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Asthma is a chronic disease related to airway hyperresponsiveness and airway remodeling. Airway remodeling is the important reason of refractory asthma and is associated with differentiation of airway epithelia into myofibroblasts via epithelial-mesenchymal transition (EMT) to increase the process of subepithelial fibrosis. There is growing evidence that autophagy modulates remodeling. However, the underlying molecular mechanisms of these effects are still unclear. In this study, we hypothesized that Follistatin-like 1 (FSTL1) promotes EMT and airway remodeling by intensifying autophagy. With the use of transmission electron microscopy (TEM), double-membrane autophagosomes were detected in the airways of patients and mice. More autophagosomes were in patients with asthma and OVA-challenged mice compared with healthy controls. The expression of FSTL1 and beclin-1 was upregulated in the airways of patients with asthma and OVA-challenged mice, accompanied by airway EMT and remodeling. In OVA-challenged Fstl1 +/- mice, the degree of airway remodeling and autophagy was decreased compared with control mice. The effects of FSTL1 on autophagy and EMT were also tested in 16HBE cells in vitro. Additionally, inhibition of autophagy by using LY-294002 and siRNA-ATG5 reduced the FSTL1-induced EMT in 16HBE cells, as measured by E-cadherin, N-cadherin, and vimentin expression. In line herewith, administration of LY-294002 reduced the expression of autophagy, EMT, and airway remodeling markers in FSTL1-challenged WT mice. Taken together, our study suggests that FSTL1 may induce EMT and airway remodeling by activating autophagy. These findings may provide novel avenues for therapeutic research targeting the autophagy and FSTL1 pathway, which may be beneficial to patients with refractory asthma., (Copyright © 2017 the American Physiological Society.)

Asthma exacerbations can be caused by a number of factors, including the fungal allergen Alternaria, which is specifically associated with severe and near-fatal attacks. The mechanisms that trigger lung responses are unclear and might vary between allergens. A comparison between Alternaria, Aspergillus, Candida, and house dust mite, all allergens in humans, showed that only Alternaria promoted immediate innate airway eosinophilia within 12 h of inhalation in nonsensitized mice. Alternaria, but not the other allergens, induced a rapid increase in airway levels of IL-33, accompanied by IL-33 receptor (IL-33R)-positive natural helper cell (NHC) production of IL-5 and IL-13. NHCs in the lung and bone marrow constitutively expressed transcription factors [GATA-3 and E26 transformation-specific sequence-1 (ETS-1)] that could allow for rapid induction of T helper type 2 (Th2) cytokines. Lung NHC numbers and proliferation (%Ki-67), but not IL-5 or GATA-3 expression, were significantly reduced in STAT6-deficient mice 3 days after one challenge with Alternaria. Alternaria induced NHC expression of the EGF receptor ligand amphiregulin (partially dependent on STAT6), as well as EGF receptor signaling in the airway epithelium. Finally, human peripheral blood NHCs (CRTH2(+)CD127(+) lineage-negative lymphocytes) from allergic individuals highly expressed GATA-3 and ETS-1, similar to lung NHCs in mice. In summary, Alternaria-induced lung NHC proliferation and expression of amphiregulin are regulated by STAT6. In addition, NHCs in mouse and humans are primed to express Th2 cytokines through constitutive expression of GATA-3 and ETS-1. Thus several transcription factor pathways (STAT6, GATA-3, and ETS-1) may contribute to NHC proliferation and Th2-type responses in Alternaria-induced asthma.

Adiponectin is a cytokine with both proinflammatory and anti-inflammatory properties that is expressed in epithelial cells in the airway in chronic obstructive pulmonary disease-emphysema (COPD-E). To determine whether adiponectin modulates levels of lung inflammation in tobacco smoke-induced COPD-E, we used a mouse model of COPD-E in which either adiponectin-deficient or wild-type (WT) mice were exposed to tobacco smoke for 6 mo. Outcomes associated with tobacco smoke-induced COPD-E were quantitated including lung inflammation [bronchoalveolar lavage (BAL) and total and differential cell count], lung mediators of inflammation (cytokines and chemokines), air space enlargement (i.e., linear intercept), and lung function (tissue elastance) in the different groups of mice. Whereas exposure of WT mice to tobacco smoke for 6 mo induced significant lung inflammation (increased total BAL cells, neutrophils, and macrophages), adiponectin-deficient mice had minimal BAL inflammation when exposed to tobacco smoke for 6 mo. In addition, whereas chronic tobacco-exposed WT mice had significantly increased levels of lung mediators of inflammation [i.e., TNF-α, keratinocyte-derived chemokine (KC), and adiponectin] as well as significantly increased air space enlargement (increased linear intercept) and decreased tissue elastance, exposure of adiponectin-deficient mice to chronic tobacco smoke resulted in no further increase in lung mediators, air space enlargement, or tissue elastance. In vitro studies demonstrated that BAL macrophages derived from adiponectin-deficient mice incubated in media containing tobacco smoke expressed minimal TNF-α or KC compared with BAL macrophages from WT mice. These studies suggest that adiponectin plays an important proinflammatory role in tobacco smoke-induced COPD-E.

