Search

Epinephrine (EPI), an endogenous catecholamine involved in the body’s fight-or-flight responses to stress, activates α1-adrenergic receptors (α1ARs) expressed on various organs to evoke a wide range of physiological functions, including vasoconstriction. In the smooth muscle of human bronchi, however, the functional role of EPI on α1ARs remains controversial. Classically, evidence suggests that EPI promotes bronchodilation by stimulating β2-adrenergic receptors (β2ARs). Conventionally, the selective β2AR agonism of EPI was thought to be, in part, due to a predominance of β2ARs and/or a sparse, or lack of α1AR activity in human airway smooth muscle (HASM) cells. Surprisingly, we find that HASM cells express a high abundance of ADRA1B (the α1AR subtype B) and identify a spontaneous “switch-like” activation of α1ARs that evokes intracellular calcium, myosin light chain phosphorylation, and HASM cell shortening. The switch-like responses, and related EPI-induced biochemical and mechanical signals, emerged upon pharmacological inhibition of β2ARs and/or under experimental conditions that induce β2AR tachyphylaxis. EPI-induced procontractile effects were abrogated by an α1AR antagonist, doxazosin mesylate (DM). These data collectively uncover a previously unrecognized feed-forward mechanism driving bronchospasm via two distinct classes of G protein-coupled receptors (GPCRs) and provide a basis for reexamining α1AR inhibition for the management of stress/exercise-induced asthma and/or β2-agonist insensitivity in patients with difficult-to-control, disease subtypes.

N-methyl-d-aspartate (NMDA) receptors are widely expressed in the central nervous system. However, their presence and function at extraneuronal sites is less well characterized. In the present study, we examined the expression of NMDA receptor subunit mRNA and protein in human pulmonary artery (HPA) by quantitative polymerase chain reaction (PCR), immunohistochemistry and immunoblotting. We demonstrate that both GluN1 and GluN2 subunit mRNAs are expressed in HPA. In addition, GluN1 and GluN2 (A–D) subunit proteins are expressed by human pulmonary artery smooth muscle cells (HPASMCs) in vitro and in vivo. These subunits localize on the surface of HPASMCs and form functional ion channels as evidenced by whole-cell patch-clamp electrophysiology and reduced phenylephrine-induced contractile responsiveness of human pulmonary artery by the NMDA receptor antagonist MK801 under hypoxic condition. HPASMCs also express high levels of serine racemase and vesicular glutamate transporter 1, suggesting a potential source of endogenous agonists for NMDA receptor activation. Our findings show HPASMCs express functional NMDA receptors in line with their effect on pulmonary vasoconstriction, and thereby suggest a novel therapeutic target for pharmacological modulations in settings associated with pulmonary vascular dysfunction.

In most living cells, the second-messenger roles for adenosine 3′,5′-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch–like actions of Gα(s) (stimulatory G protein)–activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface β(2)AR (β(2)-adrenoceptor), a G(s)-coupled GPCR (G protein–coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP](e)) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP](i)). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance–associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces β(2)AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases β-agonist–induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP](i) in HASM that may be conserved traits of the G(s)-GPCRs (G(s)-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

The soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate signaling pathway evokes vascular smooth muscle relaxation; whether this pathway mediates airway smooth muscle relaxation remains controversial. We posit that sGC activators are equi-effective as β-agonists in reversing contractile agonist-induced airway smooth muscle shortening. To provide clarity, we tested the efficacy of sGC stimulator and activator drugs, BAY 41-2272 and BAY 60-2270, respectively, in reversing bronchoconstriction of human small airways using human precision-cut lung slices (hPCLS). Both BAY drugs reversed carbachol-induced bronchoconstriction to a maximal degree comparable to that of formoterol. Moreover, the sGC drugs remained effective bronchodilators despite formoterol-induced desensitization of the airways. Analysis of the hPCLS after their activation by sGC or β2-adrenergic receptor agonist showed distinct cyclic nucleotide accumulation in the hPCLS. Collectively, these data suggest that cAMP and cyclic guanosine monophosphate pathways are equi-effective for reversing carbachol-induced bronchoconstriction in the human airway via separate and distinct second messenger pathways. This should open the door for future studies to test whether sGC-targeted drugs alone or in combination can serve as effective bronchodilators in asthma and chronic obstructive pulmonary disease.

Glucocorticoids are widely used for the suppression of inflammation, but evidence is growing that they can have rapid, non-genomic actions that have been unappreciated. Diverse cell signaling effects have been reported for glucocorticoids, leading us to hypothesize that glucocorticoids alone can swiftly increase the 3',5'-cyclic adenosine monophosphate (cAMP) production. We found that prednisone, fluticasone, budesonide, and progesterone each increased cAMP levels within 3 minutes without phosphodiesterase inhibitors by measuring real-time cAMP dynamics using the cAMP difference detector in situ assay in a variety of immortalized cell lines and primary human airway smooth muscle (HASM) cells. A membrane- impermeable glucocorticoid showed similarly rapid stimulation of cAMP, implying that responses are initiated at the cell surface. siRNA knockdown of Gαs virtually eliminated glucocorticoid-stimulated cAMP responses, suggesting that these drugs activate the cAMP production via a G protein-coupled receptor. Estradiol had small effects on cAMP levels but G protein estrogen receptor antagonists had little effect on responses to any of the glucocorticoids tested. The genomic and non-genomic actions of budesonide were analyzed by RNA-Seq analysis of 24 hours treated HASM, with and without knockdown of Gαs . A 140-gene budesonide signature was identified, of which 48 genes represent a non-genomic signature that requires Gαs signaling. Collectively, this non-genomic cAMP signaling modality contributes to one-third of the gene expression changes induced by glucocorticoid treatment and shifts the view of how this important class of drugs exerts its effects.

