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.

acute lung injury (ALI) and its extreme manifestation, acute respiratory distress syndrome (ARDS), are associated with high levels of morbidity and mortality (28, 37). Major triggers of ALI and ARDS are pneumonia, sepsis, trauma, acid aspiration, inhalation of toxic gases or smoke, hyperoxia, high pressure ventilation, heart failure, or pancreatitis. A major hallmark of ALI and ARDS is the acute increase in permeability of the pulmonary vascular and epithelial barriers, resulting in edema and severe hypoxia (9). ALI and ARDS are often associated with exaggerated inflammatory responses due to neutrophil infiltration and increased macrophage activity in the injured lung (14, 22). These inflammatory cells may aggravate injury through protease production, through generation of oxidative reactive species and proinflammatory cytokines and chemokines, and through prevention of inflammation resolution.

The ion channel transient receptor potential vanilloid 4 (TRPV4) was recently identified as a major mediator of pulmonary dysfunction in animal models of ventilator- and heart failure-induced ALI, conditions associated with dramatic increases in pulmonary and vascular pressure (17, 33). Among other locations, TRPV4 is expressed in pulmonary epithelial cells, in the vascular endothelium, and in macrophages and contributes to barrier disruption and pulmonary edema when activated by selective pharmacological agonists or by lung distension (1, 17, 39). These effects are due to TRPV4-mediated Ca²⁺-influx into epithelial and vascular endothelial cells leading to subsequent barrier dysfunction (20, 32, 39). Studies on ventilator-induced lung injury suggest that TRPV4 channels in pulmonary macrophages are crucial for induction of injury (16).

Although TRPV4 is clearly involved in mechanically (stretch or pressure) induced ALI, it remains to be established whether TRPV4 is also critical for chemically induced forms of ALI. Chemical injuries caused by the inhalation of toxic gases or smoke are frequent causes of severe lung injury (3, 11, 41). Another form of chemical lung injury with significant morbidity and mortality is acid-induced lung injury associated with gastroesophageal reflux disease or acid aspiration during surgery (5). Only limited treatment options are available for these injuries, none of them mechanism based (38).

Herein, we examined the role of TRPV4 in mouse models of acid (HCl)- and chlorine gas (Cl₂)-induced chemical ALI, mimicking acid aspiration- or toxic gas inhalation-induced injuries. Acid-exposed mice treated with TRPV4 inhibitors showed strongly diminished pulmonary inflammation, an observation recapitulated in TRPV4-deficient mice. In mice exposed to highly corrosive levels of chlorine gas TRPV4 inhibitors strongly reduced airway hyperreactivity, protein leakage, and pulmonary and systemic inflammation and prevented blood oxygen desaturation. Chemical pulmonary injury induced the production of multiple transient receptor potential (TRP) channel activators. Together, these data link TRPV4 to mechanisms of chemical lung injury and identify TRPV4 inhibitors as candidate treatments.

MATERIALS AND METHODS

TRPV4 inhibitor screen, selectivity, and pharmacokinetics.

TRPV4 inhibitor screening and selectivity assessment were performed on a fluorometric imaging plate reader (FLIPR) platform with hTRPV4-transfected HEK293 cells assessing the ability to inhibit TRPV4-mediated Ca²⁺ influx after TRPV4 activation with agonists GSK634775, GSK1016790, or hypotonicity (33). For selectivity assessment, FLIPR assays were run by using standard FLIPR protocols (Molecular Devices), and BacMam vector overexpression of the targets (TRPV1, TRPA1, TRPC3, TRPC6, TRPM8) in HEK cells stably expressing the macrophage scavenger receptor II, or U2-OS cells (NK2 and NK3). Assays were run with Fluo4 calcium indicator, except for TRPC3 and TRPC6, which were run with a membrane potential indicator. The TRPC6 channel was coexpressed with M1 muscarinic receptors that were used to indirectly open the channels. hERG and Cav1.2 were evaluated by whole-cell voltage clamp on PatchXpress 7000A. Nav1.5 responses were measured by using population patch clamp on IonWorks Quattro. The following TRP channel ligands were used: TRPV1, capsaicin; TRPA1, thymol; TRPC3 and TRPC6, carbachol; and TRPM8, icilin (33). Pharmacokinetics were determined as described (10).

Animals.

Eight- to 10-wk-old C57BL/6 male mice [20–25 g body weight (BW)] were purchased from Charles River Laboratories. They were housed in the animal unit for at least 3 days before any experimental procedure during which they were provided with mouse chow and water ad libitum. C57BL/6-backcrossed Trpv4−/− mice were provided by GlaxoSmithKline Pharmaceuticals. Sprague-Dawley rats were used for studying the pharmacological effects of newly identified TRPV4 inhibitors. All experimental protocols were approved by the Institutional Animal Care and Use Committees of GlaxoSmithKline, University of Alabama at Birmingham, and the Yale University.

