A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets.

Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged-Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

An Inhibitory Function of TRPA1 Channels in TGF-ß1-driven Fibroblast-to-Myofibroblast Differentiation.

TRPA1 (transient receptor potential ankyrin 1) is a nonselective Ca2+-permeable cation channel, which was originally cloned from human lung fibroblasts (HLFs). TRPA1-mediated Ca2+ entry is evoked by exposure to several chemicals, including allyl isothiocyanate (AITC), and a protective effect of TRPA1 activation in the development of cardiac fibrosis has been proposed. Yet the function of TRPA1 in TGF-ß1 (transforming growth factor-ß1)-driven fibroblast-to-myofibroblast differentiation and the development of pulmonary fibrosis remains elusive. TRPA1 expression and function were analyzed in cultured primary HLFs, and mRNA concentrations were significantly reduced after adding TGF-ß1. Expression of genes encoding fibrosis markers (e.g., ACTA2, SERPINE1 [plasminogen activator inhibitor 1], FN1 [fibronectin], COL1A1 [type I collagen]) was increased after siRNA-mediated downregulation of TRPA1 mRNA in HLFs. Moreover, AITC-induced Ca2+ entry in HLFs was decreased after TGF-ß1 treatment and by application of TRPA1 siRNAs, while AITC treatment alone did not reduce cell viability or enhance apoptosis. Most interestingly, AITC-induced TRPA1 activation augmented ERK1/2 (extracellular signal-regulated kinase 1/2) and SMAD2 linker phosphorylation, which might inhibit TGF-ß-receptor signaling. Our results suggest an inhibitory function of TRPA1 channels in TGF-ß1-driven fibroblast-to-myofibroblast differentiation. Therefore, activation of TRPA1 channels might be protective during the development of pulmonary fibrosis in patients.

Antiviral CD8+ T-cell immune responses are impaired by cigarette smoke and in COPD.

BACKGROUND: Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8+ T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells. METHODS: We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasome in vitro, ex vivo and in vivo using RNA and Western blot analyses. CD8+ T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8+ T-cell numbers were determined in patients' peripheral blood using tetramer technology. RESULTS: Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cells in vitro, ex vivo and in vivo. In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8+ T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8+ T-cells compared to healthy controls and asthmatics. CONCLUSION: Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8+ T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections.

Assessment of Collagen in Translational Models of Lung Research.

The extracellular matrix (ECM) plays an important role in lung health and disease. Collagen is the main component of the lung ECM, widely used for the establishment of in vitro and organotypic models of lung disease, and as scaffold material of general interest for the field of lung bioengineering. Collagen also is the main readout for fibrotic lung disease, where collagen composition and molecular properties are drastically changed and ultimately result in dysfunctional "scarred" tissue. Because of the central role of collagen in lung disease, quantification, determination of molecular properties, and three-dimensional visualization of collagen is important for both development and characterization of translational models of lung research. In this chapter, we provide a comprehensive overview on the various methodologies currently available for quantification and characterization of collagen including their detection principles, advantages, and disadvantages.

Fighting the Fiber: Targeting Collagen in Lung Fibrosis.

Organ fibrosis is characterized by epithelial injury and aberrant tissue repair, where activated effector cells, mostly fibroblasts and myofibroblasts, excessively deposit collagen into the extracellular matrix. Fibrosis frequently results in organ failure and has been estimated to contribute to at least one-third of all global deaths. Also, lung fibrosis, in particular idiopathic pulmonary fibrosis (IPF), is a fatal disease with rising incidence worldwide. As current treatment options targeting fibrogenesis are insufficient, there is an urgent need for novel therapeutic strategies. During the last decade, several studies have proposed to target intra- and extracellular components of the collagen biosynthesis, maturation, and degradation machinery. This includes intra- and extracellular targets directly acting on collagen gene products, but also such that anabolize essential building blocks of collagen, in particular glycine (Gly) and proline (Pro) biosynthetic enzymes. Collagen, however, is a ubiquitous molecule in the body and fulfills essential functions as a macromolecular scaffold, growth factor reservoir, and receptor binding site in virtually every tissue. This review summarizes recent advances and future directions in this field. Evidence for the proposed therapeutic targets and where they currently stand in terms of clinical drug development for treatment of fibrotic disease is provided. The drug targets are furthermore discussed in light of 1) specificity for collagen biosynthesis, maturation, and degradation, and 2) specificity for disease-associated collagen. As therapeutic success and safety of these drugs may largely depend on targeted delivery, different strategies for specific delivery to the main effector cells and the extracellular matrix are discussed.

