Background Cysteinyl leukotrienes contribute to asthma pathogenesis, in part through CysLT1 receptor (CysLT1R). Recently discovered lineage-negative type 2 innate lymphoid cells (ILC2) potently produce IL-5 and IL-13. Objectives We hypothesized that lung ILC2 may be activated by leukotrienes through CysLT1R. Methods ILC2 (Thy 1.2+ lineage-negative lymphocytes) and CysLT1R were detected in the lungs of WT, STAT6−/−, and RAG2−/− mice by flow cytometry. Levels of Th2 cytokines were measured in purified lung ILC2 stimulated with leukotriene D4 (LTD4) in the presence or absence of the CysLT1R antagonist montelukast. Calcium influx was measured by Fluo-4 intensity. Intranasal LTD4 and LTE4 were administered to naive mice and levels of ILC2 IL-5 production determined. Finally, LTD4 was co-administered with Alternaria repetitively to RAG2−/− mice (have ILC2) and IL-7R−/− mice (lack ILC2) and total ILC2 numbers, proliferation (Ki-67+) and BAL eosinophils measured. Results CysLT1R was expressed on lung ILC2 from WT, RAG2−/−, and STAT6−/−naïve and Alternaria-challenged mice. In vitro, LTD4 induced ILC2 to rapidly generate high levels of IL-5 and IL-13 within six hours of stimulation. Interestingly, LTD4, but not IL-33, induced high levels of IL-4 by ILC2. LTD4 administered in-vivo rapidly induced ILC2 IL-5 production that was significantly reduced by montelukast pre-treatment. Finally, LTD4 potentiated Alternaria-induced eosinophilia as well as ILC2 accumulation and proliferation. Conclusions We present novel data that CysLT1R is expressed on ILC2 and LTD4 potently induces CysLT1R-dependent ILC2 production of IL-4, IL-5, and IL-13. Additionally, LTD4 potentiates Alternaria-induced eosinophilia and ILC2 proliferation and accumulation.

Orosomucoid like 3 (ORMDL3) has been strongly linked with asthma in genetic association studies, but its function in asthma is unknown. We demonstrate that in mice ORMDL3 is an allergen and cytokine (IL-4 or IL-13) inducible endoplasmic reticulum (ER) gene expressed predominantly in airway epithelial cells. Allergen challenge induces a 127-fold increase in ORMDL3 mRNA in bronchial epithelium in WT mice, with lesser 15-fold increases in ORMDL-2 and no changes in ORMDL-1. Studies of STAT-6–deficient mice demonstrated that ORMDL3 mRNA induction highly depends on STAT-6. Transfection of ORMDL3 in human bronchial epithelial cells in vitro induced expression of metalloproteases (MMP-9, ADAM-8), CC chemokines (CCL-20), CXC chemokines (IL-8, CXCL-10, CXCL-11), oligoadenylate synthetases (OAS) genes, and selectively activated activating transcription factor 6 (ATF6), an unfolded protein response (UPR) pathway transcription factor. siRNA knockdown of ATF-6α in lung epithelial cells inhibited expression of SERCA2b, which has been implicated in airway remodeling in asthma. In addition, transfection of ORMDL3 in lung epithelial cells activated ATF6α and induced SERCA2b. These studies provide evidence of the inducible nature of ORMDL3 ER expression in particular in bronchial epithelial cells and suggest an ER UPR pathway through which ORMDL3 may be linked to asthma.