The ability of corticosteroids to reduce airway inflammation and improve lung function is significantly reduced in asthmatics who are tobacco smokers compared with asthmatics who are nonsmokers. As not only high levels of tobacco smoke exposure in active smokers, but also significantly lower levels of tobacco smoke exposure from passive environmental tobacco smoke (ETS) exposure in nonsmokers can increase both asthma symptoms and the frequency of asthma exacerbations, we utilized a mouse model to determine whether corticosteroids can reduce levels of airway inflammation, airway remodeling, and airway hyperreactivity in mice exposed to the combination of chronic ETS and ovalbumin (OVA) allergen. Chronic ETS exposure alone did not induce increases in eosinophilic airway inflammation, airway remodeling, or airway hyperreactivity. Mice exposed to chronic OVA allergen had significantly increased levels of peribronchial fibrosis, increased thickening of the smooth muscle layer, increased mucus, and increased airway hyperreactivity, which was significantly enhanced by coexposure to the combination of chronic ETS and chronic OVA allergen. Administration of corticosteroids to mice exposed to chronic ETS and OVA allergen significantly reduced levels of eosinophilic airway inflammation, mucus production, peribronchial smooth muscle thickness, airway hyperreactivity, and the number of peribronchial TGF-beta1+ cells. Overall, this study demonstrates that corticosteroids can significantly reduce levels of eosinophilic inflammation, mucus expression, airway remodeling, and airway hyperreactivity in chronic ETS-exposed mice challenged with allergen.

The contribution of CD4 T cells and other CD4+ cells to lung inflammation and airway remodeling remains unclear during bouts of chronic exposure to airborne allergen. Previously, murine models have shown that CD4 T cells are required for initiation of acute inflammation and the remodeling process. However, it is unknown whether CD4 T cells or other CD4+ cells continue to be required for remodeling during ongoing allergen challenges after the development of acute eosinophilic lung inflammation. To test this, mice were sensitized and challenged with ovalbumin (OVA). After acute airway inflammation was established, a CD4 depleting antibody was administered for 4 wk during a period of chronic exposure to intranasal OVA, resulting in effective depletion of CD4+ cells from all organs, including the lung, lung-draining lymph nodes, and spleen. In these mice, levels of peribronchial inflammation, bronchoalveolar (BAL) eosinophils, and lung CD11c+, CD8+, and Siglec-F+CD11c- cells were significantly reduced. However, mucus metaplasia, peribronchial subepithelial fibrosis, and smooth muscle mass were not affected. Additionally, depletion of CD4+ cells before the last week of chronic allergen challenges also led to significant reductions in BAL eosinophils, peribronchial inflammation, and lung CD11c+, CD8+, and Siglec-F+CD11c- cells. These results show that CD4 T cells, and other CD4+ cells including subsets of dendritic cells, iNKT cells, and LTi cells, play a role in ongoing eosinophilic lung inflammation during periods of chronic allergen challenge, but are not required for progressive airway remodeling that develops after initial acute inflammation.

In this study, we have examined the role of phosphoinositide 3 kinase gamma (PI3Kgamma), a class Ib PI3K, in contributing to airway remodeling utilizing PI3Kgamma-deficient mice exposed to chronic allergen challenge. Wild-type (WT) mice sensitized to ovalbumin (OVA) and chronically challenged with OVA for 1 mo developed significantly increased levels of eosinophilic inflammation and airway remodeling. In contrast, PI3Kgamma-deficient mice challenged with OVA had significantly reduced numbers of bronchoalveolar lavage and peribronchial eosinophils compared with WT mice. There was no significant difference in the number of bone marrow or circulating peripheral blood eosinophils when comparing WT mice and PI3Kgamma-deficient mice, suggesting that trafficking of eosinophils into the lung was reduced in PI3Kgamma-deficient mice. PI3Kgamma-deficient and WT mice had similar levels of IL-5 and eotaxin-1. The reduced eosinophil recruitment to the airway in PI3Kgamma-deficient mice challenged with OVA was associated with significantly reduced numbers of TGF-beta1+ peribronchial cells, reduced numbers of pSmad 2/3+ airway epithelial cells, and pSmad 2/3+ peribronchial cells, as well as significantly reduced levels of peribronchial fibrosis (quantitated by trichrome staining and image analysis as well as by lung collagen levels). In addition, the area of peribronchial alpha-smooth muscle staining was significantly reduced in PI3Kgamma-deficient compared with WT mice. Overall, this study demonstrates an important role for PI3Kgamma in mediating allergen-induced eosinophilic airway inflammation and airway remodeling, suggesting that PI3Kgamma may be a novel therapeutic target in asthma.

