Caspase-11 controls interleukin-1β release through degradation of TRPC1

Abstract

Caspase-11 is a highly inducible caspase that controls both inflammatory responses and cell death. Caspase-11 controls interleukin 1β (IL-1β) secretion by potentiating caspase-1 activation and induces caspase-1-independent pyroptosis downstream of noncanonical NLRP3 inflammasome activators such as lipopolysaccharide (LPS) and Gram-negative bacteria. However, we still know very little about the downstream mechanism of caspase-11 in regulating inflammation because the known substrates of caspase-11 are only other caspases. Here, we identify the cationic channel subunit transient receptor potential channel 1 (TRPC1) as a substrate of caspase-11. TRPC1 deficiency increases the secretion of IL-1β without modulating caspase-1 cleavage or cell death in cultured macrophages. Consistently, trpc1(-/-) mice show higher IL-1β secretion in the sepsis model of intraperitoneal LPS injection. Altogether, our data suggest that caspase-11 modulates the cationic channel composition of the cell and thus regulates the unconventional secretion pathway in a manner independent of caspase-1.

Figure 1. Caspase-11 Interacts with TRPC1

(A) Lysates of HEK293T cells expressing HA-TRPC1 and FLAG-Casp11 were subjected to anti-HA immunoprecipitation. The western blots were analyzed using anti-caspase-11 and anti-HA. (B) Lysates of HEK293T cells expressing HA-TRPC1 and FLAG-Casp11, FLAG-Casp11-CARD98, FLAG-Casp11-CARD103, FLAG-Casp11-p30, FLAG-Casp11-p20, or FLAG-Casp11-p10 were subjected to anti-HA immunoprecipitation. The western blots were analyzed using anti-FLAG and anti-HA. (C) Lysates of HEK293T cells expressing FLAG-Casp11 and HA-TRPC1, HA-TRPC1-N, HA-TRPC1-ΔC, HA-TRPC1-ΔN, or HA-TRPC1-TM were subjected to anti-HA immunoprecipitation. Diagrams of TRPC1 association with membrane and truncation expression constructs of TRPC1 are shown; gray sections represent transmembrane spans. The western blots were analyzed using anti-caspase-11 and anti-HA. See also Figure S1.

Figure 2. TRPC1 Is a Substrate of Caspase-11 and Degraded following LPS Treatment

(A) HEK293T cells were cotransfected with expression vectors of HA-TRPC1 and FLAG-Casp11 or Casp11^C255G catalytic mutant, and cultured for 48 hr. z-VAD.fmk (20 μM) was added as indicated for the last 24 hr of culture. The levels of HA-TRPC1 protein were assessed by western blotting using anti-HA, anti-caspase-11, and anti-actin (as a control). (B) In vitro cleavage assay of ³⁵S-labeled TRPC1 by purified recombinant caspase-11 p30. (C) TRPC1 is not a substrate of caspase-1. In vitro cleavage assay of ³⁵S-labeled TRPC1 by recombinant caspase-1 p30. (D–F) Macrophages from WT (D) or casp11^−/− or casp1^−/− casp11^−/− mice (E) were treated with LPS (100 ng/ml) for 16 hr. Macrophages from WT mice were infectedwith E. coli (J53strain, moi20) for 16hr (F). The expression levels of endogenous TRPC1 were assessed by anti-TRPC1 immunoprecipitation followed by anti-TRPC1 western blotting. See also Figure S2.

Figure 3. LPS Triggers a Caspase-11- and TRPC1-Dependent Cytosolic Ca²⁺ Decrease

(A) Macrophages from WT mice were treated with LPS (100 ng/ml) for the indicated times. Intracellular Ca²⁺ was measured through ratiometric imaging of Fura-2AM (F340/F380). Error bars indicate SE from the mean. (B) WT, trpc1^−/−, and casp11^−/− macrophages were treated with LPS (100 ng/ml, red bars) for 10 hr or left untreated (blue bars). Intracellular Ca²⁺ was measured through ratiometric imaging of Fura-2AM (F340/F380). Error bars indicate SE.

Figure 4. TRPC1 Inhibits Mature IL-1β Release

(A) Trpc1^−/− mice secrete more IL-1β than WT mice in the serum following LPS challenge. WT and trpc1^−/− mice were injected intraperitoneally with LPS (4 mg/kg). The sera were harvested after 4 hr (n = 6) or 8 hr (n = 8–9). IL-1β secretion in sera was assessed by ELISA. Error bars represent SE. Untreated mice were used as negative control (−). t test, *p < 0.05. (B) Macrophages from WT, trpc1^−/−, and casp11^−/− mice were infected with E. coli for 16 hr, and secretion of mature IL-1β in the culture supernatant was assessed by ELISA. Error bars indicate SD from the mean. (C and D) Caspase-11, caspase-1, IL-1β, IL-18 expression level, cleavage, and secretion were assessed by western blotting of cell lysates and TCA-precipitated culture media. (E) Macrophages from WT, trpc1^−/−, and casp11^−/− mice were treated with LPS (100 ng/ml, 8 hr) followed by ATP (30 min), HLLOMe (30 min), or silica (2 hr) at the indicated concentrations. Mature IL-1β in the culture supernatant was assessed by ELISA. Error bars indicate SD. (F) Caspase-11, caspase-1, IL-1β expression level, cleavage, and secretion were assessed by western blot on cell lysates and TCA-precipitated culture media. See also Figure S3.