TRPV4 activation triggers protective responses to bacterial lipopolysaccharides in airway epithelial cells

Abstract

Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca²⁺ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction.

Fig. 1

LPS stimulates mTEC in a TLR4-independent and TRPV4-dependent manner. a, b Effects of LPS (20 µg ml⁻¹), GSK1016790A (10 nM) and ATP (20 µM) on the intracellular Ca²⁺ concentration in mTEC primary cultures from wild type (a) or Tlr4 KO (b). c Traces of intracellular Ca²⁺ concentration showing the lack of response of mTEC to the TRPA1 agonist cinnamaldehyde (CA, 300 μM) and to the TRPV1 agonist capsaicin (1 μM). d mRNA expression profile of several TRP genes. Data is shown as 2^−ΔΔCT relative to Trpm7 expression. e Average amplitudes of intracellular Ca²⁺ responses to LPS (20 μg ml⁻¹), the TRPA1 agonist cinnamaldehyde (CA, 300 μM), and the TRPV1 agonist capsaicin (Caps, 1 μM) in cells responsive or irresponsive to 10 nM GSK1016790A. n = 73; *P < 0.05, Mann–Whitney U test. f Effects of LPS (20 µg ml⁻¹), GSK1016790A (10 nM) and ATP (20 µM) on the intracellular Ca²⁺ concentration in mTEC primary cultured from Trpv4 KO mice. g, h Traces of intracellular Ca²⁺ concentration in wild type (g) and Tlr4 KO (h) cells exposed to 10 µM HC067047, 20 µg ml⁻¹ LPS and later on to 10 nM GSK1016790A (GSK). i Percentage of mTEC responding to LPS (n > 46). **P < 0.01, Fisher’s exact test. HC, HC067047 (10 µM)

Fig. 2

LPS activates TRPV4. a Example of the effects of LPS on the amplitude of currents measured at +100 and −100 mV in a HEK293T cell transfected with mTRPV4. The colored data points correspond to the traces shown in (b). b Top, currents recorded in control (black trace) or in the presence of LPS (20 µg ml⁻¹, red trace). The purple trace represents the difference of the red and black traces, i.e., the current induced by LPS. Bottom, comparison of the currents induced by LPS and GSK1016790A (10 nM, blue trace). The green trace represents the GSK1016790A-evoked current normalized to the amplitude of the LPS-evoked current measured at +100 mV. c, d Average reversal potential (c) and rectification index (d) of currents induced by application of LPS or GSK1016790A (n = 11). e Average change of current amplitudes induced by the application of 20 µg ml⁻¹ LPS (n = 11) or 20 µg ml⁻¹ LPS in the presence of 10 µM HC067047 (n = 7). ^# represents the comparison to ΔCurrent = 0 pA. ^# P < 0.05; ^## P < 0.01 (n = 6), one sample t-test. *P < 0.05; **P < 0.01 (n = 6), two-tailed t-test

Fig. 3

LPS activates TRPV4 in a TLR4-independent manner. a Intracellular Ca²⁺ signals in HEK293T cells overexpressing mouse TRPV4. The black and blue traces correspond to GSK1016790A-sensitive cells responding or not to 20 μg ml⁻¹ of LPS, respectively. The red traces correspond to non-transfected cells (GSK1016790A-insensitive). b Concentration dependence of the amplitude of LPS-induced intracellular Ca²⁺ responses (n > 100 per data point). The solid line represents the fit of the data obtained in GSK1016790A-positive cells with a Hill equation. The red symbols correspond to GSK1016790A-insensitive cells. c Intracellular Ca²⁺ signals recorded in HEK293T cells pre-incubated with the TLR4 antagonist Cli-095 (1 μM). d Intracellular Ca²⁺ signals recorded in HEK293T cells treated with LPS pre-incubated with Polymixin B (PMB, 300 μg ml⁻¹). In c, d, the black and red traces represent TRPV4-transfected and non-transfected cells, respectively. LPS 20 µg ml⁻¹ and GSK1016790A (GSK) 10 nM. e Intracellular Ca²⁺ signals in response to Salmonella minnesota LPS (20 μg ml⁻¹). f Average amplitude of the intracellular Ca²⁺ responses to 20 μg ml⁻¹ LPS from E. coli and S. minnesota, and to E. coli LPS in the presence of the TLR4 inhibitor Cli-095 (1 µM, n = 77) or the LPS scavenger Polymyxin B (PMB, 300 µg ml⁻¹, n = 32). **P ≤ 0.01, two-tailed Kruskal–Wallis. g Average amplitudes of the responses to GSK1016790A (GSK) alone or mixed with Cli-095 or PMB in TRPV4-transfected HEK293T cells (n > 30 per bar). *P < 0.05, Dunn’s multiple comparison test