Validation of in vitro models for smoke exposure of primary human bronchial epithelial cells.

The bronchial epithelium is constantly challenged by inhalative insults including cigarette smoke (CS), a key risk factor for lung disease. In vitro exposure of bronchial epithelial cells using CS extract (CSE) is a widespread alternative to whole CS (wCS) exposure. However, CSE exposure protocols vary considerably between studies, precluding direct comparison of applied doses. Moreover, they are rarely validated in terms of physiological response in vivo and the relevance of the findings is often unclear. We tested six different exposure settings in primary human bronchial epithelial cells (phBECs), including five CSE protocols compared with wCS exposure. We quantified cell-delivered dose and directly compared all exposures using expression analysis of 10 well-established smoke-induced genes in bronchial epithelial cells. CSE exposure of phBECs was varied in terms of differentiation state, exposure route, duration of exposure, and dose. Gene expression was assessed by quantitative real-time PCR (qPCR) and Western Blot analysis. Cell type-specific expression of smoke-induced genes was analyzed by immunofluorescent analysis. Three surprisingly dissimilar exposure types, namely, chronic CSE treatment of differentiating phBECs, acute CSE treatment of submerged basal phBECs, and wCS exposure of differentiated phBECs performed best, resulting in significant upregulation of seven (chronic CSE) and six (acute wCS, acute submerged CSE exposure) out of 10 genes. Acute apical or basolateral exposure of differentiated phBECs with CSE was much less effective despite similar doses used. Our findings provide guidance for the design of human in vitro CS exposure models in experimental and translational lung research.

TRPM7 restrains plasmin activity and promotes transforming growth factor-ß1 signaling in primary human lung fibroblasts.

Sustained exposure of the lung to various environmental or occupational toxins may eventually lead to pulmonary fibrosis, a devastating disease with no cure. Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins such as fibronectin and collagens. The peptidase plasmin degrades the ECM, but protein levels of the plasmin activator inhibitor-1 (PAI-1) are increased in fibrotic lung tissue, thereby dampening plasmin activity. Transforming growth factor-ß1 (TGF-ß1)-induced activation of SMAD transcription factors promotes ECM deposition by enhancing collagen, fibronectin and PAI-1 levels in pulmonary fibroblasts. Hence, counteracting TGF-ß1-induced signaling is a promising approach for the therapy of pulmonary fibrosis. Transient receptor potential cation channel subfamily M Member 7 (TRPM7) supports TGF-ß1-promoted SMAD signaling in T-lymphocytes and the progression of fibrosis in kidney and heart. Thus, we investigated possible effects of TRPM7 on plasmin activity, ECM levels and TGF-ß1 signaling in primary human pulmonary fibroblasts (pHPF). We found that two structurally unrelated TRPM7 blockers enhanced plasmin activity and reduced fibronectin or PAI-1 protein levels in pHPF under basal conditions. Further, TRPM7 blockade strongly inhibited fibronectin and collagen deposition induced by sustained TGF-ß1 stimulation. In line with these data, inhibition of TRPM7 activity diminished TGF-ß1-triggered phosphorylation of SMAD-2, SMAD-3/4-dependent reporter activation and PAI-1 mRNA levels. Overall, we uncover TRPM7 as a novel supporter of TGF-ß1 signaling in pHPF and propose TRPM7 blockers as new candidates to control excessive ECM levels under pathophysiological conditions conducive to pulmonary fibrosis.

Cigarette smoke induces overexpression of active human cathepsin S in lungs from current smokers with or without COPD.

Cigarette smoking has marked effects on lung tissue, including induction of oxidative stress, inflammatory cell recruitment, and a protease/antiprotease imbalance. These effects contribute to tissue remodeling and destruction resulting in loss of lung function in chronic obstructive pulmonary disease (COPD) patients. Cathepsin S (CatS) is a cysteine protease that is involved in the remodeling/degradation of connective tissue and basement membrane. Aberrant expression or activity of CatS has been implicated in a variety of diseases, including arthritis, cancer, cardiovascular, and lung diseases. However, little is known about the effect of cigarette smoking on both CatS expression and activity, as well as its role in smoking-related lung diseases. Here, we evaluated the expression and activity of human CatS in lung tissues from never-smokers and smokers with or without COPD. Despite the presence of an oxidizing environment, CatS expression and activity were significantly higher in current smokers (both non-COPD and COPD) compared with never-smokers, and correlated positively with smoking history. Moreover, we found that the exposure of primary human bronchial epithelial cells to cigarette smoke extract triggered the activation of P2X7 receptors, which in turns drives CatS upregulation. The present data suggest that excessive CatS expression and activity contribute, beside other proteases, to the deleterious effects of cigarette smoke on pulmonary homeostasis.

