Fig 1: TRIM21 is required for inflammasome activation.a IL-1β, IL-18, TNF, and IL-6 ELISA of cleared culture supernatant (SN) from control (Ctrl) and TRIM21KO THP-1 cells primed (LPS, 1 μg mL−1, 4 h) or primed and activated with nigericin (Nig, 10 μM, 30 min). (n = 3 biological replicates, mean ± s.d., parametric two-tailed unpaired t-test). b IL-1β ELISA of cleared SN from indicated THP-1 cells primed (LPS, 1 μg mL−1, 4 h) or primed and activated with Nig (10 μM, 30 min), Calcium Pyrophosphate Dihydrate (CPPD) crystals (125 μg mL−1, 6 h), Silica (400 μg mL−1, 6 h), TcdB (2 μg mL−1, 6 h), or primed and transfected with poly(dA:dT) (2 μg mL−1, 16 h), flagellin (100 ng mL−1, 6 h) or lethal toxin (LeTx) (1 μg mL−1, 16 h). (n = 3 biological replicates, mean ± s.d., one-way ANOVA with Dunnett’s post-test). c Immunoblot of cleaved/total caspase-1 (casp1), cleaved/total gasdermin D (GSDMD), TRIM21, ASC, NLRP3, and β-tubulin from SN or total cell lysates (TCL) from indicated THP-1 cells primed (LPS, 1 μg mL−1, 4 h) or primed and activated (Nig, 10 μM, 10, 20, 30 min). d FLICA assay of active caspase-1 (AlexaFluor647) of Ctrl and TRIM21KO THP-1 cells primed (LPS, 1 μg mL−1, 4 h) or primed and activated (Nig, 10 μM, 20 min). e LDH release from Ctrl and TRIM21KO THP-1 cells primed (LPS, 1 μg mL−1, 4 h) or primed and activated (Nig, 10 μM, 10, 20, 30 min), presented as percent cytotoxicity vs maximum LDH release (n = 3 biological replicates, mean ± s.d., parametric two-tailed unpaired t-test). f Immunoprecipitation (IP) of ASC from Ctrl, TRIM21KO, and ASCKO THP-1 cells untreated or primed (LPS, 1 μg mL−1, 4 h) and activated (Nig, 10 μM, 30 min) and analyzed by immunoblot alongside TCL for ASC, NLRP3, and β-tubulin. g Immunoblot for TRIM21, GFP and β-tubulin of Ctrl, TRIM21KO, and TRIM21KO THP-1 cells restored with GFP-TRIM21, GFP-TRIM21ΔRING, GFP-TRIM21ΔbBox/CC or GFP-TRIM21ΔPRY/SPRY. TRIM21ΔbBox/CC is detected by anti-GFP and not by anti-TRIM21 antibody (*cross-reactive protein). h IL-1β, IL-18 and TNF ELISA of cleared culture SN from restored TRIM21KO as in (g) and NLRP3KO THP-1 cells that were untreated (UT), primed with Pam3CSK4 (Pam3, 1 μg mL−1, 4 h) or primed and activated (Nig, 10 μM, 30 min) (n = 3 biological replicates, mean ± s.d., one-way ANOVA with Dunnett’s post-test). Results representative of n = 3 experiments.
