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Background: Altered collagen architecture with increased “bone-type” pyridinoline collagen cross-linking, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in Idiopathic Pulmonary Fibrosis (IPF). We recently identified that hypoxia inducible factor (HIF) pathway activation promotes induction of the bone-type collagen cross-linking enzymes lysyl hydroxylase 2 and lysyl oxidase-like 2, yet the consequences of this on collagen structure-function in lung fibrosis remain unknown. Methods: Using a long term 3D in vitro model of the fibroblastic focus we cultured primary lung fibroblasts from IPF donors without or with the HIF-stabilising compound IOX2. Hydroxyproline and mature pyridinium cross-links were measured by colorimetric assays, and collagen ultrastructure assessed by electron microscopy (EM). The biomechanical effects of HIF stabilisation were investigated by parallel plate compression testing. Results: IOX2 stabilised HIF within the 3D fibroblastic focus model, promoting HIF pathway activation to disproportionately induce collagen-modifying enzymes relative to collagen fibril synthesis. After 6 weeks of culture, mature pyridinium cross-links were significantly increased by IOX2 to levels we have previously observed in IPF tissue. Ultrastructural analysis of the collagen fibrils with EM identified that IOX2 significantly reduced fibril diameter to sizes comparable with collagen fibrils enzymatically extracted from IPF tissue. IOX2 induced a greater than 3-fold increase in tissue stiffness. Conclusions: HIF pathway activation is a core regulator of bone-type collagen fibrillogenesis and altered structure-function in lung fibrosis.

Background: Maternal asthma is a risk factor for asthma and airway hyperresponsiveness (AHR) in children. The asthma susceptibility gene, ADAM33, has been associated with AHR and impaired lung function in early life. Aims and Objectives: Our aim was to study how a maternal allergic environment in pregnancy interacts with offspring-ADAM33 and the effects on their lungs after birth. We hypothesised that effects of maternal allergic airway inflammation (AAI) will be different in Adam33 knock-out (KO) compared to wild-type (WT) offspring. Methods: Heterozygous (Adam33+/-) pregnant dams were challenged with allergen to induce AAI and control dams with saline. WT and KO offspring from the same litters were studied 4 weeks post partum for AHR. Bronchoalveolar lavage and lung tissue were studied by RTqPCR, Western blots, immunostainings and precision-cut lung slices (PCLS) in the presence of agonists and antagonists. Results: Allergen-naive 4-week old WT offspring of mothers with AAI showed AHR in vivo, whereas KO offspring were protected. The pulmonary muscarinic M1 receptor was increased in all offspring of AAI dams. Ex vivo PCLS studies with methacholine and muscarinic receptor antagonists confirmed vagal reflex bronchoconstriction in WT while KO offspring were protected by a decreased airway smooth muscle constriction, as shown with a thromboxane-receptor agonist. Conclusions: Gene-environment interactions between Adam33 and maternal AAI determine AHR in early life. While maternal AAI induces AHR in WT offspring via vagal responses, Adam33 KO alters the airway smooth muscle function and suppresses AHR, pointing to a new asthma AHR therapy.

Mast cells (MCs) are immune cells classically associated with allergic diseases including asthma where they promote bronchoconstriction and Type 2 inflammation. However, they are also key immune cells against parasitic, bacterial and viral infections by releasing immune and inflammatory mediators. Rhinoviruses (RV) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of RV replication is the airway epithelium and MCs localise to the bronchial epithelium in asthma. The subsequent epithelial cell damage caused by RV leads to IL-33 release. This promotes Type 2 cytokine release by MCs contributing to airway inflammation. As IL-33 has been shown to increase MC immune responses against RSV, we hypothesised that IL-33 has a role in mediating innate immune responses in MCs against RV. The human MC cell line LAD2 was treated with IL-33 (1 - 10 ng/ml) plus RV (MOI 1) or UV-irradiated RV as control. Total RNA was extracted at 2, 4, 8 and 24 hours after treatment. Gene expression and viral copy number were quantified by RT-qPCR and protein release by MSD/ELISA. We found that IL-33 significantly induced a time and concentration dependent expression of Type 2 and pro-inflammatory cytokine genes. Furthermore, we found induction of interferon (IFN) and IFN-inducible genes suggesting activation of an innate anti-viral response. However, IL-33 was also found to induce ICAM1, the receptor for major group RV entry, and in the presence of RV, IL-33 facilitated MC infection. These results demonstrate that IL-33 is able to induce protective IFN responses in MCs, however this effect is counterbalanced by upregulation of ICAM1, rendering the MCs more susceptible to RV infection.

Mast cells (MCs) are immune cells classically associated with allergic diseases including asthma where they promote bronchoconstriction and Type 2 inflammation. However, they are also key immune cells against parasitic, bacterial and viral infections by releasing immune and inflammatory mediators. Rhinoviruses (RV) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of RV replication is the airway epithelium and MCs localise to the bronchial epithelium in asthma. The subsequent epithelial cell damage caused by RV leads to IL-33 release. This promotes Type 2 cytokine release by MCs contributing to airway inflammation. As IL-33 has been shown to increase MC immune responses against RSV, we hypothesised that IL-33 has a role in mediating innate immune responses in MCs against RV. The human MC cell line LAD2 was treated with IL-33 (1 - 10 ng/ml) plus RV (MOI 1) or UV-irradiated RV as control. Total RNA was extracted at 2, 4, 8 and 24 hours after treatment. Gene expression and viral copy number were quantified by RT-qPCR and protein release by MSD/ELISA. We found that IL-33 significantly induced a time and concentration dependent expression of Type 2 and pro-inflammatory cytokine genes. Furthermore, we found induction of interferon (IFN) and IFN-inducible genes suggesting activation of an innate anti-viral response. However, IL-33 was also found to induce ICAM1, the receptor for major group RV entry, and in the presence of RV, IL-33 facilitated MC infection. These results demonstrate that IL-33 is able to induce protective IFN responses in MCs, however this effect is counterbalanced by upregulation of ICAM1, rendering the MCs more susceptible to RV infection.

Effective treatment for severe corticosteroid refractory asthma is a significant unmet clinical need. It affects only a small percent of the asthmatic population but accounts for a disproportionate use of healthcare resources. The aim was to study IL-13 transgenic mice to test the hypothesis that a subset of IL-13 mediated airway responses are corticosteroid-unresponsive and contribute to ongoing airways symptoms. IL-13 expression in the lungs was induced using Doxycycline in Ccsp-rtTA/Otet-Il-13 (Ccsp/Il-13) mice. Where indicated, mice received intra-peritoneal injections of 3mg/kg Dexamethasone for 3-7 days. Lungs were analysed for airway hyperreactivity and inflammation. Compared to controls, Ccsp/Il-13 mice showed significantly increased airway hyperresponsiveness (AHR) to methacholine and immunohistochemistry revealed increased bronchial smooth muscle and goblet cell metaplasia. The bronchoalveolar lavage fluid contained mixed eosinophilic and neutrophilic inflammation, but neutrophils predominated. Characteristic Th2-responsive genes as well as genes associated with Th17 responses were significantly elevated. Treatment with steroids did not abrogate AHR, even though eosinophilia and the ‘Th2’ gene signature were significantly reduced. Neutrophils and the ‘Th17’ signature remained elevated. Although IL-13 promotes eosinophilic airways disease, it can also drive corticosteroid refractory inflammation characterised by persistent neutrophilia, Th17 cytokines and maintenance of AHR. The Ccsp/Il-13 mouse may be a useful model for dissecting the molecular pathways and mechanisms associated with predominant neutrophilic, corticosteroid refractory airway disease.