The Cytochrome P450 2C8*3 Variant (rs11572080) Is Associated with Improved Asthma Symptom Control in Children and Altered Lipid Mediator Production and Inflammatory Response in Human Bronchial Epithelial Cells.

This study investigated an association between the cytochrome P450 (CYP) 2C8*3 polymorphism with asthma symptom control in children and changes in lipid metabolism and pro-inflammatory signaling by human bronchial epithelial cells (HBECs) treated with cigarette smoke condensate (CSC). CYP genes are inherently variable in sequence, and while such variations are known to produce clinically relevant effects on drug pharmacokinetics and pharmacodynamics, the effects on endogenous substrate metabolism and associated physiologic processes are less understood. In this study, CYP2C8*3 was associated with improved asthma symptom control among children: Mean asthma control scores were 3.68 (n = 207) for patients with one or more copies of the CYP2C8*3 allele versus 4.42 (n = 965) for CYP2C8*1/*1 (P = 0.0133). In vitro, CYP2C8*3 was associated with an increase in montelukast 36-hydroxylation and a decrease in linoleic acid metabolism despite lower mRNA and protein expression. Additionally, CYP2C8*3 was associated with reduced mRNA expression of interleukin-6 (IL-6) and C-X-C motif chemokine ligand 8 (CXCL-8) by HBECs in response to CSC, which was replicated using the soluble epoxide hydrolase inhibitor, 12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid. Interestingly, 9(10)- and 12(13)- dihydroxyoctadecenoic acid, the hydrolyzed metabolites of 9(10)- and 12(13)- epoxyoctadecenoic acid, increased the expression of IL-6 and CXCL-8 mRNA by HBECs. This study reveals previously undocumented effects of the CYP2C8*3 variant on the response of HBECs to exogenous stimuli. SIGNIFICANCE STATEMENT: These findings suggest a role for CYP2C8 in regulating the epoxyoctadecenoic acid:dihydroxyoctadecenoic acid ratio leading to a change in cellular inflammatory responses elicited by environmental stimuli that exacerbate asthma.

Progesterone amplifies allergic inflammation and airway pathology in association with higher lung ILC2 responses.

Perimenstrual worsening of asthma occurs in up to 40% of women with asthma, leading to increased acute exacerbations requiring clinical care. The role of sex hormones during these times remains unclear. In the current study, we used a translational approach to determine whether progesterone exacerbates allergic inflammation in the traditional chicken egg ovalbumin (OVA) model in BALB/c mice. Simultaneously, we used peripheral blood mononuclear cells (PBMC) from healthy human donors to assess the effects of progesterone on circulating group 2 innate lymphoid cells (ILC2). Briefly, lungs of ovariectomized (OVX) or sham-operated female (F-Sham) controls were implanted with a progesterone (P4, 25 mg) (OVX-P4) or placebo pellet (OVX-Placebo), followed by sensitization and challenge with ovalbumin (OVA). Progesterone increased total inflammatory histologic scores, increased hyper-responsiveness to methacholine (MCh), increased select chemokines in the bronchoalveolar lavage (BAL) and serum, and increased ILC2 and neutrophil numbers, along the airways compared with F-Sham-OVA and OVX-Placebo-OVA animals. Lung ILC2 were sorted from F-Sham-OVA, OVX-Placebo-OVA and OVX-P4-OVA treated animals and stimulated with IL-33. OVX-P4-OVA lung ILC2 were more responsive to interleukin 33 (IL-33) compared with F-Sham-OVA treated, producing more IL-13 and chemokines following IL-33 stimulation. We confirmed the expression of the progesterone receptor (PR) on human ILC2, and showed that P4 + IL-33 stimulation also increased IL-13 and chemokine production from human ILC2. We establish that murine ILC2 are capable of responding to P4 and thereby contribute to allergic inflammation in the lung. We confirmed that human ILC2 are also hyper-responsive to P4 and IL-33 and likely contribute to airway exacerbations following allergen exposures in asthmatic women with increased symptoms around the time of menstruation.NEW & NOTEWORTHY There is a strong association between female biological sex and severe asthma. We investigated the allergic immune response, lung pathology, and airway mechanics in the well-described chicken egg ovalbumin (OVA) model with steady levels of progesterone delivered throughout the treatment period. We found that progesterone enhances the activation of mouse group 2 innate lymphoid cells (ILC2). Human ILC2 are also hyper-responsive to progesterone and interleukin 33 (IL-33), and likely contribute to airway exacerbations following allergen exposures in women with asthma.

Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma.

BACKGROUND: Transient receptor potential ankyrin-1 [transient receptor potential cation channel subfamily A member 1 (TRPA1)] and vanilloid-1 [transient receptor potential cation channel subfamily V member 1 (TRPV1)] detect inhaled irritants, including air pollutants and have roles in the development and exacerbation of asthma. OBJECTIVES: This study tested the hypothesis that increased expression of TRPA1, stemming from expression of the loss-of-function TRPV1 (I585V; rs8065080) polymorphic variant by airway epithelial cells may explain prior observations of worse asthma symptom control among children with the TRPV1 I585I/V genotype, by virtue of sensitizing epithelial cells to particulate materials and other TRPA1 agonists. METHODS: TRP agonists, antagonists, small interfering RNA (siRNA), a nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway inhibitor, and kinase activators and inhibitors were used to modulate TRPA1 and TRPV1 expression and function. Treatment of genotyped airway epithelial cells with particulate materials and analysis of asthma control data were used to assess consequences of TRPV1 genotype and variable TRPA1 expression on cellular responses in vitro and asthma symptom control among children as a function of voluntarily reported tobacco smoke exposure. RESULTS: A relationship between higher TRPA1 expression and function and lower TRPV1 expression and function was revealed. Findings of this study pointed to a mechanism whereby NF-κB promoted TRPA1 expression, whereas NF-κB-regulated nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 2 (NLRP2) limited expression. Roles for protein kinase C and p38 mitogen activated protein kinase were also demonstrated. Finally, the TRPV1 I585I/V genotype was associated with increased TRPA1 expression by primary airway epithelial cells and amplified responses to selected air pollution particles in vitro. However, the TRPV1 I585I/V genotype was not associated with worse asthma symptom control among children exposed to tobacco smoke, whereas other TRPA1 and TRPV1 variants were. DISCUSSION: This study provides insights on how airway epithelial cells regulate TRPA1 expression, how TRPV1 genetics can affect TRPA1 expression, and that TRPA1 and TRPV1 polymorphisms differentially affect asthma symptom control. https://doi.org/10.1289/EHP11076.

IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17ß-estradiol.

Asthmatic women tend to develop severe airway disease in their reproductive years, and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells, or ILC2, are readily detected in allergic conditions, such as rhinosinusitis, in individuals that develop nasal polyps do to allergen exposures, and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33, an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in naïve and ovalbumin (OVA) challenged, female BALB/c mice in comparison to male mice following stimulation with IL-33. Here, we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-κB signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated, and had reduced NF-κB activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2, and Nfkb1 was found among top-ranked regulatory interactions. Together, these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases.

Parenchymal and Inflammatory Cell Responses to Single and Repeated Ozone Exposure in Healthy and Surfactant Protein-C Mutant Lung.

Mutations in the alveolar epithelial-specific gene encoding for surfactant protein C (SP-C) are linked to pulmonary disease. Ozone (O3) is a ubiquitous pollutant known to exacerbate stress through oxidative injury and inflammation. To comprehend the structural, functional, and immunological impact of single and repeated O3 exposure, SP-CWT and surfactant protein-C I73T mutant (SP-CI73T) mice were exposed to air or O3 (0.8 ppm, 3 h, up to ×4 consecutive days). O3 was associated with mitochondrial and autophagic activation (PINK1, LC3B, and p62), focal remodeling, and inflammation localized at the terminal bronchiole-to-alveolar junctions. Histological damage was exacerbated by repeated exposure. Single O3 challenge resulted in transient elastin fiber loss, whereas repeated exposure resulted in marked increases in elastance in SP-CI73T mice. Flow cytometric analysis revealed increases in classical monocyte and monocyte-derived macrophages recruitment in conditions of repeated exposure, which peaked earlier (24 h) in SP-CI73T mice. Immunohistochemical analysis also showed clustering of Arg-1+ and CD206+ activated cells within regions of remodeled lung. Lymphoid cell analysis identified CX3CR1-B220+ B cells accumulating after single (24/72 h). Repeated exposure produces a switch in the phenotype of these B cells CX3CR1+ (72 h) only in SP-CWT mice. SP-CI73T mutants also displayed depletion in NK1.1+ NKp46+ natural killer cells in lung, as well as bone marrow, blood, and spleen. These results illustrate the cumulative impact of O3 on lung structure and function in healthy lung, and aberrant myeloid and lymphoid recruitment in SP-C mutants responding to challenge. Together, this work highlights the significance of modeling environmental exposure across the spectrum of genetic susceptibility, consistent with human disease.

