HUWE1 mediates inflammasome activation and promotes host defense against bacterial infection

Abstract

The mechanism by which inflammasome activation is modulated remains unclear. In this study, we identified an AIM2-interacting protein, the E3 ubiquitin ligase HUWE1, which was also found to interact with NLRP3 and NLRC4 through the HIN domain of AIM2 and the NACHT domains of NLRP3 and NLRC4. The BH3 domain of HUWE1 was important for its interaction with NLRP3, AIM2, and NLRC4. Caspase-1 maturation, IL-1β release, and pyroptosis were reduced in Huwe1-deficient bone marrow-derived macrophages (BMDMs) compared with WT BMDMs in response to stimuli to induce NLRP3, NLRC4, and AIM2 inflammasome activation. Furthermore, the activation of NLRP3, NLRC4, and AIM2 inflammasomes in both mouse and human cells was remarkably reduced by treatment with the HUWE1 inhibitor BI8622. HUWE1 mediated the K27-linked polyubiquitination of AIM2, NLRP3, and NLRC4, which led to inflammasome assembly, ASC speck formation, and sustained caspase-1 activation. Huwe1-deficient mice had an increased bacterial burden and decreased caspase-1 activation and IL-1β production upon Salmonella, Francisella, or Acinetobacter baumannii infection. Our study provides insights into the mechanisms of inflammasome activation as well as a potential therapeutic target against bacterial infection.

Keywords: Immunology; Inflammation; Innate immunity; Macrophages; Ubiquitin-proteosome system.

Figure 1. HUWE1 interacts with NLRP3, AIM2, and NLRC4.

(A and B) Immunoblot analysis of HUWE1 co-IP with FLAG-NLRP3, FLAG-AIM2 (A), and FLAG-NLRC4 (B) from lysates of HEK293T cells transfected with the indicated plasmids. (C) Illustration of AIM2, NLRP3, NLRC4, ASC, and caspase-1 domains. (D) Immunoblot analysis of HUWE1 co-IP with FLAG-AIM2-HIN, FLAG-NLRP3-NACHT, and FLAG-NLRC4-NACHT from lysates of HEK293T cells transfected with the indicated plasmids. (E) Schematic representation of fragments from HUWE1 protein. (F–H) Immunoblot analysis of FLAG-NLRP3 (F), FLAG-AIM2 (G), and FLAG-NLRC4 (H) interaction with HA-tag–fused HUWE1 fragments (F3, F4, F7, and F9) from lysates of HEK293T cells transfected with the indicated plasmids. Data are representative of 3 independent experiments.

Figure 2. HUWE1 deficiency impairs NLRP3, AIM2, and NLRC4 inflammasome activation.

(A–C) CreER-expressing Huwe1^+/Y and Huwe1^fl/Y BMDMs were treated with 4-OHT (100 nM) for 5 days to induce Huwe1 gene deletion. Immunoblot analysis of pro–caspase-1 (Pro-Casp1) and its subunit p20 (A) and analysis of IL-1β release (B) and cell death (C) in 4-OHT–treated Huwe1^+/Y and Huwe1^fl/Y BMDMs without treatment (Media) or further stimulated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 50 min) for NLRP3 inflammasome activation, or infected with F. novicida (200 MOI, 16 h) for AIM2 inflammasome activation, or Salmonella (3 MOI, 2 h) for NLRC4 inflammasome activation, or Listeria (50 MOI, 3 h) for both NLRP3 and AIM2 inflammasome activation. (D–F) Immunoblot analysis of pro-caspase-1 (Pro-Casp1) and its subunit p20 (D) and analysis of IL-1β release (E) and cell death (F) in Lyz2-Cre–expressing Huwe1^+/Y and Huwe1^fl/Y BMDMs without treatment or further stimulated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 50 min) for NLRP3 inflammasome activation; or transfected with dsDNA (0.5 μg, 8 h) for AIM2 inflammasome activation; or infected with F. novicida (200 MOI, 16 h) for AIM2 inflammasome activation, Salmonella (3 MOI, 2 h) for NLRC4 inflammasome activation, or Listeria (50 MOI, 3 h) for both NLRP3 and AIM2 inflammasome activation. Each dot represents an individual experiment (B, C, E, and F). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-sided Student’s t test without multiple-comparisons correction. Data are representative of 3 (A and D) or 4 (B, C, E, and F) independent experiments. Med, media.

Figure 3. The HUWE1 inhibitor BI8622 reduces NLRP3, AIM2, and NLRC4 inflammasome activation.

