AGER-Mediated Lipid Peroxidation Drives Caspase-11 Inflammasome Activation in Sepsis

Abstract

Inflammasome activation can trigger an inflammatory and innate immune response through the release of cytokines and induction of pyroptosis. A dysfunctional inflammasome has been implicated in the development of human pathologies, including sepsis and septic shock. Here, we show that advanced glycosylation end-product specific receptor (AGER/RAGE) is required for caspase-11 inflammasome activation in macrophages. A nuclear damage-associated molecular pattern (nDAMP) complex, including high-mobility group box 1, histone, and DNA, can promote caspase-11-mediated gasdermin D cleavage, interleukin 1β proteolytic maturation, and lactate dehydrogenase release. The inhibition of AGER-mediated lipid peroxidation via arachidonate 5-lipoxygenase (ALOX5) limits caspase-11 inflammasome activation and pyroptosis in macrophages in response to nDAMPs or cytosolic lipopolysaccharide. Importantly, the pharmacologic inhibition of the AGER-ALOX5 pathway or global depletion (Ager^-/-) or conditional depletion of AGER in myeloid cells (Ager^Mye-/-) protects against lipopolysaccharide-induced septic death in poly(I:C)-primed mice. These data identify a molecular basis for caspase-11 inflammasome activation and provide a potential strategy to treat sepsis.

Keywords: AGER; ALOX5; DAMP; LPS; caspase-11; inflammasome; lipid peroxidation; sepsis.

Figure 1

Caspase-11 is required for nuclear DAMP complex-induced pyroptosis. Indicated LPS-primed BMDMs were treated with HMGB1 (500 ng/mL), histone (500 ng/mL), and genomic DNA (500 ng/mL) for 16 h, then cytotoxicity (A), cell viability (B), IL-1β release (C), IL-18 release (D), and IL-1α release (E) were assayed. n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test.

Figure 2

GSDMD is required for nuclear DAMP complex-induced pyroptosis. (A) Western blot analysis of indicated proteins in the supernatant (SN) or cell lysate in indicated LPS-primed BMDMs after HHD treatment (500 ng/mL, 16 h). (B–G) Analysis of cytotoxicity (B), IL-1β (C), IL-18 (D), TNF (E), IL-6 (F), and IL-12 (G) release in indicated LPS-primed BMDMs after HHD treatment (500 ng/mL, 16 h) in the absence or presence of the overexpression of GSDMD WT or D275A cDNA. n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test. Western blot data represent two independent experiments.

Figure 3

AGER is required for caspase-11 inflammasome activation. (A–C) Analysis of cytotoxicity (A), IL-1β release (B), and IL-18 release (C) in LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) in the absence or presence of FPS-ZM1 (100 nM, 500 nM, and 1 μM). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05 vs. HHD group, t test. (D–F) Analysis of cytotoxicity (D), IL-1β release (E), and IL-18 release (F) in indicated LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h), LPS electroporation (LPS_e; 1 μg, 16 h), or E. coli infection (MOI = 25, 16 h). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05 vs. WT group, t test. (G) Western blot analysis of indicated proteins in the supernatant (SN) or cell lysate in LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPS_e; 1 μg, 16 h). Western blot data represent two independent experiments.

Figure 4

AGER-mediated lipid peroxidation promotes caspase-11 inflammasome activation. (A–C) Analysis of lipoxygenase activity (A), MDA level (B), and 4-HNE level (C) in indicated LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPS_e; 1 μg, 16 h) in the absence or presence of FPS-ZM1 (1 μM). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test. (D–F) Analysis of cytotoxicity (D), IL-1β release (E), and IL-18 release (F) in indicated LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPS_e; 1 μg, 16 h) in the absence or presence of zileuton (5 μM). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test. (G) Western blot analysis of indicated proteins in the supernatant (SN) or cell lysate in LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPS_e; 1 μg, 16 h). Western blot data represent two independent experiments.

Figure 5

Depletion of AGER protects against septic shock. (A) Survival of indicated mice primed with poly(I:C) (10 mg/kg, i.p.) and then challenged 6 h later with LPS (2 mg/kg, i.p.). n = 10 mice/group, *P < 0.05, Kaplan-Meier survival analysis. (B,C) In parallel to panel A, quantitation of indicated serum markers (B) or hematoxylin/eosin staining of indicated tissues (C) in poly(I:C)-primed mice challenged with LPS at +3 h (bar = 100 μM). n = 5 mice/group, *P < 0.05, ANOVA LSD test. Animal data represent two independent experiments.

Figure 6

Pretreatment of FPS-ZM1 and zileuton protects against septic shock. (A) Survival of indicated mice primed with poly(I:C) (10 mg/kg, i.p.) and then challenged 6 h later with LPS (2 mg/kg, i.p.) in the absence or presence of the administration of FPS-ZM1 (10 mg/kg, i.p.) or zileuton (30 mg/kg, p.o.) at −0.5, +12, +24, +36, and +48 h. n = 8–12 mice/group, *P < 0.05, Kaplan-Meier survival analysis. (B,C) In parallel to panel A, quantitation of indicated serum markers (B) or hematoxylin/eosin staining of indicated tissues (C) in poly(I:C)-primed mice challenged with LPS at +3 h (bar = 100 μM). n = 5 mice/group, *P < 0.05, ANOVA LSD test. Animal data represent two independent experiments.

Figure 7

Delayed administration of FPS-ZM1 and zileuton protects against septic shock. (A) Survival of indicated mice primed with poly(I:C) (10 mg/kg, i.p.) and then challenged 6 h later with LPS (2 mg/kg, i.p.) in the absence or presence of the administration of FPS-ZM1 (10 mg/kg, i.p.) or zileuton (30 mg/kg, p.o.) at +1, +12, +24, +36, and +48 h. n = 10 mice/group, *P < 0.05, Kaplan-Meier survival analysis. (B,C) In parallel to panel A, quantitation of indicated serum markers (B) or hematoxylin/eosin staining of indicated tissues (C) in poly(I:C)-primed mice challenged with LPS at +3 h (bar = 100 μM). n = 5 mice/group, *P < 0.05, ANOVA LSD test. Animal data represent two independent experiments.

Figure 8

Schematic summary of the role of the AGER-ALOX5 pathway in the regulation of caspase-11 inflammasome activation and pyroptosis. Activation of AGER by PAMP and nDMAP promotes ALOX5-dependent lipid peroxidation. Although the precise mechanism of action of lipid peroxidation remains unknown, the alteration of cellular redox status and the production of lipid peroxides may cause conformational change of CASP11 (27). CASP11 can cleave GSDMD to produce GSDMD-N to mediate pyroptotic cell death. PAMP, pathogen-associated molecular pattern; nDAMP, nuclear damage-associated molecular patterns; AGER, advanced glycosylation end-product specific receptor; ALOX5, arachidonate 5-lipoxygenase; GSDMD-N, N terminal domain of gasdermin D.