Critical Role of IL1R2-ENO1 Interaction in Inhibiting Glycolysis-Mediated Pyroptosis for Protection Against Lethal Sepsis

Abstract

Immune cell metabolic reprogramming toward glycolysis is vital for sepsis defense. While interleukin 1 receptor 2 (IL1R2) acts as a decoy receptor for IL1α/β, its potential impact on cell metabolism and death during sepsis remains unclear. This study observed elevated plasma soluble IL1R2 (sIL1R2) levels in septic patients and mice. In pyroptotic macrophages, reduced intracellular IL1R2 expression led to its release extracellularly. Proteomic screening identified enolase 1 (ENO1), a key glycolysis enzyme, as the binding partner of IL1R2 in macrophages. IL1R2 suppresses ENO1 activity to inhibit glycolysis, gasdermin D (GSDMD)-mediated pyroptosis, and inflammation in macrophages. IL1R2-deficient mice exhibited heightened susceptibility to sepsis, with increased inflammation, organ injury, and mortality. Notably, ENO1 inhibition reduced inflammation, organ injury, and improved survival rates in septic mice. The study reveals that IL1R2 interacts with ENO1 to inhibit glycolysis-mediated pyroptosis and inflammation in sepsis, suggesting the IL1R2-ENO1 interaction as a promising therapeutic target of sepsis.

Keywords: IL1R2; enolase 1; glycolysis; macrophages; pyroptosis; sepsis.

Figure 1

Soluble IL1R2(sIL1R2) is released from pyroptotic macrophages during sepsis. A) Plasma levels of sIL1R2 in patients without sepsis (non‐sepsis), as well as in patients with sepsis with or without shock. Data are expressed as mean ± SEM and compared by one‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05 versus non‐sepsis, ^# p < 0.05 versus sepsis without shock. B) Plasma levels of sIL1R2 in septic patients, comparing survivors with non‐survivors. Data are expressed as mean ± SEM and compared by Student's t test (unpaired). *p < 0.05 versus survivor. C) The correlation analysis of plasma sIL1R2 levels and SOFA score in septic patients. D) Single‐cell RNA sequencing (scRNA‐seq) data from blood immune cells of septic patients were analyzed and visualized using uniform manifold approximation and projection (UMAP) plots, with colors in parentheses indicating the identified cell clusters. E, F) UMAP representation of snRNA‐seq data from blood immune cells of health control (E) and septic patients (F), colored according to IL1R2 expression. G, H) The expression of IL1R2 on classical and non‐classical monocytes from health control and septic patients was detected by flow cytometry. Data are expressed as mean ± SEM and compared by Student's t test (unpaired). *p < 0.05 versus health. MFI, mean fluorescence intensity. I) The plasma levels of sIL1R2 were quantified by Western blotting in sham and cecal ligation and puncture (CLP) induced septic mice. Data are expressed as mean ± SEM and compared by Student's t test (unpaired). *p < 0.05 versus Sham. J, K) Peritoneal cavity (PerC) macrophages treated with or without LPS (1 µg mL⁻¹) for 3 h, followed by nigericin (5 µM) for 1 h. LN, LPS+ nigericin. (J) The levels of IL1R2 in the whole cell lysis (WCL)and culture supernatants (SN) of these macrophages were measured. Representative images from three independent experiments with similar results. (K) The treated macrophages were fixed and stained with anti‐IL1R2 Ab (red) and DNA (Hoechst33342, blue), with images were captured by confocal microscopy. Scale bar: 20 µm. Original magnification: 630×. L) Immortalized murine bone marrow‐derived macrophages (iBMDMs) were pre‐treated with disulfiram (5 µg mL⁻¹) and LPS (1 µg mL⁻¹) for 3 h, then followed with ATP (5 mM) for 0.5 h. The expressions of cleaved‐Caspase1(C‐Casp1) and IL1R2 in the supernatants, and the expression of casp1, GSDMD, and N‐GSDMD in the whole cell lysis of these iBMDMs were measured. Representative images from three independent experiments with similar results.

Figure 2

Proteomic screens elucidate IL1R2‐ENO1 interaction. A) Schematic workflow for identifying the direct binding partners of IL1R2 by affinity purification LC‐MS/MS. B) Mass spectrometry identification of ENO1 protein. C) Flag‐IL1R2 was immunoprecipitated in Flag‐IL1R2‐overexpressing iBMDMs, followed by immunoblotting with indicated antibodies (top). ENO1 was immunoprecipitated in Flag‐IL1R2‐overexpressing iBMDMs, followed by immunoblotting with indicated antibodies (bottom). Representative images from three independent experiments with similar results. D) Computational model of the interaction between mouse IL1R2 (blue) and ENO1 (light blue). E) Biacore analysis demonstrating binding of rmIL1R2 and rmENO1 with a K_D of 1.99×10⁻⁹. F) PerC macrophages treated with or without LPS (1 µg mL⁻¹) for 3 h, then followed with nigericin (5 µM) for 1 h. After stimulation, the macrophages were fixed and stained with anti‐IL1R2 Ab (red), anti‐ENO1 Ab (green), and DNA (Hoechst33342, blue), and the images were captured by confocal microscopy. Scale bar: 20 µm. Original magnification: 630×.

