Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense

Abstract

Caspases regulate cell death, immune responses, and homeostasis. Caspase-6 is categorized as an executioner caspase but shows key differences from the other executioners. Overall, little is known about the functions of caspase-6 in biological processes apart from apoptosis. Here, we show that caspase-6 mediates innate immunity and inflammasome activation. Furthermore, we demonstrate that caspase-6 promotes the activation of programmed cell death pathways including pyroptosis, apoptosis, and necroptosis (PANoptosis) and plays an essential role in host defense against influenza A virus (IAV) infection. In addition, caspase-6 promoted the differentiation of alternatively activated macrophages (AAMs). Caspase-6 facilitated the RIP homotypic interaction motif (RHIM)-dependent binding of RIPK3 to ZBP1 via its interaction with RIPK3. Altogether, our findings reveal a vital role for caspase-6 in facilitating ZBP1-mediated inflammasome activation, cell death, and host defense during IAV infection, opening additional avenues for treatment of infectious and autoinflammatory diseases and cancer.

Keywords: AAMs; NLRP3; PANoptosis; RIPK1; RIPK3; ZBP1; apoptosis; caspase-1; caspase-6; caspase-8; inflammasome; influenza A virus; necroptosis; pyroptosis.

Figure 1.. Caspase-6 Promotes IAV-Induced ZBP1-Mediated NLRP3 Inflammasome Activation

(A) Immunoblots analysis of pro- (p45, black arrow) and cleaved caspase-1 (p20, red arrow; CASP1) in LPS plus ATP-treated bone-marrow-derived macrophages (BMDMs). (B) Assessment of IL-18 release by ELISA following LPS plus ATP treatment. (C) Assessment of IL-1β release by ELISA following LPS plus ATP treatment. (D) Images of BMDMs after LPS plus ATP treatment. (E–H) Immunoblot analysis of CASP1 (E), IL-18 release (F), IL-1β release (G), and cell images (H) from BMDMs primed with LPS and then transfected with LPS. (I–L) Immunoblot analysis of CASP1 (I), IL-18 release (J), IL-1β release (K), and cell images (L) from BMDMs after influenza A virus (IAV) infection for 16 h. (M–P) Immunoblot analysis of CASP1 (M), IL-18 release (N), IL-β release (O), and cell images (P) from BMDMs after P. aeruginosa (MOI, 2) infection for 2–3 h. (Q–T) Immunoblot analysis of CASP1 (Q), IL-18 release (R), IL-1β release (S), and cell images (T) of BMDMs after F. novicida (MOI, 100) infection for 20 h. The red arrows indicate pyroptotic cells (D, H, L, P, T). NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way ANOVA). Data are representative of three independent experiments. Data are shown as mean ± SEM (B, C, F, G, J, K, N, O, R, and S). The original magnification of all images is 320. See also Figure S1.

Figure 2.. Deficiency of Caspase-6 Does Not Influence NLRP3 Priming and Viral Replication in Bone-Marrow-Derived Macrophages

(A) Immunoblot analysis of phosphorylated IκBα (pIκBα), total IκBα (tIκBα), phosphorylated ERK (pERK), total ERK (tERK), NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), pro–IL-1β, and pro- caspase-6 (pro-CASP6) in bone-marrow-derived macrophages (BMDMs) after influenza A virus (IAV) infection at the indicated time points. Actin is used as the internal control. (B) Immunoblot analysis of pro- and cleaved caspase-1 (CASP1) in BMDMs primed with or without Pam3CSK4 (PAM3) for 5 h and then infected with IAV for 16 h. (C) Immunoblot analysis of phosphorylated STAT1 (pSTAT1), total STAT1 (tSTAT1), Z-DNA binding protein 1 (ZBP1), influenza non-structural protein 1 (NS1), and influenza matrix protein 1 (M1) in BMDMs after IAV infection at the indicated time points. Actin is used as the internal control. (D) Real-time PCR analysis of IAV M1 mRNA or vRNA in BMDMs after infection at the indicated time points, presented relative to levels of the host gene actin. (E) Endpoint replication of IAV (MOI, 10) in BMDMs for 8 h. NS, not significant; *p < 0.05; ***p < 0.001 (two-way ANOVA). Data are shown as mean ± SEM (D and E). Data are representative of three independent experiments.

