IL-1/inhibitory κB kinase ε-induced glycolysis augment epithelial effector function and promote allergic airways disease

Abstract

Background: Emerging studies suggest that enhanced glycolysis accompanies inflammatory responses. Virtually nothing is known about the relevance of glycolysis in patients with allergic asthma.

Objectives: We sought to determine whether glycolysis is altered in patients with allergic asthma and to address its importance in the pathogenesis of allergic asthma.

Methods: We examined alterations in glycolysis in sputum samples from asthmatic patients and primary human nasal cells and used murine models of allergic asthma, as well as primary mouse tracheal epithelial cells, to evaluate the relevance of glycolysis.

Results: In a murine model of allergic asthma, glycolysis was induced in the lungs in an IL-1-dependent manner. Furthermore, administration of IL-1β into the airways stimulated lactate production and expression of glycolytic enzymes, with notable expression of lactate dehydrogenase A occurring in the airway epithelium. Indeed, exposure of mouse tracheal epithelial cells to IL-1β or IL-1α resulted in increased glycolytic flux, glucose use, expression of glycolysis genes, and lactate production. Enhanced glycolysis was required for IL-1β- or IL-1α-mediated proinflammatory responses and the stimulatory effects of IL-1β on house dust mite (HDM)-induced release of thymic stromal lymphopoietin and GM-CSF from tracheal epithelial cells. Inhibitor of κB kinase ε was downstream of HDM or IL-1β and required for HDM-induced glycolysis and pathogenesis of allergic airways disease. Small interfering RNA ablation of lactate dehydrogenase A attenuated HDM-induced increases in lactate levels and attenuated HDM-induced disease. Primary nasal epithelial cells from asthmatic patients intrinsically produced more lactate compared with cells from healthy subjects. Lactate content was significantly higher in sputum supernatants from asthmatic patients, notably those with greater than 61% neutrophils. A positive correlation was observed between sputum lactate and IL-1β levels, and lactate content correlated negatively with lung function.

Conclusions: Collectively, these findings demonstrate that IL-1β/inhibitory κB kinase ε signaling plays an important role in HDM-induced glycolysis and pathogenesis of allergic airways disease.

Keywords: Asthma; IL-1; glycolysis; house dust mite; inhibitor of κB kinase ε; lactate; lactate dehydrogenase A.

Figure 1.. Evaluation of glycolysis in the lung tissues of mice exposed to house dust mite (HDM).

A, Schematic depicting the dosing regimen of HDM (Detailed information is provided in the Supplemental Material). B, Lactate levels in BAL (top) and lung tissues (bottom) following a single or multiple exposures to HDM, according to the schematic in A. *P < 0.05 (ANOVA) relative to the saline group (n=5–8 per group). C, Protein expression of glycolysis enzymes in lung lysates from saline- or HDM-challenged mice harvested at the indicated times. β-Actin = loading control. D, LDHA immunohistochemistry in lung tissues of HDM-sensitized and -challenged mice harvested at Day 20 (Top: scale bar, 50 μm; Bottom: scale bar, 25 μm). Blue = LDHA. 2°control; HDM-inflamed tissue wherein primary antibody was omitted as a negative control.

Figure 2.. House dust mite (HDM)-induced T and B-cell adaptive immune responses are required for IL-1 β production and resultant increases in glycolysis in lung tissues.

A, Levels of pro-inflammatory cytokines in lung tissue of HDM-exposed mice at the times indicated. *P < 0.05 compared to saline groups (ANOVA) (n=5 per group). B-C, Lactate levels in the broncho-alveolar lavage fluid (BAL) and homogenized lung tissues (B) and Western blot analysis of HK2 and LDHA in lung tissues (C) from saline-exposed mice or HDM-exposed mice treated with vehicle (Veh) or IL-1 TRAP. Mice were harvested at day 20. *P < 0.05 compared to the saline group, †P < 0.05 compared to the HDM/Veh group (ANOVA) (n = 5 per group). Lactate levels in BAL fluid and lung tissues (D) and IL-1β levels in the lung tissues (E) from Rag^−/− mice and WT mice exposed to saline or HDM. Mice were analyzed at Day 20. *P < 0.05 compared to the saline controls, †P < 0.05 compared to the respective WT group (ANOVA, n = 4–8 per group). F, Lactate levels in BAL fluid and lung tissues from the mice 6, 24, 48, and 72 h post intranasal administration of IL-1β. *P < 0.05 compared to Veh-exposed mice (ANOVA, n=5–8 per group). G, Western blotting of HK2 and LDHA in lung tissues from mice treated with recombinant IL-1β (1 μg/mouse) for 48 h. H, Immunohistochemical analysis of LDHA in lung tissues 24 h post administration of IL-1β or vehicle (Top: scale bar, 50 μm; Bottom: scale bar, 25 μm). Blue = LDHA.

