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A defining pathological feature of human lung fibrosis is localized tissue heterogeneity, which challenges the interpretation of transcriptomic studies that typically lose spatial information. Here we investigate spatial gene expression in diagnostic tissue using digital profiling technology. We identify distinct, region-specific gene expression signatures as well as shared gene signatures. By integration with single-cell data, we spatially map the cellular composition within and distant from the fibrotic niche, demonstrating discrete changes in homeostatic and pathologic cell populations even in morphologically preserved lung, while through ligand-receptor analysis, we investigate cellular cross-talk within the fibrotic niche. We confirm findings through bioinformatic, tissue, and in vitro analyses, identifying that loss of NFKB inhibitor zeta in alveolar epithelial cells dysregulates the TGFβ/IL-6 signaling axis, which may impair homeostatic responses to environmental stress. Thus, spatially resolved deconvolution advances understanding of cell composition and microenvironment in human lung fibrogenesis.

A multichannel microfluidic platform for real-time monitoring of epithelial barrier integrity by electrical impedance has been developed. Growth and polarization of human epithelial cells from the airway or gastrointestinal tract was continuously monitored over 5 days in 8 parallel, individually perfused microfluidic chips. Electrical impedance data were continuously recorded to monitor cell barrier formation using a low-cost bespoke impedance analyser. Data was analysed using an electric circuit model to extract the equivalent transepithelial electrical resistance and epithelial cell layer capacitance. The cell barrier integrity steadily increased overtime, achieving an average resistance of 418 ± 121 Ω cm 2 (airway cells) or 207 ± 59 Ω cm 2 (gastrointestinal cells) by day 5. The utility of the polarized airway epithelial barrier was demonstrated using a 24 hour challenge with double stranded RNA to mimic viral infection. This caused a rapid decrease in barrier integrity in association with disruption of tight junctions, whereas simultaneous treatment with a corticosteroid reduced this effect. The platform is able to measure barrier integrity in real-time and is scalable, thus has the potential to be used for drug development and testing.

Background: effective treatment for severe asthma is a significant unmet need. While eosinophilic inflammation caused by Type 2 cytokines is responsive to corticosteroid and biologic therapies, many severe asthmatics exhibit corticosteroid‐unresponsive mixed granulocytic inflammation. Objective: here, we tested the hypothesis that the pro‐allergic cytokine, IL‐13, can drive both corticosteroid‐sensitive and corticosteroid‐resistant responses. Results: by integration of in vivo and in vitro models of IL‐13 driven inflammation, we identify a role for the epidermal growth factor receptor (EGFR/ERBB1) as a mediator of corticosteroid‐unresponsive inflammation and bronchial hyperresponsiveness (BHR) driven by IL‐13. Topological data analysis using human epithelial transcriptomic data from the U‐BIOPRED cohort identified severe asthma groups with features consistent with the presence of IL‐13 and EGFR/ERBB activation, with involvement of distinct EGFR ligands. Our data suggest that IL‐13 may play a dual role in severe asthma: on the one hand driving pathologic corticosteroid‐refractory mixed granulocytic inflammation, but on the other hand underpinning beneficial epithelial repair responses, which may confound responses in clinical trials. Conclusion and clinical relevance: detailed dissection of those molecular pathways that are downstream of IL‐13 and utilize the ERBB receptor and ligand family to drive corticosteroid refractory inflammation should enhance the development of new treatments that target this sub‐phenotype(s) of severe asthma, where there is an unmet need.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFβ increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

We previously reported that oxidative stress drives pseudohypoxic hypoxia-inducible factor (HIF) pathway activation to promote pathogenetic collagen structure-function in human lung fibrosis (Brereton et al., 2022). Here, through bioinformatic studies we investigate HIF pathway activation status in patients with idiopathic pulmonary fibrosis (IPF) and whether this has prognostic significance. Applying a well-established HIF gene expression signature, we classified publicly available datasets into HIF score-high and score-low groups across multiple tissue compartments. TheHIF scores in lung tissue, bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMC) were increased in IPF patients and significantly correlated with an oxidative stress signature consistent with pseudohypoxic HIF pathway activation. A high HIF score in BAL and in PBMC was a strong independent predictor of mortality in multivariate analysis. Thus, a validated HIF gene signature predicts survival across tissue compartments in IPF and merits prospective study as a non-invasive biomarker of lung fibrosis progression.