(A–C) Immunoblot analysis of pro–caspase-1 and its subunit p20 (A), analysis of IL-1β release and TNF and IL-6 production (B), and analysis of cell death (C) in control DMSO-treated or BI8622-treated WT BMDMs without treatment or further stimulated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 50 min) for NLRP3 inflammasome activation; or transfected with dsDNA (1.5 μg, 2 h) for AIM2 inflammasome activation; or infected with F. novicida (200 MOI, 16 h) for AIM2 inflammasome activation, Salmonella (3 MOI, 2 h) for NLRC4 inflammasome activation, or Listeria (50 MOI, 3 h) for both NLRP3 and AIM2 inflammasome activation. (D and E) Analysis of IL-1β release and TNF and IL-6 production (D) and cell death (E) in control DMSO-treated or BI8622-treated THP-1 cells without treatment or further stimulated with LPS (1 μg/mL, 6 h) and ATP (10 mM, 2 h); or transfected with dsDNA (1.5 μg, 10 h); or infected with Salmonella (6 MOI, 6 h). (F and G) Analysis of IL-1β release and TNF and IL-6 production (F) and cell death (G) in control DMSO-treated or BI8622-treated human PBMCs without treatment or further stimulated with LPS (1 μg/mL, 6 h) and ATP (10 mM, 2 h); or transfected with dsDNA (1.5 μg, 10 h); or infected with Salmonella (6 MOI, 6 h). Each dot represents an individual experiment. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-sided Student’s t test without multiple-comparisons correction. Data are representative of 3 (A and D–G) or 4 (B and C) independent experiments.

Figure 4. HUWE1 mediates the K27-linked polyubiquitination of NLRP3, AIM2, and NLRC4.

(A–C) Co-IP analysis of WT K27- and K27-mutant (K27R-linked) polyubiquitination of NLRP3 (A), AIM2 (B), and NLRC4 (C) mediated by HUWE1 in 293T cells transfected with the indicated plasmids. (D–F) Co-IP analysis of the polyubiquitination of WT AIM2 and AIM2 mutants (K23R, K26R, K64R, and K84R/K85R/K86R) (D), WT NLRP3 and NLRP3 mutants (K84R/K86R/K87R, K64R, K46R, K34R, and K21R/K22R/K24R) (E), and WT NLRC4 and NLRC4 mutants (K21R, K53R, K61R, and K71R) (F) mediated by HUWE1 in 293T cells transfected with the indicated plasmids. Data are representative of 3 independent experiments. Ub, ubiquitin.

Figure 5. HUWE1 increases NLRP3, AIM2, and NLRC4 inflammasome activation in 293T cells.

(A) Immunoblot analysis of pro–caspase-1 and its subunit p20, HUWE1, NLRP3, and ASC in 293T cells transfected with the indicated plasmids followed by control (Ctrl) or nigericin (Nig) stimulation (10 μg/mL) for 2 hours. Plot shows quantification analysis of the signaling intensity of p20 relative to GAPDH. (B) Immunoblot analysis of pro–caspase-1 and its subunit p20, HUWE1, NLRC4, and ASC in 293T cells transfected with the indicated plasmids followed by Salmonella (Sal) infection (3 MOI) for 2 hours. Plot shows quantification analysis of the signaling intensity of p20 relative to GAPDH. (C) Immunoblot analysis of pro–caspase-1 and its subunit p20, HUWE1, AIM2, and ASC in 293T cells stably expressing AIM2 and caspase-1, transfected with HUWE1 and ASC as indicated. Plot shows quantification analysis of the signaling intensity of p20 relative to GAPDH. (D) Immunoblot analysis of pro–caspase-1 and its subunit p20 in 293T cells transfected with WT HUWE1 or the HUWE1 C4341A mutant (C/A) and other plasmids as indicated, followed by stimulation as in A–C. Each dot represents an individual experiment (A–C). ***P < 0.001, by 2-sided Student’s t test without multiple-comparisons correction. Data are representative of 3 independent experiments.

Figure 6. K27 ubiquitin promotes HUWE1-mediated NLRP3, AIM2, and NLRC4 inflammasome activation.

(A) Immunoblot analysis of pro–caspase-1 and its subunit p20 in 293T cells transfected with WT NLRP3, AIM2, NLRC4 and their mutants in the presence of HUWE1 and other plasmids as indicated, followed by stimulation for NLRP3 (nigericin, 10 μg/mL, 2 hours), AIM2 (dsDNA, 2 μg/mL, 2 hours), and NLRC4 (Salmonella, 3 MOI, 2 hours) inflammasome activation. (B) Immunoblot analysis of pro–caspase-1 and its subunit p20 in 293T cells transfected with WT NLRP3, AIM2, and NLRC4 in the presence or absence of HUWE1 and K27 ubiquitin and other plasmids as indicated, followed by stimulation for NLRP3, AIM2, and NLRC4 inflammasome activation as in A. Data are representative of 3 independent experiments.