Airway smooth muscle is the primary cell mediating bronchomotor tone. The milieu created in the asthmatic lung modulates airway smooth muscle contractility and relaxation. Experimental findings suggest intrinsic abnormalities in airway smooth muscle derived from patients with asthma in comparison with airway smooth muscle from those without asthma. These changes to excitation-contraction pathways may underlie airway hyperresponsiveness and increased airway resistance associated with asthma.

Airway smooth muscle plays a pivotal role in modulating bronchomotor tone. Modulation of contractile and relaxation signaling is critical to alleviate the airway hyperresponsiveness (AHR) associated with asthma. Emerging studies examining the phenotype of ASM in the context of asthma provide rich avenues to develop more effective therapeutics to attenuate the AHR associated with the disease.

Obesity is emerging as a global public health epidemic. The co-morbidities associated with obesity significantly contribute to reduced quality of life, mortality, and global healthcare burden. Compared to other asthma comorbidities, obesity prominently engenders susceptibility to inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), contributes to greater disease severity and evokes insensitivity to current therapies. Unlike in other metabolic diseases associated with obesity, the mechanistic link between obesity and airway diseases is only poorly defined. Transforming growth factor-β (TGF-β) is a pleiotropic inflammatory cytokine belonging to a family of growth factors with pivotal roles in asthma. In this review, we summarize the role of TGF-β in major obesity-associated co-morbidities to shed light on mechanisms of the diseases. Literature evidence shows that TGF-β mechanistically links many co-morbidities with obesity through its profibrotic, remodeling, and proinflammatory functions. We posit that TGF-β plays a similar mechanistic role in obesity-associated inflammatory airway diseases such as asthma and COPD. Concerning the role of TGF-β on metabolic effects of obesity, we posit that TGF-β has a similar mechanistic role in obesity-associated inflammatory airway diseases in interplay with different comorbidities such as hypertension, metabolic diseases like type 2 diabetes, and cardiomyopathies. Future studies in TGF-β-dependent mechanisms in obesity-associated inflammatory airway diseases will advance our understanding of obesity-induced asthma and help find novel therapeutic targets for prevention and treatment., Graphical abstract Obesity amplifies airway hyperreactivity in asthma, potentially through TGF-β1-mediated proinflammatory, profibrotic and mitogenic mechanisms.Image 1, Highlights • Obesity is a serious global health issue, and is associated with inflammatory airways diseases. • TGF-β1 is a cytokine present in a number of inflammatory disorders. • Mechanisms underlying obesity-induced airways diseases are unclear. • The role of TGF-β1 in obesity-induced airway diseases remains largely unknown.

A subset of asthmatics develop a severe form of the disease whose etiology involves airway inflammation along with inherent drivers that remain ill-defined. To address this, we studied human airway smooth muscle cells (HASMC), whose relaxation drives airway bronchodilation and whose dysfunction contributes to airway obstruction and hypersensitivity in severe asthma. Because HASMC relaxation can be driven by the NO-soluble guanylyl cyclase (sGC)-cGMP signaling pathway, we questioned if HASMC from severe asthma donors might possess inherent defects in their sGC or in redox enzymes that support sGC function. We analyzed HASMC primary lines derived from 17 severe asthma and 16 normal donors and corresponding lung tissue samples regarding sGC activation by NO or by pharmacologic agonists, and also determined expression levels of sGC α1 and β1 subunits, supporting redox enzymes, and related proteins. We found a majority of the severe asthma donor HASMC (12/17) and lung samples primarily expressed a dysfunctional sGC that was NO-unresponsive and had low heterodimer content and high Hsp90 association. This sGC phenotype correlated with lower expression levels of the supporting redox enzymes cytochrome b5 reductase, catalase, and thioredoxin-1, and higher expression of heme oxygenases 1 and 2. Together, our work reveals that severe asthmatics are predisposed toward defective NO-sGC-cGMP signaling in their airway smooth muscle due to an inherent sGC dysfunction, which in turn is associated with inherent changes in the cell redox enzymes that impact sGC maturation and function., Graphical abstract Image 1, Highlights • The etiology of severe asthma involves airway inflammation and inherent drivers that remain ill-defined. • Airway smooth muscle cells of severe asthmatics display a NO-unresponsive and dysfunctional sGC which persists in culture. • Their inherent sGC dysfunction is associated with low CYB5R3 expression and altered expression of other redox enzymes. • That airway sGC dysfunction and redox enzyme changes cluster within severe asthma is unexpected and may help guide therapy.