Dynamic Expression of Transient Receptor Potential Vanilloid-3 and Integrated Signaling with Growth Factor Pathways during Lung Epithelial Wound Repair following Wood Smoke Particle and Other Forms of Lung Cell Injury.

Prior studies revealed increased expression of the transient receptor potential vanilloid-3 (TRPV3) ion channel after wood smoke particulate matter (WSPM) treatment of human bronchial epithelial cells (HBECs). TRPV3 attenuated pathologic endoplasmic reticulum stress and cytotoxicity mediated by transient receptor potential ankyrin-1. Here, the basis for how TRPV3 expression is regulated by cell injury and the effects this has on HBEC physiology and WSPM-induced airway remodeling in mice was investigated. TRPV3 mRNA was rapidly increased in HBECs treated with WSPM and after monolayer damage caused by tryptic disruption, scratch wounding, and cell passaging. TRPV3 mRNA abundance varied with time, and stimulated expression occurred independent of new protein synthesis. Overexpression of TRPV3 in HBECs reduced cell migration and wound repair while enhancing cell adhesion. This phenotype correlated with disrupted mRNA expression of ligands of the epidermal growth factor, tumor growth factor-ß, and frizzled receptors. Accordingly, delayed wound repair by TRPV3 overexpressing cells was reversed by growth factor supplementation. In normal HBECs, TRPV3 upregulation was triggered by exogenous growth factor supplementation and was attenuated by inhibitors of growth factor receptor signaling. In mice, subacute oropharyngeal instillation with WSPM also promoted TRPV3 mRNA expression and epithelial remodeling, which was attenuated by TRPV3 antagonist pre- and cotreatment. This latter effect may be the consequence of antagonist-induced TRPV3 expression. These findings provide insights into the roles of TRPV3 in lung epithelial cells under basal and dynamic states, as well as highlight potential roles for TRPV3 ligands in modulating epithelial damage/repair. SIGNIFICANCE STATEMENT: Coordinated epithelial repair is essential for the maintenance of the airways, with deficiencies and exaggerated repair associated with adverse consequences to respiratory health. This study shows that TRPV3, an ion channel, is involved in coordinating repair through integrated repair signaling pathways, wherein TRPV3 expression is upregulated immediately after injury and returns to basal levels as cells complete the repair process. TRPV3 may be a novel target for understanding and/or treating conditions in which airway/lung epithelial repair is not properly orchestrated.

Transient Receptor Potential Ankyrin-1 and Vanilloid-3 Differentially Regulate Endoplasmic Reticulum Stress and Cytotoxicity in Human Lung Epithelial Cells After Pneumotoxic Wood Smoke Particle Exposure.