Acid-induced pulmonary injury in mice.

Mice received a single intratracheal instillation of HCl (J. T. Baker, pH 1.5, 2 ml/kg) via a 24-gauge angiocatheter inserted into the trachea under sevoflurane anesthesia while placed on a heating pad to maintain body temperature. Control animals received saline instead of HCl in the same manner. At 5 h after instillation bronchoalveolar lavage procedure was performed and lungs were isolated and frozen in liquid nitrogen for subsequent biochemical analyses (8).

Exposure of mice to chlorine gas.

Mice were exposed to chlorine gas (Cl₂, 400 ppm) for 30 min in a cylindrical glass chamber (Specialty Glass, no. X02AI99C15A57H5). Two mass flow controllers with Kalrez seals (Scott Specialty Gases, no. 05236A1V5K) and a microprocessor control unit (Scott Specialty Gases, no. 05236E4) were used to control the flow rates of compressed air and Cl₂ (1,000 ppm Cl₂ in air; Airgas) to achieve the desired Cl₂ concentrations (400 ppm) in the exposure chamber. Continuous measurements of Cl₂ concentrations during the exposure were monitored with an Interscan (model no. RM34-20.0m) Cl₂ detector, connected to a data logger for data storage. Air and Cl₂ were mixed at a three-way junction, and they were further mixed by passing through a diffuser located inside the top lid of the exposure chamber. Gases exited the chamber via two large-bore-diameter ports in its bottom half. The exposure chamber was placed inside a chemical hood located in a negative pressure room. At the end of the exposure period (30 min) the Cl₂ gas was turned off, the chamber was vented with compressed air for 2–3 min, the two halves were separated, and the mice were removed and returned to room air.

TRPV4 inhibitor treatment.

TRPV4 inhibitor GSK2220691 (30 mg and 15 mg/kg BW) or GSK2337429A (50 mg and 25 mg/kg BW) were injected intraperitoneally or intramuscularly (into the caudal thigh) immediately after Cl₂ exposure and 8 h post-Cl₂ exposure. Animals in the acid-induced lung injury model received a single intraperitoneal dose of the TRPV4 antagonist (GSK2220691: 30 mg/kg BW; or GSK2337429A: 50 mg/kg BW) 30 min after the acid instillation.

Measurement of arterial blood oxygen saturation.

Blood arterial oxygen saturation was recorded with a rodent oximeter sensor (MouseOX; STARR Life Sciences) mounted on the tail of anesthetized (pentobarbital: 60 mg/kg BW and urethane: 1 g/kg) mice. Data were collected for a minimum of 40–60 s without any error code. Animal were kept on a heat pad during reading of the oxygen saturation levels.

Determination of chlorine-induced airway hyper-responsiveness.

The respiratory mechanics were measured using the forced oscillation technique (flexiVent, SCIREQ). Anesthetized (pentobarbital: 60 mg/kg BW and urethane: 1 g/kg) mice were intubated and mechanically ventilated by a computer controlled piston ventilator. Mice were then challenged by aerosolized bronchoconstrictor methacholine (MeCh; Sigma), at increasing concentrations (0, 12.5, 25, 50, and 100 mg/ml). At each dose, lung resistance and elastance were calculated by the single compartment model.

Collection of BALF and leukocyte analysis.

At the end of 5 h post-acid instillation or 24 h post-chlorine exposure, bronchoalveolar lavage fluid (BALF) was performed by cannulation of the trachea and gentle instillation of 1.0 ml PBS with 0.1% BSA and protease inhibitor (Roche). BALF was harvested from animals that were not subjected to pulmonary function tests. The lavage fluid was centrifuged and the supernatant was stored at −80°C for later assessment of cytokines, chemokines, myeloperoxidase (MPO), and uric acid levels. The cell pellet was treated with red blood cell lysis buffer (BD Biosciences), washed, and resuspended in 200 μl PBS. Total cell counts were determined manually with a hemocytometer. Cells were centrifuged onto cytoslides (Shandon Cytospin 4 cytocentrifuge, Thermo Electron) and stained with Diff-Quick (Dade-Behring). Differential cell counts were obtained by microscopic counting of a minimum of 200 cells/slide using standard morphological and staining criteria.

Quantification of BALF and serum cytokines.

Cytokine/chemokine levels were measured from 50 μl of cell-free BALF or serum (mouse cytokine assay; Millipore) by using a high-throughput multiplex cytokine assay system according to the manufacturer's instructions. Each sample was analyzed in duplicate on the Bio-Plex 200 system (Bio-Rad). The concentrations of analytes in these assays were quantified with a standard curve, and a five-parameter logistic regression was performed to derive an equation that was then used to predict the concentrations of the unknown samples. The data presented here excluded any number below the range of sensitivity for the particular analyte.

