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

Perimenstrual worsening of asthma occurs in up to 40% of asthmatic women, 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 OVA model in BALB/c mice. Simultaneously, we used PBMC from healthy human donors to assess the effects of progesterone on circulating ILC2. Briefly, lungs of ovarectomized (OVX) or sham-operated female (F-Sham) controls were implanted with a progesterone (P4, 25mg) (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, increased select chemokines in the BAL and serum, and increased ILC2 and neutrophil numbers, along the airways in comparison to 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 IL-33 compared to 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.

Bronchoconstriction damages airway epithelia by crowding-induced excess cell extrusion.

Asthma is deemed an inflammatory disease, yet the defining diagnostic feature is mechanical bronchoconstriction. We previously discovered a conserved process called cell extrusion that drives homeostatic epithelial cell death when cells become too crowded. In this work, we show that the pathological crowding of a bronchoconstrictive attack causes so much epithelial cell extrusion that it damages the airways, resulting in inflammation and mucus secretion in both mice and humans. Although relaxing the airways with the rescue treatment albuterol did not affect these responses, inhibiting live cell extrusion signaling during bronchoconstriction prevented all these features. Our findings show that bronchoconstriction causes epithelial damage and inflammation by excess crowding-induced cell extrusion and suggest that blocking epithelial extrusion, instead of the ensuing downstream inflammation, could prevent the feed-forward asthma inflammatory cycle.

Inhibition of TRPA1, Endoplasmic Reticulum Stress, Human Airway Epithelial Cell Damage, and Ectopic MUC5AC Expression by Vasaka (Adhatoda vasica; Malabar Nut) Tea.

This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC (MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy.

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.

CYP1B1-derived epoxides modulate the TRPA1 channel in chronic pain.

Pain is often debilitating, and current treatments are neither universally efficacious nor without risks. Transient receptor potential (TRP) ion channels offer alternative targets for pain relief, but little is known about the regulation or identities of endogenous TRP ligands that affect inflammation and pain. Here, transcriptomic and targeted lipidomic analysis of damaged tissue from the mouse spinal nerve ligation (SNL)-induced chronic pain model revealed a time-dependent increase in Cyp1b1 mRNA and a concurrent accumulation of 8,9-epoxyeicosatrienoic acid (EET) and 19,20-EpDPA post injury. Production of 8,9-EET and 19,20-EpDPA by human/mouse CYP1B1 was confirmed in vitro, and 8,9-EET and 19,20-EpDPA selectively and dose-dependently sensitized and activated TRPA1 in overexpressing HEK-293 cells and Trpa1-expressing/AITC-responsive cultured mouse peptidergic dorsal root ganglia (DRG) neurons. TRPA1 activation by 8,9-EET and 19,20-EpDPA was attenuated by the antagonist A967079, and mouse TRPA1 was more responsive to 8,9-EET and 19,20-EpDPA than human TRPA1. This latter effect mapped to residues Y933, G939, and S921 of TRPA1. Intra-plantar injection of 19,20-EpDPA induced acute mechanical, but not thermal hypersensitivity in mice, which was also blocked by A967079. Similarly, Cyp1b1-knockout mice displayed a reduced chronic pain phenotype following SNL injury. These data suggest that manipulation of the CYP1B1-oxylipin-TRPA1 axis might have therapeutic benefit.

Steady-state estradiol triggers a unique innate immune response to allergen resulting in increased airway resistance.