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic interstitial lung disease. Median survival is only 3 years, and treatment options are limited. IPF is thought to be a result of a combination of genetic and environmental factors with repetitive micro-injuries to alveolar epithelial cells playing a central role. IPF is characterised by aberrant extra cellular matrix (ECM) deposition by activated myofibroblasts. Epithelial-mesenchymal transition (EMT) is a process where polarised epithelial cells undergo molecular changes allowing them to gain a mesenchymal phenotype, with a subsequent enhanced ability to produce ECM components and increased migration and/or invasion. The source of myofibroblasts in IPF has been debated for many years, and EMT has been proposed as a source of these cells. However, lineage tracing in transgenic mice suggests the contribution of epithelial cells, which have undergone EMT, to the fibroblast population may be negligible. Instead, recent findings suggest that alveolar epithelial type II (ATII) cells undergoing EMT promote a pro-fibrotic microenvironment through paracrine signalling activating local fibroblasts. This review paper explores the contribution of ATII cells, which have undergone EMT, in the context of pulmonary fibrosis.

Significance: Idiopathic pulmonary fibrosis (IPF) patients have a poor prognosis with short lifespan following diagnosis as there are limited effective treatment options. Despite matrix stiffening being the hallmark of the disease there remains a lack of knowledge surrounding the underlying collagen alterations in the disease. Specifically, while increased collagen crosslinking has been implicated, the resulting effects on collagen macro/supramolecular changes have not been explored. Aim: We sought to determine if second-harmonic generation (SHG) microscopy could characterize differences in the collagen architecture in 3D spheroid models of IPF grown under different crosslinking modulation conditions (promotion and inhibition). Approach: We used SHG metrics based on the fiber morphology, relative SHG brightness, and macro/supramolecular structure by SHG polarization analyses to compare the structure of the IPF spheroids. Results: Comparison of the fiber morphology of the spheroids showed that the control group had the longest, straightest, and thickest fibers. The spheroids with crosslink enhancement and inhibition had the highest and lowest SHG conversion efficiencies, respectively, consistent with the resulting harmonophore density. SHG polarization analyses showed that the peptide pitch angle, alignment of collagen molecules, and overall chirality were altered upon crosslink modulation and were also consistent with reduced organization relative to the control group. Conclusions: While no single SHG signature is associated with crosslinking, we show that the suite of metrics used here is effective in delineating alterations across the collagen architecture sizescales. The results largely mirror those of human tissues and demonstrate that the combination of 3D spheroid models and SHG analysis is a powerful approach for hypothesis testing the roles of operative cellular and molecular factors in IPF.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we show that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of oxygen status (pseudohypoxia). Whilst TGFβ increased rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting ‘bone-type’ cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knock down of Factor Inhibiting HIF (FIH) or oxidative stress caused pseudohypoxic HIF activation in normal fibroblasts. In contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of IPF lung mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in airway epithelial cells for SARS-CoV-2. ACE2 binding to the SARS-CoV-2 protein spike triggers viral fusion with the cell plasma membrane, resulting in viral RNA genome delivery into the host. Despite ACE2’s critical role in SARS-CoV-2 infection, full understanding of ACE2 expression, including in response to viral infection, remains unclear. ACE2 was thought to encode five transcripts and one protein of 805 amino acids. In the present study, we identify a novel short isoform of ACE2 expressed in the airway epithelium, the main site of SARS-CoV-2 infection. Short ACE2 is substantially upregulated in response to interferon stimulation and rhinovirus infection, but not SARS-CoV-2 infection. This short isoform lacks SARS-CoV-2 spike high-affinity binding sites and, altogether, our data are consistent with a model where short ACE2 is unlikely to directly contribute to host susceptibility to SARS-CoV-2 infection.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection carries a substantial risk of severe and prolonged illness; treatment options are currently limited. We assessed the efficacy and safety of inhaled nebulised interferon beta-1a (SNG001) for the treatment of patients admitted to hospital with COVID-19.MethodsWe did a randomised, double-blind, placebo-controlled, phase 2 pilot trial at nine UK sites. Adults aged 18 years or older and admitted to hospital with COVID-19 symptoms, with a positive RT-PCR or point-of-care test, or both, were randomly assigned (1:1) to receive SNG001 (6 MIU) or placebo by inhalation via a mouthpiece daily for 14 days. The primary outcome was the change in clinical condition on the WHO Ordinal Scale for Clinical Improvement (OSCI) during the dosing period in the intention-to-treat population (all randomised patients who received at least one dose of the study drug). The OSCI is a 9-point scale, where 0 corresponds to no infection and 8 corresponds to death. Multiple analyses were done to identify the most suitable statistical method for future clinical trials. Safety was assessed by monitoring adverse events for 28 days. This trial is registered with Clinicaltrialsregister.eu (2020-001023-14) and ClinicalTrials.gov (NCT04385095); the pilot trial of inpatients with COVID-19 is now completed.FindingsBetween March 30 and May 30, 2020, 101 patients were randomly assigned to SNG001 (n=50) or placebo (n=51). 48 received SNG001 and 50 received placebo and were included in the intention-to-treat population. 66 (67%) patients required oxygen supplementation at baseline: 29 in the placebo group and 37 in the SNG001 group. Patients receiving SNG001 had greater odds of improvement on the OSCI scale (odds ratio 2·32 [95% CI 1·07-5·04]; p=0·033) on day 15 or 16 and were more likely than those receiving placebo to recover to an OSCI score of 1 (no limitation of activities) during treatment (hazard ratio 2·19 [95% CI 1·03-4·69]; p=0·043). SNG001 was well tolerated. The most frequently reported treatment-emergent adverse event was headache (seven [15%] patients in the SNG001 group and five [10%] in the placebo group). There were three deaths in the placebo group and none in the SNG001 group.InterpretationPatients who received SNG001 had greater odds of improvement and recovered more rapidly from SARS-CoV-2 infection than patients who received placebo, providing a strong rationale for further trials.FundingSynairgen Research.

