TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury

Abstract

The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging because of the lack of mechanism-based therapeutic approaches. Recent studies have shown that transient receptor potential vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure, and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Postexposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages, and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity, and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function.

Keywords: TRPV4; acute lung injury; chlorine.

Fig. 1.

Structures and pharmacological effects of newly identified TRPV4 inhibitors. A: chemical structure of TRPV4 inhibitor GSK2220691 ('691). B: chemical structure of TRPV4 inhibitor GSK2337429A ('429). C: effect of intravenous pretreatment with TRPV4 inhibitor, GSK2220691, on the increase in lung weight-to-body weight ratio (LW BW ratio) elicited by TRPV4 agonist, GSK1016790 ('790) in rats. Rats first received a 60-min infusion of vehicle or vehicle plus inhibitor, followed by coinfusion of GSK1016790 for 10 min. *P < 0.05 or **P < 0.01 vs. GSK1016790 by 1-way ANOVA Bonferroni post hoc analysis. D: effect of pretreatment by intravenous infusions of GSK2220691 on the GSK1016790-evoked ('790+Veh: 10 μg·kg⁻¹·min⁻¹ infusion) decrease in mean arterial pressure (MAP) in rats. Treatments and statistics as in A. E: effect of intravenous pretreatment with TRPV4 inhibitor, GSK2337429A, on the increase in LW/BW ratio elicited by TRPV4 agonist, GSK1016790, in rats. Protocol as in A; *P < 0.05 or **P < 0.01 vs. GSK1016790 by 1-way ANOVA Bonferroni post hoc analysis. F: effect of pretreatment by intravenous infusions of GSK2337429A on the GSK1016790-evoked ('790+Veh: 10 μg·kg⁻¹·min⁻¹ infusion) decrease in MAP in rats. Protocol as in A.

Fig. 2.

Effects of TRPV4 inhibition, or genetic deletion, on bronchoalveolar lavage fluid (BALF) inflammatory lymphocytes and myeloperoxidase (MPO) activity in HCl-induced lung injury. A: neutrophil counts in BALF extracted from C57BL6 wild-type (WT) mice 5 h after intratracheal administration of saline (Sal), or HCl, and injected intraperitoneally with drug vehicle, or TRPV4 inhibitor, GSK-2220691 (GSK-691), 30 min after HCl administration. Data are means ± SE, n = 7–14/group. B: BALF macrophage counts from the same mice as in A. C: myeloperoxidase enzymatic activity in BALF of the same mice as in A. D: comparison of BALF neutrophil counts of WT and Trpv4−/− mice [knockout (KO)], 5 h after intratracheal administration of saline or HCl; n = 4–5/group. E: BALF macrophage counts of the same mice as in D. F: myeloperoxidase enzymatic activity in BALF of the same mice as in D. G: histopathological scoring of lung sections of mice that received similar exposure and treatments in A. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. respective controls.

Fig. 3.

BALF and serum cytokine levels in HCl-exposed mice treated with TRPV4 inhibitor, and in Trpv4−/− mice. A: levels of VEGF, keratinocyte-derived chemokine (KC), and granulocyte colony-stimulating factor (GCSF) in BALF of wild-type (WT) or Trpv4−/− mice (TRPV4 KO) sampled 5 h after intratracheal administration of saline, or HCl, and injected intraperitoneally with drug vehicle or TRPV4 inhibitor GSK-2220691, 30 min after HCl administration, measured by bead-based multiplexing (Milliplex). Data are means ± SE and are representative of 3 independent experiments with 4 animals/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective controls. B: levels of IL-6, KC, and G-CSF in serum of the same mice as in A.

Fig. 4.

Effects of TRPV4 inhibitor treatment on pulmonary function, protein leakage, and hypoxemia in chlorine-exposed mice. A: pulmonary resistance (left) and elastance (right) in response to methacholine (MeCh) in mice exposed to room air (Air) or chlorine (Cl₂, 400 ppm, 30 min) and injected intraperitoneally with vehicle (0.5% methylcellulose) or TRPV4 inhibitor GSK-2220691 (+GSK-691) at 30 min (30 mg/kg) and 8 h (15 mg/kg) after end of exposure, measured by forced oscillation 24 h after exposure (flexiVent). Data are means ± SE and are representative of 3 independent experiments with n = 4–6/group. B: pulmonary resistance (left) and elastance (right) in response to methacholine in mice exposed to room air or chlorine and injected with vehicle (0.5% methylcellulose) or TRPV4 inhibitor GSK2337429A (+GSK-429) at 30 min (50 mg/kg) and 8 h (25 mg/kg) after end of exposure, measured by forced oscillation 24 h after exposure (flexiVent); n = 4–6/group. C: blood oxygen saturation levels in chlorine-exposed mice shown in A and B, measured 24 h post-chlorine exposure (400 ppm, 30 min); n = 4–6/group. D: total protein concentration in BALF in mice shown in A and B, measured at 24 h post-chlorine exposure; n = 4–6/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. air-exposed group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. chlorine-exposed group.