RATIONALE: Asthma is a chronic airway condition that occurs more often in women than men during reproductive years. Population studies have collectively shown that long-term use of oral contraceptives decreased the onset of asthma in women of reproductive age. In the current study, we hypothesized that steady-state levels of estrogen would reduce airway inflammation and airway hyperresponsiveness to methacholine challenge. METHODS: Ovariectomized BALB/c mice (Ovx) were implanted with subcutaneous hormone pellets (estrogen, OVX-E2) that deliver consistent levels of estrogen [68 ± 2 pg/mL], or placebo pellets (OVX-Placebo), followed by ovalbumin sensitization and challenge. In conjunction with methacholine challenge, immune phenotyping was performed to correlate inflammatory proteins and immune populations with better or worse pulmonary outcomes measured by invasive pulmonary mechanics techniques. RESULTS: Histologic analysis showed an increase in total cell infiltration and mucus staining around the airways leading to an increased inflammatory score in ovarectomized (OVX) animals with steady-state estrogen pellets (OVX-E2-OVA) as compared to other groups including female-sham operated (F-INTACT-OVA) and OVX implanted with a placebo pellet (OVX-Pl-OVA). Airway resistance (Rrs) and lung elastance (Ers) were increased in OVX-E2-OVA in comparison to F-INTACT-OVA following aerosolized intratracheal methacholine challenges. Immune phenotyping revealed that steady-state estrogen reduced CD3+ T cells, CD19+ B cells, ILC2 and eosinophils in the BAL across all experiments. While these commonly described allergic cells were reduced in the BAL, or airways, we found no changes in neutrophils, CD3+ T cells or CD19+ B cells in the remaining lung tissue. Similarly, inflammatory cytokines (IL-5 and IL-13) were also decreased in OVX-E2-OVA-treated animals in comparison to Female-INTACT-OVA mice in the BAL, but in the lung tissue IL-5, IL-13 and IL-33 were comparable in OVX-E2-OVA and F-INTACT OVA mice. ILC2 were sorted from the lungs and stimulated with exogenous IL-33. These ILC2 had reduced cytokine and chemokine expression when they were isolated from OVX-E2-OVA animals, indicating that steady-state estrogen suppresses IL-33-mediated activation of ILC2. CONCLUSIONS: Therapeutically targeting estrogen receptors may have a limiting effect on eosinophils, ILC2 and potentially other immune populations that may improve asthma symptoms in those females that experience perimenstrual worsening of asthma, with the caveat, that long-term use of estrogens or hormone receptor modulators may be detrimental to the lung microenvironment over time.

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.

Nicotinic Acetylcholine Receptor Partial Antagonist Polyamides from Tunicates and Their Predatory Sea Slugs.

In our efforts to discover new drugs to treat pain, we identified molleamines A-E (1-5) as major neuroactive components of the sea slug, Pleurobranchus forskalii, and their prey, Didemnum molle, tunicates. The chemical structures of molleamines were elucidated by spectroscopy and confirmed by the total synthesis of molleamines A (1) and C (3). Synthetic 3 completely blocked acetylcholine-induced calcium flux in peptidergic nociceptors (PNs) in the somatosensory nervous system. Compound 3 affected neither the α7 nAChR nor the muscarinic acetylcholine receptors in calcium flux assays. In addition to nociceptors, 3 partially blocked the acetylcholine-induced calcium flux in the sympathetic nervous system, including neurons from the superior cervical ganglion. Electrophysiology revealed a block of α3ß4 (mouse) and α6/α3ß4 (rat) nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.4 and 3.1 µM, respectively. Molleamine C (3) is a partial antagonist, reaching a maximum block of 76-82% of the acetylcholine signal and showing no partial agonist response. Molleamine C (3) may thus provide a lead compound for the development of neuroactive compounds with unique biological properties.

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.

Activation of TRPV3 by Wood Smoke Particles and Roles in Pneumotoxicity.