Fig. 4

LPS-induced activation of TRPV4 triggers NO production in mTEC. a–d Traces of intracellular Ca²⁺ signals (black traces) and normalized fluorescence of the NO-sensitive dye DAF-FM (F/F₀, red traces) recorded in mTEC harvested from wild type (a, c), Trpv4 KO (b) and Tlr4 KO (d) mice (LPS, 20 µg ml⁻¹; HC067047, 10 µM). e, f Average changes in intracellular Ca²⁺ (e) and NO production (f) induced by LPS in wild type (WT), Trpv4 KO and Tlr4 KO. In another series of experiments, WT cells were also pre-incubated with the TRPV4 inhibitor HC067047 (HC, 10 µM). The two bars on the right show the responses to GSK1016790A (GSK, 10 nM) in WT and Tlr4 KO mice. n > 40 per bar; **P < 0.01, Kruskal–Wallis test. g Western blot of mTEC lysates probed for the iNOS and nNOS isoforms. Non-stimulated splenocytes were used as negative control for NOS expression

Fig. 5

TRPV4 activation by LPS triggers NO production in human bronchial EC. a Immunoblotting of 16HBE cells lysate probed for NOS isoforms. b Left, average intracellular Ca²⁺ responses of human bronchial epithelial cells to 20 μg ml⁻¹ LPS and 10 nM GSK1016790A. The black, blue, and red solid traces correspond to data means of cells responding to LPS and GSK1016790A, GSK1016790A alone or to none of the stimuli, respectively. The thin dashed traces represent the means ± standard errors. Right, stack bar graph showing the percentages of cells responding to LPS and/or GSK1016790A (color-coded as the graph on the left; n = 116). c Immunoblotting of 16HBE cells lysate incubated in LPS (20 μg ml⁻¹) or GSK1016790A (10 nM). Where indicated, cells were previously incubated with vehicle (Veh, 0.8% DMSO) or the TRPV4 inhibitor HC067047 (+HC, 10 μM). d Average intracellular Ca²⁺ signals (black solid traces) and normalized fluorescence of the NO-sensitive dye DAF-FM (F/F₀, red solid traces) recorded in 16HBE cells (n = 52). The thin dashed traces represent the means ± standard errors. LPS, 20 µg ml⁻¹; HC067047, 10 µM; GSK1016790A, 10 nM. e Average changes in intracellular Ca²⁺ (left panel) and NO production (right panel) induced by LPS. The dark cyan bars correspond to cells pre-incubated with the TRPV4 inhibitor HC067047 (10 µM). n > 74 per bar; **P < 0.01, two-tailed t-test. f Confocal microscopy images of 16HBE cells immunostained for iNOS. The arrow head points to an aggresome structure. Scale bar, 20 μm. g iNOS aggresomes were quantified using a custom-designed software. The graph shows the area of aggresomes relative to the total green-stained area determined from images recorded from preparations fixed in control condition or 5 min after application of 20 μg ml⁻¹ LPS or 10 nM GSK1016790A. The panel on the right corresponds to cells pre-incubated with HC067047 (HC, 10 μM). Three randomly selected fields per condition were used for quantification. **P ≤ 0.01, Dunn’s multiple comparison test