Lung histology and injury scoring.

Lungs were filled in situ very slowly with 10% formalin at a constant pressure of 25 cmH₂O (∼8 ml), removed en bloc, and immersed in 10% formalin for 48 h. Fixed lungs were embedded in paraffin, sectioned at 5-μm thickness, and stained with hematoxylin and eosin. Images were obtained with a Zeiss Axio Imager Z1 microscope and analyzed by AxioVision Rel. 4.7 software (Zeiss, Munich, Germany). Histopathology of lung tissues was scored on a severity scale of 0–4 (0 = normal, 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked) in at least two sections per mouse. For scoring, the following parameters were primarily considered: airways (patency of airways, altered epithelial lining/necrotic lesions in airway passages, detachment of epithelia, loss of cilia) blood vessels (perivascular cuffing), parenchyma (atelectasis, reactive type II pneumocyte hyperplasia), and infiltration of inflammatory cells (13).

Chlorination activity of MPO.

The MPO chlorination activity of the BALF was determined as described by the manufacturer (Cayman Chemical). The assay utilizes the nonfluorescent 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]-benzoic acid, which is selectively cleaved by hypochlorite (OCl⁻) to yield the highly fluorescent compound fluorescein. Fluorescence of fluorescein is analyzed with an excitation wavelength of 480–490 nm and an emission wavelength of 515–520 nm. The kit includes a MPO-specific inhibitor for distinguishing MPO activity from MPO-independent fluorescence.

Analytical standards and reagents for lipidomics.

AEA, 2-AG, 2-linoleoyl glycerol, N-palmitoyl ethanolamine (PEA), N-stearoyl ethanolamine (SEA), N-oleoyl ethanolamine (OEA), N-linoleoyl ethanolamine (LEA), N-docosahexaenoyl ethanolamine (DHEA), N-arachidonoyl glycine (NAGly), N-linoleoyl glycine (LinGly); N-oleoyl glycine (OlGly); and NAGly-d8; and 2-AG-d8 were from Cayman Chemical. 2-Oleoyl glycerol was obtained from Avanti Polar Lipids. All additional N-acyl amides were made in house as previously described (1). HPLC-grade water and methanol were purchased from VWR International. HPLC-grade ammonium acetate was from Sigma-Aldrich. C18 solid-phase extraction and analytical (Zorbax eclipse XDB 2.1 × 50 mm reversed phase) columns were purchased from Varian.

HEK293 cell culture and Ca²⁺ imaging.

Human embryonic kidney (HEK) 293 were cultured in Dulbecco's modification of Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were transfected by Lipofectamine (Invitrogen). Ca²⁺ imaging was performed 24 h after transfection. Medium was replaced by modified standard Ringer's bath solution (in mmol/l): 140 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, and 10 HEPES, pH 7.4. Cells were loaded with fura 2-AM (10 μmol/l, Calbiochem) for 45 min and subsequently washed and imaged in standard bath solution. Ratiometric Ca²⁺ imaging was performed on an Olympus IX51 microscope with a Polychrome V monochromator (Till Photonics) and a PCO Cooke Sensicam QE CCD camera and Imaging Workbench 6 imaging software. Fura-2 emission images were obtained with exposures of 0.1 ms at 340 nm and 0.1 ms at 380 nm excitation wavelengths. The ratio of the fluorescence intensity obtained at 340 and 380 nm was used to determine the Ca²⁺ signal.

Lipid analysis.

Lipids were extracted and partially purified as previously described (7, 30). In brief, 40:1 volumes of methanol were added to each sample followed by NAGly-d8 (10 μl of 100 pM). Deuterium-labeled compounds were used as internal standards to determine the extraction efficiency. Methanolic samples were covered with parafilm and left on ice and in darkness for ∼2 h. Samples on ice were then homogenized by use of a Polytron for ∼1 min followed by centrifugation at 19,000 g at 24°C for 20 min. Supernatants were then collected and placed in polypropylene tubes. HPLC-grade water was added, making the final supernatant/water solution 25% organic. To isolate the compounds of interest partial purification of the 25% solution was performed on a Preppy apparatus assembled with 500 mg C18 solid-phase extraction columns. The columns were conditioned with 5 ml of HPLC-grade methanol immediately followed by 2.5 ml of HPLC-grade water. The supernatant/water solution was then loaded onto the C18 column, and then washed with 2.5 ml of HPLC grade water followed by 1.5 ml of 40% methanol. Elutions of 1.5 ml of 60, 75, 85, and 100% methanol were collected in individual autosampler vials and stored at −20°C until analysis by mass spectrometry.

