JanWillem Duitman

Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a proteolytic enzyme that may act on a broad spectrum of substrates, including growth factors, extracellular proteases, adhesion molecules, protease activated receptor 1, the prorenin receptor, and Corin. As a consequence of its broad-spectrum substrate activity, PCSK6 is implicated in a wide range of pathophysiological processes such as cardiovascular disease, neurological disorders, arthritis and cancer. Of special interest, PCSK6 has recently been shown to be highly expressed in patients with idiopathic pulmonary fibrosis (IPF) and PCSK6 has been associated with reduced survival of IPF patients, linking PCSK6 to pulmonary fibrosis for the first time. Of note however, PCSK6 levels in plasma were not associated with respiratory outcomes in IPF patients and higher PCSK6 levels were even associated with a lower hazard of 10% FVC decline in non-IPF ILD patients. In this review, we summarize the molecular biology of PCSK6, including its structural features and its major (potential) substrates, and outline its pathophysiological roles across various diseases. Furthermore, we propose and discuss the potential involvement of PCSK6 in pulmonary fibrosis to guide future research.

Extract Interstitial lung abnormalities (ILA) are incidental CT findings, characterised by non-dependent parenchymal abnormalities -as ground-glass opacities, reticulation, and traction bronchiectasis- affecting at least 5% of a lung zone [1]. Their detection is increasing with widespread CT use in e.g. cancer screening and cardiovascular diagnostic imaging. Prevalence ranges from 2 to 35%, occurring commonly in older individuals, smokers, and those with a family history of interstitial lung disease (ILD).

Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a proteolytic enzyme that acts on a broad spectrum of substrates, including growth factors, extracellular proteases, adhesion molecules, protease activated receptor 1, the prorenin receptor, and Corin. As a consequence of its broad spectrum substrate activity, PCSK6 is implicated in a wide range of pathophysiological processes like cardiovascular disease, neurological disorders, arthritis and cancer. Of special interest, PCSK6 has recently been shown to be highly expressed in patients with idiopathic pulmonary fibrosis (IPF) and PCSK6 was associated with reduced survival, providing the first evidence linking PCSK6 to pulmonary fibrosis. In this review, we summarize the molecular biology of PCSK6, including its structural features and major substrates, and outline its pathophysiological roles across various diseases. Furthermore, we propose and discuss the potential involvement of PCSK6 in pulmonary fibrosis to guide future research.

A recent genome-wide association study showed that the protease proprotein convertase subtilisin/kexin type 6 (PCSK6) is highly expressed in idiopathic pulmonary fibrosis (IPF) lung parenchyma and that its expression is associated with disease progression and worse survival. However, whether PCSK6 plays a role in IPF pathophysiology remains elusive. This study aimed to determine whether PCSK6 contributes to IPF pathophysiology, specifically in the cross-talk between epithelial cells and fibroblasts. A549 epithelial cells were transduced with a PCSK6-GFP or control-mCherry vector. Both proliferation (crystal violet) and cell competition assays showed that PCSK6 promoted cell proliferation. Western blot and PCSK-specific fluorogenic substrate assays showed that A549-PCSK6 conditioned medium (CM) had higher PCSK6 enzyme activity compared to mCherry control A549 CM. Human lung fibroblasts stimulated with PCSK6-CM significantly decreased collagen I protein levels as compared to fibroblasts stimulated with control A549-CM. Matrix-metalloproteinase (MMP) specific fluorogenic substrate assays subsequently showed that A549-PCSK6 CM contained higher MMP activity and that PCSK6 inhibition reduced MMP activity in A549-PCSK6 CM, suggesting that PCSK6 plays a role in the activation of MMPs that may degrade collagen type I. In conclusion, epithelial PCSK6 promotes cell proliferation and decreases collagen deposition by fibroblasts, potentially via MMP activation. These in vitro data suggest that PCSK6 could play a dual role in IPF progression and its actual role in IPF should consequently be elucidated using in vivo/ex vivo models.

Interstitial lung disease (ILD) encompasses pulmonary disorders characterised by varying degrees of inflammation and/or fibrosis. The presence and extent of these pulmonary abnormalities on (high-resolution) computed tomography (CT) have consequences for diagnosis and treatment; however, inter-observer assessment varies. Analysis of exhaled volatile organic compounds (VOCs) through gas chromatography-mass spectrometry (GC-MS) offers a noninvasive approach to biomarker discovery and pathophysiology understanding. Our study aims to explore the ability of GC-MS-driven exhaled breath analysis to differentiate ILD patients with predominant fibrotic, inflammatory, or a combination of fibrotic and inflammatory pulmonary abnormalities in a training and an external validation cohort. In a multicentre cross-sectional study, patients diagnosed with ILD were recruited. After central review of chest CT scans by independent radiologists, patients were categorised as fibrotic, inflammatory or mixed phenotype group based on the percentage of chest CT scan abnormalities. Breath samples were collected and analysed via GC-MS. Significantly different VOC fragments between groups were selected and used to differentiate groups in the training cohort with sparse partial least squares discriminant analysis. Analyses were validated with patients from an external cohort. 53 patients were included, 21 patients in the fibrotic, 14 in the inflammatory and 18 in the mixed phenotype group. Area under the curve (AUCs) for discrimination between groups ranged from 0.89–1.00 in training cohorts. An attempt to confirm these findings in our external validation cohort resulted in AUCs of 0.63–0.84. Re-evaluation of the training model led to an AUC of 0.78–0.83. This study shows that GC-MS driven exhaled breath analysis towards differentiation of ILD phenotypes is challenging. Current findings emphasise the importance of predefined validation steps during the process of biomarker discovery.

