The tetrapeptide sequence of IL-1β regulates its recruitment and activation by inflammatory caspases

Abstract

The mammalian innate immune system uses germline-encoded cytosolic pattern-recognition receptors (PRRs) to detect intracellular danger signals. At least six of these PRRs are known to form multiprotein complexes called inflammasomes which activate cysteine proteases known as caspases. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines such as IL-1β and IL-18, as well as the pore forming protein, gasdermin D (GSDMD), to induce pyroptotic cell death. In contrast, non-canonical inflammasomes, caspases-4/-5 (CASP4/5) in humans and caspase-11 (CASP11) in mice, are activated by intracellular LPS to cleave GSDMD, but their role in direct processing of inflammatory cytokines has not been established. Here we show that active CASP4/5 directly cleave IL-18 to generate the active species. Surprisingly, we also discovered that CASP4/5/11 cleave IL-1β at D27 to generate a 27 kDa fragment that is predicted to be inactive and cannot signal to the IL-1 receptor. Mechanistically, we discovered that the sequence identity of the P4-P1 tetrapeptide sequence adjacent to the caspase cleavage site (D116) regulates the recruitment and processing of IL-1β by inflammatory caspases to generate the bioactive species. Thus, we have identified new substrates of the non-canonical inflammasomes and reveal key mechanistic details regulating inflammation.

Keywords: IL-18; IL-1β; caspase-1; caspase-4; caspase-5; cytokines; non-canonical inflammasomes.

Figure 1.. The p20/10 species of human inflammatory caspases are the species that bind strongly to inflammatory substrates

(A) Schematic of human CASP1 (top), CASP4 (middle) and CASP5 (bottom) depicting the catalytic cysteines and autoproteolytic sites that give rise to the distinct caspase species. (B,D,F) HEK 293T cells stably expressing C-terminally V5-tagged GSDMD (GSDMD-V5) and Myc-tagged IL-1β (IL-1β-Myc) were transiently transfected with the indicated catalytically inactive caspase constructs. After 48 h, the cells were harvested and subjected to anti-FLAG IP followed by immunoblot analysis. GFP control was C-terminally FLAG tagged (GFP-FLAG) and the catalytically dead caspases all harbored an N-terminal 2xFLAG tag. (C,E,G) HEK 293T cells stably expressing C-terminally V5-tagged IL-18 (IL-18-V5) were transiently transfected with the indicated constructs as in B,D, and F for 48 h before the cells were harvested and subjected to anti-FLAG IP followed by immunoblotting. Data are representative of three or more independent experiments.

Figure 2.. CASP4/5 cleave IL-1β and IL-18.

(A,B) HEK 293T cells stably expressing GSDMD-V5 and IL-1β-Myc were transiently transfected with the indicated constructs. After 24 h, samples were analyzed for LDH release (A) and immunoblotting (B). (C,D) HEK 293T cells stably expressing IL-18-V5 were transiently transfected with the indicated constructs. After 24 h, samples were analyzed for LDH release (C) and immunoblotting (D). (E,F) HEK 293T cells stably expressing GSDMD-V5 and IL-1β-Myc (E) or IL-18-V5 (F) were transfected with the indicated catalytically inactive caspase constructs. After 48 h, the cells were harvested and subjected to anti-FLAG IP followed by immunoblot analysis. Data are means ± SEM of three biological replicates. ***P < 0.001, **P < 0.01 and *P < 0.05 by two-sided Student’s t-test compared with control. *Represents non-specific bands in immunoblots. Data are representative of three or more independent experiments.

Figure 3.. The P4 – P1 tetrapeptide sequence of IL-1β regulates processing by CASP1/4/5.

(A) Schematic depicting the sequence of wildtype IL-1β, IL-18, IL-1α, the caspases that were reported to cleave these substrates, and the tetrapeptide mutants. (B) HEK 293T cells stably expressing GSDMD-V5 and IL-1β-Myc were transiently co-transfected with the indicated catalytically active caspase constructs and IL-1β tetrapeptide mutants (Tet. Mut.) for 24 h prior to immunoblot analysis. (C) HEK 293T cells stably expressing IL-18-V5 were transiently co-transfected with the indicated catalytically inactive (C/A) caspase constructs and the IL-1β mutant, in which the tetrapeptide sequence was substituted for the sequence found in IL-18 (IL-1β^LESD). 48 h post transfection, samples were subjected to anti-FLAG immunoprecipitation followed by immunoblot analysis. Data are representative of two or more independent experiments.