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-β-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-β-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.

Light sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct, without any further processing or deconvolution, 3D imaging of harmonophores such as collagen in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

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.

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in the airways for SARS-CoV-2. ACE2 binding to SARS-CoV-2 protein Spike triggers viral fusion with the cell membrane, resulting in viral RNA genome delivery into the host. Despite ACE2’s critical role in SARS-CoV-2 infection, an understanding of ACE2 expression, including in response to viral infection, remains unclear.Until nowACE2was thought to encode five transcripts and one 805 amino acid protein. Here we identify a novel short isoform of ACE2. ShortACE2is expressed in the airway epithelium, the main site of SARS-CoV-2 infection; it is substantially upregulated in response to interferon stimulation and RV infection, but not in response to SARS-CoV-2 infection, and it shows differential regulation in asthma patients. This short isoform lacks SARS-CoV-2 spike glycoprotein high-affinity binding sites and altogether, our data are consistent with a model where shortACE2may influence host susceptibility to SARS-CoV-2 infection.

Airborne particulate matter (PM) is a leading cause of mortality and morbidity. However, understanding of the range and mechanisms of effects of PM components is poor. PM generated in underground railways is rich in metals, especially iron. In the ultrafine (UFPM

Hyperbaric oxygen (HBO) is widely applied to treat several hypoxia-related diseases. Previous studies have focused on the immediate effect of HBO-exposure induced oxidative stress on the lungs, but knowledge regarding the chronic effects from repetitive HBO exposure is limited, especially at the gene expression level. We found that repetitive HBO exposure did not alter the morphology of murine lungs. However, by deconvolution of RNA-seq from those mice lungs using CIBERSORTx and the expression profile matrices of 8 mesenchymal cell subtypes obtained from bleomycin-treated mouse lungs, we identify several mesenchymal cell subtype changes. These include increases in Col13a1 matrix fibroblasts, mesenchymal progenitors and mesothelial cell populations and decreases in lipofibroblasts, endothelial and Pdgfrb high cell populations. Our data suggest that repetitive HBO exposure may affect biological processes in the lungs such as response to wounding, extracellular matrix, vasculature development and immune response.

