Rationale: Glucocorticoid (GC) receptors (GR) are nuclear receptors that mediate both non-genomic and genomic effects. Upon ligand binding, the GC-GR complex translocates to the nucleus, binds specific DNA sequences, and modulates transcription. GR is a phosphoprotein, and its phosphorylation is crucial for receptor activation, affecting ligand binding, DNA interaction, subcellular localization, and biological activity. The N-terminal domain (NTD) of GR contains several phosphorylation sites, including Ser211 and Ser226. Our previous studies demonstrated that GR phosphorylation at Ser211 enhances transcriptional activity. Another antecedent related to the direct phosphorylation of Ser211 was the involvement of the cyclin-dependent kinase 5 (CDK5). However, the kinases involved in this process remain unclear. This study investigates the role of kinases in GR phosphorylation in airway smooth muscle (ASM) cells. Methods: We first sought to identify the kinases induced by glucocorticoids using the Proteome Profiler Human Phospho-Kinase Array Kit (R&D) to measure 37 kinases in human ASM cells treated with cortisol (1 µg/mL) for 1, 3, and 5 minutes. Principal component analysis (PCA) was performed with R (version 4.4.1), and statistical analysis was conducted using GraphPad Prism (version 10.4.0) with one-way ANOVA followed by Dunnett's post-test (p<0.05). In separate experiments, ASM cells were treated with the pan-CDK inhibitor Roscovitine (1, 10, and 20 µM) for 2 hours, followed by cortisol stimulation (1 µg/mL) for 1 hour. GR phosphorylation at Ser211 and total GR expression were analyzed by Western blot, and cell viability was assessed using the MTT assay. Results: Kinase profiling showed significant changes in kinase activity following cortisol treatment. After 1 minute, beta-catenin increased by 31%, PLC-γ1 (pY783) by 13%, and Src (pY419) by 24%. At 3 minutes, STAT3 (pY705) increased by 42%, and STAT6 (pY641) peaked at 5 minutes. PCA of the first two components explained 91.5% of the variation and showed a positive correlation between beta-catenin and Src (pY419) and a negative correlation with HSP27 (pS78/pS82). In pharmacological inhibition studies, Roscovitine decreased GC-induced pSer211 phosphorylation by 60%, 72%, and 77% at 1, 10, and 20 µM, respectively, without affecting cell viability. Conclusions: Our results suggest that multiple kinases, particularly the CDK family, regulate GR phosphorylation at Ser211 in human ASM cells. These insights may guide the development of therapeutic strategies targeting these pathways to modulate glucocorticoid responses, especially in glucocorticoid-resistant diseases.

Although most patients with asthma symptoms are well controlled by inhaled glucocorticoids (GCs), a subgroup of patients suffering from severe asthma respond poorly to GC therapy. Such GC insensitivity (GCI) represents a profound challenge in managing patients with asthma. Even though GCI in patients with severe asthma has been investigated by several groups using immune cells (peripheral blood mononuclear cells and alveolar macrophages), uncertainty exists regarding the underlying molecular mechanisms in non-immune cells, such as airway smooth cells (ASM) cells. In asthma, ASM cells are among the targets of GC therapy and have emerged as key contributors not only to bronchoconstriction but also to airway inflammation and remodeling, as implied by experimental and clinical evidence. We here summarize the current understanding of the actions/signaling of GCs in asthma, and specifically, GC receptor (GR) “site-specific phosphorylation” and its role in regulating GC actions. We also review some common pitfalls associated with studies investigating GCI and the inflammatory mediators linked to asthma severity. Finally, we discuss and contrast potential molecular mechanisms underlying the impairment of GC actions in immune cells versus non-immune cells such as ASM cells.

BackgroundAdiponectin is an important immunomodulatory mediator in inflammatory conditions. While we previously showed that adiponectin receptor 1 (AdipoR1) is expressed in murine regulatory T cells (Tregs), its expression in human Tregs remain unknown. Here, we examined the expression of AdipoR1 in human Tregs and whether its ligand, globular adiponectin (gAd) affects the Treg ability to secrete IL-10 and the role of Type 2 (T2) inflammation in such process.MethodsHuman Tregs from peripheral blood were analyzed by flow cytometry for AdipoR1, Helios and IL-10 expression. CD4+ T cells enriched from peripheral blood mononuclear cells (PBMCs) were cultured in the presence or the absence of gAd or the chemical adiponectin receptor agonist, AdipoRon, or in a T2 cytokine milieu. Flow cytometry was then used to assess intracellular IL-10, IL-10 secreting cells, FOXP3 and Helios expression, and phosphorylated p38 MAP kinase (MAPK). IL-10 levels in CD4+ T cell supernatants were quantified by ELISA.ResultsWe found that a subset of human Tregs expressed AdipoR1. Importantly, more Helios- cells expressed AdipoR1 than Helios+ cells. Likewise, there was a higher frequency of IL-10+ cells within Helios- AdipoR1+ Tregs compared to Helios+ AdipoR1+ Tregs. In contrast, the IL-10 mean fluorescence intensity (MFI) was higher in Helios+ AdipoR1+ Tregs compared to Helios-AdipoR1+ Tregs. When human CD4+ T cells were treated with gAd or AdipoRon, a significant increase in IL-10 secretion, FOXP3 expression, and p38 MAPK phosphorylation was observed in Helios- AdipoR1+ Tregs. Interestingly, gAd under T2 cytokine milieu significantly increased the intracellular levels of IL-10, mainly in Helios+ AdipoR1+ Tregs, and IL-10 levels in supernatants of CD4+ T cells.ConclusionsCollectively, our findings suggest that adiponectin/AdipoR1 axis promotes IL-10 release by Tregs, mainly in Helios- Tregs, and the effect was amplified by T2 inflammation in Helios+ Tregs.