Figure 4.. Dimerization of ∆CARD DmrB-CASP1/4/5/11 mediates processing of inflammatory substrates.

(A,B) HEK 293T Cells stably expressing ∆CARD DmrB-CASP1,−4, or −5 and GSDMD-V5 (A) or IL-18-V5 (B) were treated with AP20187 (1 μM) for 1 h or 24 h. Cell death was measured by LDH and samples were analyzed by immunoblotting. (C) HEK 293T cells stably expressing ∆CARD DmrB-CASP1,−4, or −5 and GSDMD-V5 were transiently transfected with the indicated IL-1β constructs for 24 h before the addition of AP20187 (1 μM) for 24 h. Cell death was measured by LDH and samples were analyzed by immunoblotting. (D) HEK 293T cells stably expressing ∆CARD Dmrb-CASP11 were transiently transfected with the indicated constructs. After 24 h, samples were treated with AP20187 (1 μM) for 24 h then analyzed for LDH release and immunoblotting. Data are means ± SEM of three biological replicates. ***P < 0.001, **P < 0.01 and *P < 0.05 by two-sided Student’s t-test compared with control. *Represents non-specific bands in immunoblot. Data are representative of two or more independent experiments.

Figure 5.. LPS activated non-canonical inflammasomes cleave IL-1β and IL-18.

(A) HEK 293T cells stably expressing IL-18-V5 and either CASP4 or CASP5 were transfected with LPS (25 μg/mL) for 24 h before samples were collected and analyzed by immunoblotting. (B,C) HEK 293T cells stably expressing 2xFLAG-CASP11 were transiently transfected with the indicated constructs. After 24 h, samples were transfected with LPS (25 μg/mL) for 24 h then analyzed for LDH release (B) and immunoblotting (C). Data are means ± SEM of three biological replicates. ***P < 0.001, **P < 0.01 and *P < 0.05 by two-sided Student’s t-test compared with control. Data are representative of two or more independent experiments. *Represents non-specific bands in immunoblot. The small m or h represents mouse or human versions of the proteins respectively.

Figure 6.. Cytosolic LPS and pathogenic infections induce non-canonical inflammasome mediated processing of inflammatory substrates in human macrophages and epithelial cells.

(A,B) THP1 cells were terminally differentiated into macrophages with phorbol 12-myristate 12-acetate (50 ng/mL) for 24 h and primed with LPS (5 μg/mL) for another 24 h. Where indicated, THP1 macrophages were treated with ZVAD (40 μM) or VX765 (40 μM) 30 minutes before LPS transfections. Cells were then transfected with LPS (25 μg/mL). 24 h after LPS transfection, samples were analyzed for LDH release (A) and immunoblotting (B). (C,D) THP1 cells were terminally differentiated into macrophages with phorbol 12-myristate 12-acetate (40 ng/mL) for 48 h. Cells were then treated with Legionella pneumophila (MOI = 20) for 7 h before the supernatants were analyzed for LDH release (C) and then combined with the lysates for immunoblotting (D). € Caco-2 cells were primed with 100 ng/ml Pam3CSK4 for 3 h, then infected with Salmonella Typhimurium (MOI = 60) for 6 h. Cells and their supernatants were collected separately, samples were precipitated, and analyzed by immunoblotting. Data are means ± SEM of three biological replicates. ***P < 0.001 and **P < 0.01 by two-sided Student’s t-test compared with control. *Represents non-specific bands in immunoblot. Data are representative of two or more independent experiments.

Figure 7.. CASP4/5 preferentially cleave IL-1β at D27 and IL-18 at D36.

(A,B) HEK 293T cells were transiently co-transfected with the indicated constructs. After 24 h, samples were harvested and analyzed by immunoblotting. (C) Schematic of inflammatory substrates and representative cleavage sites by inflammatory caspases. Data are representative of three or more independent experiments. *Represents non-specific bands in immunoblot.