Significance Odorant sensing GPCRs are the largest gene family in the human genome. We previously found multiple olfactory receptors and their obligate downstream effectors expressed in the smooth muscle of human bronchi. However, the extent to which odorant-sensing receptors (and the ligands to which they respond) on airway smooth muscle (ASM) are physiologically relevant is not established. Here we show that a monoterpene nerol activates the odorant receptor OR2W3 to relax ASM in both cell and tissue models. Surprisingly, the mechanism of action of OR2W3-mediated ASM relaxation involves paradoxical increases in [Ca2+]i that invoke a cooperative activation of TMEM16A and CFTR to compartmentalize calcium and regulate excitation-contraction coupling in human ASM cells., The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases.

Glucocorticoids (GCs) and β2-adrenergic receptor (β2AR) agonists improve asthma outcomes in most patients. GCs also modulate gene expression in human airway smooth muscle (HASM), thereby attenuating airway inflammation and airway hyperresponsiveness that define asthma. Our previous studies showed that the pro-fibrotic cytokine, transforming growth factor- β1 (TGF-β1) increases phosphodiesterase 4D (PDE4D) expression that attenuates agonist-induced levels of intracellular cAMP. Decreased cAMP levels then diminishes β2 agonist-induced airway relaxation. In the current study, we investigated whether glucocorticoids reverse TGF-β1-effects on β2-agonist-induced bronchodilation and modulate pde4d gene expression in HASM. Dexamethasone (DEX) reversed TGF-β1 effects on cAMP levels induced by isoproterenol (ISO). TGF-β1 also attenuated G protein-dependent responses to cholera toxin (CTX), a Gαs stimulator downstream from the β2AR receptor. Previously, we demonstrated that TGF-β1 treatment increased β2AR phosphorylation to induce hyporesponsiveness to a β2 agonist. Our current data shows that expression of grk2/3, kinases associated with attenuation of β2AR function, are not altered with TGF-β1 stimulation. Interestingly, DEX also attenuated TGF-β1-induced pde4d gene expression. These data suggest that steroids may be an effective therapy for treatment of asthma patients whose disease is primarily driven by elevated TGF-β1 levels.

Rationale: Th2 and Th17 cells contribute to airway remodeling, facilitate mucus secretion and increase airway hyperresponsiveness. CD6, a T cell co-stimulatory receptor, interacts with CD166 (ALCAM) and CD318 to modulate activation. We examined whether severe asthma subjects have increased CD6+ cells, the lung inflammatory environment alters CD166 or CD318 expression, and CD6 exposure of airway smooth muscle (ASM) modulates bronchomotor tone. Methods: Lungs (asthma, non-asthma, n=4/group) were lavaged and lymphocytes examined for CD6 by flow cytometry. Tissue was stained for CD166, CD318, CD3, and CD6. ASM stimulated with carbachol (Cch) or CD6-beads was examined for phosphorylation of myosin light chain (pMLC), paxillin (pPax), or cofilin (pCof). ASM were co-cultured with T cells ± IL-33 with CD166 and CD318 expression assessed. Results: Flow analysis showed no difference in CD6+ lavage cells from asthma vs. non-asthma donors. Tissue sections had large differences in lymphocyte number and CD3+/CD6+ cells, with CD166 and CD318 expression on ciliated and basal cells. ALCAM, but not cd318, expression increased in asthma ASM. CD6-coated beads increased pCof and pPax, but not pMLC, to similar levels to Cch. ASM co-cultured with differentiated Th1 cells showed decreased CD166, but not CD318 expression. Co-stimulation with Th1 cells + IL-33 markedly decreased ASM CD166 and CD318 expression. Conclusions: We demonstrate that CD6+ lymphocytes are significantly increased in fatal asthma lung tissue. Furthermore, CD6+ lymphocytes may modulate bronchomotor tone through actin cytoskeletal rearrangements in HASM, and that the inflammatory environment may influence CD6-dependent changes in bronchomotor tone.

Rhinovirus (RV) exposure has been implicated in childhood development of wheeze evoking asthma and exacerbations of underlying airways disease. Studies such as the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC) and Childhood Origins of ASThma (COAST) have identified RV as a pathogen inducing severe respiratory disease. RVs also modulate airway hyperresponsiveness (AHR), a key characteristic of such diseases. Although potential factors underlying mechanisms by which RV induces AHR have been postulated, the precise mechanisms of AHR following RV exposure remain elusive. A challenge to RV-related research stems from inadequate models for study. While human models raise ethical concerns and are relatively difficult in terms of subject recruitment, murine models are limited by susceptibility of infection to the relatively uncommon minor group (RV-B) serotypes, strains that are generally associated with infrequent clinical respiratory virus infections. Although a transgenic mouse strain that has been developed has enhanced susceptibility for infection with the common major group (RV-A) serotypes, few studies have focused on RV in the context of allergic airways disease rather than understanding RV-induced AHR. Recently, the receptor for the virulent RV-C CDHR3, was identified, but a dearth of studies have examined RV-C-induced effects in humans. Currently, the mechanisms by which RV infections modulate airway smooth muscle (ASM) shortening or excitation-contraction coupling remain elusive. Further, only one study has investigated the effects of RV on bronchodilatory mechanisms, with only speculation as to mechanisms underlying RV-mediated modulation of bronchoconstriction.

Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing β2-adrenergic receptor AC6 and another containing E prostanoid receptor AC2. We hypothesized that different PDE isozymes selectively regulate cAMP signaling in each compartment. According to RNA-sequencing data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age- and sex-matched donors with and without asthma. PDE8A was the third most abundant of the cAMP-degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked twofold greater cAMP responses to 1 μM forskolin in the presence of 3-isobutyl-1-methylxanthine. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescence sensor. PF-04957325, a PDE8-selective inhibitor, increased basal cAMP concentrations by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF-04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but it had no effect on cAMP concentrations or cell proliferation regulated by prostaglandin E2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin-1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells, where it specifically regulates β2-adrenergic receptor AC6 signaling without effects on signaling by the E prostanoid receptors 2/4-AC2 complex. In airway diseases such as asthma and chronic obstructive pulmonary disease, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.

A cardinal feature of asthma is airway hyperresponsiveness (AHR) to spasmogens, many of which activate G protein–coupled receptors (GPCRs) on airway smooth muscle (ASM) cells. Asthma subtypes associated with allergy are characterized by eosinophilic inflammation in the lung due to the type 2 immune response to allergens and proinflammatory mediators that promote AHR. The degree to which intrinsic abnormalities of ASM contribute to this phenotype remains unknown. The regulators of G protein signaling (RGS) proteins are a large group of intracellular proteins that inhibit GPCR signaling pathways. RGS2- and RGS5-deficient mice develop AHR spontaneously. Although RGS4 is upregulated in ASM from patients with severe asthma, the effects of increased RGS4 expression on AHR in vivo are unknown. Here, we examined the impact of forced RGS4 overexpression in lung on AHR using transgenic (Tg) mice. Tg RGS4 was expressed in bronchial epithelium and ASM in vivo, and protein expression in lung was increased at least 4-fold in Tg mice compared with wild-type (WT) mice. Lung slices from Tg mice contracted less in response to the m3 muscarinic receptor agonist methacholine compared with the WT, although airway resistance in live, unchallenged mice of both strains was similar. Tg mice were partially protected against AHR induced by fungal allergen challenge due to weakened contraction signaling in ASM and reduced type 2 cytokine (IL-5 and IL-13) levels in Tg mice compared with the WT. These results provide support for the hypothesis that increasing RGS4 expression and/or function could be a viable therapeutic strategy for asthma.

Author(s): Yoo, Edwin J; Cao, Gaoyuan; Koziol-White, Cynthia J; Ojiaku, Christie A; Sunder, Krishna; Jude, Joseph A; Michael, James V; Lam, Hong; Pushkarsky, Ivan; Damoiseaux, Robert; Di Carlo, Dino; Ahn, Kwangmi; An, Steven S; Penn, Raymond B; Panettieri, Reynold A | Abstract: Background and purposePI3K-dependent activation of Rho kinase (ROCK) is necessary for agonist-induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist-mediated PI3K/ROCK axis activation, however, remain unclear. Given that G12 family proteins activate ROCK pathways in other cell types, their role in M3 muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined.Experimental approachGα12 coupling was evaluated using co-immunoprecipitation and serum response element (SRE)-luciferase reporter assays. siRNA and pharmacological approaches, as well as overexpression of a regulator of G-protein signaling (RGS) proteins were applied in HASMCs. Phosphorylation levels of Akt, myosin phosphatase targeting subunit-1 (MYPT1), and myosin light chain-20 (MLC) were measured. Contraction and shortening were evaluated using magnetic twisting cytometry (MTC) and micro-pattern deformation, respectively. Human precision-cut lung slices (hPCLS) were utilized to evaluate bronchoconstriction.Key resultsKnockdown of M3 receptors or Gα12 attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα12 coimmunoprecipitated with M3 receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SRE-luciferase reporter. p115RhoGEF-RGS overexpression inhibited carbachol-induced activation of Akt, HASMC contraction, and shortening. Moreover, inhibition of RhoA blunted activation of PI3K. Lastly, RhoA inhibitors induced dilation of hPCLS.Conclusions and implicationsGα12 plays a crucial role in HASMC contraction via RhoA-dependent activation of the PI3K/ROCK axis. Inhibition of RhoA activation induces bronchodilation in hPCLS, and targeting Gα12 signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma.

