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Orosomucoid like 3 (ORMDL3), a gene localized to chromosome 17q21, has been linked in epidemiologic studies to childhood asthma and rhinovirus (RV) infections. As the single nucleotide polymorphisms linking ORMDL3 to asthma are associated with increased expression of ORMDL3, we have used hORMDL3zp3-Cre mice (which have universal increased expression of human ORMDL3) to determine whether infection of these transgenic mice with RV influences levels of airway inflammation or RV viral load. RV infection of hORMDL3zp3-Cre mice resulted in reduced RV viral load assessed by quantitative real-time PCR (lung and airway epithelium), as well as reduced airway inflammation (total bronchoalveolar lavage cells, neutrophils, macrophages, and lymphocytes) compared with RV-infected wild-type mice. Levels of the antiviral pathways including IFNs (IFN-α, IFN-ß, IFN-l) and RNAse L were significantly increased in the lungs of RV-infected hORMDL3zp3-Cre mice. Levels of the antiviral mouse oligoadenylate synthetase (mOas)1g pathway and RNAse L were upregulated in the lungs of unchallenged hORMDL3zp3-Cre mice. In addition, levels of mOas2, but not mOas1 (mOas1a, mOas1b, mOas1g), or mOas3 pathways were significantly more upregulated by IFNs (IFN-a, IFN-ß, IFN-l) in epithelial cells from hORMDL3zp3-Cre mice compared with RV-infected wild-type mouse epithelial cells. RNAse L-deficient mice infected with RV had increased RV viral load. Overall, these studies suggest that increased levels of ORMDL3 contribute to antiviral defense to RV infection in mice through pathways that may include IFNs (IFN-α, IFN-ß, IFN-l), OAS, and RNAse L. [ABSTRACT FROM AUTHOR]

In this study, we used cre-lox techniques to generate mice selectively deficient in ORMDL3 in airway epithelium (Ormdl3Δ2-3/Δ2-3/CC10) to simulate an inhaled therapy that effectively inhibited ORMDL3 expression in the airway. In contrast to the anticipated reduction in airway hyperresponsiveness (AHR), OVA allergen-challenged Ormdl3Δ2-3/Δ2-3/CC10 mice had a significant increase in AHR compared with wild-type mice. Levels of airway inflammation, mucus, fibrosis, and airway smooth muscle were no different in Ormdl3Δ2-3/Δ2-3/CC10 and wild-type mice. However, levels of sphingosine-1-phosphate (S1P) were significantly increased in Ormdl3Δ2-3/Δ2-3/CC10 mice as well as in airway epithelial cells in which ORMDL3 was inhibited with small interfering RNA. Incubation of S1P with airway smooth muscle cells significantly increased contractility. Overall, Ormdl3Δ2-3/Δ2-3/CC10 mice exhibit increased allergen-induced AHR independent of inflammation and associated with increased S1P generation. These studies raise concerns for inhaled therapies that selectively and effectively inhibit ORMDL3 in airway epithelium in asthma. [ABSTRACT FROM AUTHOR]

Chronic asthma is associated with airway remodeling and decline in lung function. In this article, we show that follistatin-like 1 (Fstl1), a mediator not previously associated with asthma, is highly expressed by macrophages in the lungs of humans with severe asthma. Chronic allergen-challenged Lys-Cretg /Fstl1Δ/Δ mice in whom Fstll is inactivated in macrophages/myeloid cells had significantly reduced airway remodeling and reduced levels of oncostatin M (OSM), a cytokine previously not known to be regulated by Fstl1. The importance of the Fstl1 induction of OSM to airway remodeling was demonstrated in murine studies in which administration of Fstl1 induced airway remodeling and increased OSM, whereas administration of an anti-OSM Ab blocked the effect of Fstl1 on inducing airway remodeling, eosinophilic airway inflammation, and airway hyperresponsiveness, all cardinal features of asthma. Overall, these studies demonstrate that the Fstl1/OSM pathway may be a novel pathway to inhibit airway remodeling in severe human asthma. [ABSTRACT FROM AUTHOR]