Fig. 6

TRPV4-induced NO exerts direct anti-bacterial effect. a Representative images of E. coli bacteria stained with SYTO9 (green, all cells) or PI (red, dead cells). Magnification, ×40. Bacteria were imaged after 2 h incubation with a monolayer of mTEC isolated from WT or Trpv4 KO mice. The TRPV4 antagonist HC067047 (10 μM) or the NOS inhibitor L-NAME (1 mM) were added 15 min prior to the bacterial challenge. Scale bar, 50 μm. b Percentage of dead cells (red/green) after co-culture with mTEC isolated from WT or Trpv4 KO mice (n ≥ 5). c Intracellular Ca²⁺ response of TRPV4-transfected HEK239T cells to the supernatant of a 0.1 OD culture of E. coli. GSK1016790A, 10 nM. d Percentage of TRPV4-expressing HEK293T cells responding to supernatant of 0.1 OD culture of E. coli. The TRPV4 inhibitor HC067047 (HC, 10 μM) was perfused 2 min prior to addition of bacterial supernatant. The LPS scavenger Polymyxin B (PMB, 300 µg ml⁻¹) was pre-incubated during 30 min with the bacterial supernatant. *P < 0.05; **P < 0.01, Fisher’s exact test

Fig. 7

TRPV4-deficient mice display exacerbated airway responses to LPS. a Time course of average enhanced pause (Penh) determined in unrestrained whole-body plethysmography experiments performed in wild type (WT, n = 10) and Trpv4 KO (n = 7) mice exposed to aerosols of LPS. The area under the curve (AUC) of Trpv4 KO traces is significantly larger than AUC from WT (144 ± 56 > 47 ± 14; P = 0.048, unpaired t-test). b, c Average number of macrophages (b) and neutrophils (c) in the bronchoalveolar lavage fluid collected 3 h after LPS exposure. **P < 0.01, two-tailed t-test. d Average Penh determined in unrestrained whole-body plethysmography experiments performed in WT mice pretreated with vehicle (0.8% DMSO, n = 7) or the TRPV4 inhibitor HC067047 (10 mg kg⁻¹ in 0.8% DMSO, n = 8). The vehicle or HC067047 were administered intraperitoneally 15 min previous to the LPS challenge. AUC_Vehicle = 23 ± 4 < AUC_HC067047 = 54 ± 14; P = 0.039, unpaired t-test. e, f Average number of macrophages (e) and neutrophils (f) in the bronchoalveolar lavage fluid collected 3 h after LPS exposure. Veh, vehicle. *P < 0.05; **P < 0.01, two-tailed t-test. g Average Penh of WT and Trpa1/Trpv1 KO mice pretreated with vehicle (0.8% DMSO, n = 6) or the TRPV4 inhibitor HC067047 (10 mg kg⁻¹ in 0.8% DMSO, n = 6). The vehicle or HC067047 were administered intraperitoneally 15 min previous to the LPS challenge. AUC_{(WT) Vehicle} = 23 ± 4 < AUC_{(Trpa1/Trpv1 dKO), Vehicle} = 88 ± 16; P = 0.0018, unpaired t-test. AUC_{(Trpa1/Trpv1 dKO), Vehicle} < AUC_{(Trpa1/Trpv1 dKO), HC067047} = 257 ± 65; P = 0.031, unpaired t-test. h Fold change of mRNA transcripts of Cxcl-1, Cxcl-2, and Il-6 in mTEC untreated (Control) or treated during 3 h with LPS (20 μg ml⁻¹). Cytokine cycle thresholds were normalized to β-actin signals. Fold change is relative to untreated cells. **P < 0.01, two-tailed t-test. In all experiments mice were challenged with aerosolized LPS (50 μg ml⁻¹) during 15 min