Dendritic cells (DCs) play a central role in regulating innate and adaptive immune responses. It is well accepted that their regulatory functions change over the life course. In order to study DCs function during early life it is important to characterize the function of neonatal DCs. However, the availability of neonatal DCs is limited due to ethical reasons or relative small samples of cord blood making it difficult to perform large-scale experiments. Our aim was to establish a robust protocol for the generation of neonatal DCs from cord blood derived CD34+ hematopoietic stem cells. For the expansion of DC precursor cells we used a cytokine cocktail containing Flt-3 L, SCF, TPO, IL-3 and IL-6. The presence of IL-3 and IL-6 in the first 2 weeks of expansion culture was essential for the proliferation of DC precursor cells expressing CD14. After 4 weeks in culture, CD14+ precursor cells were selected and functional DCs were generated in the presence of GM-CSF and IL-4. Neonatal DCs were then stimulated with Poly(I:C) and LPS to mimic viral or bacterial infections, respectively. Poly(I:C) induced a higher expression of the maturation markers CD80, CD86 and CD40 compared to LPS. In line with literature data using cord blood DCs, our Poly(I:C) matured neonatal DCs cells showed a higher release of IL-12p70 compared to LPS matured neonatal DCs. Additionally, we demonstrated a higher release of IFN-γ, TNF-α, IL-1β and IL-6, but lower release of IL-10 in Poly(I:C) matured compared to LPS matured neonatal DCs derived from cord blood CD34+ hematopoietic stem cells. In summary, we established a robust protocol for the generation of large numbers of functional neonatal DCs. In line with previous studies, we showed that neonatal DCs generated form CD34+ cord blood progenitors have a higher inflammatory potential when exposed to viral than bacterial related stimuli., Highlights • A robust protocol for the generation of high numbers of neonatal dendritic cells. • IL-3 and IL-6 are crucial for the proliferation of cord blood CD34+ progenitors. • Neonatal DCs have a higher inflammatory potential when exposed to viral stimuli. • LPS induces higher release of IL-10 in neonatal DCs compared to Poly(I:C).

Idiopathic pulmonary fibrosis (IPF) is a chronic scarring disease in which aging, environmental exposure(s) and genetic susceptibility have been implicated in disease pathogenesis, however, the causes and mechanisms of the progressive fibrotic cascade are still poorly understood. As epithelial–mesenchymal interactions are essential for normal wound healing, through human 2D and 3D in vitro studies, we tested the hypothesis that IPF fibroblasts (IPFFs) dysregulate alveolar epithelial homeostasis. Conditioned media from IPFFs exaggerated the wound-healing response of primary human Type II alveolar epithelial cells (AECs). Furthermore, AECs co-cultured with IPFFs exhibited irregular epithelialization compared with those co-cultured with control fibroblasts (NHLFs) or AECs alone, suggesting that epithelial homeostasis is dysregulated in IPF as a consequence of the abnormal secretory phenotype of IPFFs. Secretome analysis of IPFF conditioned media and functional studies identified the matricellular protein, SPARC, as a key mediator in the epithelial–mesenchymal paracrine signaling, with increased secretion of SPARC by IPFFs promoting persistent activation of alveolar epithelium via an integrin/focal adhesion/cellular-junction axis resulting in disruption of epithelial barrier integrity and increased macromolecular permeability. These findings suggest that in IPF fibroblast paracrine signaling promotes persistent alveolar epithelial activation, so preventing normal epithelial repair responses and restoration of tissue homeostasis. Furthermore, they identify SPARC-mediated paracrine signaling as a potential therapeutic target to promote the restoration of lung epithelial homoestasis in IPF patients.

Super-resolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit, however, most of the techniques are hampered by the need for fluorescent labels. Non-linear label-free techniques such as Second Harmonic Generation (SHG) provide structurally specific contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. We use the photonic nanojet (PNJ) phenomena to achieve super-resolution SHG (SR-SHG). A resolution of ~λ/6 with respect to the fundamental wavelength, that is, a ~2.3-fold improvement over conventional or diffraction-limited SHG under the same imaging conditions is achieved. Crucially we find that the polarisation properties of excitation are maintained in a PNJ. This is observed in experiment and simulations. This may have widespread implications to increase sensitivity by detection of polarisation-resolved SHG (p-SHG) by observing anisotropy in signals. These new findings allowed us to visualise biological SHG-active structures such as collagen at an unprecedented and previously unresolvable spatial scale. Moreover, we demonstrate that the use of an array of self-assembled high-index spheres overcomes the issue of a limited field of view for such a method, allowing PNJ-assisted SR-SHG to be used over a large area. Dysregulation of collagen at the nanoscale occurs in many diseases and is an underlying cause in diseases such as lung fibrosis. Here we demonstrate that pSR-SHG allows unprecedented observation of changes at the nanoscale that are invisible by conventional diffraction-limited SHG imaging. The ability to non-destructively image SHG-active biological structures without labels at the nanoscale with a relatively simple optical method heralds the promise of a new tool to understand biological phenomena and drive drug discovery.