The nongenomic mechanisms by which glucocorticoids modulate β2 agonist-induced-bronchodilation remain elusive. Our studies aimed to elucidate mechanisms mediating the beneficial effects of glucocorticoids on agonist-induced bronchodilation. Utilizing human precision-cut lung slices (hPCLS), we measured bronchodilation to formoterol, prostaglandin E2 (PGE2), cholera toxin (CTX), or forskolin in the presence and absence of budesonide. Using cultured human airway smooth muscle (HASM), intracellular cAMP was measured in live cells following exposure to formoterol, PGE2, or forskolin in the presence or absence of budesonide. We showed that simultaneous budesonide administration amplified formoterol-induced bronchodilation and attenuated agonist-induced phosphorylation of myosin light chain, a necessary signaling event mediating force generation. In parallel studies, cAMP levels were augmented by simultaneous exposure of HASM cells to formoterol and budesonide. Budesonide, fluticasone, and prednisone alone rapidly increased cAMP levels, but steroids alone had little effect on bronchodilation in hPCLS. Bronchodilation induced by PGE2, CTX, or forskolin was also augmented by simultaneous exposure to budesonide in hPCLS. Furthermore, HASM cells expressed membrane-bound glucocorticoid receptors that failed to translocate with glucocorticoid stimulation and that potentially mediated the rapid effects of steroids on β2 agonist-induced bronchodilation. Knockdown of glucocorticoid receptor-α had little effect on budesonide-induced and steroid-dependent augmentation of formoterol-induced cAMP generation in HASM. Collectively, these studies suggest that glucocorticoids amplify cAMP-dependent bronchodilation by directly increasing cAMP levels. These studies identify a molecular mechanism by which the combination of glucocorticoids and β2 agonists may augment bronchodilation in diseases such as asthma or chronic obstructive pulmonary disease.

Rhinovirus (RV) exposure evokes exacerbations of asthma that markedly impact morbidity and mortality worldwide. The mechanisms by which RV induces airway hyperresponsiveness (AHR) or by which specific RV serotypes differentially evoke AHR remain unknown. We posit that RV infection evokes AHR and inflammatory mediator release, which correlate with degrees of RV infection. Furthermore, we posit that rhinovirus C-induced AHR requires paracrine or autocrine mediator release from epithelium that modulates agonist-induced calcium mobilization in human airway smooth muscle. In these studies, we used an ex vivo model to measure bronchoconstriction and mediator release from infected airways in human precision cut lung slices to understand how RV exposure alters airway constriction. We found that rhinovirus C15 (RV-C15) infection augmented carbachol-induced airway narrowing and significantly increased release of IP-10 (IFN-γ-induced protein 10) and MIP-1β (macrophage inflammatory protein-1β) but not IL-6. RV-C15 infection of human airway epithelial cells augmented agonist-induced intracellular calcium flux and phosphorylation of myosin light chain in co-cultured human airway smooth muscle to carbachol, but not after histamine stimulation. Our data suggest that RV-C15-induced structural cell inflammatory responses are associated with viral load but that inflammatory responses and alterations in agonist-mediated constriction of human small airways are uncoupled from viral load of the tissue.

Asthma is a chronic allergic inflammatory airway disease caused by aberrant immune responses to inhaled allergens, which leads to airway hyperresponsiveness (AHR) to contractile stimuli and airway obstruction. Blocking T helper 2 (T(H)2) differentiation represents a viable therapeutic strategy for allergic asthma, and strong TCR-mediated ERK activation blocks T(H)2 differentiation. Here, we report that targeting diacylglycerol (DAG) kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protected against allergic asthma by simultaneously reducing airway inflammation and AHR though independent mechanisms. Targeted deletion of DGKζ in T cells decreased type 2 inflammation without reducing AHR. In contrast, loss of DGKζ in airway smooth muscle cells decreased AHR but not airway inflammation. T cell–specific enhancement of ERK signaling was only sufficient to limit type 2 airway inflammation, not AHR. Pharmacological inhibition of DGK diminished both airway inflammation and AHR in mice and also reduced bronchoconstriction of human airway samples in vitro. These data suggest that DGK is a previously unrecognized therapeutic target for asthma and reveal that the inflammatory and AHR components of asthma are not as interdependent as generally believed.

Background Allergens elicit host production of mediators acting on G-protein–coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. Objective We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein–coupled receptors, in asthma. Methods We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle–specific Rgs4−/− mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug–associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease–like (ptges1−/−) mice with aspirin. Results RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4−/− mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1−/− mice, respectively, in association with increased airway PGE2 levels. Conclusions RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.

Allergen immunotherapy for patients with allergies begins with weekly escalating doses of allergen under medical supervision to monitor and treat IgE mast cell-mediated anaphylaxis. There is currently no treatment to safely desensitize mast cells to enable robust allergen immunotherapy with therapeutic levels of allergen. Here, we demonstrated that liposomal nanoparticles bearing an allergen and a high-affinity glycan ligand of the inhibitory receptor CD33 profoundly suppressed IgE-mediated activation of mast cells, prevented anaphylaxis in Tg mice with mast cells expressing human CD33, and desensitized mice to subsequent allergen challenge for several days. We showed that high levels of CD33 were consistently expressed on human skin mast cells and that the antigenic liposomes with CD33 ligand prevented IgE-mediated bronchoconstriction in slices of human lung. The results demonstrated the potential of exploiting CD33 to desensitize mast cells to provide a therapeutic window for administering allergen immunotherapy without triggering anaphylaxis.