This study investigated the roles of transient receptor potential (TRP) ankyrin-1 (TRPA1) and TRP vanilloid-3 (TRPV3) in regulating endoplasmic reticulum stress (ERS) and cytotoxicity in human bronchial epithelial cells (HBECs) treated with pneumotoxic wood smoke particulate matter (WSPM) and chemical agonists of each channel. Functions of TRPA1 and TRPV3 in pulmonary epithelial cells remain largely undefined. This study shows that TRPA1 activity localizes to the plasma membrane and endoplasmic reticulum (ER) of cells, whereas TRPV3 resides primarily in the ER. Additionally, treatment of cells using moderately cytotoxic concentrations of pine WSPM, carvacrol, and other TRPA1 agonists caused ERS as a function of both TRPA1 and TRPV3 activities. Specifically, ERS and cytotoxicity were attenuated by TRPA1 inhibition, whereas inhibiting TRPV3 exacerbated ERS and cytotoxicity. Interestingly, after treatment with pine WSPM, TRPA1 transcription was suppressed, whereas TRPV3 was increased. TRPV3 overexpression in HBECs conferred resistance to ERS and an attenuation of ERS-associated cell cycle arrest caused by WSPM and multiple prototypical ERS-inducing agents. Alternatively, short hairpin RNA-mediated knockdown of TRPV3, like the TRPV3 antagonist, exacerbated ERS. This study reveals previously undocumented roles for TRPA1 in promoting pathologic ERS and cytotoxicity elicited by pneumotoxic WSPM and TRPA1 agonists, and a unique role for TRPV3 in fettering pathologic facets of the integrated ERS response. SIGNIFICANCE STATEMENT: These findings provide new insights into how wood smoke particulate matter and other transient receptor potential ankyrin-1 (TRPA1) and transient receptor potential vanilloid-3 (TRPV3) agonists can affect human bronchial epithelial cells and highlight novel physiological and pathophysiological roles for TRPA1 and TRPV3 in these cells.

Wood Smoke Particles Stimulate MUC5AC Overproduction by Human Bronchial Epithelial Cells Through TRPA1 and EGFR Signaling.

Mucus hypersecretion is a pathological feature of acute inflammatory and chronic obstructive pulmonary diseases. Exposure to air pollutants can be a cause of pathological mucus overproduction, but mechanisms by which different forms of air pollutants elicit this response are not fully understood. In this study, particulate matter (PM) generated from burning pine wood and other types of biomass was used to determine mechanisms by which these forms of PM stimulate mucin gene expression and secretion by primary human bronchial epithelial cells (HBECs). Biomass PM < 2.5 µm generated from pine wood and several other fuels stimulated the expression and secretion of the gel-forming glycoprotein MUC5AC by HBECs. Muc5ac gene induction was also observed in mouse airways following subacute oropharyngeal delivery of pine wood smoke PM. In HBECs, MUC5AC was also induced by the transient receptor potential ankyrin-1 (TRPA1) agonists' coniferaldehyde, a component of pine smoke PM, and allyl isothiocyanate, and was attenuated by a TRPA1 antagonist. Additionally, inhibition of epidermal growth factor receptor (EGFR/ErbB1) and the EGFR signaling partners p38 MAPK and GSK3ß also prevented MUC5AC overexpression. Collectively, our results suggest that activation of TRPA1 and EGFR, paired with alterations to p38 MAPK and GSK3ß activity, plays a major role in MUC5AC overproduction by bronchial epithelial cells exposed to biomass smoke PM. These results reveal specific processes for how biomass smoke PM may impact the human respiratory system and highlight potential avenues for therapeutic manipulation of lung diseases that are affected by air pollutants.

Differential Activation of TRPA1 by Diesel Exhaust Particles: Relationships between Chemical Composition, Potency, and Lung Toxicity.

Diesel exhaust particulate (DEP) causes pulmonary irritation and inflammation, which can exacerbate asthma and other diseases. These effects may arise from the activation of transient receptor potential ankyrin-1 (TRPA1). This study shows that a representative DEP can activate TRPA1-expressing pulmonary C-fibers in the mouse lung. Furthermore, DEP collected from idling vehicles at an emissions inspection station, the tailpipe of an on-road "black smoker" diesel truck, waste DEP from a diesel exhaust filter regeneration machine, and NIST SRM 2975 can activate human TRPA1 in lung epithelial cells to elicit different biological responses. The potency of the DEP, particle extracts, and selected chemical components was compared in TRPA1 over-expressing HEK-293 and human lung cells using calcium flux and other toxicologically relevant end-point assays. Emission station DEP was the most potent and filter DEP the least. Potency was related to the percentage of ethanol extractable TRPA1 agonists and was equivalent when equal amounts of extract mass was used for treatment. The DEP samples were further compared using scanning electron microscopy, energy-dispersive X-ray spectroscopy, gas chromatography-mass spectrometry, and principal component analysis as well as targeted analysis of known TRPA1 agonists. Activation of TRPA1 was attributable to both particle-associated electrophiles and non-electrophilic agonists, which affected the induction of interleukin-8 mRNA via TRPA1 in A549 and IMR-90 lung cells as well as TRPA1-mediated mucin gene induction in human lung cells and mucous cell metaplasia in mice. This work illustrates that not all DEP samples are equivalent, and studies aimed at assessing mechanisms of DEP toxicity should account for multiple variables, including the expression of receptor targets such as TRPA1 and particle chemistry.

