Shaopeng Yuan

Roles of epithelial cell-derived periostin in TGF-β activation, collagen production, and collagen gel elasticity in asthma

Periostin is considered to be a matricellular protein with expression typically confined to cells of mesenchymal origin. Here, by using in situ hybridization, we show that periostin is specifically up-regulated in bronchial epithelial cells of asthmatic subjects, and in vitro, we show that periostin protein is basally secreted by airway epithelial cells in response to IL-13 to influence epithelial cell function, epithelial–mesenchymal interactions, and extracellular matrix organization. In primary human bronchial epithelial cells stimulated with periostin and epithelial cells overexpressing periostin, we reveal a function for periostin in stimulating the TGF-β signaling pathway in a mechanism involving matrix metalloproteinases 2 and 9. Furthermore, conditioned medium from the epithelial cells overexpressing periostin caused TGF-β–dependent secretion of type 1 collagen by airway fibroblasts. In addition, mixing recombinant periostin with type 1 collagen in solution caused a dramatic increase in the elastic modulus of the collagen gel, indicating that periostin alters collagen fibrillogenesis or cross-linking and leads to stiffening of the matrix. Epithelial cell-derived periostin in asthma has roles in TGF-β activation and collagen gel elasticity in asthma.

A protective role for periostin and TGF-β in IgE-mediated allergy and airway hyperresponsiveness

The pathophysiology of asthma involves allergic inflammation and remodelling in the airway and airway hyperresponsiveness (AHR) to cholinergic stimuli, but many details of the specific underlying cellular and molecular mechanisms remain unknown. Periostin is a matricellular protein with roles in tissue repair following injury in both the skin and heart. It has recently been shown to be up‐regulated in the airway epithelium of asthmatics and to increase active TGF‐β. Though one might expect periostin to play a deleterious role in asthma pathogenesis, to date its biological role in the airway is unknown.

Alternative splicing of interleukin-33 and type 2 inflammation in asthma

Significance Type 2 inflammation occurs in a large subgroup of asthmatics and is the target of multiple novel therapies for asthma; however, the mechanisms that drive type 2 inflammation in chronic asthma are poorly understood. In this study, we identify a previously unidentified mechanism of IL-33 activity involving alternative RNA transcript splicing and provide evidence that mast cells and basophils are major cellular targets of IL-33 activity driving type 2 cytokine production in stable asthma. These data advance our understanding of the mechanisms of type 2-high asthma and guide the selection of asthmatics likely to benefit from IL-33 inhibitor therapies. Type 2 inflammation occurs in a large subgroup of asthmatics, and novel cytokine-directed therapies are being developed to treat this population. In mouse models, interleukin-33 (IL-33) activates lung resident innate lymphoid type 2 cells (ILC2s) to initiate airway type 2 inflammation. In human asthma, which is chronic and difficult to model, the role of IL-33 and the target cells responsible for persistent type 2 inflammation remain undefined. Full-length IL-33 is a nuclear protein and may function as an “alarmin” during cell death, a process that is uncommon in chronic stable asthma. We demonstrate a previously unidentified mechanism of IL-33 activity that involves alternative transcript splicing, which may operate in stable asthma. In human airway epithelial cells, alternative splicing of the IL-33 transcript is consistently present, and the deletion of exons 3 and 4 (Δ exon 3,4) confers cytoplasmic localization and facilitates extracellular secretion, while retaining signaling capacity. In nonexacerbating asthmatics, the expression of Δ exon 3,4 is strongly associated with airway type 2 inflammation, whereas full-length IL-33 is not. To further define the extracellular role of IL-33 in stable asthma, we sought to determine the cellular targets of its activity. Comprehensive flow cytometry and RNA sequencing of sputum cells suggest basophils and mast cells, not ILC2s, are the cellular sources of type 2 cytokines in chronic asthma. We conclude that IL-33 isoforms activate basophils and mast cells to drive type 2 inflammation in chronic stable asthma, and novel IL-33 inhibitors will need to block all biologically active isoforms.

FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.

Intelectin-1 Is a Prominent Protein Constituent of Pathologic Mucus Associated with Eosinophilic Airway Inflammation in Asthma

