Caffeic acid, but not ferulic acid, inhibits macrophage pyroptosis by directly blocking gasdermin D activation

Abstract

Regulated pyroptosis is critical for pathogen elimination by inducing infected cell rupture and pro-inflammatory cytokines secretion, while overwhelmed pyroptosis contributes to organ dysfunction and pathological inflammatory response. Caffeic acid (CA) and ferulic acid (FA) are both well-known antioxidant and anti-inflammatory phenolic acids, which resemble in chemical structure. Here we found that CA, but not FA, protects macrophages from both Nigericin-induced canonical and cytosolic lipopolysaccharide (LPS)-induced non-canonical pyroptosis and alleviates LPS-induced mice sepsis. It significantly improved the survival of pyroptotic cells and LPS-challenged mice and blocked proinflammatory cytokine secretion. The anti-pyroptotic effect of CA is independent of its regulations in cellular lipid peroxidation, mitochondrial function, or pyroptosis-associated gene transcription. Instead, CA arrests pyroptosis by directly associating with gasdermin D (GSDMD) and blocking its processing, resulting in reduced N-GSDMD pore construction and less cellular content release. In LPS-induced septic mice, CA inhibits GSDMD activation in peritoneal macrophages and reduces the serum levels of interleukin-1β and tumor necrosis factor-α as the known pyroptosis inhibitors, disulfiram and dimethyl fumarate. Collectively, these findings suggest that CA inhibits pyroptosis by targeting GSDMD and is a potential candidate for curbing the pyroptosis-associated disease.

Keywords: caffeic acid; ferulic acid; gasdermin D; macrophage; pyroptosis; sepsis.

FIGURE 1

Caffeic acid (CA), but not ferulic acid (FA), dose‐dependently suppressed NLRP3 inflammasome‐mediated canonical pyroptosis. After being primed with 100 ng/ml lipopolysaccharide (LPS) for 4 h, J774A.1 cells were treated with 12.5, 25, 50, 100 µg/ml CA, FA, or their combination as indicated for 2 h and then with 10 µM Nigericin for 1 h. (A–C) The concentration of LDH in supernatants was analyzed by LDH cytotoxicity assay kits. (D–F) The secretion of IL‐1β in the culture medium was detected by ELISA kits. (G) The treated cells were stained by Hoechst 33342 (blue) and PI (red) and visualized under a fluorescence microscope. Scale bar: 100 µm. (H) The relative fluorescence area of PI. (I) The structures of CA and FA. The data are means ± standard error of the mean (SEM) (n = 3). **p < 0.01 and ***p < 0.001 versus the LPS + Nig group; ^###p < 0.001 versus the Control group. CA, caffeic acid; LPS, lipopolysaccharide; Nig, nigericin; FA, ferulic acid.

FIGURE 2

Caffeic acid (CA), but not ferulic acid (FA), blocked caspase‐11 inflammasome‐mediated non‐canonical pyroptosis. After being primed with 1 µg/ml lipopolysaccharide (LPS) for 6 h, RAW264.7 cells or bone marrow‐derived macrophages (BMDMs) were pretreated with 50 µg/ml CA, 50 µg/ml FA, or their combination for 2 h and then transfected with 2 µg/ml LPS using 0.25% FuGENE (v/v) for 16 h. (A) Cellular membrane permeability was evaluated by flow cytometry after PI staining. (B) The percentage of PI‐positive RAW264.7 cells was calculated. (C) The release of LDH by RAW264.7 cells was determined using the LDH cytotoxicity assay kit. (D) The treated BMDMs were visualized and photographed under an optical microscope. Yellow triangles indicate pyroptosis cells. The scale bar is 100 µm. (E) The LDH released by BMDMs was analyzed using the LDH cytotoxicity assay kit. (F) The concentration of IL‐1β in the culture medium of BMDMs was detected by ELISA kits. The data are means ± standard error of the mean (SEM) (n = 3). *p < 0.05 and ***p < 0.001 versus the LPS‐trans group. ^###p < 0.001 versus the Control group. LPS‐trans, LPS‐transfection.

FIGURE 3

Caffeic acid (CA), but not ferulic acid (FA), alleviated lipopolysaccharide (LPS)‐induced sepsis in mice. (A and C) The schematic representations of treatment details. After pretreatments with 50 mg/kg CA, FA, or their combination by intraperitoneal injection, mice were challenged with 30 mg/kg or 10 mg/kg LPS for the indicated time. (B) After being challenged with 30 mg/kg LPS, the survival rate of mice was monitored every 6 h until 120 h. (D) After being challenged with 10 mg/kg LPS for 6 h, the peritoneal macrophages were collected and analyzed for gasdermin D (GSDMD) activation by immunoblotting. (E and F) After being challenged with 10 mg/kg LPS for 6 h, the concentrations of IL‐1β and TNF‐α in serum were analyzed by ELISA kits. Statistical analysis of the survival rate was performed using the log‐rank (Mantel‐Cox) test (n =  10). Statistical analysis of TNF‐α and IL‐1β concentration was performed using the one‐way analysis of variance (ANOVA) test (n = 6). The data are means ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the LPS group; ^###p < 0.001 versus the Control group. IP, intraperitoneal injection.