LC/MS/MS analysis and quantification.

HPLC/MS/MS methods previously described were used for each of the lipids analyzed here (7, 30). With the exception of the 2-acyl glycerol and N-acyl ethanolamine species, which were analyzed by using positive ion modes [H⁺], all other lipids were analyzed in negative ion mode [H⁺], likewise, as previously described (7). Elutions were removed from −20°C storage and allowed to warm to room temperature while covered (∼20 min), vortexed for ∼1 min before being placed into the autosampler (Agilent 1100 series autosampler), and held at 24°C for LC/MS/MS analysis. Then 10–20 μl of the eluants were injected separately for each sample to be rapidly separated by using a C18 Zorbax reversed-phase analytical column to scan for individual compounds. Gradient elution (200 μl/min) occurred under the pressure created by two Shimadzu 10AdVP pumps (Shimadzu). Next, electrospray ionization was accomplished with an Applied Biosystems/MDS Sciex API3000 triple quadrupole mass spectrometer (Applied Biosystems). Multiple reaction monitoring (MRM) was then used to analyze the level of each compound present in the sample injection. Synthetic standards were used to generate optimized MRM methods and standard curves for analysis. Additional analysis used was the product ion scan in which the parent ion was filtered in the first quadrupole and then all fragments were monitored to generate a complete fingerprint of molecular fragments.

Lipid data analysis.

All lipid analytes were identified by using synthetic standard matching chromatographic peaks coupled to matching MRM scans as previously described (7, 30). The standards provided a reference for the retention times and mass fingerprint by which the analytes were compared. They allowed the identification of the specific precursor ion and fragment ion for each analyte to enable their isolation. Quantitation of analytes was calculated by using a combination of calibration curves of the synthetic standards obtained from the Analyst software and recovery adjustments by using deuterium-labeled internal standards.

In silico analysis of TRP channel gene expression in human neutrophils.

Transcription datasets from TNF- or GM-CSF-primed human neutrophils [NCBI Gene Expression Omnibus (GEO) series accession number GSE40548] were mapped to the human genome (hg19) using Tophat and Bowtie (34, 35, 40). Mapped reads, or fragments, were assigned to transcripts by using Cufflinks and differential expression was determined by Cuffdiff. Values are displayed as fragments per kilobase of transcript per million mapped reads (FPKM).

Data analysis and statistics.

Data were analyzed with GraphPad Prism 5 (GraphPad Software) software. MPO and cytokine data were analyzed by one-way ANOVA with Tukey's multiple-comparison test. Cell differentials and airway forced oscillation data were analyzed by two-way ANOVA followed by Bonferroni post hoc tests. Error bars are SE.

RESULTS

Diminished acid-induced pulmonary inflammation in mice treated with a TRPV4 inhibitor and in TRPV4-deficient mice.

The role of TRPV4 in acid-induced lung injury was examined with a newly identified TRPV4 inhibitor, GSK2220691, and in Trpv4−/− mice (Fig. 1, Fig. 2, Tables 1 and 2). GSK2220691 represents a new class of TRPV4 inhibitors, chemically distinct from previously published inhibitors shown to prevent pulmonary edema following heart failure (Fig. 1A, Tables 1 and 2) (33). GSK2220691 inhibited pulmonary edema induced by a specific TRPV4 agonist, GSK1016790, and prevented the characteristic drop in arterial blood pressure observed following TRPV4 activation (Fig. 1, C and D). The dose selection for the present studies was based on the rat TRPV4 agonist PD model (Fig. 1) with a predicted minimum concentration (Cmin) for maximal in vivo mouse efficacy 262 ng/ml for GSK2220691.

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.

Table 1.

TRPV4 inhibitor potencies assessed by TRPV4 ortholog transduction into HEK cells and hypotonicity assessed in BHK cells

		GSK2337429	GSK2220691
Ortholog	Activator	pIC50	pIC50
Human	GSK634775	8.2 (n = 28)	8.6 (n = 14)
	GSK1016790	7.4 (n = 4)	8.2 (n = 4)
	Hypotonicity	7.6 (n = 14)	8.4 (n = 7)
Rat	GSK634775	8.8 (n = 5)	8.4 (n = 4)
	GSK1016790	8.5 (n = 2)	8.0 (n = 2)
Mouse	GSK634775	8.7 (n = 4)	8.2 (n = 6)
	GSK1016790	8.2 (n = 4)	7.7 (n = 4)
Dog	GSK634775	7.8 (n = 4)	8.1 (n = 6)
	GSK1016790	7.2 (n = 4)	7.9 (n = 4)
Monkey	GSK634775	8.0 (n = 4)	8.5 (n = 4)
	GSK1016790	7.6 (n = 3)	8.2 (n = 4)

Table 2.