In this study, we provide a comprehensive characterization of the alveolar immune landscape in patients suffering from severe acute respiratory failure, predominantly caused by pneumonia or acute respiratory distress syndrome, conditions defined by intense pulmonary inflammation and immune dysregulation. Despite diverse underlying causes, the overall composition of alveolar immune cells remained largely consistent, with neutrophils and macrophages comprising the majority of cells. However, the maturation and activation states of immune cell subsets varied significantly, not only between patients with and without pneumonia, but also among pneumonia cases stratified by pathogen type. We also observed dynamic shifts in immune cell subsets over the disease course and found that an increased proportion of CD123bright immature neutrophils and a reduction in alveolar resident macrophages were associated with increased 28-day mortality. Integration with alveolar cytokine profiles revealed tightly coordinated interactions between immune cells and the local cytokine milieu. These findings highlight the importance of assessing immune cell function, not merely abundance, through broad and longitudinal investigation to better understand the pathophysiology of acute respiratory failure and to guide precision immunomodulatory therapy.

In order to improve clinical care for chronic lung diseases, particularly personalised therapies, model systems that capture the complex cellular ecosystem of lung tissue, where diverse immune cell populations interact with stromal cells and targets such as cancer cells, viruses, or bacteria within the alveolar architecture [1, 2], are eagerly awaited. Human tissue-based approaches such as precision-cut lung slices (PCLS) from surgically resected tumour lesions retain key features of the lung tissue microenvironment, including its architecture, mechanical properties, and diverse resident cell populations [3–5] making them suitable to test novel therapeutic agents [2, 6–8]. Further development of such technologies may contribute to the advancement of understanding the underlying disease mechanisms and improving therapeutic options for the treatment of chronic lung disease patients.

Human rhinovirus 16 (HRV-16) is a major cause of common colds and can exacerbate asthma and COPD, yet no approved antiviral treatments exist. Heparin, a highly sulfated polysaccharide, is known to block viral infection of many viruses that require attachment to heparan sulfate proteoglycans (HSPGs). Here, we investigated whether heparin inhibits HRV-16 infection. HRV-16 uses ICAM-1 as its attachment receptor and lacks a confirmed HSPG-binding mechanism. Notably, heparin inhibited HRV-16 infection in vitro in a dose- and time-dependent manner. Pre-treatment of either cells or virus particles with unfractionated heparin significantly reduced HRV-16 RNA expression at 24 and 48 h post-infection. In contrast, low-molecular-weight heparins blocked infection of HRV-16 significantly less effectively compared to unfractionated heparins. Our findings suggest that the inhibitory effect of unfractionated heparin on HRV-16 infection is likely independent of specific HSPGs interactions and may be mediated by the size and highly negative charge of unfractionated heparin. Importantly, the ability of unfractionated heparin to block viruses that do not require HSPGs for attachment implies a broader antiviral potential as a prophylactic or therapeutic agent against a variety of respiratory viruses.

The clinical management of systemic sclerosis-associated interstitial lung disease (SSc-ILD) is challenging due to its heterogeneous progression. While recent studies have shown that pulmonary uptake of 18F-FDG and 68Ga-FAPI on positron emission tomography (PET) may indicate ILD activity, the spatial distribution of their molecular targets—GLUT1, GLUT3, and FAPα—in SSc-ILD lung tissue is largely unexplored. Here, we investigate the expression patterns of these markers across varying fibrosis stages in lung tissue of SSc-ILD patients and controls to improve PET interpretation. Immunohistochemistry for GLUT1, GLUT3, and FAPα was performed on lung tissue from SSc-ILD patients (n = 9) and controls (n = 8). Fibrosis stage and cell-type-specific expression of these markers were analyzed by 2 experienced ILD pathologists, and quantitative staining was assessed using QuPath software. Marker expression was analyzed in and compared between areas with varying fibrosis stages. GLUT1 positive cells were primarily erythrocytes, GLUT3 positive cells were mainly myeloid cells and FAPα positive cells were predominantly fibroblasts, endothelial cells, epithelial cells, and macrophages. All cells positive for these markers were more present in SSc-ILD compared to controls, with GLUT1 and FAPα positive cells showing highest prevalence in areas with early signs of fibrosis. The prevalence of cells expressing GLUT1 and FAPα is increased in areas with early signs of fibrosis and the expression of GLUT1, GLUT3 and FAPα is observed in different cell types suggesting that 18F-FDG and 68Ga-FAPI PET each may be useful to visualize different aspects of ILD activity.