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Aberrant expansion of KRT5+basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5+cells in IPF have not been delineated. Here, we reveal a potential mechanism by which KRT5+cells migrate within the fibrotic lung, navigating regional differences in collagen topography.In vitro, KRT5+cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry-based proteomics revealed compositional differences in ECM components secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrix restricts KRT5+cell migrationin vitro. Together, our findings demonstrate how changes to the ECM in IPF directly influence KRT5+cell behaviour and function contributing to remodelling events in the fibrotic niche.

Airway macrophages (AMs) play key roles in the maintenance of lung immune tolerance. Tissue tailored, highly specialised and strategically positioned, AMs are critical sentinels of lung homoeostasis. In the last decade, there has been a revolution in our understanding of how metabolism underlies key macrophage functions. While these initial observations were made during steady state or using in vitro polarised macrophages, recent studies have indicated that during many chronic lung diseases (CLDs), AMs adapt their metabolic profile to fit their local niche. By generating reactive oxygen species (ROS) for pathogen defence, utilising aerobic glycolysis to rapidly generate cytokines, and employing mitochondrial respiration to fuel inflammatory responses, AMs utilise metabolic reprogramming for host defence, although these changes may also support chronic pathology. This review focuses on how metabolic alterations underlie AM phenotype and function during CLDs. Particular emphasis is given to how our new understanding of AM metabolic plasticity may be exploited to develop AM-focused therapies.

Summary Interferon regulatory factor 5 (IRF5) is a master regulator of macrophage phenotype and a key transcription factor involved in expression of proinflammatory cytokine responses to microbial and viral infection. Here, we show that IRF5 controls cellular and metabolic responses. By integrating ChIP sequencing (ChIP‐Seq) and assay for transposase‐accessible chromatin using sequencing (ATAC)‐seq data sets, we found that IRF5 directly regulates metabolic genes such as hexokinase‐2 (Hk2). The interaction of IRF5 and metabolic genes had a functional consequence, as Irf5−/− airway macrophages but not bone marrow‐derived macrophages (BMDMs) were characterized by a quiescent metabolic phenotype at baseline and had reduced ability to utilize oxidative phosphorylation after Toll‐like receptor (TLR)‐3 activation, in comparison to controls, ex vivo. In a murine model of influenza infection, IRF5 deficiency had no effect on viral load in comparison to wild‐type controls but controlled metabolic responses to viral infection, as IRF5 deficiency led to reduced expression of Sirt6 and Hk2. Together, our data indicate that IRF5 is a key component of AM metabolic responses following influenza infection and TLR‐3 activation., Here, we provide novel data showing that the transcription factor Interferon regulatory factor 5 (IRF5) controls cellular and metabolic responses to influenza infection and TLR3 activation in vivo. Upon pro‐inflammatory stimulation IRF5 co‐localises with open chromatin regions at metabolism‐associated genes; these interactions led to a functional consequences as IRF5 deficient airway macrophages (AMs) had an impaired ability to utilize oxidative phosphorylation. Our data highlight a novel role for IRF5 in the regulation of AM metabolic responses.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive and inevitably fatal condition for which there are a lack of effective biomarkers to guide therapeutic decision making. OBJECTIVES: To determine the relationship between serum concentrations of the cytokeratin fragment CYFRA 21-1 and disease progression and mortality in individuals with IPF enrolled in the Prospective Observation of Fibrosis in the Lung Clinical Endpoints (PROFILE) study. METHODS: CYFRA 21-1 was identified by immunohistochemistry in samples of human lung obtained at surgery. Concentrations of CYFRA 21-1 were measured using an ELISA-based assay in serum samples collected at baseline, 1 month, and 3 months from 491 individuals with an incident diagnosis of IPF who were enrolled in the PROFILE study and from 100 control subjects at baseline. Study subjects were followed for a minimum of 3 years after their first blood draw. MEASUREMENTS AND MAIN RESULTS: CYFRA 21-1 localizes to hyperplastic epithelium in IPF lung tissue. Peripheral CYFRA 21-1 concentrations were significantly higher in subjects with IPF than in healthy control subjects in both the discovery (n = 132) (control: 0.96 ± 0.81 ng/ml; vs. IPF: 2.34 ± 2.15 ng/ml; P

Rationale: Airway macrophages (AMs) are key regulators of the lung environment and are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal respiratory disease with no cure. However, knowledge about the epigenetics of AMs in IPF is limited. Objectives: To assess the role of epigenetic regulation of AMs during lung fibrosis. Methods: We undertook DNA methylation (DNAm) profiling by using Illumina EPIC (850k) arrays in sorted AMs from healthy donors (n = 14) and donors with IPF (n = 30). Cell-type deconvolution was performed by using reference myeloid-cell DNA methylomes. Measurements and Main Results: Our analysis revealed that epigenetic heterogeneity was a key characteristic of IPF AMs. DNAm "clock" analysis indicated that epigenetic alterations in IPF AMs were not associated with accelerated aging. In differential DNAm analysis, we identified numerous differentially methylated positions (n = 11) and differentially methylated regions (n = 49) between healthy and IPF AMs, respectively. Differentially methylated positions and differentially methylated regions encompassed genes involved in lipid (LPCAT1 [lysophosphatidylcholine acyltransferase 1]) and glucose (PFKFB3 [6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3]) metabolism, and importantly, the DNAm status was associated with disease severity in IPF. Conclusions: Collectively, our data identify that changes in the epigenome are associated with the development and function of AMs in the IPF lung.