Figure 3.. Caspase-6 Promotes IAV-Induced Pyroptosis, Apoptosis, and Necroptosis

(A) Real-time analysis of cell death in bone-marrow-derived macrophages (BMDMs) using the IncuCyte imaging system and SYTOX Green nucleic acid staining after infection with influenza A virus (IAV) for 12 h. The original magnification is ×20. The red denotes the cells counted as dead in the analysis. (B) Quantification of the cell death observed in (A). (C) Immunoblot analysis of caspase-6 (CASP6) in mouse embryonic fibroblasts (MEFs) following CRISPR-directed deletion. (D) Microscopic analysis of cell death in MEFs infected with IAV for 24 h. The original magnification is ×10. (E) Quantification of the cell death observed in (D). (F) Immunoblot analysis of the pro-and cleaved forms of caspase-3 (CASP3), caspase-7 (CASP7), and gasdermin D (GSDMD) in BMDMs after IAV infection for 9 h. Actin is used as the internal control. (G) Immunoblot analysis of the pro- and cleaved forms of CASP3 and CASP7 in MEFs after IAV infection for 24 h. Actin is used as the internal control. (H) Immunoblot analysis of pro- and cleaved caspase-8 (CASP8) in BMDMs after IAV infection for 9 h. Actin is used as the internal control. (I) Immunoblot analysis of pro- and cleaved CASP8 in MEFs after IAV infection for 24 h. Actin is used as the internal control. (J) Immunoblot analysis of phosphorylated mixed lineage kinase domain-line (pMLKL) and total MLKL (tMLKL) in BMDMs after IAV infection at the indicated time points. Actin is used as the internal control. ****p < 0.0001. Analysis was performed using the Student’s t test (B) or one-way ANOVA (E). Data are shown as mean ± SEM (B and E). See also Figure S2.

Figure 4.. Caspase-6 Contributes to Host Protection against IAV Infection In Vivo

(A) Body weight of 6 to 8-week-old wild-type (WT) and Casp6^−/− mice infected intranasally with 50 plaque forming units (PFUs) of influenza A virus (IAV). (B) Survival of 6- to 8-week-old WT and Casp6^−/− mice infected intranasally with 50 PFUs of IAV. (C) Survival of 6- to 8-week-old littermate Casp6^+/− and Casp6^−/− mice infected intranasally with 125 PFUs of IAV. (D) Immunohistochemistry staining of viral nucleoprotein (NP) in the lungs collected from WT and Casp6^−/− mice at day 5 post-infection. (E and F) Lung viral titers in WT and Casp6^−/− mice infected with IAV for 3 days (E) or 7 days (F). (G and H) IL-1β levels in the bronchoalveolar lavage fluid (BALF) from WT and Casp6^−/− mice infected with IAV for 3 days (G) or 7 days (H). (I) Quantification of the percentage of the lung lesioned in WT and Casp6^−/− mice infected with IAV for 5 days. (J) Microscopic analysis of Arg1+ cells in the lung tissue of WT and Casp6^−/− mice infected with IAV for 7 days. The original magnification is ×10 and ×40 as indicated. NS, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001. Analysis was performed using the Student’s t test (E–I), two-way ANOVA (A), and log-rank test (B and C). Data are shown as mean ± SEM (A and E–I). Data are pooled from 4 independent experiments (A and B).

Figure 5.. Caspase-6 Is Critical for Enhancing the Interaction between RIPK3 and ZBP1

(A) Immunoblot analysis of pro- and cleaved forms of caspase-6 (CASP6), −8 (CASP8), −7 (CASP7), −3 (CASP3), and gasdermin D (GSDMD) in bone-marrow-derived macrophages (BMDMs) after influenza A virus (IAV) infection for 12 h. Actin is used as the internal control. (B) Immunoblot analysis of phosphorylated mixed lineage kinase domain-like (pMLKL) and total MLKL (tMLKL) in BMDMs after IAV infection at the indicated time points. Actin is used as the internal control. (C) Schematic depiction of the relationship between receptor-interacting protein homotypic interaction motif (RHIM) domain-containing proteins in the Z-DNA binding protein 1 (ZBP1)-initiated cell death complex and caspase-6 after IAV infection. The red boxes indicate the RHIM domains. (D) Immunoblot analysis of pro- and cleaved forms of CASP6, CASP8, CASP7, CASP3, and GSDMD in BMDMs after IAV infection for 9 h. Actin is used as the internal control. (E) Immunoblot analysis of pMLKL and tMLKL in BMDMs after IAV infection at the indicated time points. Actin is used as the internal control. (F) Immunoprecipitates and total lysates from 293T cells after co-transfection of FLAG-CASP6 with receptor-interacting protein kinase (RIPK) 1, RIPK3-GFP, ZBP1, or CASP8 for 30 h. (G) Immunoprecipitates and total lysates from 293T cells after co-transfection with RIPK1, RIPK3-GFP, and ZBP1 in the absence or presence of FLAG-CASP6 for 30 h. (H) Immunoprecipitates and total lysates from BMDMs with IAV infection for 16 h. Data are representative of three independent experiments. See also Figures S3, S4, and S5.

Figure 6.. Both N- and C-Terminal Domains of Caspase-6 Are Critical for the Binding of RIPK3 to ZBP1

(A) Schematic depiction of the domains in mouse CASP6 and RIPK3 in the full-length construct (F), N-terminal construct (N), and C-terminal construct (C). The aspartic acid sites shown denote the mouse CASP6 cleavage sites, and the cysteine denotes the catalytic site. L, large subunit; RHIM, receptor-interacting protein homotypic interaction motif; S, small subunit. (B) Immunoprecipitates and total lysates from 293T cells after co-transfection with the indicated forms of CASP6 and RIPK3 for 30 h. (C) Immunoprecipitates and total lysates from 293T cells after co-transfection with RIPK1, RIPK3-GFP, and Z-DNA binding protein 1 (ZBP1) in the absence or presence of full-length or truncated CASP6 for 30 h. Data are representative of three independent experiments. CA, CASP6-C146A. See also Figure S6.