Figure 3

IL1R2 suppresses ENO1‐mediated glycolysis in macrophages after LPS stimulation. A–C) WT or IL1R2^−/‐ primary PerC macrophages were stimulated without or with LPS (1 µg/mL) for 12 h. Extracellular acidification rate (ECAR) in macrophages as assessed by Seahorse assay. (A) Real‐time changes in the ECAR of macrophages after treatment with glucose, Oligomycin, and 2‐DG. Glycolysis capacity (double‐headed arrow) is shown in macrophages. (B) Glycolysis of macrophages was measured by real‐time changes in ECAR. (C) Glycolysis capacity of macrophages measured by real‐time changes in ECAR. Data are expressed as mean ± SEM and compared by one‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05 versus WT+LPS (‐), ^# p < 0.05 versus WT+LPS (+). D–F) IL1R2^−/‐ primary PerC macrophages were stimulated without or with LPS and treated with Enoblock (10 µM) for 12 h. ECAR in macrophages as assessed by Seahorse assay. (D) Real‐time changes in the ECAR of IL1R2^−/‐ macrophages after treatment with glucose, Oligomycin, and 2‐DG. Glycolysis capacity (double‐headed arrow) is shown in macrophages. (E) Glycolysis of IL1R2^−/‐ macrophages measured by real‐time changes in ECAR. (F) Glycolysis capacity of IL1R2^−/‐ macrophages measured by real‐time changes in ECAR. Data are expressed as mean ± SEM and compared by Student's t test (unpaired). *p < 0.05 versus LPS (+), Eno (‐). G) WT and IL1R2^−/‐ PerC macrophages treated with or without LPS (1 µg mL⁻¹) for 3 h, then followed with ATP (5 mM) and Enoblock (10 µM) for 0.5 h. The expression of ENO1 in the whole cell lysis of these macrophages was measured by WB. Representative images from three independent experiments with similar results. H) The activity of ENO1 in these macrophages was measured by ENO1 activity kit. All experiments were repeated three times with similar results. Data are expressed as mean ± SEM and compared by two‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05 versus WT LPS (‐) ATP (‐) Enob (‐), #p < 0.05 versus IL1R2^−/− LPS (‐) ATP (‐) Enob (‐), &p < 0.05 versus IL1R2^−/− LPS (+) ATP (+) Enob (‐).

Figure 4

IL1R2 suppresses GSDMD‐mediated pyroptosis in macrophages through ENO1. A–E) iBMDMs were transfected with a flag‐IL1R2 overexpression plasmid (oe‐IL1R2) or a negative control (NC). After transfection, the cells were treated with LPS (1 µg/mL) for 3 h, then followed by ATP (5 mM) for 0.5 h. (A) The expressions of cleaved‐Caspase1(C‐Casp1) and IL1β in the supernatants (SN), and the expression of casp1, GSDMD, N‐GSDMD, and IL1R2 (Flag) in the whole cell lysis (WCL) of these iBMDMs were measured. Representative images from three independent experiments with similar results. (B) The levels of LDH in the culture supernatants of iBMDMs were measured. (C) The percentage of cell death (PI⁺ cells) was checked in these iBMDMs. (D, E) The levels of IL1α and IL1β in the culture supernatants of iBMDMs were measured by ELISA kits. Data are expressed as mean ± SEM and compared by two‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05 versus NC+LPS, ^#p < 0.05 versus NC+LA. F) iBMDMs were transfected with flag‐IL1R2 overexpression plasmid (oe‐IL1R2) or negative control (NC). After transfection, the cells were treated with LPS (1 µg/mL) for 6 h, followed by LPS (5 µg mL⁻¹) for 6 h. The expressions of Caspase11(Casp11), GSDMD, N‐GSDMD, and IL1R2 (Flag) in the whole cell lysis of these iBMDMs were measured. Representative images from three independent experiments with similar results. G–L) WT and IL1R2^−/− PerC macrophages treated with LPS (1 µg/mL) for 3 h, then followed with ATP (5 mM) and Enoblock (10 µM) for 0.5 h. (G) The expression of cleaved‐caspase1(C‐Casp1) and IL1β in the supernatants (SN), and the expression of casp1, pro‐IL1β, GSDMD, N‐GSDMD, and ENO1 in the WCL of these macrophages were measured. Representative images from three independent experiments with similar results. (H) The levels of LDH in the culture supernatants of macrophages were measured by kits. (I) The percentage of cell death (PI⁺ cells) was checked in these macrophages. (J‐L) The levels of IL1α (J), IL1β (K), and TNFα (L) in the culture supernatants of macrophages were measured by ELISA kits. Data are expressed as mean ± SEM and compared by two‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05 versus WT+LA, ^# p < 0.05 versus IL1R2^−/−+LA. LA, LPS with ATP. M) WT PerC macrophages treated with LPS (1 µg mL⁻¹) for 3 h, then followed with nigericin (5 µM) and Enoblock (10 µM) for 0.5 h. The expressions of cleaved‐caspase1(C‐Casp1) and IL1R2 in the supernatants, and the expression of IL1R2 and β‐actin in the WCL of these macrophages were measured. Representative images from three independent experiments with similar results.