Orosomucoid-like (ORMDL)3 has been strongly linked with asthma in genetic association studies. Because allergen challenge induces lung ORMDL3 expression in wild-type mice, we have generated human ORMDL3 zona pellucida 3 Cre (hORMDL3zp3-Cre) mice that overexpress human ORMDL3 universally to investigate the role of ORMDL3 in regulating airway inflammation and remodeling. These hORMDL3zp3-Cre mice have significantly increased levels of airway remodeling, including increased airway smooth muscle, subepithelial fibrosis, and mucus. hORMDL3zp3-Cre mice had spontaneously increased airway responsiveness to methacholine compared to wild-type mice. This increased airway remodeling was associated with selective activation of the unfolded protein response pathway transcription factor ATF6 (but not Ire1 or PERK). The ATF6 target gene SERCA2b, implicated in airway remodeling in asthma, was strongly induced in the lungs of hORMDL3zp3-Cre mice. Additionally, increased levels of expression of genes associated with airway remodeling (TGF-b1, ADAM8) were detected in airway epithelium of these mice. Increased levels of airway remodeling preceded increased levels of airway inflammation in hORMDL3zp3-Cre mice. hORMDL3zp3-Cre mice had increased levels of IgE, with no change in levels of IgG, IgM, and IgA. These studies provide evidence that ORMDL3 plays an important role in vivo in airway remodeling potentially through ATF6 target genes such as SERCA2b and/or through ATF6-independent genes (TGF-β1, ADAM8). [ABSTRACT FROM AUTHOR]

Sialic acid-binding, Ig-like lectin (Siglec)-F is highly expressed on mouse eosinophils and plays an important role in regulating levels of eosinophilic lung inflammation. In this study we investigated the mechanism of constitutive and inducible Siglec-F ligand expression by lung airway epithelial cells and inflammatory cells in wild-type (WT) and genetically altered mice (ST3Gal-III heterozygotes, Fuc-TIV/VII double null, STAT6 null). Flow cytometry demonstrated that Siglec-F ligands are constitutively expressed in vitro and in vivo in selected lung cell types (epithelial cells, eosinophils, macrophages, and mast cells, but not CD4, CD8, or B cells) and are induced in response to divergent stimuli, including innate stimuli (TLR ligands, Alternaria), Th2 cytokines (IL-4, IL-13), and adaptive immune stimuli (OVA allergen). Furthermore, studies of deficient mice demonstrated the greater importance of the sialyltransferase ST3Gal-III compared with fucosyltransferases Fuc-TIV/VII in the synthesis of the constitutive and inducible Siglec-F ligands by lung epithelial and nonepithelial cells. In keeping with this, ST3Gal-III heterozygote mice (deficient in expression of Siglec-F ligands) also had significantly enhanced OVA-induced eosinophilic airway inflammation associated with reduced eosinophil apoptosis. Reduced eosinophil apoptosis in the lung of ST3Gal-III-deficient mice is likely mediated by reduced epithelial expression of Siglec-F ligands as WT eosinophils (which highly express Siglec-F) cultured with ST3Gal-III-deficient epithelial cells (which do not express Siglec-F ligand) showed reduced eosinophil apoptosis compared with WT eosinophils cultured with WT epithelial cells. Overall, these studies demonstrate that ST3Gal-III plays an important role in Siglec-F ligand formation and eosinophil apoptosis with resultant effects on eosinophilic inflammation in the lung. [ABSTRACT FROM AUTHOR]

Lung fibrosis and tissue remodeling are features of chronic diseases such as severe asthma, idiopathic pulmonary fibrosis, and systemic sclerosis. However, fibrosis-targeted therapies are currently limited. We demonstrate in mouse models of allergenand bleomycin-driven airway inflammation that neutralization of the TNF family cytokine TL1A through Ab blocking or genetic deletion of its receptor DR3 restricted increases in peribronchial smooth muscle mass and accumulation of lung collagen, primary features of remodeling. TL1Awas found as a soluble molecule in the airways and expressed on the surface of alveolar macrophages, dendritic cells, innate lymphoid type 2 cells, and subpopulations of lung structural cells. DR3 was found on CD4 T cells, innate lymphoid type 2 cells, macrophages, fibroblasts, and some epithelial cells. Suggesting in part a direct activity on lung structural cells, administration of recombinant TL1A into the naive mouse airways drove remodeling in the absence of other inflammatory stimuli, innate lymphoid cells, and adaptive immunity. Correspondingly, human lung fibroblasts and bronchial epithelial cells were found to express DR3 and responded to TL1A by proliferating and/or producing fibrotic molecules such as collagen and periostin. Reagents that disrupt the interaction of TL1Awith DR3 then have the potential to prevent deregulated tissue cell activity in lung diseases that involve fibrosis and remodeling. [ABSTRACT FROM AUTHOR]

