Glucocorticoid Insensitivity in Virally Infected Airway Epithelial Cells Is Dependent on Transforming Growth Factor-ß Activity.

Asthma and chronic obstructive pulmonary disease (COPD) exacerbations are commonly associated with respiratory syncytial virus (RSV), rhinovirus (RV) and influenza A virus (IAV) infection. The ensuing airway inflammation is resistant to the anti-inflammatory actions of glucocorticoids (GCs). Viral infection elicits transforming growth factor-ß (TGF-ß) activity, a growth factor we have previously shown to impair GC action in human airway epithelial cells through the activation of activin-like kinase 5 (ALK5), the type 1 receptor of TGF-ß. In the current study, we examine the contribution of TGF-ß activity to the GC-resistance caused by viral infection. We demonstrate that viral infection of human bronchial epithelial cells with RSV, RV or IAV impairs GC anti-inflammatory action. Poly(I:C), a synthetic analog of double-stranded RNA, also impairs GC activity. Both viral infection and poly(I:C) increase TGF-ß expression and activity. Importantly, the GC impairment was attenuated by the selective ALK5 (TGFßRI) inhibitor, SB431542 and prevented by the therapeutic agent, tranilast, which reduced TGF-ß activity associated with viral infection. This study shows for the first time that viral-induced glucocorticoid-insensitivity is partially mediated by activation of endogenous TGF-ß.

Plasminogen-stimulated inflammatory cytokine production by airway smooth muscle cells is regulated by annexin A2.

Plasminogen has a role in airway inflammation. Airway smooth muscle (ASM) cells cleave plasminogen into plasmin, a protease with proinflammatory activity. In this study, the effect of plasminogen on cytokine production by human ASM cells was investigated in vitro. Levels of IL-6 and IL-8 in the medium of ASM cells were increased by incubation with plasminogen (5-50 µg/ml) for 24 hours (P < 0.05; n = 6-9), corresponding to changes in the levels of cytokine mRNA at 4 hours. The effects of plasminogen were attenuated by α2-antiplasmin (1 µg/ml), a plasmin inhibitor (P < 0.05; n = 6-12). Exogenous plasmin (5-15 mU/ml) also stimulated cytokine production (P < 0.05; n = 6-8) in a manner sensitive to serine-protease inhibition by aprotinin (10 KIU/ml). Plasminogen-stimulated cytokine production was increased in cells pretreated with basic fibroblast growth factor (300 pM) in a manner associated with increases in urokinase plasminogen activator expression and plasmin formation. The knockdown of annexin A2, a component of the putative plasminogen receptor comprised of annexin A2 and S100A10, attenuated plasminogen conversion into plasmin and plasmin-stimulated cytokine production by ASM cells. Moreover, a role for annexin A2 in airway inflammation was demonstrated in annexin A2-/- mice in which antigen-induced increases in inflammatory cell number and IL-6 levels in the bronchoalveolar lavage fluid were reduced (P < 0.01; n = 10-14). In conclusion, plasminogen stimulates ASM cytokine production in a manner regulated by annexin A2. Our study shows for the first time that targeting annexin A2-mediated signaling may provide a novel therapeutic approach to the treatment of airway inflammation in diseases such as chronic asthma.

Pro-inflammatory and immunomodulatory functions of airway smooth muscle: emerging concepts.

Airway smooth muscle (ASM) is the main regulator of bronchomotor tone. Extensive studies show that in addition to their physical property, human airway smooth muscle (ASM) cells can participate in inflammatory processes modulating the initiation, perpetuation, amplification, and perhaps resolution of airway inflammation. Upon stimulation or interaction with immune cells, ASM cells produce and secrete a variety of inflammatory cytokines and chemokines, cell adhesion molecules, and extracellular matrix (ECM) proteins. These released mediators can, in turn, contribute to the inflammatory state, airway hyperresponsiveness, and airway remodeling present in asthma. As our knowledge of ASM myocyte biology improves, novel bioactive factors are emerging as potentially important regulators of inflammation. This review provides an overview of our understanding of some of these molecules, identifies rising questions, and proposes future studies to better define their role in ASM cell modulation of inflammation and immunity in the lung and respiratory diseases.

ACE2 Expression in Organotypic Human Airway Epithelial Cultures and Airway Biopsies.

Coronavirus disease 2019 (COVID-19) caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an acute respiratory disease with systemic complications. Therapeutic strategies for COVID-19, including repurposing (partially) developed drugs are urgently needed, regardless of the increasingly successful vaccination outcomes. We characterized two-dimensional (2D) and three-dimensional models (3D) to establish a physiologically relevant airway epithelial model with potential for investigating SARS-CoV-2 therapeutics. Human airway basal epithelial cells maintained in submerged 2D culture were used at low passage to retain the capacity to differentiate into ciliated, club, and goblet cells in both air-liquid interface culture (ALI) and airway organoid cultures, which were then analyzed for cell phenotype makers. Airway biopsies from non-asthmatic and asthmatic donors enabled comparative evaluation of the level and distribution of immunoreactive angiotensin-converting enzyme 2 (ACE2). ACE2 and transmembrane serine proteinase 2 (TMPRSS2) mRNA were expressed in ALI and airway organoids at levels similar to those of native (i.e., non-cultured) human bronchial epithelial cells, whereas furin expression was more faithfully represented in ALI. ACE2 was mainly localized to ciliated and basal epithelial cells in human airway biopsies, ALI, and airway organoids. Cystic fibrosis appeared to have no influence on ACE2 gene expression. Neither asthma nor smoking status had consistent marked influence on the expression or distribution of ACE2 in airway biopsies. SARS-CoV-2 infection of ALI cultures did not increase the levels of selected cytokines. Organotypic, and particularly ALI airway cultures are useful and practical tools for investigation of SARS-CoV-2 infection and evaluating the clinical potential of therapeutics for COVID-19.

