TRPV4 Regulates the Macrophage Metabolic Response to Limit Sepsis-induced Lung Injury

Abstract

Sepsis is a systemic inflammatory response that requires effective macrophage metabolic functions to resolve ongoing inflammation. Previous work showed that the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), mediates macrophage phagocytosis and cytokine production in response to lung infection. Here, we show that TRPV4 regulates glycolysis in a stiffness-dependent manner by augmenting macrophage glucose uptake by GLUT1. In addition, TRPV4 is required for LPS-induced phagolysosome maturation in a GLUT1-dependent manner. In a cecal slurry mouse model of sepsis, TRPV4 regulates sepsis-induced glycolysis as measured by BAL fluid (BALF) lactate and sepsis-induced lung injury as measured by BALF total protein and lung compliance. TRPV4 is necessary for bacterial clearance in the peritoneum to limit sepsis-induced lung injury. It is interesting that BALF lactate is increased in patients with sepsis compared with healthy control participants, supporting the relevance of lung cell glycolysis to human sepsis. These data show that macrophage TRPV4 is required for glucose uptake through GLUT1 for effective phagolysosome maturation to limit sepsis-induced lung injury. Our work presents TRPV4 as a potential target to protect the lung from injury in sepsis.

Keywords: TRPV4; glycolysis; lung injury; macrophage; sepsis.

Figure 1.

Transient receptor potential vanilloid 4 (TRPV4) enhances LPS-induced macrophage phagolysosome maturation in a stiffness-dependent manner. Wild-type (WT) and Trpv4^−/− bone marrow–derived macrophages (BMDMs) were plated on polyacrylamide gels of pathophysiologic-range lung stiffness (1 kPa = normal lung, 25 kPa = injured/stiffened lung) treated or not treated with 100 ng/ml LPS for 24 hours with or without pHrodo Zymosan A bioparticles. (A) Phagolysosome maturation was imaged by confocal microscopy with a representative image shown. Original magnification, 63×. Scale bars, 25 μm. (B) Phagolysosome maturation was increased by 186.8% in WT BMDMs on LPS treatment in a stiffness-dependent manner that was abrogated in Trpv4^−/− BMDMs as measured by integrated density per cell. n = 3 biological replicates; each dot represents one high-powered field. *P ⩽ 0.01 denotes increase in LPS versus UT. ^†P ⩽ 0.01 denotes difference between WT and KO at 25 kPa by Kruskal-Wallis test and Dunn’s multiple comparisons test. KO = knockout; UT = untreated.

Figure 2.

TRPV4 enhances glycolysis in LPS-treated macrophages in a stiffness-dependent manner. WT and Trpv4^−/− BMDMs were plated on polyacrylamide gels of pathophysiologic-range lung stiffness (1 kPa = normal lung; 25 kPa = injured/stiffened lung) or standard tissue-culture conditions with or without 100 ng/ml LPS treatment for 24 hours. Glycolysis and glycolytic capacity were measured using extracellular acidification rate (ECAR) in mpH per minutes per cell. (A) Glycolysis is stiffness dependent in LPS-treated macrophages as measured by ECAR. (B) Glycolysis and glycolytic capacity is increased in a stiffness-dependent manner. *P ⩽ 0.0001 denotes 1 kPa versus 25 and 10⁶ kPa by Kruskal-Wallis test with Dunn’s multiple comparisons tests. ^†P ⩽ 0.0001 denotes 1 kPa versus 25 and 10⁶ kPa by one-way ANOVA with Newman-Keuls multiple comparisons test. ^‡P ⩽ 0.001 denotes 25 kPa versus 10⁶ kPa by one-way ANOVA with Newman-Keuls multiple comparisons test. (C) Glycolysis is increased in WT compared with Trpv4^−/− LPS-treated macrophages on pathophysiologic-range lung stiffness (25 kPa) as measured by ECAR. (D) Glycolysis and glycolytic capacity have a greater induction in response to LPS in WT BMDMs compared with Trpv4^−/− BMDMs at 25 kPa. n = 3 biological replicates. *P ⩽ 0.0001 denotes WT versus KO by t test. ^†P ⩽ 0.001 denotes WT versus KO by t test. mpH = milli pH; Norm. = normal; Oligo = Oligomycin; Rot/Ant = Rotenone/Antimycin A.

Figure 3.