To the Editor: Intelectin-1 (ITLN-1) is an epithelial cell protein that is up-regulated in asthma (1). ITLN-1 is a pleotropic adipokine (also known as omentin-1) with roles in the gut ranging from host defense against pathogenic bacteria to promotion of insulin-stimulated glucose uptake (2–4). The host defense roles of ITLN-1 may result from its ability to bind structures expressed by microorganisms in a carbohydrate-dependent manner (5). ITLN-1 is also a binding partner for lactoferrin (6), and ITLN-1 may cooperate with lactoferrin in host defense (6, 7). Little is known about the function of ITLN-1 in human asthma. One possibility is that it participates in pathways of inflammation downstream of IL-13 (1). Indeed, studies in a mouse model of asthma suggest that ITLN-1 mediates IL-13–induced monocyte chemotactic protein-1 and -3 production in epithelial cells (8). Another possibility is that ITLN-1 is a component of airway mucus and contributes to pathologic mucus gel formation in disease. Supporting this possibility are studies in the gastrointestinal tract showing that ITLN-1 is a goblet cell protein that is secreted with mucus into the intestinal lumen (9). In addition, other studies in the intestine have suggested mucin–intelectin interactions that could alter the biophysical properties of mucus (10). Some of the results of these studies have been previously reported in the form of an abstract (11) Because mucus pathology causes mucus plugging and airway occlusion (12, 13), especially in fatal asthma (14), we set out to determine if ITLN-1 is a component of pathologic mucus in acute asthma. We first immunostained lung tissue sections from cases of fatal asthma and found prominent ITLN-1 immunostaining in the pathologic mucus plugs that occlude the airways (Figures 1A–1C). The cellular source of the ITLN-1 appears to be goblet cells (Figure 1C). We next measured ITLN-1 protein in sputum from 11 patients with acute severe asthma and two control groups (35 subjects with chronic stable asthma and 11 healthy control subjects) (Table 1). We found that ITLN-1 protein levels in the subgroup of patients with asthma in exacerbation were significantly higher than in stable asthma and in healthy control subjects (Figure 1D). We also noted that the increase in ITLN-1 in acute asthma was driven by the subgroup with increased sputum eosinophils (>2%) (Figure 1E), a finding that is consistent with ITLN-1’s regulation by IL-13 in airway epithelial cells (1). ITLN-1 up-regulation is thus a feature of “Th2-high” asthma, and the known pathologic characteristics of this disease endotype can be extended to include high ITLN-1 protein concentrations in mucus forming during disease exacerbations. Figure 1. Intelectin-1 (ITLN-1) protein in airway biospecimens and binding of ITLN-1 to airway mucins and lactoferrin. Sections of lung tissue from lungs of patients with fatal asthma were stained with an anti–ITLN-1 antibody or peptide blocking control. ... Table 1: Subject Characteristics The prominent immunostaining for ITLN-1 in mucus plugs in fatal asthma and the high concentrations of ITLN-1 in sputum in acute severe asthma prompted us to explore if ITLN-1 can bind to human airway mucins. ITLN-1 is a lectin with known specificity for galactosyl structures, especially the galactofuranosyl sugars expressed by microorganisms (5). To determine if ITLN-1 binds to human airway mucin glycans, we developed a plate-based binding assay using high-molecular-weight mucin preparations that we purified from induced sputum samples from subjects with chronic stable asthma (“mucin study”; Table 1). Specifically, we used biotinylated recombinant ITLN-1 to probe mucin coated on microtiter plates (see online supplement). Biotinylated jacalin, a tetrameric plant seed lectin with specificity for galactose, was used as a positive control. Although jacalin showed binding to mucin, ITLN-1 did not (Figure 1F). It is possible that ITLN-1 cannot recognize the pyranosyl forms of galactose in human mucin, but another possibility is that mucin glycans prevent binding through steric hindrance. It could also be that the plate assay is suboptimal for measuring ITLN-1 binding to mucin because other proteins or cofactors involved in an ITLN-1–mucin interaction in vivo are not represented in vitro. Because ITLN-1 has been characterized as the lactoferrin receptor (6, 7), we considered if ITLN-1 interacts with lactoferrin in airway mucus in acute asthma. We found that lactoferrin levels in sputum from patients with acute severe asthma are significantly higher than in control samples (Figure 1G). Notably, the concentration of lactoferrin ranged from 500 to 1,000 μg/ml in some of these sputum samples, a 1,000-fold higher concentration than ITLN-1. This large amount of lactoferrin in asthmatic mucus may bind and concentrate ITLN-1 in mucus. To examine the binding of ITLN-1 to lactoferrin, we used a plate-binding assay similar to the one we used for mucin-ITLN-1 binding. In this way, we found that biotinylated ITLN-1 binds avidly to immobilized lactoferrin (Figure 1H). This binding was inhibited by heparin, suggesting a charge-based interaction between ITLN-1 and lactoferrin’s basic N-terminal region (15), and increased by methyl galactofuranoside. Galactofuranoside is found in many microbial polysaccharides and is recognized as a preferred glycan ligand for ITLN-1 (7, 16). Our data suggest that ITLN-1 binding to galactofuranosyl residues on microorganisms might improve its ability to bind lactoferrin and target it to regions of high microorganism burden. We conclude that ITLN-1 is a prominent protein component of pathologic mucus in fatal asthma and in acute severe asthma, especially in the context of eosinophilic airway inflammation. The binding of ITLN-1 to lactoferrin is increased by galactofuranoside providing a mechanism by which ITLN-1 can cooperate with lactoferrin to defend against microbes.