Effects of fuel components and combustion particle physicochemical properties on toxicological responses of lung cells.

The physicochemical properties of combustion particles that promote lung toxicity are not fully understood, hindered by the fact that combustion particles vary based on the fuel and combustion conditions. Real-world combustion-particle properties also continually change as new fuels are implemented, engines age, and engine technologies evolve. This work used laboratory-generated particles produced under controlled combustion conditions in an effort to understand the relationship between different particle properties and the activation of established toxicological outcomes in human lung cells (H441 and THP-1). Particles were generated from controlled combustion of two simple biofuel/diesel surrogates (methyl decanoate and dodecane/biofuel-blended diesel (BD), and butanol and dodecane/alcohol-blended diesel (AD)) and compared to a widely studied reference diesel (RD) particle (NIST SRM2975/RD). BD, AD, and RD particles exhibited differences in size, surface area, extractable chemical mass, and the content of individual polycyclic aromatic hydrocarbons (PAHs). Some of these differences were directly associated with different effects on biological responses. BD particles had the greatest surface area, amount of extractable material, and oxidizing potential. These particles and extracts induced cytochrome P450 1A1 and 1B1 enzyme mRNA in lung cells. AD particles and extracts had the greatest total PAH content and also caused CYP1A1 and 1B1 mRNA induction. The RD extract contained the highest relative concentration of 2-ring PAHs and stimulated the greatest level of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNFα) cytokine secretion. Finally, AD and RD were more potent activators of TRPA1 than BD, and while neither the TRPA1 antagonist HC-030031 nor the antioxidant N-acetylcysteine (NAC) affected CYP1A1 or 1B1 mRNA induction, both inhibitors reduced IL-8 secretion and mRNA induction. These results highlight that differences in fuel and combustion conditions affect the physicochemical properties of particles, and these differences, in turn, affect commonly studied biological/toxicological responses.

Characterization of Transient Receptor Potential Vanilloid-1 (TRPV1) Variant Activation by Coal Fly Ash Particles and Associations with Altered Transient Receptor Potential Ankyrin-1 (TRPA1) Expression and Asthma.

Transient receptor potential (TRP) channels are activated by environmental particulate materials. We hypothesized that polymorphic variants of transient receptor potential vanilloid-1 (TRPV1) would be uniquely responsive to insoluble coal fly ash compared with the prototypical soluble agonist capsaicin. Furthermore, these changes would manifest as differences in lung cell responses to these agonists and perhaps correlate with changes in asthma symptom control. The TRPV1-I315M and -T469I variants were more responsive to capsaicin and coal fly ash. The I585V variant was less responsive to coal fly ash particles due to reduced translation of protein and an apparent role for Ile-585 in activation by particles. In HEK-293 cells, I585V had an inhibitory effect on wild-type TRPV1 expression, activation, and internalization/agonist-induced desensitization. In normal human bronchial epithelial cells, IL-8 secretion in response to coal fly ash treatment was reduced for cells heterozygous for TRPV1-I585V. Finally, both the I315M and I585V variants were associated with worse asthma symptom control with the effects of I315M manifesting in mild asthma and those of the I585V variant manifesting in severe, steroid-insensitive individuals. This effect may be due in part to increased transient receptor potential ankyrin-1 (TRPA1) expression by lung epithelial cells expressing the TRPV1-I585V variant. These findings suggest that specific molecular interactions control TRPV1 activation by particles, differential activation, and desensitization of TRPV1 by particles and/or other agonists, and cellular changes in the expression of TRPA1 as a result of I585V expression could contribute to variations in asthma symptom control.