Figure 3.. IL-1α/β increase lactate production, glycolysis gene expression, glucose usage, and glycolytic flux rate in primary mouse tracheal epithelial (MTE) cells.

A, Lactate levels in the cell-culture supernatants of MTE cells following 24 h stimulation with IL-1α, IL-1β, IL-6, IL-13, IL-33, TGF-β1, TNFα, IL-17, LPS, or HDM. *P < 0.05 compared to the sham group (ANOVA). Representative results from one out three independent experiments are shown. B, mRNA expression of glycolysis-related genes in MTE cells treated with or without IL-1β (10 ng/mL). P values from Student’s t test are indicated. C, ECAR and OCR of IL-1β- or sham-treated MTE cells, measured via a Seahorse Extracellular Flux (XF24) Analyzer. Glucose, oligomycin, and 2-DG were injected sequentially marked by the vertical lines. *P < 0.05 compared to the sham group (Student’s t test). Representative results out three independent experiments were shown. D, glucose consumption (left) and uptake (right) in MTE cells 24 h post stimulation with IL-1 β. *P < 0.05 compared to the sham group (Student’s t test).

Figure 4.. Importance of glycolysis for IL-1β-induced pro-inflammatory responses and the IL-1β-mediated augmentation of HDM-induced innate cytokine responses in primary mouse tracheal epithelial (MTE) cells.

A-D, Lactate (A&C) and levels of proinflammatory mediators (B&D) in the cell culture supernatants of MTE cells. MTE cells were pre-treated with 2-Deoxyglucose (2-DG, 10 mM) (A-B), or oxamate (10 mM) (C-D), followed by stimulation with IL-1β (10 ng/mL) for 24 h. E-F, Importance of glycolysis in the IL-1β- mediated augmentation of HDM (50 μg/ml)-induced KC, CCL20, TSLP, and GM-CSF levels in culture supernatants. *P < 0.05 compared to non-HDM exposed sham group, †P < 0.05 compared to respective non-IL-1β treated vehicle group (Veh.), and ‡P < 0.05 relative to non-2DG or non-oxamate treated control group (Ctrl) (two-way ANOVA).

Figure 5.. A causal role for Inhibitory kappa B kinase ε (IKKε) in HDM- and IL-1β-mediated increases in glycolysis and the pathogenesis of allergic airways diseases.

A, Western blot analyses of IKKs in lung tissues of WT mice subjected to the HDM regimen for the indicated times. WT or Ikbke −/− mice were exposed as described in Fig. 1. Mice were euthanized at day 20 for assessment of IKKε in lung tissue via Western blot analysis (B), lactate levels in lung tissues (C), total and differential cell counts in BAL fluid (D), and AHR (E). *P < 0.05 compared to the saline control group, †P < 0.05 compared to respective wildtype (WT) (ANOVA, n = 5–10 per group). F, Assessment of mucus metaplasia in WT or Ikbke^−/− mice exposed to HDM or saline (scale bar, 50 μm) (Top). Quantification of airway mucus staining (PAS) intensity (Bottom). Data are expressed as means (±SEM) from five mice per group. *P < 0.05 compared with respective saline controls. †P < 0.05 compared with WT HDM groups (Kruskal-Wallis). Levels of Muc5AC (G) and pro-inflammatory mediators (H) in lung tissues of WT and Ikbke^−/− mice exposed to HDM as described in B-E. I, BAL and lung lactate levels in WT and Ikbke^−/− mice exposed to IL-1β for 24 h. *P < 0.05 relative to Veh (vehicle) control group, †P < 0.05 relative to the respective wild-type (WT) group (ANOVA, n = 5–10 per group).