Wood/biomass smoke particulate materials (WBSPM) are pneumotoxic, but the mechanisms by which these materials affect lung cells are not fully understood. We previously identified transient receptor potential (TRP) ankyrin-1 as a sensor for electrophiles in WBSPM and hypothesized that other TRP channels expressed by lung cells might also be activated by WBSPM, contributing to pneumotoxicity. Screening TRP channel activation by WBSPM using calcium flux assays revealed TRPV3 activation by materials obtained from burning multiple types of wood under fixed conditions. TRPV3 activation by WBSPM was dependent on the chemical composition, and the pattern of activation and chemical components of PM agonists was different from that of TRPA1. Chemical analysis of particle constituents by gas chromatography-mass spectrometry and principal component analysis indicated enrichment of cresol, ethylphenol, and xylenol analogues, plus several other chemicals among the most potent samples. 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-xylenol, 2-, 3-, and 4-ethylphenol, 2-methoxy-4-methylphenol, and 5,8-dihydronaphthol were TRPV3 agonists exhibiting preferential activation versus TRPA1, M8, V1, and V4. The concentration of 2,3- and 3,4-xylenol in the most potent samples of pine and mesquite smoke PM (<3 µm) was 0.1-0.3% by weight, while that of 5,8-dihydronaphthol was 0.03%. TRPV3 was expressed by several human lung epithelial cell lines, and both pine PM and pure chemical TRPV3 agonists found in WBSPM were more toxic to TRPV3-over-expressing cells via TRPV3 activation. Finally, mice treated sub-acutely with pine particles exhibited an increase in sensitivity to inhaled methacholine involving TRPV3. In summary, TRPV3 is activated by specific chemicals in WBSPM, potentially contributing to the pneumotoxic properties of certain WBSPM.

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.

Activation of Human Transient Receptor Potential Melastatin-8 (TRPM8) by Calcium-Rich Particulate Materials and Effects on Human Lung Cells.

To better understand how adverse health effects are caused by exposure to particulate materials, and to develop preventative measures, it is important to identify the properties of particles and molecular targets that link exposure with specific biologic outcomes. Coal fly ash (CFA) is a by-product of coal combustion that can affect human health. We report that human transient receptor potential melastatin-8 (TRPM8) and an N-terminally truncated TRPM8 variant (TRPM8-Δ801) are activated by CFA and calcium-rich nanoparticles and/or soluble salts within CFA. TRPM8 activation by CFA was potentiated by cold temperature involving the phosphatidylinositol 4,5-bisphosphate binding residue (L1008), but was independent of the icilin and menthol binding site residue Y745 and, essentially, the N-terminal amino acids 1-800. CFA, calcium nanoparticles, and calcium salts also activated transient receptor potential vanilloid-1 (TRPV1) and transient receptor potential ankyrin-1 (TRPA1), but not TRPV4. CFA treatment induced CXCL1 and interleukin-8 mRNA in BEAS-2B and primary human bronchial epithelial cells through activation of both TRPM8 and TRPV1. However, neither mouse nor rat TRPM8 was activated by these materials, and Trpm8 knockout had no effect on cytokine induction in the lungs of CFA-instilled mice. Amino acids S921 and S927 in mouse Trpm8 were identified as important for the lack of response to CFA. These results imply that TRPM8, in conjunction with TRPV1 and TRPA1, might sense selected forms of inhaled particulate materials in human airways, shaping cellular responses to these materials, and improving our understanding of how and why certain particulate materials elicit different responses in biologic systems, affecting human health.

Inhaled Remimazolam Potentiates Inhaled Remifentanil in Rodents.

BACKGROUND: Remimazolam is an ester-based short-acting benzodiazepine currently in clinical trials for IV administration. This study explored the feasibility of delivering remimazolam alone and as an adjunct to remifentanil via inhalation in rodent models. METHODS: Mice were exposed to remimazolam via inhalation; sedation was assessed using time to movement outside a set perimeter. Rats were also exposed to remimazolam aerosol alone and in combination with inhaled remifentanil, and analgesia was quantified by using a tail flick meter. Pulmonary injury was assessed in mice using mechanics measurements. RESULTS: Mice showed significantly increased time to movement outside a set perimeter after 5-minute exposure to increasing concentrations (10-25 mg/mL solutions) of inhaled remimazolam aerosols. Differences in mean (95% confidence interval) time to movement from pretest baseline group (0.05 [0.01-0.09] minutes) were 11 (4-18), 15 (5-26), 30 (19-41), and 109 (103-115) minutes after exposure to remimazolam aerosol of 10, 15, 20, and 25 mg/mL, respectively (P = .007 - P < .0001). Exposure of rats to remimazolam aerosols alone failed to produce sedation or analgesia after a 5-minute exposure. When remimazolam (10 or 25 mg/mL) was administered in combination with 250 µg/mL remifentanil, there was a significant difference in time to tail flick (P < .0001) consistent with a strong analgesic effect. Mean (95% confidence interval) differences in time to tail flick from the pretest baseline group (3.2 [2.5-3.9] seconds) were 14 (10-18) seconds when 250 µg/mL remifentanil was administered with either 10 or 25 mg/mL remimazolam. Remimazolam alone or in combination with remifentanil did not cause lung irritation, bronchospasm, or other adverse pulmonary events to the respiratory tract of mice as assessed by Flexi-Vent pulmonary function tests. CONCLUSIONS: Remimazolam can significantly potentiate the analgesic effect of remifentanil when concurrently delivered via inhalation.