TRPV4 inhibitor TRP selectivity profiles

	GSK2337429		GSK2220691
Target	pIC50	EC50, nM	IC50, nM	EC50, nM
TRPV1	<4.6 (n = 2)		<4.6 (n = 6)
TRPA1	<4.6 (n = 2)	<4.6 (n = 2)	<4.6 (n = 4)	<4.6 (n = 4)
TRPC3	<4.6 (n = 4)	<4.6 (n = 4)	<4.6 (n = 4)	<4.6 (n = 4)
TRPC6	<4.6 (n = 4)	<4.6 (n = 4)	<4.6 (n = 6)	<4.6 (n = 4)
TRPM5	<4.6 (n = 3)	<4.6 (n = 3)	<4.6 (n = 3)	<4.6 (n = 3)
TRPM8			<4.6 (n = 2)

A single intraperitoneal injection of GSK2220691 (30 mg/kg) was administered 30 min after induction of injury by intratracheal administration of HCl (pH 1.5, 2 ml/kg), and inflammatory parameters were analyzed after 5 h. BALF of GSK2220691-treated mice contained much smaller numbers of neutrophils and macrophages, and less MPO activity than BALF of vehicle-injected mice (Fig. 2, A–C). These effects were recapitulated in HCl-exposed Trpv4−/− mice that showed almost complete absence of neutrophils in BALF and strongly diminished macrophage counts and MPO activity (Fig. 2, D–F). Histopathological analyses of HCl-exposed lungs revealed a strong reduction in injury scores in both Trpv4−/− and GSK2220691-treated mice (Figs. 2G and 5). Multiplex peptide analyses of inflammatory cytokines and chemokines in BALF revealed that TRPV4 inhibition completely suppressed HCl-induced increases in key factors such as VEGF, keratinocyte-derived chemokine (KC; CXCL1), and granulocyte colony-stimulating factor (GCSF) (Fig. 3A). GSK2220691 treatment also reduced serum markers of pulmonary injury to almost baseline levels (Fig. 3B). These anti-inflammatory effects in the HCl-induced model were recapitulated in Trpv4−/− mice (Fig. 3, A and B).

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. 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.

Improved airway mechanics, oxygen saturation and diminished protein leakage in chlorine-exposed mice treated with TRPV4 inhibitors.

Encouraged by the observed therapeutic effects of TRPV4 inhibition in the HCl-induced lung injury model, we examined the effects of TRPV4 inhibitors in a mouse model of severe ALI, induced by exposure to Cl₂. Exposures to chlorine and other corrosive gases induce reactive airways dysfunction syndrome (31) and edema, characterized by airway hyperreactivity, protein leakage, and alveolar damage leading to hypoxia. Following exposure to chlorine (400 ppm for 30 min), mice were injected either intraperitoneally or intramuscularly with GSK2220691 (30 mg/kg) or with GSK2337429A (50 mg/kg), another newly identified TRPV4 inhibitor, chemically unrelated to GSK2220691 and similar to GSK2193874 (33) (Fig. 1, B, E, and F). TRPV4 inhibitors were injected again 8 h after the beginning of exposure (15 and 25 mg/kg, respectively), and lung mechanics were assessed 24 h after exposure by forced oscillation procedures (flexiVent). Cl₂-exposed mice developed severe airway hyperreactivity toward the bronchoconstrictor MeCh (Figs. 4, A and B, and 7A). Cl₂ also caused a dramatic increase in lung elastance, indicating alveolar damage (Fig. 4, A and B). In Cl₂-exposed mice treated with the TRPV4 inhibitors, airway reactivity remained at baseline levels and lung elastance was strongly reduced (Figs. 4, A and B, and 7A). Whereas Cl₂-exposed control mice showed a large drop in blood oxygen saturation 24 h after exposure, blood oxygen saturation in Cl₂-exposed and GSK-222069-treated mice remained at baseline equivalent to uninjured mice breathing air and dropped by a lesser extent in Cl₂-exposed mice treated with GSK2337429A (Fig. 4C). Chlorine exposure caused a dramatic increase in BALF protein content, an indicator of epithelial and vascular leakage associated with edema formation (Fig. 4D). Both TRPV4 inhibitors strongly reduced protein leakage in Cl₂-exposed mice (Fig. 4D).

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. 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.

Anti-inflammatory effects of TRPV4 inhibitors in chlorine-injured mice.