Background and purpose Asthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyper-reactivity (AHR). Spleen tyrosine kinase (Syk) mediates allergen-induced mast cell degranulation, a central component of allergen-induced inflammation and AHR. However, the role of Syk in IgE-mediated constriction of human small airways remains unknown. In this study, we addressed whether selective inhibition of Syk attenuates IgE-mediated constriction and mast cell mediator release in human small airways. Experimental Approach Human precision cut lung slices (hPCLS) ex vivo derived from non-asthmatic donors were incubated overnight with human IgE, dexamethasone, montelukast, antihistamines or a selective Syk inhibitor (SYKi). FceRI activation by anti-IgE cross-linking was performed, and constriction and mediator release measured. Airway constriction was normalized to that induced by maximal carbachol stimulation. Syk expression (determined by qPCR and immunoblot) was also evaluated in human primary airway smooth muscle (HASM) cells to determine whether Syk directly modulates HASM function. Key Results While dexamethasone had little effect on FceR-mediated contraction, montelukast or antihistamines partially attenuated the response. SYKi completely abrogated anti-IgE-mediated contraction and suppressed the release of mast cell or basophil mediators from the IgE-treated hPCLS. In contrast, SYKi had little effect on the non-allergic contraction induced by carbachol. Syk mRNA and protein were undetectable in HASM cells. Conclusions and Implications Our data demonstrate that a selective Syk inhibitor, but not corticosteroids, abrogated FceR-mediated contraction in ex vivo human small airways. The mechanism involves FceRI receptor activation on mast cells or basophils that degranulate causing airway constriction, rather than direct actions on HASM.

Formaldehyde, a common indoor air pollutant, exacerbates asthma and synergizes with allergen to induce airway hyperresponsiveness (AHR) in animal models. The mechanisms mediating formaldehyde-induced AHR remain poorly understood. We posit that formaldehyde modulates agonist-induced contractile response of human airway smooth muscle (HASM) cells to elicit AHR. HASM cells were exposed to formaldehyde or vehicle and agonist-induced intracellular Ca2+ ([Ca2+]i) and myosin light-chain phosphatase (MYPT1) phosphorylation were determined. Air–liquid interface–differentiated human bronchial epithelial (HBE) cells were exposed to formaldehyde or vehicle and cocultured with HASM cells. Agonist-induced [Ca2+]i and MYPT1 phosphorylation were determined in the cocultured HASM cells. Precision-cut human lung slices were exposed to PBS or varying concentrations of formaldehyde, and then carbachol-induced airway narrowing was determined 24 hours after exposure. HASM cells were transfected with nontargeting or nuclear factor erythroid-derived 2, like 2 (Nrf-2)-targeting small interfering RNA and exposed to formaldehyde or vehicle, followed by determination of antioxidant response (quinone oxido-reductase 1 and thioredoxin 1) and basal and agonist-induced MYPT1 phosphorylation. Formaldehyde enhanced the basal Rho-kinase activity and MYPT1 phosphorylation with little effect on agonist-induced [Ca2+]i in HASM cells. Formaldehyde induced Nrf-2–dependent antioxidant response in HASM cells, although the MYPT1 phosphorylation was independent of Nrf-2 induction. Although HBE cells exposed to formaldehyde had little effect on agonist-induced [Ca2+]i or MYPT1 phosphorylation in cocultured HASM cells, formaldehyde enhanced carbachol-induced airway responsiveness in precision-cut human lung slices. In conclusion, formaldehyde induces phosphorylation of the regulatory subunit of MYPT1, independent of formaldehyde-induced Nrf-2 activation in HASM cells. The findings suggest that the Rho kinase-dependent Ca2+ sensitization pathway plays a role in formaldehyde-induced AHR.

Author(s): Koziol-White, Cynthia J; Yoo, Edwin J; Cao, Gaoyuan; Zhang, Jie; Papanikolaou, Eleni; Pushkarsky, Ivan; Andrews, Adam; Himes, Blanca E; Damoiseaux, Robert D; Liggett, Stephen B; Di Carlo, Dino; Kurten, Richard C; Panettieri, Reynold A | Abstract: Background and purposeAsthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyperresponsiveness (AHR). Although the molecular mechanisms remain unclear, activation of specific PI3K isoforms mediate inflammation and AHR. We aimed to determine whether inhibition of PI3Kδ evokes dilation of airways and to elucidate potential mechanisms.Experimental approachHuman precision cut lung slices from non-asthma donors and primary human airway smooth muscle (HASM) cells from both non-asthma and asthma donors were utilized. Phosphorylation of Akt, myosin phosphatase target subunit 1 (MYPT1) and myosin light chain (MLC) were assessed in HASM cells following either PI3K inhibitor or siRNA treatment. HASM relaxation was assessed by micro-pattern deformation. Reversal of constriction of airways was assessed following stimulation with PI3K or ROCK inhibitors.Key resultsSoluble inhibitors or PI3Kδ knockdown reversed carbachol-induced constriction of human airways, relaxed agonist-contracted HASM and inhibited pAkt, pMYPT1 and pMLC in HASM. Similarly, inhibition of Rho kinase also dilated human PCLS airways and suppressed pMYPT1 and pMLC. Baseline pMYPT1 was significantly elevated in HASM cells derived from asthma donors in comparison with non-asthma donors. After desensitization of the β2 -adrenoceptors, a PI3Kδ inhibitor remained an effective dilator. In the presence of IL-13, dilation by a β agonist, but not PI3K inhibitor, was attenuated.Conclusion and implicationsPI3Kδ inhibitors act as dilators of human small airways. Taken together, these findings provide alternative approaches to the clinical management of airway obstruction in asthma.