Iron is an essential nutrient for numerous cellular processes. However, excess iron in the lung (e.g. inhaled in pollution/cigarette smoke) can be harmful, acting as a catalyst in the formation of free radicals. Pulmonary iron content is therefore tightly regulated and alterations in iron metabolism have been associated with chronic lung disease. In particular, patients with idiopathic pulmonary fibrosis have been reported to have numerous aspects of dysfunctional iron metabolism in the lung, including increased iron levels, presence of iron-laden macrophages and iron-induced oxidative stress. In a recent issue of The Journal of Pathology, Ali et al showed a mechanistic link between iron accumulation and pulmonary fibrosis pathology. Using mouse models of iron overload, the authors showed that increased iron levels resulted in reduced lung function and worse pulmonary fibrosis upon lung injury by bleomycin. Treatment with inhaled iron chelator deferoxamine ameliorated pulmonary fibrosis and prevented lung function decline in vivo. This study highlights the importance of iron homeostasis in the lung and provides evidence of pulmonary iron overload contributing to the development and progression of pulmonary fibrosis. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background: Asthma is a chronic disease of the airways characterised by airway remodelling, inflammation and mucus production. Airway macrophages (AMs) form a first line of defence against inhaled pathogen. In steady state and in response to pathogen, AM phenotype and function are underpinned by changes in cellular metabolism. The TCA cycle-derived metabolite itaconic acid (IA), synthesised by the enzyme aconitate decarboxylase (ACOD)-1, is a master regulator of macrophage function. We recently showed that IA limits pulmonary fibrosis in human and in murine models. However, its role in regulating asthma and allergic airway disease (AAD) is unknown. Methods: To study the kinetics of the Acod1/IA pathway in AAD, we analysed the metabolic profile of AMs ex vivo cultured with the aeroallergen house dust mite (HDM). Next, we measured airway inflammation, lung function and bronchoalveolar lavage (BAL) metabolite levels in WT and Acod1-/- mice. Finally, to investigate the role of IA in asthma, we measured metabolite levels in response to allergen challenge in sputum of mild asthmatics. Results:Ex vivo culture of AMs with HDM resulted in increased Acod1 expression and a more glycolytic phenotype. Continuous HDM exposure of WT mice led to increased lung Acod1 expression accompanied by augmented IA levels in BAL. Upon exposure to aeroallergen Acod1-/- mice showed a lower eosinophil-to-neutrophil ratio compared to more eosinophilic WT controls, which was rescued by inhaled IA. Finally, analysis of sputum from people with mild asthma revealed an inverse relationship between neutrophil numbers and IA levels. Conclusion: Our data indicate that the Acod1/IA pathway is highly induced during AAD and regulates the balance between neutrophil and eosinophil recruitment in response to inhaled allergen.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic lung disease of unknown aetiology. There is growing evidence that the lung microbiota may play a role in IPF. However, no study has investigated the functional impact of the short-chain fatty acids (SCFAs) on primary bronchial epithelial cells (PBECs) and disease pathogenesis. Therefore, we investigated the influence of acetate, propionate, and butyrate on PBECs from healthy controls and subjects with IPF. Subjects diagnosed with IPF (n=201) and healthy controls (n=40) were prospectively recruited and underwent bronchoalveolar lavage. Bacterial DNA was isolated and 16S rRNA gene sequencing undertaken to characterise bacterial communities. Untargeted 1H nuclear magnetic resonance spectroscopy-based metabolomics and targeted gas chromatography-mass spectrometry captured the metabolic profile of these samples. PBECs from healthy controls and subjects with IPF were differentiated at air-liquid interface (ALI) and either left untreated or exposed to the SCFAs. The IPF microbiota was less diverse (P Subjects with IPF display an altered microbiome which is associated with a distinct metabolic signature in the lower airways. Differences in specific bacterial genera and an increased bacterial burden in IPF results in changes in the SCFAs in the airways. In vitro work demonstrates the potential of these SCFAs to shape immunological responses in the lung, mediating the pathogenesis of fibrosis.

Airway macrophages (AMs) are key regulators of the lung environment and are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal respiratory disease with no cure. However, the epigenetics of AMs development and function in IPF are limited. Here, we characterised the DNA-methylation (DNAm) profile of AMs from IPF (n=30) and healthy (n=14) donors. Our analysis revealed epigenetic heterogeneity was a key characteristic of IPF AMs. DNAm ‘clock’ analysis indicated epigenetic alterations in IPF-AMs was not associated with accelerated ageing. In differential DNAm analysis, we identified numerous differentially methylated positions (DMPs, n=11) and regions (DMRs, n=49) between healthy and IPF AMs respectively. DMPs and DMRs encompassed genes involved in lipid (LPCAT1) and glucose (PFKB3) metabolism and importantly, DNAm status was associated with disease severity in IPF. Collectively, our data identify that profound changes in the epigenome underpin the development and function of AMs in the IPF lung.