Functional expression of IgG-Fc receptors in human airway smooth muscle cells.

IgE-Fc receptors and IgG-Fc receptors are expressed on hematopoietic cells, but some evidence suggests that these receptors are also found on nonhematopoietic cells, including human airway smooth muscle (hASM) cells. Our study characterizes the expression of IgE-Fc receptors (FcεRI/CD23) and IgG-Fc receptors (FcγRs-I, -II, and -III) in cultured hASM cells by flow cytometry and Western blotting, and the functional activity of receptors was determined through quantification of cell proliferation and released cytokines. Expression of Fc receptor-linked intracellular signaling proteins and phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 and p38(MAPK) in hASM cells was examined by Western blotting. Expression of FcεRI and CD23 was not detectable in hASM cells. However, FcγRI and FcγRII were shown to be expressed on these cells. Specific antibodies, validated using transfected cell lines, revealed that the inhibitory IgG receptor, FcγRIIb, was the most abundant Fc receptor subtype expressed. Although cross-linking FcγR with heat-aggregated γ globulin (HAGG) did not induce detectable cell stimulation, pretreating hASM cells with HAGG significantly inhibited IL-1α-induced increases in cytokine levels and basic fibroblast growth factor-induced cell proliferation. This inhibitory effect of HAGG was abrogated by preincubation of cells with an anti-FcγRIIb antigen-binding fragment (Fab). Expression of proteins involved in the canonical FcγRIIb inhibitory signaling pathway was established in hASM cells. Pretreatment of hASM cells with HAGG significantly inhibited IL-1α- and basic fibroblast growth factor-induced extracellular signal-regulated kinase 1/2 and p38(MAPK) phosphorylation. This study identifies functional expression of FcγRIIb in hASM cells, with the potential to suppress their remodeling and immunomodulatory roles.

A Non-canonical Pathway with Potential for Safer Modulation of Transforming Growth Factor-ß1 in Steroid-Resistant Airway Diseases.

Impaired therapeutic responses to anti-inflammatory glucocorticoids (GC) in chronic respiratory diseases are partly attributable to interleukins and transforming growth factor ß1 (TGF-ß1). However, previous efforts to prevent induction of GC insensitivity by targeting established canonical and non-canonical TGF-ß1 pathways have been unsuccessful. Here we elucidate a TGF-ß1 signaling pathway modulating GC activity that involves LIM domain kinase 2-mediated phosphorylation of cofilin1. Severe, steroid-resistant asthmatic airway epithelium showed increased levels of immunoreactive phospho-cofilin1. Phospho-cofilin1 was implicated in the activation of phospholipase D (PLD) to generate the effector(s) (lyso)phosphatidic acid, which mimics the TGF-ß1-induced GC insensitivity. TGF-ß1 induction of the nuclear hormone receptor corepressor, SMRT (NCOR2), was dependent on cofilin1 and PLD activities. Depletion of SMRT prevented GC insensitivity. This pathway for GC insensitivity offers several promising drug targets that potentially enable a safer approach to the modulation of TGF-ß1 in chronic inflammatory diseases than is afforded by global TGF-ß1 inhibition.

Human mast cell line-1 (HMC-1) cells transfected with FcεRIα are sensitive to IgE/antigen-mediated stimulation demonstrating selectivity towards cytokine production.

Mast cells play important roles in allergic and inflammatory diseases. Efforts to better understand human mast cell activation and develop novel inhibitory agents have been hampered by the lack of suitable human mast cell lines. The HMC-1 mast cell line has been extensively used, but lacks native expression of the human high-affinity IgE receptor FcεRI limiting its applications. We have stably transfected HMC-1 cells with the IgE-binding α-subunit of FcεRI to generate HMCα cells that are antigen-responsive. We have used flow cytometry, cell signaling assays, pharmacological pathway inhibitors and cell functional assays to characterize the properties of HMCα cells. IgE/antigen responses were compared with those of the adenosine receptor agonist NECA. Surface expression of FcεRI in HMCα cells was demonstrated and was enhanced by prior sensitization with IgE. Activation of HMCα cells with IgE/antigen did not produce degranulation, but did lead to release of numerous cytokines. Whilst there was no measurable increase of intracellular Ca(2+) or marked general changes in protein tyrosine phosphorylation, IgE/antigen stimulation of HMCα cells enhanced phosphorylation of p38(MAPK) and Erk. Inhibitors of these pathways, as well as the src kinase inhibitor PP2, attenuated IgE/antigen-induced cytokine release. In summary, we have generated and characterized HMCα cells and show that they are a useful and relevant human mast cell model to examine FcεRI stabilization, signaling and mediator release. We envisage that HMCα cells will have utility in understanding the importance of mast cells in human allergic disease and in assessing the activity of novel anti-allergic compounds.