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.

Inhaled Remifentanil in Rodents.

BACKGROUND: Remifentanil is an injectable opioid that is metabolized rapidly at a constant rate by plasma esterases. This supports its use as an analgesic for short-term, but painful, procedures in a wide range of patients. The aim of this study was to explore the feasibility and safety of administering remifentanil via inhalation. Our hypothesis was that inhaled remifentanil would be absorbed rapidly, pharmacologically active, rapidly cleared, and noninjurious to rodent airways and lungs. METHODS: Rats were exposed to remifentanil aerosol (100-2000 µg/mL) for varying times (1-5 minutes). Analgesia was quantified as a function of dose and time by measuring time to tail flick in response to a painful stimulus. Remifentanil was measured in blood using liquid chromatography-tandem mass spectrometry. Pulmonary mechanics and histology were assessed in mice for the evidence of adverse effects after acute and repeated (subacute) dosing. RESULTS: Exposure of rats to remifentanil aerosols produced dose-dependent analgesia within 2 minutes, which was sustained for the exposure period. Subsequently, the rats experienced rapid and complete recovery with a return to baseline tail flick response to a painful stimulus within 5 minutes. Analgesia mirrored the concentration profile of remifentanil in blood, and the animals were not affected adversely by repeated dosing. Pulmonary mechanics measurements in mice indicated that remifentanil was nonirritating and that the nasal and respiratory tissues of rats were free of significant morphological changes. CONCLUSIONS: Remifentanil delivered by inhalation is rapidly absorbed, pharmacologically active, rapidly cleared, and noninjurious to respiratory tissues in rodents.

Activation of Transient Receptor Potential Ankyrin-1 by Insoluble Particulate Material and Association with Asthma.

Inhaled irritants activate transient receptor potential ankyrin-1 (TRPA1), resulting in cough, bronchoconstriction, and inflammation/edema. TRPA1 is also implicated in the pathogenesis of asthma. Our hypothesis was that particulate materials activate TRPA1 via a mechanism distinct from chemical agonists and that, in a cohort of children with asthma living in a location prone to high levels of air pollution, expression of uniquely sensitive forms of TRPA1 may correlate with reduced asthma control. Variant forms of TRPA1 were constructed by mutating residues in known functional elements and corresponding to single-nucleotide polymorphisms in functional domains. TRPA1 activity was studied in transfected HEK-293 cells using allyl-isothiocynate, a model soluble electrophilic agonist; 3,5-ditert butylphenol, a soluble nonelectrophilic agonist and a component of diesel exhaust particles; and insoluble coal fly ash (CFA) particles. The N-terminal variants R3C and R58T exhibited greater, but not additive, activity with all three agonists. The ankyrin repeat domain-4 single nucleotide polymorphisms E179K and K186N exhibited decreased response to CFA. The predicted N-linked glycosylation site residues N747A and N753A exhibited decreased responses to CFA, which were not attributable to differences in cellular localization. The pore-loop residue R919Q was comparable to wild-type, whereas N954T was inactive to soluble agonists but not CFA. These data identify roles for ankyrin domain-4, cell surface N-linked glycans, and selected pore-loop domain residues in the activation of TRPA1 by insoluble particles. Furthermore, the R3C and R58T polymorphisms correlated with reduced asthma control for some children, which suggest that TRPA1 activity may modulate asthma, particularly among individuals living in locations prone to high levels of air pollution.