intrinsically have the ability to promote the efficient generation of iT reg cells via TGF-. In the present study, our data reveal that tissue-resident lung MØs in unsensitized mice constitutively express TGF- and retinal dehydrogenases (RALDH1 and RALDH2), the enzymes which regulate retinoic acid production. These MØs can take up inhaled antigen and present to naive and activated T cells in a tolerogenic manner without any exogenous stimuli, resulting in the development of Foxp3 + iT reg cells. We also show that these MØs retain expression of TGF- and RALDH when allergens are inhaled, but they lose their antiinflammatory activity and ability to induce iT reg cells, correlating with a loss of tolerance. Clinical therapy for allergic asthma is limited at present, and insight into mechanisms that induce tolerance to allergens could lead to new treatment strategies. Our data suggest that knowledge of ways to specifically target this lung MØ and maintain its T reg cell-inducing activity or promote the accumulation of these MØs may be applicable for therapy of allergic airway disease. RESULTS Phenotypic characterization of tissue-resident MØs and DCs in the naive murine lungAs our prior data identified TGF--inducible Foxp3 + T reg cells as mediating tolerance after inhalation of soluble antigen in naive mice (Duan et al., 2008(Duan et al., , 2011, we sought to identify a resident APC in the noninflamed lung that might possess the ability to promote these antigen-specific iT reg cells. We focused on pulmonary tissue MØs and DCs, reasoning they would be immediately available to take up and process inhaled antigens. We gated on CD11c + CD45 + cells that should encompass all DC and MØ populations and assessed samples from naive murine lungs that were perfused and lavaged to exclude circulating cells and bronchoalveolar cells including alveolar MØs (Fig. 1 A). CD11c + CD45 + cells contained two distinct subpopulations in terms of autofluorescence (AF), with the majority being high AF. CD11c + high-AF cells were MHC II lo , whereas CD11c + low-AF cells were MHC II hi . In addition, CD11c + high-AF cells expressed high levels of F4/80 and Siglec F (a sialic acid-recognizing lectin), whereas the low-AF cells were negative for these markers (Fig. 1 A). This combined phenotype corresponds to previous designations used to discriminate pulmonary-resident MØs and DCs (Vermaelen and Pauwels, 2004;Stevens et al., 2007;Zasłona et al., 2009). Further phenotypic analysis showed that CD11c + high-AF F4/80 + Siglec F + cells had little expression of CD11b and the C-type lectin CD205 and low-level expression of CD24 (HSA) but were positive for CD68, an MØ/monocyte marker. In contrast, CD11c + low-AF F4/80  Siglec F  cells highly expressed CD11b, CD205, and CD24 but did not express CD68 ( Fig. 1 B). Sorting of these cells from perfused and lavaged naive lungs into CD11c + high-AF Siglec F + MHC II lo versus CD11c + low-AF Siglec F  MHC II hi populations confirmed morphological features of MØs and DCs, respectively (Fig. 1 C).of thi...

ORMDL3 (orosomucoid like 3) has been strongly linked with asthma in genetic association studies. As allergen challenge induces lung ORMDL3 expression in WT 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 spontaneous increased AHR to methacholine compared to WT 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. In addition, increased levels of expression of genes associated with airway remodeling (TGF-β1, 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).

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.

Individuals with chronic asthma show a progressive decline in lung function that is thought to be due to structural remodeling of the airways characterized by subepithelial fibrosis and smooth muscle hyperplasia. Here we show that the tumor necrosis factor (TNF) family member LIGHT is expressed on lung inflammatory cells after allergen exposure. Pharmacological inhibition of LIGHT using a fusion protein between the IgG Fc domain and lymphotoxin β receptor (LTβR) reduces lung fibrosis, smooth muscle hyperplasia and airway hyperresponsiveness in mouse models of chronic asthma, despite having little effect on airway eosinophilia. LIGHT-deficient mice also show a similar impairment in fibrosis and smooth muscle accumulation. Blockade of LIGHT suppresses expression of lung transforming growth factor-β (TGF-β) and interleukin-13 (IL-13), cytokines implicated in remodeling in humans, whereas exogenous administration of LIGHT to the airways induces fibrosis and smooth muscle hyperplasia, Thus, LIGHT may be targeted to prevent asthma-related airway remodeling.

Cerebral and pulmonary injuries are often associated with maxillofacial fractures in severely injured trauma patients. Knowledge of these associated injuries provides useful strategies for patient care and prevention of further complications. A multidisciplinary and coordinated approach is important for optimum stabilization and ongoing treatment of patients with facial fractures.

Gasdermin B (GSDMB) on chromosome 17q21 demonstrates a strong genetic linkage to asthma, but its function in asthma is unknown. Here we identified that GSDMB is highly expressed in lung bronchial epithelium in human asthma. Overexpression of GSDMB in primary human bronchial epithelium increased expression of genes important to both airway remodeling [TGF-β1, 5-lipoxygenase (5-LO)] and airwayhyperresponsiveness (AHR) (5-LO). Interestingly, hGSDMBZp3-Cre mice expressing increased levels of the human GSDMB transgene showed a significant spontaneous increase in AHR and a significant spontaneous increase in airway remodeling, with increased smooth muscle mass and increased fibrosis in the absence of airway inflammation. In addition, hGSDMB Zp3-Cre mice showed increases in the same remodeling and AHR mediators (TGF-β1, 5-LO) observed in vitro in GSDMBoverexpressing epithelial cells. GSDMB induces TGF-β1 expression via induction of 5-LO, because knockdown of 5-LO in epithelial cells overexpressing GSDMB inhibited TGF-β1 expression. These studies demonstrate that GSDMB, a gene highly linked to asthma but whose function in asthma is previously unknown, regulates AHR and airway remodeling without airway inflammation through a previously unrecognized pathway in which GSDMB induces 5-LO to induce TGF-β1 in bronchial epithelium.GSDMB | asthma | airway-hyperresponsiveness | remodeling | inflammation