Airway Macrophages (AMs) are important in the pathogenesis of lung fibrosis. Inflammasomes (NLRP3,AIM2,NLRC4) are cytosolic receptors of the innate immune system responsible for cleavage of IL-1b, a proinflammatory,profibrotic cytokine. Inflammasomes respond to local microbiota changes. In IPF the bacterial burden is elevated and its composition is altered. AIM2 is increased and linked to exacerbation of fibrosis after Streptococcal infection (Cho,Thorax 2019). In IPF exacerbations, top upregulated genes in the BAL are IL-1b and NLRP3. MtROS drives NLRP3 activation and is elevated in IPF AMs (Tsitoura,RespRes 2019). We hypothesised that changes in lung microbiota in IPF may drive Inflammasome activation. As such, we studied the capacity of AMs to activate inflammasomes in relation to microbial burden and mtROS. We prospectively enrolled healthy and IPF subjects and isolated AMs from BAL. AMs were treated with specific inflammasome stimuli; ATP for NLRP3, dsDNA for AIM2, flagellin for NLRC4. IL-1b release was measured by ELISA, BAL microbial burden by 16S rRNA qPCR and mtROS by flow cytometry. The samples are sent for lung microbiome characterisation. Upon stimulation, IPF AMs over activate NLPR3 and AIM2 but not NLRC4. Baseline IL-1b release was similar between groups. Excessive inflammasome activation was linked to higher baseline mtROS. Microbial burden tended to be increased in IPF. Microbial burden correlated with baseline IL1b release. There was a strong association between the levels of 16S rRNA and relative AIM2 mRNA. Our results reveal a possible novel role of inflammasomes’ response to local microbiota changes which could be associated with the pathogenesis of IPF acute exacerbations.

RATIONALE: Chronic hypersensitivity pneumonitis (CHP) is a condition that arises following repeated exposure and sensitisation to inhaled antigens. The lung microbiome is increasingly implicated in respiratory disease but to date, no study has investigated the composition of microbial communities in the lower airways in CHP. OBJECTIVE: To characterise and compare the airway microbiome in subjects with CHP, idiopathic pulmonary fibrosis (IPF) and controls. METHODS: We prospectively recruited individuals diagnosed with CHP (n=110), IPF (n=45) and controls (n=28). Subjects underwent bronchoalveolar lavage and bacterial DNA was isolated, quantified by qPCR and the 16S rRNA gene was sequenced to characterise the bacterial communities in the lower airways. MAIN MEASUREMENTS AND RESULTS: Distinct differences in the microbial profiles were evident in the lower airways of subjects with CHP and IPF. At the phylum level, the prevailing microbiota of both IPF and CHP subjects included Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria. However, in IPF, Firmicutes dominated while the percentage of reads assigned to Proteobacteria in the same group was significantly lower compared to CHP subjects. At the genus level, Staphylococcus was increased in CHP and Actinomyces and Veillonella in IPF. The lower airway bacterial burden in CHP subjects was higher than controls but lower than those with IPF. In contrast to IPF, there was no association between bacterial burden and survival in CHP. CONCLUSIONS: The microbial profile of the lower airways in subjects with CHP is distinct from that of IPF and, notably, bacterial burden in individuals with CHP fails to predict survival.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease in which airway macrophages (AMs) play a key role. Itaconate has emerged as a mediator of macrophage function, but its role during fibrosis is unknown. Here, we reveal that itaconate is an endogenous antifibrotic factor in the lung. Itaconate levels are reduced in bronchoalveolar lavage, and itaconate-synthesizing cis-aconitate decarboxylase expression (ACOD1) is reduced in AMs from patients with IPF compared with controls. In the murine bleomycin model of pulmonary fibrosis, Acod1-/- mice develop persistent fibrosis, unlike wild-type (WT) littermates. Profibrotic gene expression is increased in Acod1-/- tissue-resident AMs compared with WT, and adoptive transfer of WT monocyte-recruited AMs rescued mice from disease phenotype. Culture of lung fibroblasts with itaconate decreased proliferation and wound healing capacity, and inhaled itaconate was protective in mice in vivo. Collectively, these data identify itaconate as critical for controlling the severity of lung fibrosis, and targeting this pathway may be a viable therapeutic strategy.

Increasing bacterial burden in the lower airways of patients with idiopathic pulmonary fibrosis confers an increased risk of disease progression and mortality. However, it remains unclear whether this increased bacterial burden directly influences progression of fibrosis or simply reflects the magnitude of the underlying disease extent or severity. We prospectively recruited 193 patients who underwent bronchoscopy and received a multidisciplinary diagnosis of idiopathic pulmonary fibrosis. Quantification of the total bacterial burden in bronchoalveolar lavage fluid was performed by 16S rRNA gene qPCR. Imaging was independently evaluated by two readers assigning quantitative scores for extent, severity and topography of radiographic changes and relationship of these features with bacterial burden was assessed. Increased bacterial burden significantly associated with disease progression (HR 2.1; 95% CI 1.287–3.474; p=0.0028). Multivariate stepwise regression demonstrated no relationship between bacterial burden and radiological features or extent of disease. When specifically considering patients with definite or probable usual interstitial pneumonia there was no difference in bacterial burden between these two groups. Despite a postulated association between pleuroparenchymal fibroelastosis and clinical infection, there was no relationship between either the presence or extent of pleuroparenchymal fibroelastosis and bacterial burden. We demonstrate that bacterial burden in the lower airways is not simply secondary to the extent of the underlying architectural destruction of the lung parenchyma seen in idiopathic pulmonary fibrosis. The independent nature of this association supports a relationship with the underlying pathogenic mechanisms and highlights the urgent need for functional studies., Elevated bacterial burden predicts mortality in IPF and is independent of both radiological features and extent of disease http://bit.ly/2RaDbdv