Fig 2: TRIM21 is co-released with polymerized ASC during inflammasome activation.a Flow cytometric detection of polymerized TRIM21 in indicated THP-1 cells untreated (UT), primed (Pam3CSK4, Pam3, 1 μg mL−1, 4 h), primed and activated (nigericin, Nig, 10 μM, 30 min). Percent of decreased width(W):area(A) TRIM21 signal ratio represents activated cells, also presented as histogram. b, c PMA-differentiated EGFP-TRIM21/mScarlet-ASC-expressing THP-1 cells untreated, primed (LPS, 0.5 μg mL−1, 2 h), primed and activated (Nig, 5 μM, 5–45 min). Representative microscopy images (30 min) (b) and quantification of ASC+ and ASC+TRIM21+ aggregates at all timepoints shown in Supplementary Fig. 5 (c) (n = 10 frames/sample, mean ± s.d., one-way ANOVA with Dunnett’s post-test). Nuclei/DNA were stained with DAPI. Scale bar: 10 μm. d Manders’ (left) and Pearson’s (right) co-localization coefficients calculated within the speck (speck) and cell area outside the speck (cellΔspeck) (n = 10 frames, mean ± s.d., parametric two-tailed unpaired t-test). e, f EGFP-TRIM21 fluorescence intensity in cleared supernatants (SN) from EGFP-TRIM21-expressing THP-1 cells pre-treated with vehicle, PYR-41 (25 μM, 4 h) (e) or DSF (40 μM, 1 h) (f) followed or not by LPS (0.5 μg mL−1, 2.5 h) priming and Nig (10 μM, 30 min) activation. (n = 3 biological replicates, mean ± s.d., parametric two-tailed unpaired t-test). g TRIM21 ELISA of cleared SN from indicated THP-1 cells untreated, primed (LPS, 1 μg mL−1, 2 h) or primed and activated (Nig, 10 μM, 45 min). (n = 3 biological replicates, mean ± s.d., one-way ANOVA with Dunnett’s post-test). h Immunoblot of TRIM21, ASC, NLRP3, total/cleaved caspase-1 (casp1), β-tubulin using ASC particles purified from SN alongside TCL from indicated THP-1 cells untreated, primed (LPS, 0.5 μg mL−1, 2 h), primed and activated (Nig, 10 μM, 30 min). i ASC immunoprecipitation (IP) from SN and immunoblot of TRIM21, ASC, NLRP3 alongside TCL from indicated THP-1 cells untreated, primed (LPS, 1 μg mL−1, 2 h), primed and activated (Nig, 10 μM, 30 min). HC heavy-chain. j Flow cytometric detection of polymerized mScarlet-ASC+ and mScarlet-ASC+EGFP-TRIM21+ particles released from mScarlet-ASC/EGFP-TRIM21-expressing THP-1 cells untreated, primed (LPS, 0.5 μg mL−1, 2 h), primed and activated (Nig, 10 μM, 30 min), presented as particle number. k IL-1β ELISA of cleared SN from Ctrl, TRIM21KO, NLRP3KO THP-1 cells untreated, primed (LPS, 0.5 μg mL−1, 3 h) and activated with 2000(+)/5,000(++)/10,000(+++) sorted mScarlet-ASC+EGFP-TRIM21+ particles for 12 h (n = 3 biological replicates, mean ± s.d., one-way ANOVA with Dunnett’s post-test). Results representative of n = 3 experiments.
Fig 3: TRIM21 promotes ASC polymerization and release.a Flow cytometric detection of polymerized ASC in indicated THP-1 cells left untreated, primed with Pam3CSK4 (Pam3, 1 μg mL−1, 4 h) or primed and activated with nigericin (Nig, 10 μM, 30 min). Histograms represent ASC width(W):area(A) ratio. Percent of decreased W:A ASC signal represents activated cells, which is also presented as bar graph (bottom). b Immunoblot of ASC from total cell lysates (TCL) and DSS-crosslinked TCL from indicated THP-1 cells left untreated, primed with LPS (1 μg mL−1, 2.5 h) or primed and activated with Nig (10 μM, 15 min). c, d Fluorescence intensity of mScarlet-ASC in the cleared culture supernatant (SN) from mScarlet-ASC expressing THP-1 cells pre-treated with vehicle or with PYR-41 (25 μM, 4 h) (c) or DSF (40 μM, 1 h) (d), followed or not by LPS (0.5 μg mL−1, 2.5 h) priming and Nig (10 μM, 30 min) activation. (n = 3 biological replicates, mean ± s.d., parametric two-tailed unpaired t-test). e Flow cytometric detection of polymerized ASC particles released from untreated Ctrl THP-1 cells and Ctrl, TRIM21KO, ASCKO, and NLRP3KO THP-1 cells primed with LPS (0.5 μg mL−1, 2 h) and activated with Nig (10 μM, 30 min) and presented as histograms. Unstained baseline is presented in black. Results representative of n = 3 experiments.