Figure 5

IL1R2 deficiency exacerbates organ damage in septic mice. A–J) WT and IL1R2^−/‐ mice were assigned to sham or CLP‐induced sepsis. After 20 h, blood and lung tissue were collected. (A‐D) The plasma levels of AST (A), ALT (B), Cre (C), and BUN (D) were measured by commercial kits. (E) The plasma levels of lactate were measured using by commercial kit. (F‐H) The plasma levels of IL1β (F), IL6 (G), and TNFα (H) were measured by ELISA kits. Experiments were performed three times, and all data were used for analysis. (I, J) Lungs were embedded in paraffin, sectioned, and analyzed for histologic injury using H&E staining and analyzed using lung injury scoring. Data are expressed as mean ± SEM and compared by two‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05 versus sham WT, ^# p < 0.05 versus sham IL1R2^−/−, ^& p < 0.05 versus CLP WT. K) WT or IL1R2^−/‐ mice subjected to CLP‐induced sepsis and monitored for 7 days for humane endpoints, and differences in survival were determined using Kaplan‐Meier survival plots and a log‐rank test. n = 12 mice/group. Hazard ratios (HR): WT, 0.36; IL1R2^−/−, 2.75. *p < 0.05 versus WT.

Figure 6

ENO1 inhibition improves sepsis in CLP‐induced septic mice. A–F) WT mice were subjected to CLP‐induced sepsis with Enoblock treatment (5 mg kg⁻¹) or vehicle (volume‐equivalent). After 20 h, blood and lung tissue were collected for respective analyses. (A, B) The plasma levels of AST (A) and ALT (B) were measured by commercial kits. (C) The plasma levels of lactate were measured using by commercial kit. (D‐F) The plasma levels of TNFα (D), IL6 (E), and IL1β (F) were measured by ELISA kits. Experiments were performed 3 times, and all data were used for analysis. Data are expressed as mean ± SEM and compared by one‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05 versus SV, ^# p < 0.05 versus CV. SV, Sham+vehicle; CV, CLP+vehicle; SE, Sham+Enoblock; CE, CLP+ Enoblock. G) WT mice subjected to CLP‐induced sepsis with Enoblock treatment (5 mg/kg) or vehicle (volume equivalent) and monitored for 7 days for humane endpoints, and differences in survival were determined using Kaplan‐Meier survival plots and a log‐rank test. n = 20 mice/group. Hazard ratios: CL+vehicle, 2.51; CLP+ Enoblock, 0.40. *p < 0.05 versus CLP vehicle. H–J) WT PerC macrophages were treated with LPS (1 µg/mL) for 3 h, then followed with nigericin (5 µM) and indicated dose of Enoblock for 1 h. (H) The expression of cleaved‐caspase1(C‐Casp1), caspase1, GSDMD, and N‐GSDMD, in the whole cell lysis of these macrophages, was measured by WB. Representative images from three independent experiments with similar results. (I) The levels of LDH in the culture supernatants of macrophages were measured by kits. (J) The levels of IL1β in the culture supernatants of macrophages were measured by ELISA kits. Data are expressed as mean ± SEM and compared by one‐way ANOVA and Tukey's multiple comparisons test. * p < 0.05 versus LPS+ nigericin (‐) Enob (‐), ^# p < 0.05 versus LPS+ nigericin (+) Enob (+). Enob, Enobock.

Figure 7

Summary of findings. We discovered that intracellular IL1R2 negatively regulates inflammatory cell death and inflammation by inhibiting glycolysis in sepsis. Soluble IL1R2 is released from macrophages undergoing pyroptosis. IL1R2 acts as a novel negative regulator of glycolysis by interacting with ENO1, thereby inhibiting GSDMD‐mediated pyroptosis and inflammation. The pharmacologic and genetic inhibition of ENO1 suppressed GSDMD‐mediated pyroptosis by downregulating glycolysis‐mediated caspase‐1 activation. These results suggest that IL1R2‐ENO1 interaction holds potential as a therapeutic intervention for sepsis.