Background: Airway macrophages (AMs) are key sentinels of lung homeostasis and responses to inhaled allergens and pathogens. Recent work has shown that macrophage activation and plasticity are under metabolic control. Interferon regulatory factor 5 (IRF5) is a key transcription factor involved in the induction of pro-inflammatory cytokine responses to infection. Previous studies identified IRF5 as a master regulator of macrophage phenotype in vitro, however the role of IRF5 in directing AM metabolic responses to infection is not known. Aim: We hypothesise that IRF5 directs AM phenotype by metabolic reprogramming in response to infection. Methods: To investigate the role of IRF5 in the regulation of AM metabolism, disease pathology and inflammation, we infected WT and Irf5-/- mice with influenza. Moreover, bone marrow derived macrophages (BMDMs) and primary AMs were stimulated with TLR-agonists. To analyse the metabolic capacity of AMs, expression of pro-inflammatory/metabolic genes were assessed as well as the metabolic profile using an extracellular flux (Seahorse) assay. Results: Influenza-infected Irf5-/- mice had impaired myeloid cell responses in comparison to WT controls, characterised by decreased neutrophilia and increased eosinophilia. Analysis of IRF5 ChiP-Seq data and open chromatin regions (OCRs) revealed frequent binding of IRF5 to OCRs of pro-inflammatory mediator and metabolism genes. Moreover, Irf5-/- AMs but not BMDMs showed reduced mitochondrial oxygen consumption and extracellular acidification rate upon TLR3 stimulation or viral infection in comparison to WT controls. Conclusion: In summary, our data reveal a critical role of IRF5 in the regulation of AM metabolism in response to infection.

Chronic hypersensitivity pneumonitis (CHP) is a scarring lung disease that arises following repeated exposure to antigens. While the lung microbiome is increasingly implicated in respiratory disease, to date, no study has investigated the composition of microbial communities and metabolites in the lower airways in CHP. We prospectively recruited individuals diagnosed with CHP (n=110), idiopathic pulmonary fibrosis (IPF) (n=45) and controls (n=28). Subjects underwent bronchoalveolar lavage with differential cell counts. Bacterial DNA was isolated, quantified by qPCR and 16S rRNA gene sequencing performed to characterise bacterial communities. 1H Nuclear Magnetic Resonance spectroscopy was utilised to capture the metabolic profile of these individuals. Lymphocyte and neutrophil proportions were higher in CHP and IPF, respectively. Evident alterations in the composition and microbial community structure (β-diversity) of the respiratory microbiome were observed in CHP compared to IPF; Staphylococcus was increased in CHP and Actinomyces and Veillonella in IPF. There was an increased bacterial burden in cases compared to controls. Individuals with CHP had significantly reduced α-diversity and bacterial load compared to subjects with IPF. Metabolomics analysis revealed decreased levels of the short-chain fatty acids acetate, propionate and butyrate in CHP compared to IPF. Subjects with CHP and IPF displayed an altered microbiome and a distinct metabolic signature in the lower airways. Collectively, differences in specific bacterial genera may provide a low-level antigenic stimulus for repetitive alveolar injury. Further elucidation of these differences will improve our understanding of crosstalk between microbe and host and its role in the progression of fibrosis.

Introduction a Objectives: Airway macrophages (AMs) are implicated in the dysregulated wound healing response in idiopathic pulmonary fibrosis (IPF). The endogenous metabolite itaconate, synthesised by immune-response gene 1 (IRG1), has been shown to influence macrophage function via regulation of succinate dehydrogenase. Here we investigated the role of itaconate in pulmonary fibrosis. Methods: To assess the expression pattern of Irg1 in the lung, we analysed gene expression in AMs from IPF patients or controls. To investigate the role of itaconate in the bleomycin model of pulmonary fibrosis we utilised WT and Irg1-/- mice, in addition to therapeutic dosing of exogenous itaconate. To determine the role of secreted itaconate on the stromal compartment in IPF, primary lung fibroblasts were cultured with itaconate in vitro and proliferation/wound healing was assessed. Results: Irg1 expression was reduced in IPF AMs compared to controls. In the bleomycin model, Irg1-/- mice had decreased survival, worsened lung function and increased collagen deposition at the resolution time point (42d post bleomycin). Tissue-resident AMs (Tr-AMs) upregulated the expression of fibrosis-related genes in Irg1-/- mice and showed impaired Oxidative Phosphorylation. Inhaled itaconate during the fibrotic phase of the bleomycin model improved lung function and decreased gene expression of type IV collagen and fibronectin. In vitro culture of primary human lung fibroblasts with itaconate decreased proliferation and wound healing capacity. Conclusions: Overall these data indicate that itaconate is essential for the resolution of lung fibrosis and that targeting this pathway may be a viable therapeutic strategy in IPF.