Activation of transient receptor potential ankyrin-1 (TRPA1) in lung cells by wood smoke particulate material.

Cigarette smoke, diesel exhaust, and other combustion-derived particles activate the calcium channel transient receptor potential ankyrin-1 (TRPA1), causing irritation and inflammation in the respiratory tract. It was hypothesized that wood smoke particulate and select chemical constituents thereof would also activate TRPA1 in lung cells, potentially explaining the adverse effects of wood and other forms of biomass smoke on the respiratory system. TRPA1 activation was assessed using calcium imaging assays in TRPA1-overexpressing HEK-293 cells, mouse primary trigeminal neurons, and human adenocarcinoma (A549) lung cells. Particles from pine and mesquite smoke were less potent agonists of TRPA1 than an equivalent mass concentration of an ethanol extract of diesel exhaust particles; pine particles were comparable in potency to cigarette smoke condensate, and mesquite particles were the least potent. The fine particulate (PM < 2.5 µm) of wood smoke were the most potent TRPA1 agonists and several chemical constituents of wood smoke particulate, 3,5-ditert-butylphenol, coniferaldehyde, formaldehyde, perinaphthenone, agathic acid, and isocupressic acid, were TRPA1 agonists. Pine particulate activated TRPA1 in mouse trigeminal neurons and A549 cells in a concentration-dependent manner, which was inhibited by the TRPA1 antagonist HC-030031. TRPA1 activation by wood smoke particles occurred through the electrophile/oxidant-sensing domain (i.e., C621/C641/C665/K710), based on the inhibition of cellular responses when the particles were pretreated with glutathione; a role for the menthol-binding site of TRPA1 (S873/T874) was demonstrated for 3,5-ditert-butylphenol. This study demonstrated that TRPA1 is a molecular sensor for wood smoke particulate and several chemical constituents thereof, in sensory neurons and A549 cells, suggesting that TRPA1 may mediate some of the adverse effects of wood smoke in humans.

Contributions of TRPV1, endovanilloids, and endoplasmic reticulum stress in lung cell death in vitro and lung injury.

Endogenous agonists of transient receptor potential vanilloid-1 (TRPV1) (endovanilloids) are implicated as mediators of lung injury during inflammation. This study tested the hypothesis that endovanilloids produced following lipopolysaccharide (LPS) treatment activate TRPV1 and cause endoplasmic reticulum stress/GADD153 expression in lung cells, representing a mechanistic component of lung injury. The TRPV1 agonist nonivamide induced GADD153 expression and caused cytotoxicity in immortalized and primary human bronchial, bronchiolar/alveolar, and microvascular endothelial cells, proportional to TRPV1 mRNA expression. In CF-1 mice, Trpv1 mRNA was most abundant in the alveoli, and intratracheal nonivamide treatment promoted Gadd153 expression in the alveolar region. Treatment of CF-1 mice with LPS increased Gadd153 in the lung, lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid, and lung wet-to-dry weight ratio. Cotreating mice with LPS and the TRPV1 antagonist LJO-328 reduced Gadd153 induction and LDH in BAL but did not inhibit increases in lung wet-to-dry ratio. In Trpv1(-/-) mice treated with LPS, Gadd153 induction and LDH in BAL were reduced relative to wild-type mice, and the wet-to-dry weight ratios of lungs from both wild-type and Trpv1(-/-) mice decreased. Organic extracts of blood collected from LPS-treated mice were more cytotoxic to TRPV1-overexpressing cells compared with BEAS-2B cells and extracts from control mice, however, most pure endovanilloids did not produce cytotoxicity in a characteristic TRPV1-dependent manner. Collectively, these data indicate a role for TRPV1, and endogenous TRPV1 agonists, in ER stress and cytotoxicity in lung cells but demonstrate that ER stress and cytotoxicity are not essential for pulmonary edema.