Fig. 5.

Representative histopathological lung sections from mice exposed to chlorine and treated with TRPV4 inhibitors. Groups and treatments as in Fig. 2 and 4.

Fig. 6.

Pulmonary lymphocytes and myeloperoxidase activity in chlorine-exposed mice. A: macrophage counts in BALF extracted from C57BL6 WT mice 24 h after exposure to chlorine (400 ppm, 30 min), and injected intraperitoneally with drug vehicle (0.5% methylcellulose), or TRPV4 inhibitors, GSK-2220691 [30 min (30 mg/kg) and 8 h (15 mg/kg) after exposure, GSK691] or GSK2337429A [at 30 min (50 mg/kg) and 8 h (25 mg/kg) after exposure, +GSK-429]. Data are means ± SE, n = 12/group. B: BALF neutrophil counts from the same mice as in A. C: myeloperoxidase enzymatic activity in BALF of the same mice as in A. D: histopathological scoring of lung sections of mice that received similar exposure and treatments as those in A. ***P < 0.001, ****P < 0.0001 vs. respective controls.

Fig. 7.

Effects of intramuscular TRPV4 inhibitor administration on markers of chlorine-induced inflammation. A: pulmonary resistance in response to methacholine (MeCh) in mice exposed to room air (Air) or chlorine (Cl₂, 400 ppm, 30 min), and injected intramuscularly with drug vehicle (0.5% methylcellulose), or TRPV4 inhibitor, GSK-2220691 [30 min (30 mg/kg) and 8 h (15 mg/kg) after exposure, GSK691], measured by forced oscillation 24 h after exposure (flexiVent). Data are means ± SE, n = 5/group. B: macrophage counts in BALF extracted from the same mice as in A. C: BALF neutrophil counts from the same mice as in A. D: myeloperoxidase chlorination activity in BALF of the same mice as in A. E: cytokines and chemokine levels in BALF of the same mice as in A, extracted 24 h after chlorine exposure (400 ppm, 30 min), measured by bead multiplex analysis. **P < 0.01, ***P < 0.001 vs. air-exposed group; #P < 0.05, ###P < 0.001 vs. chlorine-exposed group.

Fig. 8.

Inflammatory cytokines and chemokines in BALF and serum, markers of vascular injury in BALF of chlorine-exposed mice A: cytokine and chemokine concentrations in BALF extracted from C57BL6 WT mice 24 h after exposure to chlorine (400 ppm, 30 min), and injected intraperitoneally with drug vehicle (0.5% methylcellulose), or TRPV4 inhibitors, GSK-2220691 [30 min (30 mg/kg) and 8 h (15 mg/kg) after exposure, GSK691] or GSK2337429A [at 30 min (50 mg/kg) and 8 h (25 mg/kg) after exposure, +GSK-429]. Data are means ± SE, n = 12/group. B: cytokine and chemokine concentrations in serum of the same mice as in A. C: concentration of SAP, fibrinogen, adiponectin, and sVCAM-1 in BALF of mice as in A, measured by bead multiplex analysis. **P < 0.01, ***P < 0.001 vs. respective controls.

Fig. 9.

Low-pH extracellular solution and NaOCl have no effect on TRPV4 expressed in HEK293 cells, measured by ratiometric Ca²⁺ imaging. Representative Ca²⁺ imaging traces showing the effect of 0.05% NaOCl (A) or pH 3.0 extracellular solution (B) on mouse TRPV4 (mTRPV4) transiently expressed in HEK293 cells, followed by application of GSK1016790A (GSK, 30 nM) and ionomycin (Iono, 1 μM). GSK1016790A was used as a positive control to activate TRPV4, and ionomycin was used to determine the maximal Ca²⁺ responses in the end. Cells were loaded with fura-2 AM. 20–30 cells were included in each graph. C: summarized data showing the increases of fluorescence ratio (340/380) of mTRPV4 expressed HEK293 cells responding to vehicle (veh), NaOCl, low-pH extracellular solution, and GSK; n = 30–40 cells/group. **P < 0.01; NS, no significance.

Fig. 10.

Transient receptor potential (TRP) channel gene expression in primed human neutrophils. A: transcription analysis of TRP ion channel genes in untreated (solid), TNF-primed (open), and GM-CSF-primed (shaded) human neutrophils derived from RNAseq datasets (NCBI GEO Series accession number GSE40548) (40). Reads were mapped to the human genome (hg19) by using Tophat and Bowtie. Mapped reads, or fragments, were assigned to transcripts by use of Cufflinks and differential expression was determined by using Cuffdiff. Values are displayed as fragments per kilobase of transcript per million mapped reads (FPKM). B: comparison of TRP ion channel transcript levels in untreated human neutrophils with those of other ion channel genes involved in leukocyte activation (OraI1 and 2, P2X1), STIM1 (associated with OraI), and the cell adhesion molecule ICAM1. Analysis as in A.