Chlorine-induced lung injury is associated with strong pulmonary inflammation driven by macrophages and neutrophils, levels of which were highly increased in BALF 24 h after exposure (Fig. 6, A and B; Fig. 7, B and C; Fig. 5). Both TRPV4 inhibitors, GSK222069 and GSK2337429A, diminished the increase in macrophages and neutrophils, with BALF cell counts reduced to almost baseline levels 24 h after induction of injury (Figs. 5, 6, A and B, and 7, B and C). Both inhibitors also reduced MPO activity in BALF of the treated mice (Figs. 6C and 7D). The chlorine-induced cellular inflammatory response was accompanied by dramatic increases in BALF levels of key proinflammatory factors such as KC (CXCL1), GCSF, and VEGF (Figs. 7E and 8A). Both TRPV4 inhibitors strongly suppressed the increases in these factors to levels near baseline when sampled 24 h after chlorine exposure (Figs. 7E and 8A).

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. 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.

Diminished vascular damage in chlorine exposed mice treated with TRPV4 inhibitors.

In mice exposed to high levels of chlorine, injury is not restricted to the respiratory system but also affects the cardiovascular system and other organ systems (19). In chlorine-exposed mice some of the same proinflammatory factors we identified in BALF were also present at high levels in serum, including KC, GCSF, and IL-6 (Fig. 8B). Chlorine caused significant vascular damage as evidenced by the high levels of vascular damage markers in BALF, including serum amyloid P component (SAP), fibrinogen, adiponectin, and soluble vascular cell adhesion molecule 1 (sVCAM-1). The levels of these vascular damage markers were strongly reduced by both TRPV4 inhibitors (Fig. 8C).

Formation of endogenous TRP channel agonists during ALI.

Some TRP ion channels are directly activated by the acidic (TRPV1) or oxidizing (TRPA1) stimuli we used to induce pulmonary injury (4, 24). However, these stimuli neither activated TRPV4 channels directly when heterologously expressed in HEK293 cells, nor in BEAS-2B cells, a pulmonary epithelial cell line natively expressing TRPV4 channels that readily responded to GSK1016790A, a selective TRPV4 agonist (Fig. 9) (25). We therefore speculated that pulmonary injury would induce the production of endogenous agonists of TRPV4. In our present study we focused on N-acyl amines, fatty acid-derived products related to anandamide and similar endocannabinoids (6). N-acyl amides are known to activate TRP ion channels, including TRPV4 (6, 29). Exposure to chlorine, or intratracheal administration of HCl, resulted in increases of almost all N-acyl amides we tested for in both BALF and total lung tissue, suggesting that multiple TRP channel agonists are produced following ALI (Table 3).

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.

Table 3.

Concentrations of fatty acid acylamides in lungs of chlorine or HCl-exposed mice

Acylamide	Air	Chlorine
N-stearoyl ethanolamine	2.34E-10 ± 1.67E-11 (n = 4)	5.11E-10 ± 5.72E-11 (n = 4)‡
N-palmitoyl glycine	2.03E-11 ± 2.00E-12 (n = 4)	3.04E-11 ± 3.39E-12 (n = 4)*
N-docosahexaenoyl glycine	3.76E-12 ± 1.1E-13 (n = 4)	6.99E-12 ± 4.47E-13 (n = 4)‡
N-docosahexaenoyl ethanolamine	2.53E-12 ± 2.62E-13 (n = 4)	5.87E-12 ± 9.15E-13 (n = 4)†
N-linoleoyl glycine	5.38E-12 ± 4.79E-13 (n = 4)	8.67E-12 ± 2.87E-13 (n = 4)‡
N-stearoyl ethanolamine	2.34E-10 ± 1.67E-11 (n = 4)	5.11E-10 ± 5.72E-11 (n = 4)‡
N-archidonoyl phenylalanine	0 (n = 4)	4.75E-13 ± 6.47E-14 (n = 4)‡
N-archidonoyl serine	1.25E-13 ± 1.25E-13 (n = 4)	1.87E-12 ± 2.8E-13 (n = 4)‡
N-oleoyl valine	2.55E-11 ± 1.09E-11 (n = 4)	7.47E-11 ± 4.38E-12 (n = 4)‡

Acylamide	Saline	HCl
N-stearoyl tryptophan	1.76E-12 ± 1.95E-13 (n = 8)	3.48E-12 ± 5.87E-13 (n = 8)†
N-palmytoyl tyrosine	1.1E-12 ± 1.27E-13 (n = 8)	3.08E-12 ± 6.88E-13 (n = 8)†
N-stearoyl tyrosine	1.37E-12 ± 1.86E-13 (n = 8)	4.06E-12 ± 1.04E-12 (n = 8)†
N-oleoyl tyrosine	1.75E-13 ± 1.14E-13 (n = 8)	2.37E-12 ± 6.14E-13 (n = 8)‡

Lipid extracts from lung homogenates were analyzed by LC/MS/MS from C57BL6 mice exposed to Air or chlorine (400 ppm, 30 min) and Saline or HCl (pH 1.5, 2 ml/kg). Data are presented as means ± SD, n = 4/group in chlorine-exposed mice and n = 8/group in HCl-treated mice.

