Lung extracellular matrix modulates KRT5+ basal cell activity in pulmonary fibrosis.

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 migration in 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.

CYFRA 21-1 Predicts Progression in Idiopathic Pulmonary Fibrosis: A Prospective Longitudinal Analysis of the PROFILE Cohort.

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 < 0.0001) and validation (n = 359) (control: 2.21 ± 1.54 ng/ml; and IPF: 4.13 ± 2.77 ng/ml; P < 0.0001) cohorts. Baseline concentrations of CYFRA 21-1 were able to distinguish individuals at risk of 12-month disease progression (C-statistic, 0.70; 95% confidence interval, 0.61-0.79; P < 0.0001) and were predictive of overall mortality (hazard ratio, 1.12 [95% confidence interval, 1.06-1.19] per 1 ng/ml increase in CYFRA 21-1; P = 0.0001). Furthermore, 3-month change in concentrations of CYFRA 21-1 separately predicted 12-month and overall survival in both the discovery and validation cohorts. Conclusions: CYFRA 21-1, a marker of epithelial damage and turnover, has the potential to be an important prognostic and therapeutic biomarker in individuals with IPF.

A glutamine 'tug-of-war': targets to manipulate glutamine metabolism for cancer immunotherapy.

Within the tumour microenvironment (TME), there is a cellular 'tug-of-war' for glutamine, the most abundant amino acid in the body. This competition is most evident when considering the balance between a successful anti-tumour immune response and the uncontrolled growth of tumour cells that are addicted to glutamine. The differential effects of manipulating glutamine abundance in individual cell types is an area of intense research and debate. Here, we discuss some of the current strategies in development altering local glutamine availability focusing on inhibition of enzymes involved in the utilisation of glutamine and its uptake by cells in the TME. Further studies are urgently needed to complete our understanding of glutamine metabolism, to provide critical insights into the pathways that represent promising targets and for the development of novel therapeutic strategies for the treatment of advanced or drug resistant cancers.

DNA Methylome Alterations Are Associated with Airway Macrophage Differentiation and Phenotype during Lung Fibrosis.

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.

The Respiratory Microbiome in Chronic Hypersensitivity Pneumonitis Is Distinct from That of Idiopathic Pulmonary Fibrosis.

Rationale: Chronic hypersensitivity pneumonitis (CHP) is a condition that arises after repeated exposure and sensitization 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.Objectives: To characterize and compare the airway microbiome in subjects with CHP, subjects with idiopathic pulmonary fibrosis (IPF), and control subjects.Methods: We prospectively recruited individuals with a CHP diagnosis (n = 110), individuals with an IPF diagnosis (n = 45), and control subjects (n = 28). Subjects underwent BAL and bacterial DNA was isolated, quantified by quantitative PCR and the 16S ribosomal RNA gene was sequenced to characterize the bacterial communities in the lower airways.Measurements and Main 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 subjects with IPF and subjects with CHP included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. However, in IPF, Firmicutes dominated, whereas the percentage of reads assigned to Proteobacteria in the same group was significantly lower than the percentage found in subjects with CHP. At the genus level, the Staphylococcus burden was increased in CHP, and Actinomyces and Veillonella burdens were increased in IPF. The lower airway bacterial burden in subjects with CHP was higher than that in control subjects but lower than that of 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, the bacterial burden in individuals with CHP fails to predict survival.

Macrophage metabolic reprogramming during chronic lung disease.

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.

Itaconate controls the severity of pulmonary fibrosis.

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.

Lung fibrosis enters the iron age†.

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.

Bacterial burden in the lower airways predicts disease progression in idiopathic pulmonary fibrosis and is independent of radiological disease extent.

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.

Modelling Forced Vital Capacity in Idiopathic Pulmonary Fibrosis: Optimising Trial Design.

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).

Interleukin-11 is a therapeutic target in idiopathic pulmonary fibrosis.

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.

The Transferrin Receptor CD71 Delineates Functionally Distinct Airway Macrophage Subsets during Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating progressive disease with limited therapeutic options. Airway macrophages (AMs) are key components of the defense of the airways and are implicated in the pathogenesis of IPF. Alterations in iron metabolism have been described during fibrotic lung disease and in murine models of lung fibrosis. However, the role of transferrin receptor 1 (CD71)-expressing AMs in IPF is not known. Objectives: To assess the role of CD71-expressing AMs in the IPF lung. Methods: We used multiparametric flow cytometry, gene expression analysis, and phagocytosis/transferrin uptake assays to delineate the role of AMs expressing or lacking CD71 in the BAL of patients with IPF and of healthy control subjects. Measurements and Main Results: There was a distinct increase in proportions of AMs lacking CD71 in patients with IPF compared with healthy control subjects. Concentrations of BAL transferrin were enhanced in IPF-BAL, and furthermore, CD71- AMs had an impaired ability to sequester transferrin. CD71+ and CD71- AMs were phenotypically, functionally, and transcriptionally distinct, with CD71- AMs characterized by reduced expression of markers of macrophage maturity, impaired phagocytosis, and enhanced expression of profibrotic genes. Importantly, proportions of AMs lacking CD71 were independently associated with worse survival, underlining the importance of this population in IPF and as a potential therapeutic target. Conclusions: Taken together, these data highlight how CD71 delineates AM subsets that play distinct roles in IPF and furthermore show that CD71- AMs may be an important pathogenic component of fibrotic lung disease.

