Ion channel Piezo1 activation promotes aerobic glycolysis in macrophages

Abstract

Altered microenvironmental stiffness is a hallmark of inflammation. It is sensed by the mechanically activated cation channel Piezo1 in macrophages to induce subsequent immune responses. However, the mechanism by which the mechanosensitive signals shape the metabolic status of macrophages and tune immune responses remains unclear. We revealed that Piezo1-deficient macrophages exhibit reduced aerobic glycolysis in resting or liposaccharide (LPS)-stimulated macrophages with impaired LPS-induced secretion of inflammatory cytokines in vitro. Additionally, pretreatment with the Piezo1 agonist, Yoda1, or cyclical hydrostatic pressure (CHP) upregulated glycolytic activity and enhanced LPS-induced secretion of inflammatory cytokines. Piezo1-deficient mice were less susceptible to dextran sulfate sodium (DSS)-induced colitis, whereas Yoda1 treatment aggravated colitis. Mechanistically, we found that Piezo1 activation promotes aerobic glycolysis through the Ca²⁺-induced CaMKII-HIF1α axis. Therefore, our study revealed that Piezo1-mediated mechanosensitive signals Piezo1 can enhance aerobic glycolysis and promote the LPS-induced immune response in macrophages.

Keywords: HIF1 alpha; Piezo1; colitis; glycolysis; macrophage.

Figure 1

Piezo1 activation promotes LPS-induced expression and secretion of inflammatory cytokines. (A) A hierarchical differentiation tree depicting the expression of Piezo1 using BloodSpot (online software). (B) The expression of Piezo1 mRNA in the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox and Piezo1 ^flox/flox control mice relative to β-actin (n = 6). (C) IL6 and TNF-α levels in the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice treated with 10 ng/ml LPS for 24 h; IL-1β level in the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice treated with 1 µg/ml LPS for 4 h and 25 µM nigericin for 30 min, measured by ELISA (n = 6). (D) IL6 and TNF-α levels in the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice treated with 10 ng/ml LPS under cyclical hydrostatic pressure cycling once per second from 45 mmHg to 60 mmHg with 5% CO₂ at 37°C for 6 h; IL-1β level in the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice treated with 1 µg/ml LPS for 4 h and 25 µM nigericin for 30 min, measured by ELISA under CHP (n = 6). (E) IL6 and TNF-α levels in the BMDMs with 10 ng/ml LPS for 24 h pretreated with 10 µM Yoda1 or DMSO for 30 min; IL-1β level in the BMDMs with 1 µg/ml LPS for 4 h and 25 µM nigericin for 30 min pretreated with 10 µM Yoda1 or DMSO for 30 min (n = 6). (F) Relative gene expression of IL6, TNF-α, and IL-1β in the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice incubated with 10 ng/ml LPS for 2 h or 1 ug/ml LPS for 4 h and 25 µM nigericin for 30 min (n = 6). (G) Relative gene expression of IL6 and TNF-α in the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice treated with 10 ng/ml LPS for 2 h under cyclical hydrostatic pressure; relative gene expression of IL-1β in the BMDMs treated with 1 ug/ml LPS for 4 h and 25 µM nigericin for 30 min (n = 6). (H) The expression of Piezo1 mRNA in BMDMs from Csf1r ^creERT2; Piezo1 ^flox/flox and Piezo1 ^flox/flox control mice relative to β-actin (n = 6). (I) IL6, TNF-α, and IL-1β secretion in the BMDMs from Csf1r ^creERT2; Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice incubated with 10 ng/ml LPS for 24 h, 1 µg/ml LPS for 4 h, and 25 µM nigericin for 30 min under static pressure or cyclical hydrostatic pressure (n = 6). Statistical significances were calculated using (C, I) one-way ANOVA, Tukey’s multiple comparisons test; (B, D–H) two-tailed Student t test. Data are expressed as the mean ± SD. **** P < 0.0001, ns, not significant. BMDMs, bone marrow-derived macrophages; Lyz2 ^cre/+ Piezo1 ^flox/flox, conditionally Piezo1-deficient mice; IL, interleukin; TNF-α, tumor necrosis factor α; LPS, liposaccharide; CHP, cyclical hydrostatic pressure.

Figure 2

Piezo1 is essential for glycolic reprogramming in macrophages. (A) A t-SNE plot of myeloid gene clusters of myeloid cells harvested from the bone marrow of Lyz2 ^cre/+ Piezo1 ^flox/flox mice and littermate controls, analyzed by scRNA-seq. (B) Violin plot showing expression levels of Aldoa, Pkm, Eno1, Gapdh, Ldha, and Gpi1. (C) GO analysis of the top five enriched pathways based on differentially expressed genes (DEGs). (D, E) Glycolytic rate assay profile and glycolytic analysis of BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice (n = 7). (F–H) Glycolytic rate assay profile and glycolytic analysis of BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice treated with or without LPS (10 ng/ml) for 24 h (n = 7). (I) IL6 and TNF-α levels in BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice treated with or without LPS (10 ng/ml) and 2-DG (1 mM) for 24 h (n = 6). (J, K) Quantitative proteomics was performed to assess metabolites related to glucose metabolism in the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice under static pressure or cyclical hydrostatic pressure (n = 3). Statistical significances were calculated using (G–I) one-way ANOVA, Tukey’s multiple comparisons test; (E, K) two-tailed Student t test. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; **** P < 0.0001, ns, not significant. BMDMs, bone marrow-derived macrophages; Lyz2 ^cre/+ Piezo1 ^flox/flox, conditionally Piezo1-deficient mice; IL, interleukin; TNF-α, tumor necrosis factor α; LPS, liposaccharide.