^*P ≤0.05,

^†P ≤0.01,

^‡P ≤0.001.

In silico examination of TRPV4 expression in neutrophils.

TRPV4 is highly expressed in the pulmonary epithelium and vasculature, and also in alveolar macrophages, where it was found to be essential for the production of proinflammatory oxidative mediators and cytokines in the ventilator-induced lung injury model (15, 16, 26, 33). In our present study we observed that treatment of chlorine- and acid-exposed mice with TRPV4 inhibitors strongly reduced BALF macrophage numbers, thereby diminishing inflammation. TRPV4 inhibitors, or deletion of the TRPV4 gene, also strongly reduced BALF neutrophil levels and MPO activity, suggesting that TRPV4 may control neutrophil function during injury. Since TRPV4 expression in neutrophils has not been reported previously, we analyzed RNAseq neutrophil transcriptome datasets, comparing TRPV4 gene transcript numbers with those of other TRP ion channels and of other ion channels and cell surface molecules critical for neutrophil function (34, 35, 40). In all examined datasets from unstimulated and TNF-α- and GM-CSF-stimulated neutrophils, TRPV4 transcripts were represented at very low levels, between 0.013 and 0.029 FPKM (Fig. 10, A and B). In contrast, TRPC6, TRPM2, TRPM6, TRPM7, TRPV1, TRPV2, and TRPV5 transcripts were detected at much higher frequency, with TRPM6 at >5 FKPM, and TRPV2 at >3 FPKM (Fig. 10A). Transcripts of the gene encoding for OraI, the capacitative calcium entry channel, were present at FPKM>20, and transcripts of the ATP-gated ion channel, P₂X₁, were present at FPKM>30 (Fig. 10B). Transcripts of cell adhesion molecules were present at frequencies much higher than those of ion channels (ICAM: >100; l-selectin >1,200).

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.

DISCUSSION

The treatment of ALI following chemical inhalational exposures remains highly challenging because of the limited availability of mechanism-based therapeutic approaches. The present study identifies the ion channel TRPV4 as a crucial element of injury pathways activated by pulmonary chemical exposures. Selective TRPV4 inhibitors showed efficacies in two models of acute chemical lung injury, induced by intratracheal administration of hydrochloric acid or inhalational exposure to chlorine gas. Inhibition of TRPV4 reduced critical hallmarks of chemical lung injury, including airway hyperreactivity and increase in lung elastance, protein leakage, neutrophil and macrophage infiltration of the lungs, and the associated production of oxidative mediators and inflammatory chemokines and cytokines. In the acid-induced model, the crucial role of TRPV4 was validated in TRPV4-deficient mice that presented outcomes similar to TRPV4 inhibitor-treated animals. In chlorine-exposed mice, we used two TRPV4 inhibitors, GSK222069 and GSK2337429A, derived from distinct chemical classes of TRPV4 inhibitors that produced almost identical therapeutic effects.

Exposures to chlorine gas or chlorine bleach aerosol occur frequently because of occupational or domestic accidents, and transportation accidents such as train derailments have led to the release of large amounts of chlorine with subsequent fatalities and injuries in the affected surrounding communities (2, 3, 36, 41). Chlorine has also been used as a chemical weapon and in terrorist incidents, posing a significant threat to the population (3, 41). We observed that TRPV4 inhibitor treatment prevented blood oxygen desaturation caused by chlorine exposures, a critical parameter associated with mortality and chronic morbidity, suggesting that inhibition of TRPV4 may provide a survival benefit and improved outcomes. Recent studies have shown that chlorine inhalation, in addition to pulmonary injury, causes severe systemic damage, including a strong drop in blood pressure and vascular dysfunction (19). We observed that inhibition of TRPV4 reduced levels of critical inflammatory chemokines and cytokines in serum, and of markers of vascular damage, including SAP, fibrinogen, adiponectin, and soluble VCAM. These findings suggest that TRPV4 inhibitors protect both pulmonary and systemic vascular function in chlorine-injured animals. In addition to the intraperitoneal route, TRPV4 inhibitors were also injected intramuscularly into the caudal thigh, with similar therapeutic effects. This route may allow the rapid deployment of treatment in exposure situations with large numbers of casualties.