We characterized monocyte/macrophage populations from infants to older adults and lineage traced these cells using sex-mismatched lung transplant recipients. Classical monocytes peak in adulthood and decline in older adults. Importantly, our data show that after transplant, donor human airway macrophages (AMs) are readily replaced by recipient-derived monocytes., The ontogeny of airway macrophages (AMs) in human lung and their contribution to disease are poorly mapped out. In mice, aging is associated with an increasing proportion of peripherally, as opposed to perinatally derived AMs. We sought to understand AM ontogeny in human lung during healthy aging and after transplant. We characterized monocyte/macrophage populations from the peripheral blood and airways of healthy volunteers across infancy/childhood (2–12 yr), maturity (20–50 yr), and older adulthood (>50 yr). Single-cell RNA sequencing (scRNA-seq) was performed on airway inflammatory cells isolated from sex-mismatched lung transplant recipients. During healthy aging, the proportions of blood and bronchoalveolar lavage (BAL) classical monocytes peak in adulthood and decline in older adults. scRNA-seq of BAL cells from lung transplant recipients indicates that after transplant, the majority of AMs are recipient derived. These data show that during aging, the peripheral monocyte phenotype is consistent with that found in the airways and, furthermore, that the majority of human AMs after transplant are derived from circulating monocytes.

Introduction and objectives Idiopathic pulmonary fibrosis (IPF) is a devastating disease with limited therapeutic options. Airway macrophages (AMs) are key components of airway defence and are implicated in the dysregulated wound healing underlying IPF. Itaconate is an endogenous metabolite with antimicrobial and anti-inflammatory potential. Synthesis of itaconate is catalysed by immune-response gene 1 (Irg1) and Irg1/itaconate are increased in macrophages upon stimulation with LPS1. We hypothesised that the expression of Irg1/itaconate in AMs is involved in the pathogenesis/resolution of pulmonary fibrosis and manipulation of this pathway could ameliorate disease. Methods To assess the distribution and expression pattern of Irg1 in healthy/IPF lung, we employed gene expression analysis in AMs, bronchial epithelial cells and lung fibroblasts from IPF patients or controls. To mechanistically interrogate the role of Irg1 in the bleomycin model of pulmonary fibrosis we utilised mice expressing, or lacking Irg1, in addition to therapeutic dosing of exogenous itaconate. Finally, to determine the role of secreted itaconate on the stromal compartment in IPF, primary lung fibroblasts were cultured with itaconate in vitro and proliferation/wound healing was assessed. Results Irg1 was expressed in AMs, but not epithelial cells or fibroblasts from healthy controls/IPF patients; interestingly, IPF AMs showed reduced expression of Irg1 compared to controls. In the bleomycin model, Irg1-/- mice had decreased survival, worsened lung function, and increased collagen deposition at the resolution time point (42d post bleomycin) compared to WT mice. Monocyte-recruited AMs (Mo-AMs) showed higher expression of Irg1 compared to tissue-resident AMs (Tr-AMs). Tr-AMs significantly upregulated the expression of fibrosis-related genes in Irg1-/- compared to WT, while the functional phenotype of monocyte-recruited AMs was not affected by Irg1-/-. Treatment with itaconate during the fibrotic phase of the bleomycin model improved lung function and decreased gene expression of type IV collagen and fibronectin. In vitro culture of primary human lung fibroblasts with itaconate decreased proliferation and wound healing capacity. Conclusions Taken together these data indicate that Irg1-expressing Mo-AMs are essential for the resolution of lung fibrosis and that targeting this pathway may be a viable therapeutic strategy in IPF. Reference Lampropoulou, Vicky, et al. ( 2016) Cell metabolism.

Introduction Forced vital capacity is the only registrational endpoint in idiopathic pulmonary fibrosis clinical trials. As most new treatments will be administered on top of standard of care, estimating treatment response will become more challenging. We developed a simulation model to quantify variability associated with forced vital capacity decline. Methods The model is based on publicly available clinical trial summary and home spirometry data. A single, illustrative trial setting is reported. Model assumptions are 400 subjects randomised 1:1 to investigational drug or placebo over 52 weeks, 50% of each group receiving standard of care (all-comer population), and a 90-mL treatment difference in annual forced vital capacity decline. Longitudinal profiles were simulated and the impact of varying clinical scenarios evaluated. Results Power to detect a significant treatment difference was 87–97%, depending on the analysis method. Repeated measures analysis generally outperformed analysis of covariance and mixed linear models, particularly with missing data (as simulated data were non-linear). A 15% yearly random dropout rate led to 0.6–5% power loss. Forced vital capacity decline-related dropout introduced greater power loss (up to 12%), as did subjects starting/stopping standard of care or investigational drug. Power was substantially lower for a 26-week trial due to the smaller assumed treatment effect at week 26 (sample size would need doubling to reach a power similar to that of a 52-week trial). Conclusions Our model quantifies forced vital capacity decline and associated variability, with all the caveats of background therapy, permitting robust power calculations to inform future idiopathic pulmonary fibrosis clinical trial design. Funding Galapagos NV (Mechelen, Belgium). Electronic supplementary material The online version of this article (10.1007/s12325-019-01093-3) contains supplementary material, which is available to authorized users.

Interferon regulatory factor 5 (IRF5) is a key transcription factor involved in the control of the expression of pro-inflammatory cytokine and responses to infection, however its role in regulating pulmonary immune responses to allergen is unknown. We used genetic ablation, adenoviral vector-driven overexpression and adoptive transfer approaches to interrogate the role of IRF5 in pulmonary immunity and during challenge with the aero-allergen, house dust mite. Global IRF5 deficiency resulted in impaired lung function and extracellular matrix (ECM) deposition. IRF5 was also essential for effective responses to inhaled allergen, controlling airway hyper- responsiveness, mucus secretion and eosinophilic inflammation. Adoptive transfer of IRF5- deficient alveolar macrophages into the WT pulmonary milieu was sufficient to drive airway hyper-reactivity, at baseline or following antigen challenge. These data identify IRF5-expressing macrophages as a key component of the immune defence of the airways. Manipulation of IRF5 activity in the lung could therefore be a viable strategy for the redirection of pulmonary immune responses and thus, the treatment of lung disorders.