TRPV4 is associated with increased glucose uptake and cellular metabolites in LPS-treated macrophages. LPS-treated WT and Trpv4^−/− BMDMs were plated on polyacrylamide gels of injured lung stiffness (25 kPa). Metabolites were measured using mass spectrometry. (A) Simplified schematic of glycolysis pathway. (B–F) Glycolytic metabolites are reduced in LPS-treated Trpv4^−/− BMDMs compared with WT on 25 kPa (injured/stiffened lung) as measured by mass spectrometry, including (B) glucose (P ⩽ 0.05), (C) fructose-1–6-bisphosphate (P = 0.056), (D) DHAP/GADP (P ⩽ 0.01), (E) phosphoenolpyruvate (P ⩽ 0.05), and (F) lactate (P = 0.081). n = 5 biological replicates. *P ⩽ 0.05 denotes WT versus KO by t test. ^†P ⩽ 0.01 denotes WT versus KO by t test.

Figure 4.

Glucose uptake, lactate production, and phagolysosome maturation in LPS-treated macrophages require GLUT1 and Ca²⁺. (A) LPS-induced glucose uptake and (B) lactate production are abrogated with GLUT1 inhibition and in Ca²⁺-free conditions in WT BMDMs. *P < 0.0001 denotes UT versus LPS. ^†P < 0.001 denotes +Ca²⁺ and −Ca²⁺ conditions. ^‡P < 0.0001 denotes LPS versus BAY/LPS by one-way ANOVA with Tukey and Sidak’s multiple comparisons tests or Kruskal-Wallis test as appropriate. (C) WT and Trpv4^−/− BMDMs were incubated with or without LPS and with or without BAY 876 (GLUT1 inhibitor, 24 h) with pHrodo Zymosan A bioparticles and imaged by means of confocal microscopy. Scale bars, 25 μm. (D) Representative images are shown and quantified. GLUT1 inhibition abrogated phagolysosome maturation. n = 3 biological replicates. Each dot represents one high-powered field, *P ⩽ 0.05 denotes UT versus LPS. ^†P ⩽ 0.05 denotes LPS versus BAY/LPS. ^‡P ⩽ 0.05 denotes WT LPS versus Trpv4^−/− LPS by one-way ANOVA with Tukey and Sidak’s multiple comparisons tests or Kruskal-Wallis test as appropriate.

Figure 5.

Increased GLUT1 expression rescues phagolysosome maturation in Trpv4^−/− macrophages. WT and TRPV4 BMDMs were transfected with GLUT1 LV or EV and treated or not treated with 100 ng/ml LPS on standard tissue-culture conditions. Phagolysosome maturation was measured with pHrodo Zymosan A bioparticles. (A) GLUT1 overexpression increased LPS-induced phagolysosome maturation in Trpv4^−/− BMDMs. Scale bars, 25 μm. (B) Representative images are shown and quantified. n = 3 biological replicates. Each dot represents a high-powered field, 63×. EV = empty vector; LV = lentivirus vector. *P ⩽ 0.05 denotes WT versus KO upon treatment with EV. ^†P ⩽ 0.05 denotes KO^+/− treatment with GLUT1LV.

Figure 6.

TRPV4 augments BAL lactate and protects the lungs against sepsis-induced lung injury in vivo. WT and Trpv4^−/− mice were infected with intraperitoneal cecal slurry (CS) or glycerol vehicle control. (A) BALF lactate is reduced in Trpv4^−/− mice. *P < 0.05 denotes WT versus KO by t test. (B) BALF protein was increased in Trpv4^−/− mice compared with WT mice (P ⩽ 0.01). ^†P < 0.01 denotes WT versus KO by t test. (C) Static compliance of the lung was unchanged in WT mice but reduced by 20.4% in Trpv4^−/− mice (P ⩽ 0.05). ^‡P ⩽ 0.05 denotes KO^+/− CS by one-way ANOVA with Sidak’s multiple comparisons test. (D) Bacterial clearance from the peritoneal cavity was impaired in Trpv4^−/− mice compared with WT (P ⩽ 0.05); n = 6 mice that received glycerol, and n = 9 mice that received CS per genotype. *P < 0.05 denotes WT versus KO by t test, ^†P < 0.05 denotes KO^+/− CS by one-way ANOVA with Sidak’s multiple comparisons test. (E) BAL fluid lactate is elevated in patients with sepsis versus healthy control participants (HCs). n = 5 HCs, and n = 5 patients with sepsis. * and † denotes WT mice that received CS versus KO mice that received CS by t test, *KO UT versus KO CS by one-way ANOVA with Sidak’s multiple comparisons test. *P ⩽ 0.05 HC versus sepsis by t test.

Figure 7.

Schematic of the proposed mechanism of TRPV4 mediated metabolism in macrophages. TRPV4 regulates GLUT1 glucose uptake function after LPS to induce glycolysis and phagolysosome maturation in macrophages in a stiffness-dependent manner, protecting against sepsis-induced lung injury. Figure was created with BioRender.com.