The fungal allergen, Alternaría, is specifically associated with severe asthma, including life-threatening exacerbations. To better understand the acute innate airway response to Alternaría, naive wild-type (WT) mice were challenged once intranasally with Alternaría. Naive WT mice developed significant bronchoalveolar lavage eosinophilia following Alternaría challenge when analyzed 24 h later. In contrast to Alternaria, neither Aspergillus nor Candida induced bronchoalveolar lavage eosinophilia. Gene microarray analysis of airway epithelial cell brushings demonstrated that Afterwana-challenged naive WT mice had a >20-fold increase in the level of expression of found in inflammatory zone 1 (FIZZl/Retnla), a resistin-like molecule. Lung immunostaining confirmed strong airway epithelial FIZZ1 expression as early as 3 h after a single Alternaría challenge that persisted for 5 d and was significantly reduced in STAT6-deficient, but not protease-activated receptor 2-deficient mice. Bone marrow chimera studies revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, whereas STAT6 present in bone marrow-derived cells contributed to airway eosinophilia. Studies investigating which cells in the nonchallenged lung bind FIZZ1 demonstrated that CD45+CDllc+ cells (macrophages and dendritic cells), as well as collagen-1-producing CD45- cells (fibroblasts), can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naive WT mice led to airway eosinophilia, peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaría induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight into the uniquely pathogenic aspects of After/iaria-associated asthma. [ABSTRACT FROM AUTHOR]

Orosomucoid like 3 (ORMDL3), a gene localized to chromosome 17q21, has been linked in epidemiologic studies to childhood asthma and rhinovirus (RV) infections. As the single nucleotide polymorphisms linking ORMDL3 to asthma are associated with increased expression of ORMDL3, we have used hORMDL3 zp3-Cre mice (which have universal increased expression of human ORMDL3) to determine whether infection of these transgenic mice with RV influences levels of airway inflammation or RV viral load. RV infection of hORMDL3 zp3-Cre mice resulted in reduced RV viral load assessed by quantitative real-time PCR (lung and airway epithelium), as well as reduced airway inflammation (total bronchoalveolar lavage cells, neutrophils, macrophages, and lymphocytes) compared with RV-infected wild-type mice. Levels of the antiviral pathways including IFNs (IFN-α, IFN-β, IFN-λ) and RNAse L were significantly increased in the lungs of RV-infected hORMDL3 zp3-Cre mice. Levels of the antiviral mouse oligoadenylate synthetase (mOas)1g pathway and RNAse L were upregulated in the lungs of unchallenged hORMDL3 zp3-Cre mice. In addition, levels of mOas2, but not mOas1 (mOas1a, mOas1b, mOas1g), or mOas3 pathways were significantly more upregulated by IFNs (IFN-α, IFN-β, IFN-λ) in epithelial cells from hORMDL3 zp3-Cre mice compared with RV-infected wild-type mouse epithelial cells. RNAse L-deficient mice infected with RV had increased RV viral load. Overall, these studies suggest that increased levels of ORMDL3 contribute to antiviral defense to RV infection in mice through pathways that may include IFNs (IFN-α, IFN-β, IFN-λ), OAS, and RNAse L., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Sialic acid-binding, Ig-like lectin (Siglec)-F is highly expressed on mouse eosinophils and plays an important role in regulating levels of eosinophilic lung inflammation. In this study we investigated the mechanism of constitutive and inducible Siglec-F ligand expression by lung airway epithelial cells and inflammatory cells in wild-type (WT) and genetically altered mice (ST3Gal-III heterozygotes, Fuc-TIV/VII double null, STAT6 null). Flow cytometry demonstrated that Siglec-F ligands are constitutively expressed in vitro and in vivo in selected lung cell types (epithelial cells, eosinophils, macrophages, and mast cells, but not CD4, CD8, or B cells) and are induced in response to divergent stimuli, including innate stimuli (TLR ligands, Alternaria), Th2 cytokines (IL-4, IL-13), and adaptive immune stimuli (OVA allergen). Furthermore, studies of deficient mice demonstrated the greater importance of the sialyltransferase ST3Gal-III compared with fucosyltransferases Fuc-TIV/VII in the synthesis of the constitutive and inducible Siglec-F ligands by lung epithelial and nonepithelial cells. In keeping with this, ST3Gal-III heterozygote mice (deficient in expression of Siglec-F ligands) also had significantly enhanced OVA-induced eosinophilic airway inflammation associated with reduced eosinophil apoptosis. Reduced eosinophil apoptosis in the lung of ST3Gal-III-deficient mice is likely mediated by reduced epithelial expression of Siglec-F ligands as WT eosinophils (which highly express Siglec-F) cultured with ST3Gal-III-deficient epithelial cells (which do not express Siglec-F ligand) showed reduced eosinophil apoptosis compared with WT eosinophils cultured with WT epithelial cells. Overall, these studies demonstrate that ST3Gal-III plays an important role in Siglec-F ligand formation and eosinophil apoptosis with resultant effects on eosinophilic inflammation in the lung.