Figure 3

Yoda1 induces glycolysis in macrophages. (A, B) Glycolytic rate assay profile and glycolytic analysis of BMDMs pretreated with 10 µM Yoda1 or DMSO, incubated in the indicated conditions (blank; 10 ng/ml LPS for 24 h; 10 ng/ml IL4 for 24 h) (n = 4) (C) The mRNA expression of glycolysis-related genes treated with 10 µM Yoda1 or DMSO for 30 min (n = 3). (D) The mRNA expression of glycolysis-related genes pretreated with 10 µM Yoda1 or DMSO for 30 min and 10 ng/ml LPS for 2 h (n = 3). (E, F) Glycolytic rate assay profile and glycolytic analysis of the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice and Piezo1 ^flox/flox control mice pretreated with or without 10 µM Yoda1 for 30min (n = 7). Statistical significances were calculated using (B, D, F) one-way ANOVA, Tukey’s multiple comparisons test; (C) two-tailed Student t test. Data expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; **** P < 0.0001, ns, not significant. BMDMs, bone marrow-derived macrophages; Lyz2 ^cre/+ Piezo1 ^flox/flox, conditionally Piezo1-deficient mice; IL, interleukin; TNF-α, tumor necrosis factor α; LPS, liposaccharide.

Figure 4

Piezo1 promotes a metabolic switch in macrophages via the Ca²⁺-CaMKII-HIF1α axis. (A)The mRNA expression of Glut1, Pkm, and Ldha treated with 10 µM Yoda1 or DMSO for 30 min in calcium-rich or calcium-free media. (n=3, one-way ANOVA, Tukey’s multiple comparisons test) (B) Glycolytic rate assay profile of BMDMs treated with blank (DMSO); KN93 (10uM KN93 for 24 h); Yoda1 (10uM Yoda1 for 30 min and overnight culture); Yoda1+KN93 (10uM Yoda1 for 30 min and 10uM KN93 for 24 h) (C) Immunofluorescence of HIF1α in the BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mouse and Piezo1 ^flox/flox control mice treated with or without LPS (10 ng/ml) for 24 h. (D, E) HIF1α, phospho-CaMKII, total-CaMKII, P300, β-actin, and LaminA/C, nuclear HIF1α expression in BMDMs in the indicated conditions (10 ng/ml LPS for 0 or 6 h, 10 µM KN93 for 0 or 6 h, static pressure or cyclical hydrostatic pressure for 0 or 6 h). (F) Glycolytic rate assay profile of BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice or Piezo1 ^flox/flox control mice treated with siNC or siHIF1α. (G) Glycolytic rate assay profile of BMDMs from Lyz2 ^cre/+ Piezo1 ^flox/flox mice or Piezo1 ^flox/flox control mice treated with DMSO or 10 uM KN93 for 24 h. Data are expressed as the mean ± SD. *P < 0.05, ***P < 0.001; ns, not significant. BMDMs, bone marrow-derived macrophages; Lyz2 ^cre/+ Piezo1 ^flox/flox, conditionally Piezo1-deficient mice; HIF1α, hypoxia-inducible factor 1α.

Figure 5

Piezo1 deficiency ameliorates murine ulcerative colitis. Lyz2 ^cre/+ Piezo1 ^flox/flox and Piezo1 ^flox/flox control mice were challenged with water or 3% DSS in drinking water for seven days (n = 7). (A) Body weight loss, (B, C) colon length, (D) representative hematoxylin and eosin (H&E)-stained colon cross-sections, and histological scores were measured. (E) mRNA expression of IL-1β, IL6, and TNF-α in colon tissue. (F) mRNA expression of Glut1, Pkm, and Ldha in macrophages in the lamina propria. Mice were challenged with 3% DSS in drinking water for 7 d, to which 0.4 mg/kg Yoda1 or DMSO were added on day 1 and day 4 (n = 5). (G) Body weight loss, (H, I) colon length, (J) representative H&E-stained colon cross-sections, and histological scores were measured. (K) mRNA expression of IL-1β, IL6, and TNF-α in the colon tissue. (L) mRNA expression of Glut1, Pkm, and Ldha in macrophages of the lamina propria. Statistical significances were calculated using (A) two way ANOVA, *P < 0.05; **** P < 0.0001 (DSS Piezo1 ^flox/flox versus DSS Lyz2 ^cre/+ Piezo1 ^flox/flox); (G) two way ANOVA, *P < 0.05 (DSS+DMSO versus DSS+Yoda1); ^#P < 0.05, ^{# #}P < 0.01, ^####P < 0.0001 (H₂O+DMSO; DSS+DMSO); (B–F, H–L) one-way ANOVA, Tukey’s multiple comparisons test. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; **** P < 0.0001, ns, not significant. Lyz2 ^cre/+ Piezo1 ^flox/flox, conditionally Piezo1-deficient mice; DSS, dextran sulfate sodium; IL, interleukin; TNF-α, tumor necrosis factor α.