Pulmonary Epithelial Cell-Derived Cytokine TGF-ß1 Is a Critical Cofactor for Enhanced Innate Lymphoid Cell Function.

Epithelial cells orchestrate pulmonary homeostasis and pathogen defense and play a crucial role in the initiation of allergic immune responses. Maintaining the balance between homeostasis and inappropriate immune activation and associated pathology is particularly complex at mucosal sites that are exposed to billions of potentially antigenic particles daily. We demonstrated that epithelial cell-derived cytokine TGF-ß had a central role in the generation of the pulmonary immune response. Mice that specifically lacked epithelial cell-derived TGF-ß1 displayed a reduction in type 2 innate lymphoid cells (ILCs), resulting in suppression of interleukin-13 and hallmark features of the allergic response including airway hyperreactivity. ILCs in the airway lumen were primed to respond to TGF-ß by expressing the receptor TGF-ßRII and ILC chemoactivity was enhanced by TGF-ß. These data demonstrate that resident epithelial cells instruct immune cells, highlighting the central role of the local environmental niche in defining the nature and magnitude of immune reactions.

IRF5 controls both acute and chronic inflammation.

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.

Pulmonary macrophages: key players in the innate defence of the airways.

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.

Lung macrophages contribute to house dust mite driven airway remodeling via HIF-1α.

UNLABELLED: HIF-1α is a transcription factor that is activated during hypoxia and inflammation and is a key regulator of angiogenesis in vivo. During the development of asthma, peribronchial angiogenesis is induced in response to aeroallergens and is thought to be an important feature of sustained chronic allergic inflammation. Recently, elevated HIF-1α levels have been demonstrated in both the lung tissue and bronchoalveolar lavage of allergic patients, respectively. Therefore, we investigated the role of HIF-1α on the development of angiogenesis and inflammation following acute and chronic allergen exposure. Our data shows that intranasal exposure to house dust mite (HDM) increases the expression of HIF-1α in the lung, whilst reducing the expression of the HIF-1α negative regulators, PHD1 and PHD3. Blockade of HIF-1α in vivo, significantly decreased allergic inflammation and eosinophilia induced by allergen, due to a reduction in the levels of IL-5 and Eotaxin-2. Importantly, HIF-1α blockade significantly decreased levels of VEGF-A and CXCL1 in the lungs, which in turn led to a profound decrease in the recruitment of endothelial progenitor cells and a reduction of peribronchial angiogenesis. Furthermore, HDM or IL-4 treatment of primary lung macrophages resulted in significant production of both VEGF-A and CXCL1; inhibition of HIF-1α activity abrogated the production of these factors via an up-regulation of PHD1 and PHD3. These findings suggest that novel strategies to reduce the expression and activation of HIF-1α in lung macrophages may be used to attenuate allergen-induced airway inflammation and angiogenesis through the modulation of VEGF-A and CXCL1 expression. CLINICAL RELEVANCE: This study provides new insights into the role of HIF-1α in the development of peribronchial angiogenesis and inflammation in a murine model of allergic airway disease. These findings indicate that strategies to reduce activation of macrophage derived HIF-1α may be used as a target to improve asthma pathology.

IRF5 is a specific marker of inflammatory macrophages in vivo.

Macrophages are an integral part of the innate immune system and key players in pathogen clearance and tissue remodelling. Both functions are accomplished by a pivotal network of different macrophage subtypes, including proinflammatory M1 and anti-inflammatory M2 macrophages. Previously, our laboratory identified the transcription factor interferon regulatory factor 5 (IRF5) as the master regulator of the M1 macrophage polarisation. IRF5 was found to be highly expressed in human M1 compared to M2 macrophages. Furthermore, IRF5 dictates the expression of proinflammatory genes such as IL12b and IL23a whilst repressing anti-inflammatory genes like IL10. Here we show that murine bone marrow derived macrophages differentiated in vitro with GM-CSF are also characterised by high levels of IRF5 mRNA and protein and express proinflammatory cytokines upon LPS stimulation. These macrophages display characteristic expression of M1-marker MHC II but lack the M2-marker CD206. Significantly, we develop intracellular staining of IRF5- expressing macrophages and utilise it to recapitulate the in vitro results in an in vivo model of antigen-induced arthritis, emphasising their physiological relevance. Thus, we establish the species-invariant role of IRF5 in controlling the inflammatory macrophage phenotype both in vitro and in in vivo.

Three clinical studies showing the anti-aging benefits of sodium salicylate in human skin.

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 naïve 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 < 0.01). In the third study reductions in wrinkle depth were observed by expert assessment at both 4 and 8 weeks for the SS-containing formulation compared to its placebo (P < 0.05). Equally, non-expert graders recorded the SS formulation superior to its placebo. CONCLUSION: Although the mechanism of action is not completely understood, we believe the benefits of SS are derived from its intrinsic stratum corneum exfoliation effects. All three studies demonstrate the significant anti-aging effects of SS that are especially suitable for subjects with sensitive skin.