Siglec-F is a sialic acid-binding Ig superfamily receptor that is highly expressed on eosinophils. We have investigated whether administration of an anti-Siglec-F Ab to OVA-challenged wild-type mice would reduce levels of eosinophilic inflammation and levels of airway remodeling. Mice sensitized to OVA and challenged repetitively with OVA for 1 mo who were administered an anti-Siglec-F Ab had significantly reduced levels of peribronchial eosinophilic inflammation and significantly reduced levels of subepithelial fibrosis as assessed by either trichrome staining or lung collagen levels. The anti-Siglec-F Ab reduced the number of bone marrow, blood, and tissue eosinophils, suggesting that the anti-Siglec-F Ab was reducing the production of eosinophils. Administration of a F(ab')(2) fragment of an anti-Siglec-F Ab also significantly reduced levels of eosinophilic inflammation in the lung and blood. FACS analysis demonstrated increased numbers of apoptotic cells (annexin V(+)/CCR3(+) bronchoalveolar lavage and bone marrow cells) in anti-Siglec-F Ab-treated mice challenged with OVA. The anti-Siglec-F Ab significantly reduced the number of peribronchial major basic protein(+)/TGF-beta(+) cells, suggesting that reduced levels of eosinophil-derived TGF-beta in anti-Siglec-F Ab-treated mice contributed to reduced levels of peribronchial fibrosis. Administration of the anti-Siglec-F Ab modestly reduced levels of periodic acid-Schiff-positive mucus cells and the thickness of the smooth muscle layer. Overall, these studies suggest that administration of an anti-Siglec-F Ab can significantly reduce levels of allergen-induced eosinophilic airway inflammation and features of airway remodeling, in particular subepithelial fibrosis, by reducing the production of eosinophils and increasing the number of apoptotic eosinophils in lung and bone marrow.

We screened bronchoalveolar lavage (BAL) fluids from COPD-E (chronic obstructive pulmonary disease-Emphysema) and control subjects using a 120 Ab cytokine array and demonstrated that adiponectin was highly expressed in BAL in COPD-E. An adiponectin ELISA confirmed that adiponectin was highly expressed in BAL in COPD-E compared with smokers and healthy control subjects. Immunohistochemistry studies of lung sections from subjects with COPD-E demonstrated that airway epithelial cells expressed significant levels of adiponectin and adiponectin receptor (AdipoR) 1 but not AdipoR2. In vitro studies with purified populations of human lung A549 epithelial cells demonstrated that they expressed both adiponectin and AdipoR1 (but not AdipoR2) as assessed by RT-PCR, Western blot, and immunohistochemistry. Lung A549 epithelial AdipoR1were functional as incubation with adiponectin induced release of IL-8, which was inhibited by small interfering RNA to AdipoR1. Using a mouse model of COPD, tobacco smoke exposure induced both evidence of COPD as well as increased levels of adiponectin in BAL fluid and increased adiponectin expression by airway epithelial cells. As adiponectin expression in adipocytes is dependent upon NF-kappaB we determined levels of adiponectin in tobacco smoke exposed CC10-Cre(tg)/Ikkbeta(Delta/Delta) mice (deficient in the ability to activate NF-kappaB in airway epithelium). These studies demonstrated that CC10-Cre(tg)/Ikkbeta(Delta/Delta) and wild-type mice had similar levels of BAL adiponectin and airway epithelial adiponectin immunostaining. Overall, these studies demonstrate the novel observation that adiponectin and functional AdipoR1are expressed by lung epithelial cells, suggesting a potential autocrine and/or paracrine pathway for adiponectin to activate epithelial cells in COPD-E.

Intracellular signaling pathways that converge on Smad 3 are used by both TGF-beta and activin A, key cytokines implicated in the process of fibrogenesis. To determine the role of Smad 3 in allergen-induced airway remodeling, Smad 3-deficient and wild-type (WT) mice were sensitized to OVA and challenged by repetitive administration of OVA for 1 mo. Increased levels of activin A and increased numbers of peribronchial TGF-beta1(+) cells were detected in WT and Smad 3-deficient mice following repetitive OVA challenge. Smad 3-deficient mice challenged with OVA had significantly less peribronchial fibrosis (total lung collagen content and trichrome staining), reduced thickness of the peribronchial smooth muscle layer, and reduced epithelial mucus production compared with WT mice. As TGF-beta and Smad 3 signaling are hypothesized to mediate differentiation of fibroblasts to myofibroblasts in vivo, we determined the number of peribronchial myofibroblasts (Col-1(+) and alpha-smooth muscle actin(+)) as assessed by double-label immunofluorescence microscopy. Although the number of peribronchial myofibroblasts increased significantly in WT mice following OVA challenge, there was a significant reduction in the number of peribronchial myofibroblasts in OVA-challenged Smad 3-deficient mice. There was no difference in levels of eosinophilic airway inflammation or airway responsiveness in Smad 3-deficient compared with WT mice. These results suggest that Smad 3 signaling is required for allergen-induced airway remodeling, as well as allergen-induced accumulation of myofibroblasts in the airway. However, Smad 3 signaling does not contribute significantly to airway responsiveness.

