APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

Figures

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  Fig. 1. APIP upregulates the NLRP3 inflammasome activity and pyroptosis in macrophages. a, b APIP expression in BMDMs from Apipfl/fl (control) and Lyz2-cre; Apipfl/fl (Apip cKO) mice, untreated (NT) or LPS-stimulated (200 ng/ml, 3 h), analyzed by western blotting (a) and qRT-PCR (b). c–e IL-1β secretion and caspase-1 activation in control and Apip cKO BMDMs after LPS (200 ng/ml, 3 h) and ATP (3 mM, 30 min) treatment. L + A; LPS + ATP. Supernatants (Sup) and lysates (Cell) were analyzed by western blotting (c), and IL-1β (d) and caspase-1 (e) signals quantified. Data represent mean ± SD (n = 4 independent cultures; panels b, d, e). f, g APIP expression in WT and APIPTG/+ BMDMs analyzed by western blotting (f) and qRT-PCR (g). Data represent mean ± SD (n = 3 independent cultures). h–j IL-1β secretion and caspase-1 activation in WT and APIPTG/+ BMDMs after LPS (500 ng/ml, 3 h) and ATP (3 mM, 15 min or 30 min) treatment (WT, n = 5; APIPTG/+, n = 6 independent cultures) (h, i). Cells treated with LPS (1 μg/ml, 3 h), and ATP (3 mM, 30 min), and FAM-YVAD-FMK were stained with Hoechst and observed under a fluorescence microscope (j). FAM-YVAD-FMK–positive cells were quantified from 4 (NT) or 10…

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  Fig. 2. APIP promotes the NLRP3 inflammasome formation upon stimulation. a–c Stimulatory effect of APIP on ASC oligomerization. Control and Apip cKO BMDMs were untreated (NT) or treated with LPS (200 ng/ml, 3 h) and ATP (3 mM, 30 min) (a, b). WT and APIPTG/+ BMDMs were treated with LPS (1 μg/ml, 3 h), Takinib (10 μM, 30 min), and ATP (3 mM, 30 min) (c). Cell lysates were incubated with DSS (2 mM, 1 h), and ASC pellets and lysates were analyzed by western blotting (a, c). Pellet signals were quantified and normalized to ASC levels in lysates (b). Data represent mean ± SD (n = 3 independent cultures). d Co-IP analysis of the ASC–NLRP3 interaction. WT and APIPTG/+ BMDMs treated as in (c) were subjected to Co-IP using an anti-ASC antibody. Input, 5%. e, f Stimulatory effect of APIP on ASC speck formation. Control and Apip cKO BMDMs were treated with LPS (1 μg/ml, 3 h) and ATP (3 mM, 30 min) (e). WT and APIPTG/+ BMDMs were treated with LPS (500 ng/ml, 3 h) and ATP (3 mM, 30 min) (f). Cells and nuclei were stained with anti-ASC antibody and Hoechst 33342, respectively. ASC speck–positive cells (magenta arrowheads; representative) were quantified from 2 (f, NT), 4 (e, NT), or 10 (LPS + ATP)…

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  Fig. 3. APIP promotes the caspase-11 non-canonical inflammasome activation by intracellular LPS. a–f Effect of APIP on non-canonical inflammasome activation. Control and Apip cKO BMDMs (a–c), or WT and APIPTG/+ BMDMs (d–f) were treated with Pam3CSK4 (1 μg/ml, 4 h) and MCC950 (10 μM, 1 h), and transfected with LPS (2.5 μg/ml, 16 h). Supernatants were analyzed by LDH assay (a, d). Cell lysates were analyzed by western blotting (b, e), and blot signals quantified by densitometry (c, f). LPS (tf), LPS transfection; NT-GSDMD, N-terminal GSDMD. Data represent mean ± SD. (n = 4 independent cultures; c n = 5). g–l Effect of APIP on caspase-11 expression. Control and Apip cKO BMDMs (g–i), or WT and APIPTG/+ BMDMs (j–l) were treated with LPS (1 μg/ml, 3 h). Cell lysates were analyzed by western blotting (g, j), and signals of pro-CASP11 (p38, arrow) and APIP were quantified by densitometry (h, i, k, l). Data represent mean ± SD. (n = 4 independent cultures; h n = 3). Statistical tests: two-way ANOVA with Tukey’s multiple comparison test (a, h, k) or unpaired two-tailed Student’s t-test (c, d, f, i, l). ns, non-significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Exact p values are in…