Fig 4: TRIM21 is required for inflammasome activation in vivo.a IL-1β, IL-18, and TNF ELISA of cleared culture supernatant (SN) from Trim21fl/+ and Trim21fl/+ CreL BMDM left untreated, primed with LPS (1 μg mL−1, 4 h), or primed and activated with nigericin (Nig, 5 or 10 μM as indicated, 30 min). (n = 3 biological replicates, mean ± s.d., parametric two-tailed unpaired t-test). b Immunoblot of cleaved and total caspase-1 (casp1), cleaved and total gasdermin D (GSDMD), TRIM21, and β-tubulin loading control from SN or total cell lysate (TCL) from Trim21fl/+ and Trim21fl/+ CreL BMDM left untreated, primed with Pam3CSK4 (Pam3, 1 μg mL−1, 4 h) or primed and activated with Nig (15 μM, 20 min). c LDH release from Trim21fl/+ and Trim21fl/+ CreL BMDM left untreated, primed with LPS (1 μg mL−1, 4 h) or primed and activated with Nig (15 μM, 20 min) is presented as percent cytotoxicity compared to maximum LDH release (n = 6 biological replicates, mean ± s.d., parametric two-tailed unpaired t-test). d, e In vivo imaging of myeloperoxidase (MPO) (d) and serum IL-1β ELISA (e) in Trim21fl/+ and Trim21fl/+ CreL mice i.p.-injected with LPS (2.5 mg kg−1, 4 h) (n = 4–6 mice, mean ± s.d., parametric two-tailed unpaired t-test). f Survival of male Trim21fl/+ and Trim21fl/+ CreL mice following i.p. injection of LPS (20 mg kg−1) presented as Kaplan–Meier estimate and Log-rank (Mantel–Cox) test (n = 5 mice). Results representative of n = 2 (d–f) or n = 3 (a–c) experiments.
Fig 5: TRIM21 deficiency alleviates autoinflammation in vivo.a Representative pictures of Nlrp3A350V/+ CreL and Nlrp3A350V/+ Trim21fl/+ CreL pups at day 3 and day 8 after birth. b Body weight (left) and survival analysis (right) of wild-type (WT), Nlrp3A350V/+ CreL and Nlrp3A350V/+ Trim21fl/+ CreL pups (n = 6−11 pups, linear regression analysis [left] and Log-rank (Mantel–Cox) test [right]). c, d Flow cytometric detection and quantification of polymerized ASC+, TRIM21+ and ASC+TRIM21+ particles in the serum of Nlrp3A350V/+ and Nlrp3A350V/+ CreL pups at day 17 (c) and Mefv+/+ and MefvV726A/V726A mice at day 30–90 (d). (c: n = 6–11 pups; d: n = 7–12 pups, parametric two-tailed unpaired t-test). e Relative mRNA expression of TRIM21 as determined by RNAseq of total human blood cells (GEO accession GSE57253)36 in healthy donors (HD) and Neonatal Onset Multisystem Inflammatory Disease (NOMID) patients before and after treatment with Anakinra (n = 5–7 patients, mean ± s.d.). f, g Plasma from HD and patients with active flares of Familial Cold Autoinflammatory Syndrome (FCAS), and/or Muckle–Wells Syndrome (MWS), or NOMID was analyzed for ASC and TRIM21 by ELISA and presented as HD versus CAPS (f) and each patient sample and corresponding mutation grouped by disease (g). (n = 11 human plasma samples, f parametric two-tailed unpaired t-test; g linear regression analysis of ASC and TRIM21 present in HD, FCAS, MWS, and NOMID). h, i Sera from WT and NLRP3D301N/+CreL mice at day 10 and 25 after birth (h) and plasma from HD and CAPS patients (i) were analyzed for TRIM21 IgG by ELISA. (h: n = 3–4 pups, parametric two-tailed unpaired t-test, mean ± s.d.), (i: n = 18 human plasma samples). j Sera from NLRP3D301N/+CreL mice were analyzed for TRIM21 IgG by ELISA using full-length (FL) TRIM21 and domain-deleted TRIM21 as antigen and presented as relative units (RU) (n = 4 pups, mean ± s.d.). k Plasma samples from HD and CAPS patients were analyzed for TRIM21 IgG by ELISA using full-length TRIM21 and domain-deleted TRIM21 as antigen and presented as fold increase compared to HD. Results are representative of n = 2 experiment(s).
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