Introduction: Cases of anaphylaxis in children are often not appropriately managed by caregivers. We aimed to develop and to test the effectiveness of an education tool to help pediatric patients and their families better understand anaphylaxis and its management and to improve current knowledge and treatment guidelines adherence. Methods: The GEAR (Guidelines and Educational programs based on an Anaphylaxis Registry) is an initiative that recruits children with food-induced anaphylaxis who have visited the ED at the Montreal Children's Hospital and at The Children's Clinic located in Montreal, Quebec. The patients and parents, together, were asked to complete six questions related to the triggers, recognition and management of anaphylaxis at the time of presentation to the allergy clinic. Participants were automatically shown a 5-minute animated video addressing the main knowledge gaps related to the causes and management of anaphylaxis. At the end of the video, participants were redirected to same 6 questions to respond again. To test long-term knowledge retention, the questionnaire will be presented again in one year's time. A paired t-test was used to compare the difference between the baseline score and the follow-up score based on percentage of correct answers of the questionnaire. Results: From June to November 2019, 95 pediatric patients with diagnosed food-induced anaphylaxis were recruited. The median patient age was 4.5 years (Interquartile Range (IQR): 1.6–7.4) and half were male (51.6%). The mean questionnaire baseline score was 0.77 (77.0%, standard deviation (sd): 0.16) and the mean questionnaire follow-up score was 0.83 (83.0%, sd: 0.17). There was a significant difference between the follow-up score and baseline score (difference: 0.06, 95% CI: 0.04, 0.09). There were no associations of baseline questionnaire scores and change in scores with age and sex. Conclusion: Our video teaching method was successful in educating patients and their families to better understand anaphylaxis. The next step is to acquire long-term follow up scored to determine retention of knowledge.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease where invasive pulmonary myofibroblasts secrete collagen and destroy lung integrity. Here, we show that interleukin-11 (IL11) is up-regulated in the lung of patients with IPF, associated with disease severity, and IL-11 is secreted from IPF fibroblasts. In vitro, IL-11 stimulates lung fibroblasts to become invasive actin alpha 2, smooth muscle-positive (ACTA2+), collagen-secreting myofibroblasts in an extracellular signal-regulated kinase (ERK)-dependent, posttranscriptional manner. In mice, fibroblast-specific transgenic expression or administration of murine IL-11 induces lung myofibroblasts and causes lung fibrosis. IL-11 receptor subunit alpha-1 (Il11ra1)-deleted mice, whose lung fibroblasts are unresponsive to profibrotic stimulation, are protected from fibrosis in the bleomycin mouse model of pulmonary fibrosis. We generated an IL-11-neutralizing antibody that blocks lung fibroblast activation downstream of multiple stimuli and reverses myofibroblast activation. In therapeutic studies, anti-IL-11 treatment diminished lung inflammation and reversed lung fibrosis while inhibiting ERK and SMAD activation in mice. These data prioritize IL-11 as a drug target for lung fibrosis and IPF.

A histamine metabolite, imidazole acetic acid (IAA), recapitulates key histamine-driven biology, including recruitment of eosinophils, induction of itch, and induction of anaphylaxis. IAA may perpetuate anaphylactic and other allergic responses after the initial release and metabolism of histamine.

Airway hyperresponsiveness (AHR) is a critical feature of wheezing and asthma in children, but the initiating immune mechanisms remain unconfirmed. We demonstrate that both recombinant interleukin-33 (rIL-33) and allergen [ house dust mite (HDM) or Alternaria alternata] exposure from day 3 of life resulted in significantly increased pulmonary IL-13(+)CD4(+) T cells, which were indispensable for the development of AHR. In contrast, adult mice had a predominance of pulmonary Lin(neg)CD45(+)CD90(+)IL-13(+) type 2 innate lymphoid cells (ILC2s) after administration of rIL-33. HDM exposure of neonatal IL-33 knockout (KO) mice still resulted in AHR. However, neonatal CD4(cre)IL-13 KO mice (lacking IL-13(+)CD4(+) T cells) exposed to allergen from day 3 of life were protected from AHR despite persistent pulmonary eosinophilia, elevated IL-33 levels, and IL-13(+) ILCs. Moreover, neonatal mice were protected from AHR when inhaled Acinetobacter lwoffii (an environmental bacterial isolate found in cattle farms, which is known to protect from childhood asthma) was administered concurrent with HDM. A. lwoffii blocked the expansion of pulmonary IL-13(+)CD4(+) T cells, whereas IL-13(+) ILCs and IL-33 remained elevated. Administration of A. lwoffii mirrored the findings from the CD4(cre)IL-13 KO mice, providing a translational approach for disease protection in early life. These data demonstrate that IL-13(+)CD4(+) T cells, rather than IL-13(+) ILCs or IL-33, are critical for inception of allergic AHR in early life.

