TREM-1 governs NLRP3 inflammasome activation of macrophages by firing up glycolysis in acute lung injury

Abstract

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a pro-inflammatory immune receptor potentiating acute lung injury (ALI). However, the mechanism of TREM-1-triggered inflammation response remains poorly understood. Here, we showed that TREM-1 blocking attenuated NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome activation and glycolysis in LPS-induced ALI mice. Then, we observed that TREM-1 activation enhanced glucose consumption, induced glycolysis, and inhibited oxidative phosphorylation in macrophages. Specifically, inhibition of glycolysis with 2-deoxyglucose diminished NLRP3 inflammasome activation of macrophages triggered by TREM-1. Hypoxia-inducible factor-1α (HIF-1α) is a critical transcriptional regulator of glycolysis. We further found that TREM-1 activation facilitated HIF-1α accumulation and translocation to the nucleus via the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. Inhibiting mTOR or HIF-1α also suppressed TREM-1-induced metabolic reprogramming and NLRP3/caspase-1 activation. Overall, the mTOR/HIF-1α/glycolysis pathway is a novel mechanism underlying TREM-1-governed NLRP3 inflammasome activation. Therapeutic targeting of the mTOR/HIF-1α/glycolysis pathway in TREM-1-activated macrophages could be beneficial for treating or preventing inflammatory diseases, such as ALI.

Keywords: Acute lung injury; HIF-1α; NLRP3 inflammasome; TREM-1; glycolysis; macrophages.

Figure 1

Blockade of TREM-1 reduced intrapulmonary inflammation and limited glycolysis in LPS-induced ALI mice. C57BL/6J mice were intravenously injected with LR12 (5 mg/kg) 2 h before the LPS administration (5 mg/kg, i.t.). (A) Six hours later, mouse lungs were excised, and lung histopathology was performed with H&E staining. One representative picture of six mice in each group is shown. (B) Inflammation score was measured, n=6 mice/group. (C) Pro-IL-1β, IL-1β p17, and iNOS in the lung lysates were assessed by western blot with β-actin as a loading control. (D-F) The western blot results were quantitated using Image Lab, n=8 mice per group. (G-H) Lactate concentration in BALF and serum was assayed, n=4-9 mice/group. (I) Expression of Hk2, Pfkfb3, and Hif-1α mRNA in the lungs was detected by real-time PCR. Data was normalized to housekeeping gene β-actin, n=5-10 mice/group. (J) Glycolysis-associated proteins of HK2, p-mTOR, mTOR, and HIF-1α in the lung lysates were assessed by western blot with α-tubulin as a loading control. (K-N) Quantification of indicated protein levels in (J), n=6-8 mice/group. In all cases, the experiment was repeated twice. Dots represent individual animal values. Statistical analysis was performed using One-way ANOVA adjusted by Tukey's multiple comparison test for Control vs. ALI or ALI vs. LR12+ALI. Error bars indicate mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001. Original western blots represented in graphs are available in Figure 1-source data.

Figure 2

TREM-1 activation instigated glucose metabolic reprogramming in macrophages. Macrophages were stimulated with 10 μg/mL of an agonist anti-TREM-1 mAb. (A) Six hours later, the lactate in the supernatant was assayed, n=3. (B) Glucose consumption in the supernatant in control or 24 h TREM-1-activated macrophages, n=3. (C) Glut-1, Hk2, Pfkfb3, Pkm2, Ldha, and Hif-1α mRNA in control or 6 h TREM-1-activated macrophages. Data was normalized to housekeeping gene β-actin, n=3-5. (D-F) Western blot and quantification of the glycolytic enzymes (HK2 and LDHA) in the control or 24 h TREM-1-activated macrophages, n=3. (G) OXPHOS-related proteins: ATP5A, MTCO1, UQCRC2, SDHB, and NDUFB8, in the macrophages were detected by western blot. (H-J) Quantification of indicated protein levels in (G), n=3. Macrophages were administrated with LR12 (25 μg/mL) 30 min before the LPS stimulation (1 ng/mL). Twenty-four hours later, (K) the lactate and (L) relative glucose consumption in the supernatant were assayed, n=3. (M) Expression of Glut-1, Ldha, and Hif-1α mRNA in macrophages was detected by real-time PCR, n=3. n represents experiments performed on different macrophages from separate mice. Bar graphs represent mean ± SD. Student's t-test (two-tailed, unpaired) was used to compare Mab1187 and Control in (A-J): * P < 0.05, ** P < 0.01, and *** P < 0.001. One-way ANOVA adjusted by Tukey's multiple comparison test was used in (K-M): * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 3

TREM-1 triggered the NLRP3 inflammasome activation in macrophages. Macrophages were incubated with plate-bound isotype-matched control or plate-bound anti-TREM-1 mAb (10 μg/mL). (A-C) Six hours later, Nlrp3, Pro-caspase-1, and Pro-il-1β mRNA expressions in macrophages were measured using qPCR. Data was normalized to housekeeping gene β-actin, n=3. (D) Twenty-four hours later, protein expression of NLRP3, pro-caspase-1, and pro-IL-1β in the cell lysate was detected by western blot with α-tubulin as a loading control. (E-G) Quantification of indicated protein levels in (D), n=3. (H-J) Caspase-1 p10 and IL-1β p17 in the supernatant were detected by western blot, n=3. (K) IL-1β contents in the supernatants were analyzed with ELISA, n=5. n represents experiments performed on different macrophages from separate mice. Bar graphs represent mean ± SD. Student's t-test (two-tailed, unpaired) was used to compare Control and Mab1187: * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 4