Figure 7. HUWE1 interacts with endogenous NLRP3, AIM2, and NLRC4 and regulates their ubiquitination.

(A) Co-IP analysis of endogenous HUWE1 and NLRP3, AIM2, and NLRC4 in Lyz2-Cre–expressing Huwe1^+/Y and Huwe1^fl/Y BMDMs with or without NLRP3 inflammasome stimulation (LPS, 500 ng/mL, 4 h and ATP, 5 mM, 10 min), AIM2 inflammasome stimulation (F. novicida, 100 MOI, 12 h), or NLRC4 inflammasome stimulation (Salmonella, 3 MOI, 1 h). (B) Co-IP analysis of polyubiquitination of endogenous NLRP3, AIM2, and NLRC4 in Lyz2-Cre–expressing Huwe1^+/Y and Huwe1^fl/Y BMDMs stimulated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 10 min) or infected with F. novicida (100 MOI, 12 h) or Salmonella (3 MOI, 1 h) as indicated. Data are representative of 3 independent experiments.

Figure 8. HUWE1 promotes inflammasome assembly and ASC speck formation.

(A) Immunoblot analysis of NLRP3 oligomerization in Nlrp3^–/– and Lyz2-Cre–expressing Huwe1^+/Y and Huwe1^fl/Y BMDMs treated with DSS for 30 minutes at the indicated concentrations. (B) Confocal microscopic analysis of NLRP3 and HUWE1 in Lyz2-Cre–expressing Huwe1^+/Y and Huwe1^fl/Y BMDMs without treatment or stimulated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 20 min). Arrows indicate the distribution of large puncta. Scale bars: 10 μm. (C) Confocal microscopic analysis of ASC speck formation in Lyz2-Cre–expressing Huwe1^+/Y and Huwe1^fl/Y BMDMs without treatment or stimulated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 20 min); or transfected with dsDNA (1.5 μg, 30 min); or infected with F. novicida (100 MOI, 12 h) or Salmonella (3 MOI, 1 h) as indicated. Arrows indicate ASC specks. Scale bars: 10 μm. (D) Co-IP analysis of caspase-1 that coimmunoprecipitated with ASC in Lyz2-Cre–expressing Huwe1^+/Y and Huwe1^fl/Y BMDMs without treatment or stimulated with LPS (500 ng/mL, 4 h) and nigericin (10 μg/mL, 30 min) or infected with F. novicida (100 MOI, 12 h) or Salmonella (3 MOI, 1 h) as indicated. Data are representative of 3 independent experiments.

Figure 9. HUWE1 deficiency increases host susceptibility to Salmonella, F. novicida, and A. baumannii infection.

Huwe1^fl/fl-CreER and Huwe1^+/+-CreER mice were injected with tamoxifen (2 mg/100 μL per mouse) for 5 consecutive days. Five days after the last injection, mice were infected with bacterial pathogens, and the bacterial burden and host immune responses were analyzed. (A and B) Tamoxifen-treated Huwe1^fl/fl-CreER and Huwe1^+/+-CreER mice were infected intraperitoneally with 5000 CFU Salmonella, and body weight change after infection (A) and bacterial burden in the spleen and liver on day 2 after infection (B) were measured. (C and D) Tamoxifen-treated Huwe1^fl/fl-CreER and Huwe1^+/+-CreER mice were infected subcutaneously with 1.5 × 10⁵ CFU F. novicida, and (C) body weight change after infection and (D) bacterial burden in the spleen and liver on day 2 after infection were measured. (E) H&E staining of liver sections from uninfected and Salmonella- or F. novicida–infected mice in B and D. Dashed outlines indicate infiltrated immune cells. Scale bars: 100 μm. (F) Tamoxifen-treated Huwe1^fl/fl-CreER and Huwe1^+/+-CreER mice were infected intranasally with 5.0 × 10⁸ CFU A. baumannii, and bacterial burden in the lungs on day 1 after infection was measured. (G) H&E staining of lung sections from uninfected and A. baumannii–infected mice in F. Scale bars: 100 μm. (H and I) Immunoblot analysis of pro–caspase-1 and its subunit p20 in the liver, spleen, and lungs of uninfected, Salmonella-infected (H), F. novicida–infected, and A. baumannii–infected (I) mice. GAPDH was used as a loading control. (J) ELISA analysis of IL-1β, TNF, and IL-6 in sera from uninfected and bacteria-infected mice in B, D, and F. Each dot represents an individual mouse (B, D, F, and J). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-sided Student’s t test without multiple-comparisons correction. Data are representative of 2 independent experiments.