Idiopathic pulmonary fibrosis (IPF) remains a progressive disease despite best medical management. We previously identified IL-11 as a critical factor for cardiovascular fibrosis and examine here its role in pulmonary fibrosis.IL-11is consistently upregulated in IPF genomic datasets, which we confirmed by histology. Pulmonary fibroblasts stimulated with IL-11 transform into invasive myofibroblasts whereas fibroblasts fromIl11radeleted mice did not respond to pro-fibrotic stimuli. In the mouse, injection of recombinant Il-11 or fibroblast-specific expression of Il-11 caused pulmonary fibrosis. We then generated a neutralising IL-11 binding antibody that blocks lung fibroblast activation across species. In a mouse model of IPF, anti-IL-11 therapy attenuated lung fibrosis and specifically blocked Erk activation. These data prioritise IL-11 as an accessible drug target in IPF.One Sentence SummaryNon-canonical IL-11 signalling is a central hallmark of idiopathic pulmonary fibrosis and represents a novel target for antibody therapies.

Macrophages are the most numerous immune-cells present in the lung environment under homoeostatic conditions and are ideally positioned to dictate the innate defence of the airways. Pulmonary macrophage populations are heterogeneous and demonstrate remarkable plasticity, owing to variations in origin, tissue residency and environmental influences. Lung macrophage diversity facilitates considerable specialisation, aids efficient responses to environmental signals and allows rapid alterations in phenotype and physiology in response to a plethora of cytokines and microbial signals. This review describes pulmonary macrophage origins, phenotypes, roles in diseases of the airways and implications for the treatment of respiratory disease.

Blazek et al. demonstrate that treatment with IL-28A reduces inflammation in collagen-induced arthritis by restricting the recruitment of IL-1β+ neutrophils., The most studied biological role of type III interferons (IFNs) has so far been their antiviral activity, but their role in autoimmune and inflammatory diseases remains largely unexplored. Here, we show that treatment with IFN-λ2/IL-28A completely halts and reverses the development of collagen-induced arthritis (CIA) and discover cellular and molecular mechanisms of IL-28A antiinflammatory function. We demonstrate that treatment with IL-28A dramatically reduces numbers of proinflammatory IL-17–producing Th17 and γδ T cells in the joints and inguinal lymph nodes, without affecting T cell proliferative responses or levels of anticollagen antibodies. IL-28A exerts its antiinflammatory effect by restricting recruitment of IL-1b–expressing neutrophils, which are important for amplification of inflammation. We identify neutrophils as cells expressing high levels of IFN-λ receptor 1 (IFNLR1)–IL-28 receptor α (IL28RA) and targeted by IL-28A. Our data highlight neutrophils as contributors to the pathogenesis of autoimmune arthritis and present IFN-λs or agonists of IFNLR1–IL28RA as putative new therapeutics for neutrophil-driven inflammation.

Pulmonary fibrosis (PF) is a growing clinical problem which can result in breathlessness or respiratory failure and has an average life expectancy of 3 years from diagnosis. Therapeutic options for PF are limited and there is therefore a significant unmet clinical need. The recent resurgent interest in macrophage biology has led to a new understanding of lung macrophage origins, biology, and phenotypes. In this review we discuss fibrotic mechanisms and focus on the role of macrophages during fibrotic lung disease. Data from both human and murine studies are reviewed, highlighting novel macrophage-orientated biomarkers for disease diagnosis and potential targets for future anti-fibrotic therapies.

Summary Background The pathophysiology of cellulite involves changes in the subcutaneous adipose layer and the extracellular matrix (ECM) that supports it together with overlying dermal layer. Cellular mechanisms governing cellulite are not fully understood. However, it is accepted that changes include enhanced lipogenesis, decreased lipolysis, and increased lipid storage within the adipocytes as well as changes in the dermal architecture. Aim In our studies the ability of cosmetic agents Furcellaria lumbricalis, Fucus vesiculosus, retinoid, conjugated linoleic acid (CLA), and a glaucine mixture to stimulate in vitro1) lipolysis in human adipocytes and 2) production of pro-collagen I by fibroblasts was investigated in vitro. The ability of these ingredients to improve cellulite condition in vivo was also determined. Patients/Methods Mature adipocytes and 'aged' fibroblasts were used for in vitro studies. The assessment of cellulite in vivo was performed by dermatological grading and ultrasound measurements. Results Mature adipocytes treated with combined actives resulted in a significant synergistic increase in free glycerol release. On “aged” fibroblasts, combined treatment of F. vesiculosus and F. lumbricalis stimulated pro-collagen I production. CLA increased pro-collagen I production, but the glaucine mixture had no effect. The clinical study demonstrated a significant improvement in cellulite grading by a dermatologist after 8 and 12 weeks vs. vehicle, and ultrasound imaging showed a significant decrease in fat thickness compared with placebo after 12 weeks. Conclusions Our studies revealed a potent cocktail of ingredients that when combined together can act in vitro to markedly improve lipolysis mechanisms and by way of stimulating pro-collagen I can also have an effect on the surrounding extracellular matrix. The in vitro actions of the ingredients were translated in vivo, where a clinical improvement of cellulite condition was observed.

In an investigation of 4-amino-3,4-dihydronaphthalen-1(2H)-ones as novel modulators of allergic and inflammatory phenomena, we have investigated a series of cyclic analogues. Tertiary amines of structural types 9, 10, 20 and 21 were synthesised and evaluated for mast cell stabilising activity. In vitro and in vivo studies showed that of these compounds, the cyclohexenylamino derivatives of tetralone and benzosuberone of series 20 and 21 exhibited interesting activity both in vitro and in vivo.