Glucocorticoids, commonly used asthma controller medications, decrease symptoms in most patients, but some remain symptomatic despite high-dose treatment. The physiological basis underlying the glucocorticoid response, especially in asthma patients with severe, refractory disease, is not fully understood. We sought to identify differences between the transcriptomic response of airway smooth muscle (ASM) cells derived from donors with fatal asthma and donors without asthma to glucocorticoid exposure and to compare ASM-specific changes with those observed in other cell types. In cells derived from nine donors with fatal asthma and eight donors without asthma, RNA sequencing was used to measure ASM transcriptome changes after exposure to budesonide (100 nM 24 h) or control vehicle (DMSO). Differential expression results were obtained for this dataset, as well as 13 publicly available glucocorticoid-response transcriptomic datasets corresponding to seven cell types. Specific genes were differentially expressed in response to glucocorticoid exposure (7,835 and 6,957 in ASM cells derived from donors with fatal asthma and donors without asthma, respectively; adjusted

Epidemiologic studies have demonstrated an association between acetaminophen (APAP) use and the development of asthma symptoms. However, few studies have examined relationships between APAP-induced signaling pathways associated with the development of asthma symptoms. We tested the hypothesis that acute APAP exposure causes airway hyper-responsiveness (AHR) in human airways.Precision cut lung slice (PCLS) airways from humans and mice were used to determine the effects of APAP on airway bronchoconstriction and bronchodilation and to assess APAP metabolism in lungs.APAP did not promote AHR in normal or asthmatic human airways ex vivo. Rather, high concentrations mildly bronchodilated airways pre-constricted with carbachol (CCh), histamine (His), or immunoglobulin E (IgE) cross-linking. Further, the addition of APAP prior to bronchoconstrictors protected the airways from constriction. Similarly, in vivo treatment of mice with APAP (200 mg/kg IP) resulted in reduced bronchoconstrictor responses in PCLS airways ex vivo. Finally, in both mouse and human PCLS airways, exposure to APAP generated only low amounts of APAP-protein adducts, indicating minimal drug metabolic activity in the tissues.These findings indicate that acute exposure to APAP does not initiate AHR, that high-dose APAP is protective against bronchoconstriction, and that APAP is a mild bronchodilator. Epidemiologic studies have demonstrated an association between acetaminophen (APAP) use and the development of asthma symptoms. However, few studies have examined relationships between APAP-induced signaling pathways associated with the development of asthma symptoms. We tested the hypothesis that acute APAP exposure causes airway hyper-responsiveness (AHR) in human airways. Precision cut lung slice (PCLS) airways from humans and mice were used to determine the effects of APAP on airway bronchoconstriction and bronchodilation and to assess APAP metabolism in lungs. APAP did not promote AHR in normal or asthmatic human airways ex vivo. Rather, high concentrations mildly bronchodilated airways pre-constricted with carbachol (CCh), histamine (His), or immunoglobulin E (IgE) cross-linking. Further, the addition of APAP prior to bronchoconstrictors protected the airways from constriction. Similarly, in vivo treatment of mice with APAP (200 mg/kg IP) resulted in reduced bronchoconstrictor responses in PCLS airways ex vivo. Finally, in both mouse and human PCLS airways, exposure to APAP generated only low amounts of APAP-protein adducts, indicating minimal drug metabolic activity in the tissues. These findings indicate that acute exposure to APAP does not initiate AHR, that high-dose APAP is protective against bronchoconstriction, and that APAP is a mild bronchodilator.

Glucocorticoid (GC) anti-inflammatory effects generally require a prolonged onset of action and involve genomic processes. Because of the rapidity of some of the GC effects, however, the concept that non-genomic actions may contribute to GC mechanisms of action has arisen. While the mechanisms have not been completely elucidated, the non-genomic effects may play a role in the management of inflammatory diseases. For instance, we recently reported that GCs 'rapidly' enhanced the effects of bronchodilators, agents used in the treatment of allergic asthma. In this review article, we discuss (i) the non-genomic effects of GCs on pathways relevant to the pathogenesis of inflammatory diseases and (ii) the putative role of the membrane GC receptor. Since GC side effects are often considered to be generated through its genomic actions, understanding GC non-genomic effects will help design GCs with a better therapeutic index.

The asthmatic airways are highly susceptible to inflammatory injury by air pollutants such as ozone (O3), characterized by enhanced activation of eosinophilic granulocytes and a failure of immune protective mechanisms. Eosinophil activation during asthma exacerbation contributes to the proinflammatory oxidative stress by high levels of nitric oxide (NO) production and extracellular DNA release. Surfactant protein-D (SP-D), an epithelial cell product of the airways, is a critical immune regulatory molecule with a multimeric structure susceptible to oxidative modifications. Using recombinant proteins and confocal imaging, we demonstrate here that SP-D directly bound to the membrane and inhibited extracellular DNA trap formation by human and murine eosinophils in a concentration and carbohydrate-dependent manner. Combined allergic airway sensitization and O3 exposure heightened eosinophilia and nos2 mRNA (iNOS) activation in the lung tissue and S-nitrosylation related de-oligomerisation of SP-D in the airways. In vitro reproduction of the iNOS action led to similar effects on SP-D. Importantly, S-nitrosylation abolished the ability of SP-D to block extracellular DNA trap formation. Thus, the homeostatic negative regulatory feedback between SP-D and eosinophils is destroyed by the NO-rich oxidative lung tissue environment in asthma exacerbations. Demonstration that specific carbohydrate-dependent inhibition of murine and human eosinophil extracellular DNA trap release is abolished by s-nitrosylation of the SP-D molecule.