Environmental tobacco smoke (ETS) can increase asthma symptoms and the frequency of asthma attacks. However, the contribution of ETS to airway remodeling in asthma is at present unknown. In this study, we have used a mouse model of allergen-induced airway remodeling to determine whether the combination of chronic exposure to ETS and chronic exposure to OVA allergen induces greater levels of airway remodeling than exposure to either chronic ETS or chronic OVA allergen alone. Mice exposed to chronic ETS alone did not develop significant eosinophilic airway inflammation, airway remodeling, or increased airway hyperreactivity to methacholine. In contrast, 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. Mice coexposed to chronic ETS and chronic OVA allergen had significantly increased levels of eotaxin-1 expression in airway epithelium which was associated with increased numbers of peribronchial eosinophils, as well as increased numbers of peribronchial cells expressing TGF-beta1. These studies suggest that chronic coexposure to ETS significantly increases levels of allergen-induced airway remodeling (in particular smooth muscle thickness) and airway responsiveness by up-regulating expression of chemokines such as eotaxin-1 in airway epithelium with resultant recruitment of cells expressing TGF-beta1 to the airway and enhanced airway remodeling.

To determine whether immunostimulatory sequences of DNA (ISS) can reverse established airway remodeling, mice that had developed airway remodeling following 3 mo of repetitive OVA challenges, were treated with ISS for 1-3 mo. Systemic administration of ISS to mice that had already developed established airway remodeling significantly reduced the degree of airway collagen deposition (assessed by lung collagen content, peribronchial trichrome staining, and immunostaining with anticollagen type III and type V Abs). ISS reduced bronchoalveolar lavage and lung levels of TGF-beta1 and reduced the number of TGF-beta1-positive eosinophils and TGF-beta1-positive mononuclear cells recruited to the airway. In vitro studies demonstrated that ISS inhibited TGF-beta1 expression by macrophages (RAW 264.7 cell line and bone marrow-derived macrophages). In addition, ISS significantly reduces lung levels of expression of the chemokine thymus- and activation-regulated chemokine, as well as the number of peribronchial CD4(+) lymphocytes that express Th2 cytokines that promote peribronchial fibrosis. Overall, these studies demonstrate that ISS can reverse features of airway collagen deposition by reducing levels of lung TGF-beta1, as well as by reducing levels of the chemokine thymus- and activation-regulated chemokine and the numbers of peribronchial CD4(+) lymphocytes that drive the ongoing Th2 immune response.

Few peribronchial mast cells are noted either in the lungs of naive mice or in the lungs of OVA-sensitized mice challenged acutely with OVA by inhalation. In this study, we demonstrate that OVA-sensitized mice exposed to repetitive OVA inhalation for 1-6 mo have a significant accumulation of peribronchial mast cells. This accumulation of peribronchial mast cells is associated with increased expression of the Th2 cell-derived mast cell growth factors, including IL-4 and IL-9, but not with the non-Th2 cell-derived mast cell growth factor, stem cell factor. Pretreating mice with immunostimulatory sequences (ISS) of DNA containing a CpG motif significantly inhibited the accumulation of peribronchial mast cells and the expression of IL-4 and IL-9. To determine whether mast cells express Toll-like receptor-9 (TLR-9; the receptor for ISS), TLR-9 expression by mouse bone marrow-derived mast cells (MBMMCs) was assessed by RT-PCR. MBMMCs strongly expressed TLR-9 and bound rhodamine-labeled ISS. However, incubation of MBMMCs with ISS in vitro neither inhibited MBMMC proliferation nor inhibited Ag/IgE-mediated MBMMC degranulation, but they did induce IL-6. Overall these studies demonstrate that mice exposed to repetitive OVA challenge, but not acute OVA challenge, have an accumulation of peribronchial mast cells and express increased levels of mast cell growth factors in the lung. Although mast cells express TLR-9, ISS does not directly inhibit mast cell proliferation in vitro, suggesting that ISS inhibits accumulation of peribronchial mast cells in vivo by indirect mechanism(s), which include inhibiting the lung expression of Th2 cell-derived mast cell growth factors.