Figure 6.. Inhibitory kappa B kinase ε (IKKε) is required for IL-1β-mediated increases in glycolysis, and the IL-1β-mediated augmentation of HDM-induced innate cytokine responses in MTE cells.

A, Immunofluorescence analysis of IKKε in the lungs from HDM- or IL-1β-exposed mice. Red: IKKε, Blue: DAPI counterstain (scale bar, 50 μm). B, mRNA expression of Ikbke in MTE cells exposed to IL-1β. *P < 0.05 relative to sham control (Student’s t test). C, Lactate levels in supernatants of WT or Ikbke^−/− MTE cells stimulated with IL-1β for 24 h. *P < 0.05 compared to sham controls, †P < 0.05 relative to respective WT (ANOVA). D, Lactate levels in cell culture supernatants of MTE cells treated with vehicle or amlexanox, at the indicated concentrations. *P < 0.05 compared to sham controls (Student’s t test). E, Attenuation of IL-1β-induced expression of glycolysis genes in MTE cells pre-treated with 100 μΜ amlexanox. *P < 0.05 relative to the veh/sham group, †P < 05 relative to Veh/IL-1β (ANOVA). F MTE cells were pre-treated with 100 μΜ amlexanox, followed by stimulation of IL-1β for 24 h prior to exposure to HDM (50 μg/ml) for an additional 2 h according to the indicated schematic. KC, CCL20, TSLP and GM-CSF in the cell culture supernatants of mouse tracheal epithelial cells. G KC, CCL20, TSLP and GM- CSF levels in supernatants of WT or Ikbke−/− MTE cells sequentially exposed to IL-1β and HDM according to the schematic. *P < 0.05 relative to non-HDM exposed sham group, †P < 0.05 compared to respective non-IL-1β treated vehicle group (Veh.), and ‡P < 0.05 relative to respective non-amlexanox treated control group (Figure F) or wt group (Figure G) (two-way ANOVA).

Figure 7.. siRNA-mediated knockdown of Ldha attenuates HDM-mediated increase in glycolysis, airway inflammation, and airways hyperresponsiveness.

A, Schematic depicting the dosing regimen of HDM, control (Ctrl) and Ldha siRNAs. At day 40, Salineexposed mice or HDM-exposed mice treated with Ctrl siRNA or Ldha siRNA were harvested for the assessment of LDHA protein levels in the lung tissues via Western blot analyses (B) levels of lactate in BAL and lung tissue (C), total and differential cell counts in the BAL (D), levels of IL-1β, CCL20, IL-33, TSLP, GM-CSF, and IL-13 in the lung tissue (E). *P < 0.05 relative to the naive group, †P < 0.05 relative to the HDM/Ctrl siRNA group (ANOVA). F, Periodic acid Schiff (PAS) staining of airway mucus in saline- or HDM-exposed mice treated with Ctrl siRNA or Ldha siRNA (scale bar, 50 μm) (Left). Quantification of airway mucus staining (PAS) intensity (Right). Data are expressed as means (±SEM) from five-six mice per group. *P < 0.05 compared with naive mice. †P < 0.05 compared to HDM/ctrl siRNA group (Kruskal Wallis) G, Measurement of muc5AC levels in the BAL from mice described in A-E. H, Assessment of AHR. *P < 0.05 relative to naive group, †P < 0.05 relative to HDM/Ctrl siRNA group (ANOVA).

Figure 8.. Evidence of increases in glycolysis in human asthma.

A, Western blot analysis of PKM2 and LDHA, in saline or HDM-treated nasal cells isolated from asthmatics or healthy individuals. Data are representative of 6 healthy subjects, and 6 asthmatics B, Lactate content in culture supernatants of cells shown in A. *P < 0.05 compared to cells from healthy controls not exposed to HDM, †P < 0.05 compared to cells from healthy controls exposed to HDM, (ANOVA). C-F, Lactate (C) and IL-1β levels (E) in the sputum supernatants from healthy subjects (n=20) or asthmatics (n=94). Correlations between lactate content and forced expiratory volume in 1 s percentage predicted (FEV1%) (D) or IL-1β levels (F) in asthmatics and healthy subjects. Correlation analyses were performed via Spearman rank correlation coefficients.