Matrix metalloproteinases (MMPs) are a family of extracellular proteases that are responsible for the degradation of the extracellular matrix during tissue remodeling. We have used a mouse model of allergen-induced airway remodeling to determine whether MMP-9 plays a role in airway remodeling. MMP-9-deficient and wild-type (WT) mice were repetitively challenged intranasally with ovalbumin (OVA) antigen to develop features of airway remodeling including peribronchial fibrosis and increased thickness of the peribronchial smooth muscle layer. OVA-challenged MMP-9-deficient mice had less peribronchial fibrosis and total lung collagen compared with OVA-challenged WT mice. There was no reduction in mucus expression, smooth muscle thickness, or airway responsiveness in OVA-challenged MMP-9-deficient compared with OVA-challenged WT mice. OVA-challenged MMP-9-deficient mice had reduced levels of bronchoalveolar lavage (BAL) regulated on activation, normal T cell expressed, and secreted (RANTES), as well as reduced numbers of BAL and peribronchial eosinophils compared with OVA-challenged WT mice. There were no significant difference in levels of BAL eotaxin, thymus- and activation-regulated chemokine (TARC), or macrophage-derived chemokine (MDC) in OVA-challenged WT compared with MMP-9-deficient mice. Overall, this study demonstrates that MMP-9 may play a role in mediating selected aspects of allergen-induced airway remodeling (i.e., modest reduction in levels of peribronchial fibrosis) but does not play a significant role in mucus expression, smooth muscle thickness, or airway responsiveness.

At present there are conflicting results from studies investigating the role of corticosteroids in inhibiting airway remodeling in asthma. We have used a mouse model to determine whether administration of corticosteroids prevents the development of allergen-induced structural features of airway remodeling. Mice treated with corticosteroids were subjected to repetitive ovalbumin (OVA) challenge for 3 mo, at which time levels of peribronchial fibrosis and the thickness of the peribronchial smooth muscle layer were assessed by immunohistology, levels of transforming growth factor (TGF)-beta1 by ELISA, and the number of alpha-smooth muscle actin+/Col-1+ peribronchial myofibroblasts by immunohistochemistry. Corticosteroids significantly reduced allergen-induced increases in peribronchial collagen deposition and levels of total lung collagen but did not reduce allergen-induced increases in the thickness of the peribronchial smooth muscle layer. Levels of lung TGF-beta1 were significantly reduced in mice treated with systemic corticosteroids, and this was associated with a significant decrease in the number of peribronchial inflammatory cells that expressed TGF-beta1, including eosinophils and mononuclear cells. Corticosteroids also significantly reduced the number of peribronchial myofibroblasts. Overall, these studies demonstrate that administration of corticosteroids significantly reduces levels of allergen-induced peribronchial fibrosis. The reduction in peribronchial fibrosis mediated by corticosteroids is likely to be due to several mechanisms including inhibition of expression of TGF-beta1, a reduction in the number of peribronchial inflammatory cells expressing TGF-beta1 (eosinophils, macrophages), as well as by corticosteroids reducing the accumulation of peribronchial myofibroblasts that contribute to collagen expression.

We have investigated the importance of cell-surface serine- and/or threonine-linked oligosaccharide adhesion molecules synthesized by the Golgi enzyme core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GlcNAcT) in mediating eosinophil trafficking to the lung in studies utilizing C2GlcNAcT-I-deficient mice. The number of bronchoalveolar eosinophils, the number of lung eosinophils, and airway responsiveness to methacholine were not significantly different in C2GlcNAcT-I-deficient compared with wild-type mice sensitized and challenged by inhalation with ovalbumin. C2GlcNAcT-I-deficient mice do not demonstrate defects in neutrophil trafficking to the lung in response to lipopolysaccharide (LPS). In contrast, ragweed-sensitized C2GlcNAcT-I-deficient mice exhibit significantly reduced eosinophil trafficking to the peritoneal cavity in response to ragweed peritoneal challenge. C2GlcNAcT-I-deficient mice also have significantly reduced neutrophil trafficking to the peritoneal cavity in response to LPS challenge. Overall, these studies demonstrate an important role for serine/threonine-linked oligosaccharides synthesized by the Golgi enzyme C2GlcNAcT-I in eosinophil and neutrophil trafficking to the peritoneum but not for eosinophil or neutrophil trafficking to the lung.