Pentraxins are soluble pattern recognition receptors that play a major role in regulating innate immune responses. Through their interaction with complement components, Fcγ receptors, and different microbial moieties, Pentraxins cause an amplification of the inflammatory response. Pentraxin-3 is of particular interest since it was identified as a biomarker for several immune-pathological diseases. In allergic asthma, pentraxin-3 is produced by immune and structural cells and is up-regulated by pro-asthmatic cytokines such as TNFα and IL-1β. Strikingly, some recent experimental evidence demonstrated a protective role of pentraxin-3 in chronic airway inflammatory diseases such as allergic asthma. Indeed, reduced pentraxin-3 levels have been associated with neutrophilic inflammation, Th17 immune response, insensitivity to standard therapeutics and a severe form of the disease. In this review, we will summarize the current knowledge of the role of pentraxin-3 in innate immune response and discuss the protective role of pentraxin-3 in allergic asthma.

Glucocorticoids (GCs) act via the GC receptor (GR), a receptor ubiquitously expressed in the body where it drives a broad spectrum of responses within distinct cell types and tissues, which vary in strength and specificity. The variability of GR-mediated cell responses is further extended by the existence of GR isoforms, such as GRα and GRβ, generated through alternative splicing mechanisms. While GRα is the classic receptor responsible for GC actions, GRβ has been implicated in the impairment of GRα-mediated activities. Interestingly, in contrast to the popular belief that GRβ actions are restricted to its dominant-negative effects on GRα-mediated responses, GRβ has been shown to have intrinsic activities and "directly" regulates a plethora of genes related to inflammatory process, cell communication, migration, and malignancy, each in a GRα-independent manner. Furthermore, GRβ has been associated with increased cell migration, growth, and reduced sensitivity to GC-induced apoptosis. We will summarize the current knowledge of GRβ-mediated responses, with a focus on the GRα-independent/intrinsic effects of GRβ and the associated non-canonical signaling pathways. Where appropriate, potential links to airway inflammatory diseases will be highlighted.

The mechanisms driving corticosteroid insensitivity in asthma are still unclear although evidence points toward a potential role of lung mast cells. Indeed, a number of in vitro studies using various cell types showed that different mediators produced by activated mast cells, including cytokines, have the capacity to interfere with the therapeutic action of corticosteroids. In patients with severe allergic refractory asthma, the anti-IgE monoclonal antibody (mAb), Omalizumab, has been shown to be associated with a marked reduction in inhaled and systemic use of corticosteroids, further suggesting a key role of mast cells in the poor response of patients to these drugs. The present chapter will discuss the possible underlying mechanisms by which mast cells could contribute to reducing corticosteroid sensitivity seen in patients with severe asthma.

The mechanisms underlying corticosteroid insensitivity in severe asthma have not been elucidated although some indirect clinical evidence points toward a role of mast cells. Here, we tested the hypothesis that mast cells can drive corticosteroid insensitivity in airway smooth muscle cells, a key player in asthma pathogenesis. Conditioned media from resting or FcεR1-activated human lung mast cells were incubated with serum-deprived ASM cells (1:4 dilution, 24 h) to determine their impact on the anti-inflammatory action of fluticasone on ASM cell chemokine expression induced by TNFα (10 ng/ml). Conditioned media from FcεR1-activated mast cells (but not that from non-activated mast cells or control media) significantly reduced the ability of 100 nM fluticasone to suppress ASM TNFα-dependent CCL5 and CXCL10 production at both mRNA and protein levels. In contrast, fluticasone inhibition of CXCL-8 production by TNFα was still preserved in the presence of activated mast cell conditioned media. Transcriptomic analysis validated by individual qPCR assays revealed that activated mast cell conditioned media dramatically reduced the number of anti-inflammatory genes induced by fluticasone in ASM cells. Our study demonstrates for the first time that conditioned media from FcεR1-activated mast cells blunt the anti-inflammatory action of corticosteroids in ASM cells by altering their transactivation properties. Because infiltration of mast cells within the ASM bundles is a defining feature of asthma, mast cell-derived mediators may contribute to the glucocorticoid insensitivity present in severe asthma.