Upregulation and Nuclear Location of MMP28 in Alveolar Epithelium of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive aging-associated disease of unknown etiology. A growing body of evidence indicates that aberrant activated alveolar epithelial cells induce the expansion and activation of the fibroblast population, leading to the destruction of the lung architecture. Some matrix metalloproteinases (MMPs) are upregulated in IPF, indicating that they may be important in the pathogenesis and/or progression of IPF. In the present study, we examined the expression of MMP28 in this disease and evaluated its functional effects in two alveolar epithelial cell lines and in human primary bronchial epithelial cells. We found that the enzyme is expressed in bronchial (apical and cytoplasmic localization) and alveolar (cytoplasmic and nuclear localization) epithelial cells in two different groups of patients with IPF. In vitro MMP28 epithelial silencing decreased the proliferation rate and delayed wound closing, whereas overexpression showed opposite effects, protecting from apoptosis and enhanced epithelial-mesenchymal transition. Our findings demonstrate that MMP28 is upregulated in epithelial cells from IPF lungs, where it may play a role in increasing the proliferative and migratory phenotype in a catalysis-dependent manner.

FK506-binding protein 10 (FKBP10) regulates lung fibroblast migration via collagen VI synthesis.

BACKGROUND: In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration. METHODS: Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-ß1 (TGF-ß1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays. RESULTS: FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration. CONCLUSIONS: These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF.

A Novel Antifibrotic Mechanism of Nintedanib and Pirfenidone. Inhibition of Collagen Fibril Assembly.

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix, in particular, collagens. Two IPF therapeutics, nintedanib and pirfenidone, decelerate lung function decline, but their underlying mechanisms of action are poorly understood. In this study, we sought to analyze their effects on collagen synthesis and maturation at important regulatory levels. Primary human fibroblasts from patients with IPF and healthy donors were treated with nintedanib (0.01-1.0 µM) or pirfenidone (100-1,000 µM) in the absence or presence of transforming growth factor-ß1. Effects on collagen, fibronectin, FKBP10, and HSP47 expression, and collagen I and III secretion, were analyzed by quantitative polymerase chain reaction and Western blot. The appearance of collagen fibrils was monitored by scanning electron microscopy, and the kinetics of collagen fibril assembly was assessed using a light-scattering approach. In IPF fibroblasts, nintedanib reduced the expression of collagen I and V, fibronectin, and FKBP10 and attenuated the secretion of collagen I and III. Pirfenidone also down-regulated collagen V but otherwise showed fewer and less pronounced effects. By and large, the effects were similar in donor fibroblasts. For both drugs, electron microscopy of IPF fibroblast cultures revealed fewer and thinner collagen fibrils compared with untreated controls. Finally, both drugs dose-dependently delayed fibril formation of purified collagen I. In summary, both drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in down-regulating profibrotic gene expression and collagen secretion. Importantly, both drugs inhibited collagen I fibril formation and caused a reduction in and an altered appearance of collagen fibril bundles, representing a completely novel mechanism of action for both drugs.

An ex vivo model to induce early fibrosis-like changes in human precision-cut lung slices.

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic interstitial lung disease (ILD) characterized by lung tissue scarring and high morbidity. Lung epithelial injury, myofibroblast activation, and deranged repair are believed to be key processes involved in disease onset and progression, but the exact molecular mechanisms behind IPF remain unclear. Several drugs have been shown to slow disease progression, but treatments that halt or reverse IPF progression have not been identified. Ex vivo models of human lung have been proposed for drug discovery, one of which is precision-cut lung slices (PCLS). Although PCLS production from IPF explants is possible, IPF explants are rare and typically represent end-stage disease. Here we present a novel model of early fibrosis-like changes in human PCLS derived from patients without ILD/IPF using a combination of profibrotic growth factors and signaling molecules (transforming growth factor-ß, tumor necrosis factor-α, platelet-derived growth factor-AB, and lysophosphatidic acid). Fibrotic-like changes of PCLS were qualitatively analyzed by histology and immunofluorescence and quantitatively by water-soluble tetrazolium-1, RT-qPCR, Western blot analysis, and ELISA. PCLS remained viable after 5 days of treatment, and fibrotic gene expression (FN1, SERPINE1, COL1A1, CTGF, MMP7, and ACTA2) increased as early as 24 h of treatment, with increases in protein levels at 48 h and increased deposition of extracellular matrix. Alveolar epithelium reprogramming was evident by decreases in surfactant protein C and loss of HOPX In summary, using human-derived PCLS, we established a novel ex vivo model that displays characteristics of early fibrosis and could be used to evaluate novel therapies and study early-stage IPF pathomechanisms.