In the version of this Article originally published, in Fig. 1a, all cells in the top schematic were missing, and in the bottom-left schematic showing multiple pattern shapes, two cells were missing in the bottom-right corner. This figure has now been updated in all versions of the Article.

As cells with aberrant force-generating phenotypes can directly lead to disease, cellular force-generation mechanisms are high-value targets for new therapies. Here, we show that single-cell force sensors embedded in elastomers enable single-cell force measurements with ~100-fold improvement in throughput than was previously possible. The microtechnology is scalable and seamlessly integrates with the multi-well plate format, enabling highly parallelized time-course studies. In this regard, we show that airway smooth muscle cells isolated from fatally asthmatic patients have innately greater and faster force-generation capacity in response to stimulation than healthy control cells. By simultaneously tracing agonist-induced calcium flux and contractility in the same cell, we show that the calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, by quantifying phagocytic forces in thousands of individual human macrophages, we show that force initiation is a digital response (rather than a proportional one) to the proper immunogen. By combining mechanobiology at the single-cell level with high-throughput capabilities, this microtechnology can support drug-discovery efforts for clinical conditions associated with aberrant cellular force generation.

Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing β

RationaleRespiratory viral infections can result in the establishment of chronic lung diseases. Understanding the early innate immune mechanisms that participate in the development of chronic postviral lung disease may reveal new targets for therapeutic intervention. The intracellular viral sensor protein melanoma differentiation-associated protein 5 (MDA5) sustains the acute immune response to Sendai virus, a mouse pathogen that causes chronic lung inflammation, but its role in the development of postviral chronic lung disease is unknown.ObjectivesTo establish the role of MDA5 in the development of chronic lung disease.MethodsMDA5-deficient or control mice were infected with Sendai virus. The acute inflammatory response was evaluated by profiling chemokine and cytokine expression and by characterizing the composition of the cellular infiltrate. The impact of MDA5 on chronic lung pathology and function was evaluated through histological studies, degree of oxygen saturation, and responsiveness to carbachol.Measurements and main resultsMDA5 deficiency resulted in normal virus replication and in a distinct profile of chemokines and cytokines that associated with acute lung neutropenia and enhanced accumulation of alternatively activated macrophages. Diminished expression of neutrophil-recruiting chemokines was also observed in cells infected with influenza virus, suggesting a key role of MDA5 in driving the early accumulation of neutrophils at the infection site. The biased acute inflammatory response of MDA5-deficient mice led to an enhanced chronic lung inflammation, epithelial cell hyperplasia, airway hyperreactivity, and diminished blood oxygen saturation.ConclusionsMDA5 modulates the development of chronic lung inflammation by regulating the early inflammatory response in the lung.

Allergic asthma, a chronic respiratory disorder marked by inflammation and recurrent airflow obstruction, is associated with elevated levels of IL-5 family cytokines and elevated numbers of eosinophils (EOS). IL-5 family cytokines elongate peripheral blood EOS (EOSPB) viability, recruit EOSPB to the airways, and, at higher concentrations, induce degranulation and reactive oxygen species generation. Although airway EOS (EOSA) remain signal ready in that GM-CSF treatment induces degranulation, treatment of EOSA with IL-5 family cytokines no longer confers a survival advantage. Because the IL-5 family receptors have common signaling capacity, but are uncoupled from EOSA survival, whereas other IL-5 family induced endpoints remain functional, we tested the hypothesis that EOSA possess a JAK/STAT-specific regulatory mechanism (because JAK/STAT signaling is critical to EOS survival). We found that IL-5 family–induced STAT3 and STAT5 phosphorylation is attenuated in EOSA relative to blood EOS from airway allergen–challenged donors. However, IL-5 family–induced ERK1/2 phosphorylation is not altered between EOSA and EOS from airway allergen–challenged donors. These observations suggest EOSA possess a regulatory mechanism for suppressing STAT signaling distinct from ERK1/2 activation. Furthermore, we found, in EOSPB, IL-5 family cytokines induce members of the suppressors of cytokine signaling (SOCS) genes, CISH and SOCS1. Additionally, following allergen challenge, EOSA express significantly more CISH and SOCS1 mRNA and CISH protein than EOSPB counterparts. In EOSPB, long-term pretreatment with IL-5 family cytokines, to varying degrees, attenuates IL-5 family–induced STAT5 phosphorylation. These data support a model in which IL-5 family cytokines trigger a selective downregulation mechanism in EOSA for JAK/STAT pathways.