Background and objectives Enhanced airway smooth muscle (ASM) mass is a hallmark feature of remodeled airways in asthma and results from increased ASM proliferation. Glucocorticoid (GC) is a treatment of choice in asthma where most of its effects are mediated by GC receptor α isoform (GRα). As a result of alternative splicing mechanisms, another GR isoform, GRβ, has been described. While most of GRβ effects have been reported to be GRα-dependent and inhibit GRα-dependent transcriptional activities, recent evidence from cancer cells showed some GRαindependent effects of GRβ where GRβ “directly” modulates cell proliferation by binding to the promoter of phosphatase and tensin homolog, which increased Akt1 guided proliferation. We here sought to determine whether GRβ modulates cell proliferation in ASM. Methods Growtharrested primary human ASM cells from healthy subjects were treated with PDGF (10 ng/ml) at different time points, then total proteins were extracted and GRβ expression was assessed by western blot. ASM cell proliferation was examined using CyQuant assay where cells were treated with various doses of PDGF for 24-72 hr in the presence or absence of fluticasone propionate (FP) (100 nM) added 2 hr before. To examine the role of GRβ in cell proliferation, GRβ expression was downregulated by transfecting cells with siRNA GRβ (10-100 nM) for 48 hr prior to the proliferation assay. The specificity of siRNA GRβ transfection was confirmed by western blot. Results We found that PDGF induced an early but transient increase of GRβ expression (at 5 and 10 min). GRβ expression augmented again at 1 hr of PDGF treatment and was sustained up to 6 hr before declining at 24 hr. When ASM proliferation was measured, doseresponse and time course analysis showed that 2 ng/ml of PDGF induced an optimal increase of cell proliferation (+20%) after 72 hr. The addition of FP markedly inhibited such increase by 30%. Interestingly, siRNA GRβ-transfected ASM cells showed a reduced proliferation (-20%) as compared with scrambled siRNA-transfected cells. Notably, PDGF-induced proliferation was completely abrogated in siRNA GRβ-transfected cells and the addition of FP did no have any further effects. Conclusions: Collectively, we showed for the first time that i) PDGF increased GRβ expression and ii) GRβ directly modulate ASM growth suggesting a novel role of GRβ in airway remodeling. Further studies are still needed to determine the mechanisms underlying GRβ effects on ASM proliferation in cells derived from healthy and asthmatic patients.

Glucocorticoids (GCs) are the treatment of choice for chronic inflammatory diseases such as asthma. Despite proven effective anti-inflammatory and immunosuppressive effects, long-term and/or systemic use of GCs can potentially induce adverse effects. Strikingly, some recent experimental evidence suggests that GCs may even exacerbate some disease outcomes. In asthma, airway smooth muscle (ASM) cells are among the targets of GC therapy and have emerged as key contributors not only to bronchoconstriction, but also to airway inflammation and remodeling, as implied by experimental and clinical evidence. We here will review the beneficial effects of GCs on ASM cells, emphasizing the differential nature of GC effects on pro-inflammatory genes and on other features associated with asthma pathogenesis. We will also summarize evidence describing how GCs can potentially promote pro-inflammatory and remodeling features in asthma with a specific focus on ASM cells. Finally, some of the possible solutions to overcome these unanticipated effects of GCs will be discussed.

Background: Around 70% of asthmatics have a reduced therapeutic response to β2AR agonists. Multiple factors (cytokines, growth factors, respiratory viruses, allergens) and mechanisms (changes in receptor expression, and/or coupling) have been associated with β2AR dysfunction in airway smooth muscle (ASM) cells. Aims: To determine whether β2AR function was impaired in ASM cells derived from severe asthma and in healthy ASM cells treated with cytokines. Methods: ASM cells from severe asthmatic patients (n=6) were treated with 10 ng/ml TNFα for 24 hours alone or in the presence of 10-10 to 10-5 M albuterol. Also, ASM cells from healthy subjects (n=6) were treated with TNFα alone or in combination with 25 ng/ml IFNγ. ASM cell responsiveness to β2AR agonists was investigated by their ability to inhibit cytokine-induced chemokine production assessed in ASM cell supernatants by ELISA. Results: TNFα produced higher amount of CCL5 and CXCL-10 in healthy ASM cells when compared to levels in severe asthmatic ASM cells (6133 ± 561 vs 1704 ±95 pg/ml, P= 0.0015 and 5772 ± 109 vs 2248 ± 229 pg/ml, P=.0002, respectively). Pre-treatment of healthy ASM cells with albuterol dose-dependently inhibited CCL5 (LogIC50= -8.25±0.44) and CXCL-10 (LogIC50=-7.89±0.52) production induced by TNFα alone, reaching a maximum inhibition of 55% (p<0.01) and 40% (P<0.01) at 10-5 M, respectively. In contrast, albuterol failed to inhibit chemokine production in severe asthmatic ASM cells treated with TNFα alone or in healthy ASM cells treated with both TNFα and IFNγ. Conclusion: ASM cells from severe asthmatic subjects have an impaired response to β2AR agonists.