BackgroundSevere asthma is associated with multiple comorbidities, including gastro-oesophageal reflux disease (GORD), which can contribute to exacerbation frequency and poor quality of life. Since epithelial dysfunction is an important feature in asthma, we hypothesised that in severe asthma the bronchial epithelium is more susceptible to the effects of acid reflux.MethodsWe developed an in vitro model of GORD using differentiated bronchial epithelial cells (BECs) from normal or severe asthmatic donors exposed to a combination of pepsin, acid pH and bile acids using a multiple challenge protocol (MCP-PAB). In addition, we analysed bronchial biopsies and undertook RNA sequencing of bronchial brushings from controls and severe asthmatics without or with GORD.ResultsExposure of BECs to the MCP-PAB caused structural disruption, increased permeability, interleukin (IL)-33 expression, inflammatory mediator release and changes in gene expression for multiple biological processes. Cultures from severe asthmatics were significantly more affected than those from healthy donors. Analysis of bronchial biopsies confirmed increased IL-33 expression in severe asthmatics with GORD. RNA sequencing of bronchial brushings from this group identified 15 of the top 37 dysregulated genes found in MCP-PAB treated BECs, including genes involved in oxidative stress responses.Conclusions and clinical implicationBy affecting epithelial permeability, GORD may increase exposure of the airway submucosa to allergens and pathogens, resulting in increased risk of inflammation and exacerbations. These results suggest the need for research into alternative therapeutic management of GORD in severe asthma.

Rationale: ADAMs (proteinases with a disintegrin and a metalloproteinase domain) have not been well studied in chronic obstructive pulmonary disease (COPD).Objectives: To investigate whether ADAM9 ...

Background/Aim: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. Materials and Methods: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). Results: The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of highly organized extracellular matrix (ECM) interspersed with fibroblastic cells separated by a basement membrane. EM analysis of the mucosal construct after repetitive exposure of to CSE revealed epithelial damage, loss of cilia, and ECM remodeling, as occurs in vivo.Conclusions: We have developed a robust bronchial mucosal model. The structural changes observed following CSE exposure suggest the model should have utility for drug discovery and preclinical testing, especially those targeting airway remodeling.

Objective To assess risks and costs of hospital admission associated with short term exposure to fine particulate matter with diameter less than 2.5 µm (PM2.5) for 214 mutually exclusive disease groups. Design Time stratified, case crossover analyses with conditional logistic regressions adjusted for non-linear confounding effects of meteorological variables. Setting Medicare inpatient hospital claims in the United States, 2000-12 (n=95 277 169). Participants All Medicare fee-for-service beneficiaries aged 65 or older admitted to hospital. Main outcome measures Risk of hospital admission, number of admissions, days in hospital, inpatient and post-acute care costs, and value of statistical life (that is, the economic value used to measure the cost of avoiding a death) due to the lives lost at discharge for 214 disease groups. Results Positive associations between short term exposure to PM2.5 and risk of hospital admission were found for several prevalent but rarely studied diseases, such as septicemia, fluid and electrolyte disorders, and acute and unspecified renal failure. Positive associations were also found between risk of hospital admission and cardiovascular and respiratory diseases, Parkinson’s disease, diabetes, phlebitis, thrombophlebitis, and thromboembolism, confirming previously published results. These associations remained consistent when restricted to days with a daily PM2.5 concentration below the WHO air quality guideline for the 24 hour average exposure to PM2.5. For the rarely studied diseases, each 1 µg/m3 increase in short term PM2.5 was associated with an annual increase of 2050 hospital admissions (95% confidence interval 1914 to 2187 admissions), 12 216 days in hospital (11 358 to 13 075), US$31m (£24m, €28m; $29m to $34m) in inpatient and post-acute care costs, and $2.5bn ($2.0bn to $2.9bn) in value of statistical life. For diseases with a previously known association, each 1 µg/m3 increase in short term exposure to PM2.5 was associated with an annual increase of 3642 hospital admissions (3434 to 3851), 20 098 days in hospital (18 950 to 21 247), $69m ($65m to $73m) in inpatient and post-acute care costs, and $4.1bn ($3.5bn to $4.7bn) in value of statistical life. Conclusions New causes and previously identified causes of hospital admission associated with short term exposure to PM2.5 were found. These associations remained even at a daily PM2.5 concentration below the WHO 24 hour guideline. Substantial economic costs were linked to a small increase in short term PM2.5.