Summary Background Anti-aging effects of high concentrations of salicylic acid (SA) peels are commonly known. Like all acids, SA can produce somatosensory and visible irritation to the skin and as such may be unsuitable for subjects with sensitive skin. Aims To provide evidence that sodium salicylate (SS) obtained from neutralization of 1% SA by sodium hydroxide can deliver significant anti-aging benefits. Methods The effects of SS were examined using three approaches: (1) evaluating its effects on stimulating the synthesis of fibrillin and collagen-1 in vivo; (2) examining its efficacy by using Fast Optical in vivo Topometry (FOITS) in a double-blind, placebo-controlled clinical study; (3) determining its effects on both expert and naive grader assessement of wrinkles in a double-blind, placebo-controlled study. Results In the first study SS produced significant increases of the fibrillin and collagen-1 anti-aging biomarkers compared with the untreated skin control. A commercially available retinol cream delivered similar effects to SS. In the second study using FOITS we showed that the SS formulation significantly reduced wrinkle depth (Rz) and skin roughness (Ra) after 4 and 8 weeks of daily application vs. placebo (Rz: −8.2 ± 1.40% and −11.4 ± 1.07%; Ra: −7.8 ± 1.33% and −11.9 ± 0.61%; P

Basement membranes are thin structures present in the extracellular matrix that provide a supporting framework on which epithelial and endothelial cells reside. Type IV collagen is present ubiquitously in all basement membranes and plays an important role in cell adhesion, migration differentiation, and growth. These are especially important at the dermoepidermal junction (DEJ) in skin. A reduction in the levels of DEJ proteins occurs in photodamaged skin and especially Type IV collagen at the base of a wrinkle. In these studies, the ability of a triple peptide complex (TPC) to stimulate the production of collagen IV in human skin fibroblasts and its effects on photoaged skin was investigated. Fibroblasts, matured to represent "aged" cells, were stimulated for 72 h with the TPC as well as the three individual peptides constituting the complex, and collagen IV production by the fibroblasts was determined immunochemically. The results show that stimulation with the individual peptides at doses found in 1% (v/v) of the TPC did not result in soluble collagen IV production above levels detected by the non-stimulated cells. However, after stimulation with 1% (v/v) of the TPC, collagen IV was produced by the cells (1.4 ng/ng total protein +/- 0.4 SD, n = 5) when compared to control un-stimulated cells (0.32 ng/ng total protein +/- 0.1 SD, n = 5). This indicates that the combination of the individual peptides is necessary to synergistically stimulate collagen IV production. These findings suggest that the TPC could play a role in the strengthening of the DEJ through its ability to produce collagen IV. In order to determine whether these results translated into significant effects in vivo, we performed two studies. In the first four-week study, a double blind, placebo-controlled and fully randomized clinical study on 22 healthy Caucasian volunteers displaying moderate periorbital wrinkles, a significant reduction in wrinkle parameters determined by profilometry was observed over the 4-week period in comparison to the placebo. This result was reproduced in a 12-week monadic study which also showed improvements in expertly graded wrinkle scores. Collectively, these results effectively demonstrate the anti-aging applications of the TPC.

Whereas the importance of macrophages in chronic inflammatory diseases is well recognized, there is an increasing awareness that neutrophils may also play an important role. In addition to the well-documented heterogeneity of macrophage phenotypes and functions, neutrophils also show remarkable phenotypic diversity among tissues. Understanding the molecular pathways that control this heterogeneity should provide abundant scope for the generation of more specific and effective therapeutics. We have shown that the transcription factor IFN regulatory factor 5 (IRF5) polarizes macrophages toward an inflammatory phenotype. IRF5 is also expressed in other myeloid cells, including neutrophils, where it was linked to neutrophil function. In this study we explored the role of IRF5 in models of acute inflammation, including antigen-induced inflammatory arthritis and lung injury, both involving an extensive influx of neutrophils. Mice lacking IRF5 accumulate far fewer neutrophils at the site of inflammation due to the reduced levels of chemokines important for neutrophil recruitment, such as the chemokine (C-X-C motif) ligand 1. Furthermore we found that neutrophils express little IRF5 in the joints and that their migratory properties are not affected by the IRF5 deficiency. These studies extend prior ones suggesting that inhibiting IRF5 might be useful for chronic macrophage-induced inflammation and suggest that IRF5 blockade would ameliorate more acute forms of inflammation, including lung injury.

Background Current data concerning maternal paracetamol intake during pregnancy, or intake during infancy and risk of wheezing or asthma in childhood is inconclusive based on epidemiological studies. We have investigated whether there is a causal link between maternal paracetamol intake during pregnancy and lactation and the development of house dust mite (HDM) induced allergic airways disease (AAD) in offspring using a neonatal mouse model.\ud \ud Methods Pregnant mice were administered paracetamol or saline by oral gavage from the day of mating throughout pregnancy and/or lactation. Subsequently, their pups were exposed to intranasal HDM or saline from day 3 of life for up to 6 weeks. Assessments of airway hyper-responsiveness, inflammation and remodelling were made at weaning (3 weeks) and 6 weeks of age.\ud \ud Results Maternal paracetamol exposure either during pregnancy and/or lactation did not affect development of AAD in offspring at weaning or at 6 weeks. There were no effects of maternal paracetamol at any time point on airway remodelling or IgE levels.\ud \ud Conclusions Maternal paracetamol did not enhance HDM induced AAD in offspring. Our mechanistic data do not support the hypothesis that prenatal paracetamol exposure increases the risk of childhood asthma.