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 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.

Pentraxin-3 (PTX3) is a multifunctional protein involved in both innate and adaptive immunity. Glucocorticoid (GC) is the first-line therapy to mitigate airway inflammation in asthma. Previous pieces of evidence showed that GC has divergent effects on PTX3 production in various cell types. The molecular mechanisms controlling PTX3 expression in HASMC are, however, not yet characterized. In this study, we demonstrate that the synthetic GC, dexamethasone (DEX) increases the expression of PTX3 both at the protein and mRNA levels. We also found that such an effect of DEX was dependent on de novo protein synthesis and the GC receptor (GR). While DEX increases PTX3 mRNA stability, it did not affect its promoter activity. Interestingly, HASMC pre-treated with p42/p44 ERK inhibitor, but not with p38 or JNK-MAPK inhibitors, significantly interfered with DEX-induced PTX3 secretion. Taken together, our data suggest that GC regulates PTX3 expression in HASMC through transcriptional and post-transcriptional mechanisms in a GR and ERK-dependent manner.

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 remodeling in asthma manifests, in part, as enhanced airway smooth muscle (ASM) mass, due to myocyte proliferation. While the anti-proliferative effects of glucocorticoid (GC) were investigated in normal ASM cells (NASMC), little is known about such effects in ASM cells derived from asthma subjects (AASMC). We posit that GC differentially modulates mitogen-induced proliferation of AASMC and NASMC. Cells were cultured, starved, then treated with Epidermal growth factor (EGF) (10 ng/ml) and Platelet-derived growth factor (PDGF) (10 ng/ml) for 24 h and/or fluticasone propionate (FP) (100 nM) added 2 h before. Cell counts and flow cytometry analyses showed that FP failed to decrease the cell number of and DNA synthesis in AASMC irrespective of mitogens used. We also examine the ability of Insulin Growth Factor Binding Protein-1 (IGFBP-1), a steroid-inducible gene that deters cell growth in other cell types, to inhibit proliferation of AASMC where FP failed. We found that FP increased IGFBP1 mRNA and protein levels. Interestingly, the addition of IGFBP1 (1 μg/ml) to FP completely inhibited the proliferation of AASMC irrespective to the mitogens used. Further investigation of different signaling molecules involved in ASM growth and GC receptor functions (Protein kinase B (PKB/AKT), Mitogen-activated protein kinases (MAPKs), Focal Adhesion Kinase (FAK)) showed that IGFBP-1 selectively decreased mitogen-induced p38 phosphorylation in AASMC. Collectively, our results show the insensitivity of AASMC to the anti-proliferative effects of GC, and demonstrate the ability of IGFBP1 to modulate AASMC growth representing, hence, a promising strategy to control ASM growth in subjects with GC insensitive asthma.

Among patients with asthma, heterogeneity exists regarding the pattern of airway inflammation and response to treatment, prompting the necessity of recognizing specific phenotypes. Based on the analysis of inflammatory cell counts in induced sputum, asthmatic patients can be classified into 4 unique phenotypes: eosinophilic asthma, neutrophilic asthma, mixed granulocytic asthma, and paucigranulocytic asthma (PGA). PGA is an asthma phenotype with no evidence of increased numbers of eosinophils or neutrophils in sputum or blood and in which anti-inflammatory therapies are ineffective at controlling symptoms. Although underinvestigated, PGA is the most common asthma phenotype in patients with stable asthma. However, PGA is sometimes underestimated because of the exclusive reliance on induced sputum cell counts, which are variable among cohorts of studies, prompting the necessity of developing improved biomarkers. Importantly, investigators have reported that inhaled corticosteroids had a limited effect on airway inflammatory markers in patients with PGA and therefore defining PGA as a potentially "steroid-insensitive" phenotype that requires exploration of alternative therapies. PGA manifests as an uncoupling of airway obstruction from airway inflammation that can be driven by structural changes within the airways, such as airway smooth muscle tissue hypertrophy. Animal models provide evidence that processes evoking airway hyperresponsiveness and airway smooth muscle thickening occur independent from inflammation and might be a consequence of a loss of negative homeostatic processes. Collectively, further understanding of PGA with a focus on the characterization, prevalence, clinical significance, and pathobiology derived from animal studies will likely provide precision therapies that will improve PGA clinical outcomes.