Background Maternal allergic asthma is a strong risk factor for the development of asthma and airway hyperresponsiveness (AHR) in children. ADAM33, an asthma susceptibility gene, has been associated with AHR and impaired lung function in early life. Our aim was to investigate how the maternal allergic environment during pregnancy interacts with the ADAM33 status of their offspring, and the effects this has on the lungs of offspring after birth. We hypothesised that the effects of maternal allergy will be different in Adam33 knock-out (KO) compared to wild-type (WT) offspring Methods Allergic airway inflammation (AAI) during pregnancy was induced in heterozygous (Adam33±) mice through intranasal house dust mite (HDM) challenges. Control mice were challenged with saline. WT and KO (Adam33-/-) offspring from the same litters were studied 4 weeks post partum (pp). Lung function was measured in response to increasing doses of methacholine. Bronchoalveolar lavage fluid (BALF) and lung tissue were obtained for RTqPCR, Western Blots and immunostainings. Precision-cut lung slices (PCLS) from 4-weeks old offspring were investigated for airway contraction in response to different agonists and antagonists in vitro. Results Allergen-naive WT offspring of allergic mothers showed AHR 4 weeks pp compared to those of control mothers, whereas KO offspring from the same litter were protected. Expression of the muscarinic M1 receptor was elevated in both KO and WT offspring lungs of HDM-challenged dams. Experiments using muscarinic receptor antagonists and methacholine in PCLS confirmed that maternal AAI causes increased bronchoconstriction through vagal reflexes in WT offspring. KO offspring were protected from this effect due to decreased sensitivity of airway smooth muscle, suggested by a delayed response to a thromboxane-receptor agonist in PCLS. Conclusions Our studies show how gene-environment interactions between Adam33 and maternal AAI determine development of AHR in early life. While the AAI of the mother leads to an increased pulmonary muscarinic M1 receptor expression, the absence of Adam33 alters the airway smooth muscle function in the offspring. Together these changes manifest in AHR only in WT offspring, but not in KO offspring. Further studies are needed to determine how ADAM33 KO changes smooth muscle function in the lungs.

Sarcoidosis is a multisystem disease of unknown aetiology. Up to 90% of sarcoidosis patients have lung involvement including pulmonary fibrosis. This leads to impaired quality of life and sometimes mortality. To better understand the nature of the lung matrix we generated novel 3D multicellular foci from individual patient cells. Primary lung fibroblasts were isolated from explant tissue from patients with sarcoidosis. These were then grown in optimal conditions to promote 3D multicellular foci formation for a total of six weeks, treated +/- transforming growth factor-beta-1 (TGFβ1). Alpha smooth muscle actin (aSMA) and collagen mRNA expression were assess­ed by RTqPCR and gross mechanical stiffness of the resulting multicellular foci was assessed by parallel plate compression testing. Total collagen was assessed by hydroxyproline assay (ELISA), pyridinoline (PYD) concentrations were measured by ELISA and LC-MS to assess collagen cross-linking. Treatment of sarcoidosis fibroblasts with 2.5ng/mL TGFβ1 caused a 6-fold increase in collagen-1 (p TGFβ1 increases fibrotic markers in sarcoidosis fibroblasts cultured in this long-term cell culture model.

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.

Background: Altered collagen architecture, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in idiopathic pulmonary fibrosis (IPF), with the collagen cross-linking enzymes lysyl hydroxylase 2 (LH2) and lysyl oxidase-like 2 (LOXL2) promoting pathological bone-type collagen crosslinking and increased tissue stiffness (Jones et al eLife. 2018;7:e36354). However, the upstream mechanisms regulating this process remain unknown. Objective: To determine the cellular provenance of LOXL2 and LH2 in IPF tissue and identify the mechanisms promoting their dysregulation. Methods: Cellular provenance of LH2 and LOXL2 was analysed using mRNA in-situ hybridisation. IPF lung fibroblasts were cultured under a range of conditions that have been associated with IPF pathogenesis and their effects on LH2 and LOXL2 induction assessed by RTqPCR and Western Blotting. Small-interfering RNAs (siRNA) were transfected into IPF fibroblasts to investigate signalling pathways of interest and their role in regulating LH2 and LOXL2 expression. Results: LH2 and LOXL2 were co-expressed within fibroblastic foci in IPF tissue. Hypoxia-inducible factor (HIF) pathways were the strongest inducers of both LOXL2 and LH2 in IPF fibroblasts. siRNA mediated knockdown of HIF-1α but not HIF-2α prevented LH2 induction, whilst knockdown of both HIF-1α and HIF-2α was required to prevent LOXL2 induction. Conclusions: Our results suggest that HIF pathway activation via LH2/LOXL2 may be a core regulator of bone-type collagen fibrillogenesis in IPF.