FIGURE 4

The anti‐pyroptotic effect of caffeic acid (CA) is independent of its regulatory activities on pyroptosis‐associated gene transcription, mitochondrion protection, and lipid peroxidation. (A‐H) J774A.1 cells primed with 100 ng/ml lipopolysaccharide (LPS) for 4 h were treated with 50 µg/ml CA, ferulic acid (FA), or their combination for 2 h, and then stimulated with 10 µM Nigericin for 1 h. (A–D) The mRNA expressions of caspase‐1, caspase‐11, GSDMD, and IL‐1β in treated J774A.1 cells were quantified by qRT‐PCR analysis. (E, F) Cellular lipid peroxidation was detected by BODIPY 581⁄591 C11 using flow cytometry and expressed as the ratio of red (reduced) to green (oxidized). (G and H) Mitochondrial membrane potential was detected by JC‐1 using flow cytometry and indicated by the ratio of green (JC‐1 monomers) to red (JC‐1 aggregates). (I) J774A.1 cells primed with 100 ng/ml LPS for 4 h were treated with the indicated concentration of Zileuton or CA for 2 h and then stimulated with 10 µM Nigericin for 1 h. The LDH release in the supernatant was measured by the LDH cytotoxicity assay kit. The data are means ± standard error of the mean (SEM) (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the LPS + Nig group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 versus the Control group. ZL, Zileuton.

FIGURE 5

Caffeic acid (CA) inhibited macrophage pyroptosis by blocking the formation of N‐gasdermin D (GSDMD) pores. (A, B) Lipopolysaccharide (LPS)‐primed bone marrow‐derived macrophages (BMDMs) were treated with 50 µg/ml CA or ferulic acid (FA) for 2 h by replacing the culture medium and then were transfected with 2 µg/ml LPS using 0.25% FuGENE (v/v) for 16 h. LPS‐primed J774A.1 cells were treated with 50 µg/ml CA or FA for 2 h before 10 µM Nigericin stimulation for 1 h. The cell pellets were collected and the activation of critical proteins was analyzed by western blot assay. (C) The cell micromorphology was observed and pictured under the standard error of the mean (SEM). (D) The aggregation of N‐GSDMD (green) was photographed under a laser confocal microscope using the immunofluorescence assay. White arrows indicate the aggregation of N‐GSDMD in the cell membrane. (E) The binding poses of CA or FA to GSDMD were calculated by Autodock. The ligand‐receptor interaction interfaces were highlighted by red dotted boxes. The close‐up views of them were shown aside, in which the hydrogen bonds were indicated as yellow dotted lines, and the interacted residues were colored in blue. (F) The GSDMD‐CA binding was confirmed by a biotin affinity pull‐down assay. The pulled‐down proteins were detected by immunoblotting. (G and H) LPS‐primed J774A.1 cells were treated with 7.5 µg/ml DSF, 3.6 µg/ml DMF, or 50 µg/ml CA for 2 h before 10 µM Nigericin stimulation. The cytotoxicity was determined by the LDH cytotoxicity assay kit. The cleaved IL‐1β in the cytoplasm and supernatant were analyzed by western blot assay. ***p < 0.001 versus the LPS + Nig group; ^###p < 0.001 versus the Control group. DSF, disulfiram; DMF, dimethyl fumarate.

FIGURE 6

Caffeic acid (CA), like DSF and DMF, can protect mice from lipopolysaccharide (LPS)‐induced sepsis by inhibiting macrophage pyroptosis. (A, C) The schematic representation of treatment details. After 12 h pretreatments with 50 mg/kg CA, DSF, or DMF by intraperitoneal injection, mice were challenged with 30 mg/kg or 10 mg/kg LPS for the indicated time. (B) After being challenged with 30 mg/kg LPS, the survival rate of mice was monitored every 6 h until 120 h. (D) After being challenged with 10 mg/kg LPS for 6 h, the peritoneal macrophages were collected and analyzed for gasdermin D (GSDMD) activation by immunoblotting. (E, F) After being challenged with 10 mg/kg LPS for 6 h, the concentrations of IL‐1β and TNF‐α in serum were analyzed by ELISA kits. Statistical analysis of the survival rate was performed using the log‐rank (Mantel‐Cox) test (n =  10). Statistical analysis of TNF‐α and IL‐1β concentration was performed using the one‐way analysis of variance (ANOVA) test (n = 6). The data are means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the LPS group; ^###p < 0.001 versus the Control group. IP, intraperitoneal injection.

FIGURE 7

The schematic overview of the proposed anti‐pyroptotic mechanism of caffeic acid (CA). Canonical NLRP3 inflammasome can be activated by a wide range of stimuli that induce potassium ions efflux, lysosomal disruption, and mitochondrial dysfunction. As a potassium‐proton ionophore antibiotic, Nigericin is a potent NLRP3 inflammasome inducer, which leads to the assembly of NLRP3 inflammasome and activation of caspase‐1. Active caspase‐1 directly converts the pro‐inflammatory cytokines, pro‐IL‐1β and pro‐IL‐18, into their mature forms. Additionally, caspase‐1 cleaves gasdermin D (GSDMD) and liberates the pore‐forming N‐GSDMD to execute pyroptotic cell death and release of cellular contents. In comparison to NLRP3 inflammasome, non‐canonical inflammasome is specifically activated by cytosolic lipopolysaccharide (LPS), which promotes the activation of caspase‐11. Caspase‐11 also cleaves GSDMD and mediates lytic cell death, while it does not directly process pro‐inflammatory cytokines. Even so, in most macrophages, caspase‐11‐mediated GSDMD pore formation can lead to NLPR3‐dependent maturation and secretion of IL‐1β and IL‐18 by inducing potassium ions efflux. CA inhibits both canonical and non‐canonical pyroptotic cell death and IL‐1β secretion by blocking the activation of GSDMD, the key pyroptosis executor protein.