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  Fig. 4. APIP stimulates the priming of NLRP3 inflammasome activation. a–f qRT-PCR analysis of LPS-induced inflammation. J774A.1 cells were transfected with negative control siRNA (NC) or siApip (100 nM, 48 h) and left untreated (NT) or treated with LPS (500 ng/ml, 3 h) (a–d). Control and Apip cKO BMDMs were treated with LPS (200 ng/ml, 3 h) (e, f). Total RNAs were analyzed by qRT-PCR. Data represent mean ± SD (n = 3 independent cultures). g–k Western blot analysis of LPS-induced inflammation. J774A.1 cells were transfected with NC or siApip (100 nM, 48 h) and treated with LPS (500 ng/ml, 3 h) (g, h). RAW264.7 cells were transfected with Mock or APIP for 24 h and treated with LPS (1 μg/ml) alone or with Takinib (10 μM) for 3 h (i–k). Cell lysates were analyzed by western blotting (g, i), and blot signals quantified by densitometry (h, j, k). Data represent mean ± SD (n = 4 independent cultures; h n = 5 for pro-IL-1β and n = 3 for p-IκBα). Statistical tests: two-way ANOVA with Tukey’s multiple comparison test (a–d, j), unpaired two-tailed Student’s t-test (e, f), multiple unpaired t-test with Holm–Sidak’s method (h), or one-way ANOVA with Tukey’s multiple comparison test (k). ns,…

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  Fig. 5. APIP interacts with TRAF6 via its N-terminus to increase TRAF6 activity. a, b Endogenous interaction between APIP and TRAF6 in macrophages. J774A.1 cells left untreated (NT) (a) or treated with LPS (1 μg/ml, 10 or 30 min) (b), were subjected to Co-IP using an anti-APIP antibody. Input, 5%; arrows, TRAF6; asterisks, non-specific signals. c Requirement of the N-terminal region of APIP for TRAF6 binding. HEK293T cells transfected with APIP WT and deletion (∆) mutants for 24 h (upper) were analyzed by Co-IP using an anti-TRAF6 antibody (lower). Input, 5%; asterisks, light chains. d–f Effect of APIP WT and ΔN60 mutant on pro-IL-1β and p-NF-κB. RAW264.7 cells transfected with Mock, APIP WT, or ΔN60 mutant for 24 h, treated with LPS (500 ng/ml, 3 h), were analyzed by western blotting (d). Signals of pro-IL-1β (e) and p-NF-κB (f) quantified by densitometry. Data represent mean ± SD (n = 4 independent cultures). g, h Effect of APIP on TRAF6 auto-ubiquitination. RAW264.7 cells transfected with FLAG-TRAF6 alone or with APIP for 24 h (g), and J774A.1 cells transfected with NC or siApip (100 nM, 48 h) (h), were treated with MG132 (5 μM, 4 h) and LPS (1 μg/ml, 30 min). Cells were analyzed by…

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  Fig. 6. APIP exacerbates systemic inflammation in mice. a–d Effects of APIP conditional-KO on systemic inflammation. Control and Apip cKO mice (10–11-weeks-old, n = 13 mice per group) were intraperitoneally injected with LPS (E. coli O111:B4, 15 mg/kg). Survival (a) and body temperature (b) were monitored. Control and Apip cKO mice (female, 10–12 weeks) were injected with LPS (15 mg/kg, 8 h), and serum IL-1β and TNF were quantified by ELISA (c, d). e–h Effects of APIP overexpression on systemic inflammation. WT and APIPTG/+ mice (male, 9 weeks, n = 6 mice per group) were injected with LPS (E. coli O55:B5, 20 mg/kg). Survival (e) and body temperature (f) were monitored. WT and APIPTG/+ mice (10–12 weeks) were injected with LPS (15 mg/kg, 8 h) alone or with C25-140 (14 mg/kg, three doses at 12 h intervals, last dose 4 h before LPS) (g, h). MCC950 (10 mg/kg) was administered 1 h before and 3.5 h after LPS, followed by 4.5 h incubation (g). Serum IL-1β (g) and TNF (h) were quantified by ELISA. i Serum IL-1β levels in mice co-injected with LPS and MSU. WT, control, and Apip cKO mice (10–12 weeks) were injected with LPS (1.5 mg/kg), followed 4 h later by MSU crystals (50 mg/kg). After 12 h,…

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  Fig. 7. A working model illustrating the role of APIP in regulating inflammasomes and systemic inflammation. APIP binds to TRAF6 and enhances its activity upon LPS exposure. This interaction promotes the NF-κB pathway, driving transcriptional priming of the NLRP3 inflammasome and caspase-11/4 non-canonical inflammasomes, and the JNK pathway, contributing to non-transcriptional priming of the NLRP3 inflammasome. Together, these processes amplify pyroptosis in response to danger signals and exacerbate systemic inflammation in mice. This figure was created in BioRender. Kwangmin (2025) https://BioRender.com/i6tv231.