the following bone marrow chimeras. After 8 wk, the chimeric mice were immunized to induce EAM (n = 5 per group). Data are representative of at least two independent experiments. (A) A schematic representation of the generation of bone marrow chimeras is shown. (B–D) By day 21 after immunization, mice were killed to evaluate EAM. (B) Representative images of H&E staining (Top) and Sirius Red staining (Bottom). (C) Pathological scores for each group, including inflammation score and fibrosis score. (D) qPCR analysis of Tnfα, Il6, and Cxcl1 in hearts and Gapdh normalization. (E) Representative IF images showing TRIM21 (red) and α-SMA (green) in EAM hearts at days 0, 14, 21, and 35 postimmunization. Fluorescence intensity calculated by ImageJ (n = 5 per time point). Error bars represent group median, and each data point represents an individual mouse. Data in C–E (Right) were analyzed by one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

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  Fig. 4.. TRIM21 is induced by IL-17A and amplifies IL-17A signaling in cardiac fibroblasts (CFs). (A) Primary CMs and CFs were isolated to detect Il17ra, Il17rb, and Il17rc mRNA expression levels with expression normalized to the level of Il17rc (n = 6). (B) TRIM21 expression in CFs from WT (Left) and il17ra KO (Right) mice treated with heart tissue homogenates from EAM or control mice, analyzed by western blot. (C and D) Primary CFs isolated from WT mice were cultured and stimulated with murine IL-17A (100 ng/mL) for 0, 12, 24, 36, and 48 h. qPCR analysis of Trim21 mRNA levels (C, n = 6) and western blot analysis of protein level (D, n = 3). (E) Representative IF images showing TRIM21 (red), α-SMA (green), and DAPI (blue) in IL-17A-treated and control L929 cells (n = 6). (F) CFs from WT and Trim21 KO mice were treated with murine IL-17A (100 ng/mL) for 24 h. qPCR analysis of inflammatory and fibrotic genes (n = 6) and Gapdh normalization. (G) Western blot of IL-17A-stimulated signaling in CFs from WT and Trim21 KO mice for the indicated times (100 ng/mL, n = 3) including phosphorylated JNK, ERK, p38, and p65 with corresponding total proteins. (H–K) WT and Trim21 KO mice were…

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  Fig. 5.. TRIM21 enhances IL-17A-mediated signaling by interacting with TRAF3. (A) Myc-TRIM21 expression plasmids were transfected into HEK293T cells alongside Flag-tagged TRAF2, TRAF3, TRAF5, and TRAF6 to examine potential interactors of TRIM21. (B) CFs were transfected with EGFP-TRIM21 or EGFP-NC AAV (MOI = 150) and then treated with TRAF6 inhibitor, followed by stimulation with IL-17A (100 ng/mL). 24 h later, cells were subjected to qPCR (n = 6). (C) WT and Trim21 KO CFs were transfected with si-Traf3 and then treated with IL-17A (100 ng/mL). 24 h later, cells were subjected to qPCR (n = 6). (D) Cells expressing TRIM21 and Flag-TRAF3 were lysed and subjected to immunoprecipitation using anti-Myc beads (Top) or anti-Flag beads (Bottom), followed by western blot detection with the indicated antibodies. (E) Co-IP analysis of endogenous TRIM21 with endogenous TRAF3 was performed on WT CFs (Left) or L929 cells (Right) stimulated with IL-17A (100 ng/mL) for 24 h. (F) Molecular docking visualized the TRIM21–TRAF3 interaction site. (G) HEK293T cells were transfected with TRIM21 or its truncation mutants alongside Flag-TRAF3 for 36 h, followed by IP with anti-Flag beads and western blot…

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  Fig. 6.. TRIM21 promotes lysosomal degradation of TRAF3. (A) TRAF3 level in HEK293T cells transfected with Flag-TRAF3 and increasing doses of Myc-TRIM21, detected by western blot. (B) TRAF3 level in L929 cells transfected with Myc-TRIM21 at increasing doses and stimulated with IL-17A (100 ng/mL), detected by western blot. (C) Relative Traf3 mRNA levels in CFs or L929 cells infected with EGFP-Trim21 or EGFP-NC AAV and stimulated with IL-17A, detected by qPCR (n = 6). (D) HEK293T cells transfected with Flag-TRAF3 and Myc-TRIM21 or control vectors for 18 h were treated with the protein synthesis inhibitor CHX (10 μM) for the indicated times. Flag-TRAF3 levels were detected by western blot (n = 3). (E) TRAF3 levels in HEK293T cells treated with MG132 (10 μM), CQ (10 μM), or NH4Cl (10 mM) posttransfection with Flag-TRAF3 and Myc-TRIM21. Flag-TRAF3 levels were detected by western blot (n = 3). (F) TRIM21 or its truncation mutants were transfected into HEK293T cells alongside Flag-TRAF3 for 24 h and were subjected to immunoblotting with the indicated antibodies. (G and H) Endogenous TRAF3 ubiquitination assessed in TRIM21 overexpressing L929 cells or Trim21 KO primary CFs. (I and J) HEK293T…

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  Fig. 7.. TRIM21 knockdown in CFs attenuates myocarditis and fibrosis in mice. (A) Schematic representation of the CF-specific Trim21 knockdown strategy. (B) Mice received AAV9-Postn-NC-RNAi or AAV9-Postn-Trim21-RNAi at day 21 post-EAM and were killed at day 45 (n = 3 per group). TRIM21 protein levels in α-SMA-positive CFs isolated from these hearts were detected by western blot. (C–F) Mice received AAV9-Postn-NC-RNAi or AAV9-Postn-Trim21-RNAi at day 21 post-EAM and were evaluated at day 45 (n = 6 per group). (C) Serum CK-MB activity. (D) Echocardiography (EF, FS). (E) Gross heart morphology; H&E with inflammation score; Sirius Red with fibrosis score. (F) qPCR of Tnfα, Cxcl1, Il6, Csf2, Cxcl5, and Cxcl10 in whole hearts and Gapdh normalization. Error bars represent group means ± SD. Each data point represents an individual mouse. Statistical analysis: Data were analyzed by two-way ANOVA with Sidak multiple comparisons test. Significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.