Blockade of glycolysis partially inhibited TREM-1-mediated NLRP3 inflammasome activation in macrophages. (A) Macrophages (1×10⁶ cells/well) were premixed with PBS control or 2-DG (5 mM) and then plated into 12-well plates with agonist anti-TREM-1 mAb (10 μg/mL). After 24 h, supernatants were analyzed for lactate production, n=3. (B, D) Macrophages were treated as in (A), and 6 h later, Tnf‐α and Pro-il-1β mRNA levels in macrophages were measured using qPCR, n=3. (C, E) Macrophages were treated as in (A). Twenty-four hours later, TNF‐α and IL-1β production in the supernatant was measured by ELISA, n=3. (F) NLRP3, pro-IL-1β, IL-1β p17, and caspase-1 p10 protein in cell lysate were detected by western blot, n=3. (G-J) Quantification of indicated protein levels in (F). n=3 biological replicates. Data are expressed as the mean ± SD. One-way ANOVA adjusted by Tukey's multiple comparison test was used. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 5

TREM-1 activation induced HIF-1α accumulation and translocation to the nucleus in macrophages. Macrophages were incubated with anti-TREM-1 mAb (10 μg/mL) in a normoxia condition. (A) Six hours later, Hif‐1α gene levels were measured using qPCR, n=4-6. (B-C) Twenty-four hours later, the protein of HIF‐1α was performed by western blot with GAPDH as a loading control, n=3. (D) Macrophages were cultured on anti-TREM-1 for 24 h and then subjected to immunofluorescence examination to analyze the HIF-1α accumulation and translocation to the nucleus (scale bar, 50 μm). (E) Average fluorescent intensity was calculated by HIF-1α⁺ fluorescence intensity (IntDen)/area of the region (Area) using ImageJ, n=6. (F-G) 1×10⁶ cells/well were premixed with PBS control or PX-478 (25 μM) for 30 min, then plated into 12-well plates with agonist anti-TREM-1 mAb (10 μg/mL). HK2 protein levels were measured after an additional incubation for 24 h, n=3. n represents experiments performed on different macrophages from separate mice. Data are expressed as the mean ± SD. Student's t-test (two-tailed, unpaired) was used to compare Mab1187 and Control in (A-E): * P < 0.05, ** P < 0.01, and *** P < 0.001. One-way ANOVA adjusted by Tukey's multiple comparison test was used in G: *** P < 0.001.

Figure 6

HIF-1α droves TREM-1-mediated NLRP3 inflammasome activation. Macrophages were premixed with PBS control or PX-478 (25 μM) before incubating with plate-bound agonistic anti-TREM-1 mAb (10 μg/mL). (A) Twenty-four hours later, lactate level in supernatant was assayed, n=3. (B-C) The concentration of TNF‐α and IL-1β in the supernatant was assayed using ELISA, n=3. (D) Protein expression of NLRP3, pro-IL-1β, IL-1β p17, and caspase-1 p10 in macrophage lysate was detected by western blot. (E-H) Quantification of indicated protein levels in (D), n=3 biological replicates. Statistical analysis was performed using One-way ANOVA adjusted by Tukey's multiple comparison test. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 7

TREM-1 activation stimulated HIF-1α accumulation via PI3K/AKT/mTOR signaling. 1×10⁶ macrophages/well were plated into 12-well plates with agonist anti-TREM-1 mAb (10 μg/mL). (A) p-PI3K p85α^T607, total PI3K, p-AKT^s473, AKT, p-mTOR^s2448, and mTOR protein levels in control or 24 h TREM-1-activated macrophages. (B-D) Quantification of p-PI3K p85α^T607, p-AKT^s473 and p-mTOR^s2448 in (A), n=3 biological replicates. Statistical analysis was performed using Student's t-test (two-tailed, unpaired). (E) Lactate secretion, (F) glucose consumption in the supernatant, and (G-H) HIF-1α protein levels in control or 24 h TREM-1-activated macrophages co-treated with or without LY294002 (25 μM). n=3 biological replicates. Statistical analysis was performed using One-way ANOVA. (I) Lactate secretion, (J) glucose consumption in the supernatant, and (K-L) HIF-1α protein levels in control or 24 h TREM-1-activated macrophages co-treated with or without Rapamycin (100 nM). n=3 biological replicates. Statistical analysis was performed using One-way ANOVA adjusted by Tukey's multiple comparison test. Data were expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 8

TREM-1 triggered the NLRP3 inflammasome activation by enhancing mitochondrial fission in macrophages. Macrophages were stimulated with 10 μg/mL of an agonist anti-TREM-1 mAb. (A-B) Western blot and quantification of p-DRP1^ser616 and DRP1 in control or 24 h TREM-1-activated macrophages. n=3 biological replicates. Statistical analysis was performed using Student's t-test (two-tailed, unpaired). Macrophages were premixed with PBS control or Mdivi-1 (100 nM) before incubating with plate-bound agonistic anti-TREM-1 mAb for 24 h. (C) Protein levels of NLRP3, pro-IL-1β, IL-1β p17, and caspase-1 p10 in macrophages were detected by western blot. (D-G) Quantification of NLRP3, pro-IL-1β, IL-1β p17, and caspase-1 p10 (C), n=3 biological replicates. Statistical analysis was performed using One-way ANOVA. Data were expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 9

Schematic illustration. TREM-1 activation stimulates HIF-1α induced glucose metabolic reprogramming via PI3K/AKT/mTOR pathway. HIF-1α-induced glycolysis promotes TREM-1-governed NLRP3 inflammasome activation, facilitating intrapulmonary inflammation in ALI.