The role of TRPV4 in lung injury has been studied previously in animal models in which injury was induced by mechanical stress or pressure, including a ventilator-induced lung injury model, a model of heart failure induced by aortic banding, and a model of myocardial infarction induced by arterial ligation (17, 21, 33). Ventilator-induced lung injury is associated with subsequent inflammation driven by macrophages, and it was suggested that macrophage-expressed TRPV4 channels play a crucial role in activating these cells (16). Although previous studies have shown that TRPV4 transcripts and protein are highly expressed in lung epithelial and vascular cells and in alveolar macrophages; it has remained unclear whether TRPV4 is also expressed in neutrophils (15, 16, 26, 33). In our present study we observed that TRPV4 inhibition, or genetic deletion, strongly reduced neutrophil numbers in the BALF, suggesting that, similar to macrophages, TRPV4 plays a critical role in cell recruitment to the injured lung. We therefore carried out a systematic analysis of neutrophil RNAseq datasets, comparing the frequency of TRPV4 transcript fragments with those of other TRP ion channels and of membrane protein genes critical for neutrophil function. In all datasets, either from noninduced or induced neutrophils, the frequency of TRPV4 transcripts was very low. Transcripts of other TRP ion channel transcripts, especially those of TRPM6 and TRPV2, were represented at much higher frequencies. The rank order of TRP channel transcript frequencies in the neutrophil transcriptome determined in the present study is very similar to data produced in previous studies using quantitative PCR, supporting the validity of our approach (18). In summary, transcriptome analysis suggests that, in contrast to macrophages, neutrophil TRPV4 expression is minimal. It is therefore likely that TRPV4 affects neutrophil function indirectly, possibly through control of cell extravasation from the pulmonary vasculature or control of attractant factors by pulmonary tissue or macrophages.

In ex vivo lung preparations and in models in which TRPV4 agonists were administered, activation of TRPV4 induced severe edema and cardiovascular depression, suggesting that TRPV4 channels are crucial for the control of epithelial and endothelial barrier function (1, 33, 39). These findings were confirmed by in vitro studies showing that TRPV4 inhibition prevented calcium influx into particulate-exposed pulmonary epithelial cells, inhibited ciliary beating in pulmonary epithelial cells, and reduced cytokine-induced permeability in an organotypic lung on a chip model (20, 25, 27). Moreover, TRPV4 expression was found in endothelial cells and in pulmonary arterial smooth muscle where these channels are involved in calcium signaling (33, 42).

The therapeutic effects of TRPV4 inhibitors are at least equal to or exceed the efficacies of previously examined anti-inflammatory treatments such as corticosteroids (23). The key strength of TRPV4 inhibitors is that, in addition to suppressing inflammation, they protect vascular and epithelial function preventing TRPV4-activated vasodilation and leakage leading to edema. This assumption is supported by our data demonstrating that TRPV4 is essential for the control of both cellular inflammatory responses and tissue barriers in the lung. We observed that TRPV4 inhibition suppressed macrophage and neutrophil infiltration in both acid- and chlorine-exposed mice. At the same time, both pulmonary resistance, a function of smooth muscle resistance in the airways, and elastance were diminished, a parameter correlating with alveolar damage.

The systematic lipid analysis in the present study demonstrates that pulmonary injury is associated with production of fatty acid acylamides, a novel finding not previously reported. Fatty acid acylamides are a group of lipid derivatives related to endogenous cannabinoid mediators. Some fatty acid acylamides were shown to activate TRP ion channels, including TRPV1 and TRPV4 (6, 29). Although the injury-induced concentrations of individual injury-induced fatty acid acylamides in BALF or lung tissue samples were low, we found almost all tested species (>10) to be increased. It is possible that their cumulative effects may be sufficient to activate TRPV4, or that these molecules are enriched in the previously identified TRPV4-containing lipid microdomains that were found to be crucial for vascular control (12). Recent studies have shown that very small increases in TRPV4 activity are sufficient to cause profound physiological effects due to the strong amplification of TRPV4-mediated cellular calcium signals (12, 32). Pulmonary injury is also known to produce other lipid-derived TRPV4 agonists such as eicosanoid epoxides (21). Osmotic gradients created during edema formation may further increase TRPV4 activity.

In summary, the present study identifies TRPV4 inhibitors as potential therapeutic agents for the treatment of pulmonary injury following highly reactive chemical exposures, including acid and the oxidant gas chlorine. Our findings lend further support for a critical role of TRP ion channels in mechanisms of tissue injury and as pharmacological targets for tissue protection.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

S.B., M.C., H.S.E., G.Y., R.N.W., K.S.T., H.B.B., S.M., and S.-E.J. conception and design of research; S.B., W.S., S.F.D., B.L., Z.Y., A.S., and E.L. performed experiments; S.B., S.A., M.M.K., H.S.E., and H.B.B. analyzed data; S.B., K.S.T., H.B.B., and S.-E.J. interpreted results of experiments; S.B. and M.M.K. prepared figures; S.B. and S.-E.J. drafted manuscript; S.B., M.C., G.Y., K.S.T., S.M., and S.-E.J. edited and revised manuscript; S.B., R.N.W., K.S.T., S.M., and S.-E.J. approved final version of manuscript.