FK506-Binding Protein 10, a Potential Novel Drug Target for Idiopathic Pulmonary Fibrosis.

RATIONALE: Increased abundance and stiffness of the extracellular matrix, in particular collagens, is a hallmark of idiopathic pulmonary fibrosis (IPF). FK506-binding protein 10 (FKBP10) is a collagen chaperone, mutations of which have been indicated in the reduction of extracellular matrix stiffness (e.g., in osteogenesis imperfecta). OBJECTIVES: To assess the expression and function of FKBP10 in IPF. METHODS: We assessed FKBP10 expression in bleomycin-induced lung fibrosis (using quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence), analyzed microarray data from 99 patients with IPF and 43 control subjects from a U.S. cohort, and performed Western blot analysis from 6 patients with IPF and 5 control subjects from a German cohort. Subcellular localization of FKBP10 was assessed by immunofluorescent stainings. The expression and function of FKBP10, as well as its regulation by endoplasmic reticulum stress or transforming growth factor-ß1, was analyzed by small interfering RNA-mediated loss-of-function experiments, quantitative reverse transcriptase-polymerase chain reaction, Western blot, and quantification of secreted collagens in the lung and in primary human lung fibroblasts (phLF). Effects on collagen secretion were compared with those of the drugs nintedanib and pirfenidone, recently approved for IPF. MEASUREMENTS AND MAIN RESULTS: FKBP10 expression was up-regulated in bleomycin-induced lung fibrosis and IPF. Immunofluorescent stainings demonstrated localization to interstitial (myo)fibroblasts and CD68(+) macrophages. Transforming growth factor-ß1, but not endoplasmic reticulum stress, induced FKBP10 expression in phLF. The small interfering RNA-mediated knockdown of FKBP10 attenuated expression of profibrotic mediators and effectors, including collagens I and V and α-smooth muscle actin, on the transcript and protein level. Importantly, loss of FKBP10 expression significantly suppressed collagen secretion by phLF. CONCLUSIONS: FKBP10 might be a novel drug target for IPF.

Different localization of P2X4 and P2X7 receptors in native mouse lung - lack of evidence for a direct P2X4-P2X7 receptor interaction.

Introduction: P2X receptors are a family of homo- and heterotrimeric cation channels gated by extracellular ATP. The P2X4 and P2X7 subunits show overlapping expression patterns and have been involved in similar physiological processes, such as pain and inflammation as well as various immune cell functions. While formation of P2X2/P2X3 heterotrimers produces a distinct pharmacological phenotype and has been well established, functional identification of a P2X4/P2X7 heteromer has been difficult and evidence for and against a physical association has been found. Most of this evidence stems, however, from in vitro model systems. Methods: Here, we used a P2X7-EGFP BAC transgenic mouse model as well as P2X4 and P2X7 knock-out mice to re-investigate a P2X4-P2X7 interaction in mouse lung by biochemical and immunohistochemical experiments as well as quantitative expression analysis. Results: No detectable amounts of P2X4 could be co-purified from mouse lung via P2X7-EGFP. In agreement with these findings, immuno-histochemical analysis using a P2X7-specific nanobody revealed only limited overlap in the cellular and subcellular localizations of P2X4 and P2X7 in both the native lung tissue and primary cells. Comparison of P2X4 and P2X7 transcript and protein levels in the respective gene-deficient and wild type mice showed no mutual interrelation between their expression levels in whole lungs. However, a significantly reduced P2rx7 expression was found in alveolar macrophages of P2rx4 -/- mice. Discussion: In summary, our detailed analysis of the cellular and subcellular P2X4 and P2X7 localization and expression does not support a physiologically relevant direct association of P2X4 and P2X7 subunits or receptors in vivo.

Assessing Heterogeneity in the N-Telopeptides of Type I Collagen by Mass Spectrometry.

Collagen cross-links created by the lysyl oxidase and lysyl hydroxylase families of enzymes are a significant contributing factor to the biomechanical strength and rigidity of tissues, which in turn influence cell signaling and ultimately cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach involving chemical digestion for protein solubilization coupled with mass spectrometry allows for the identification of the NTX cross-links in a range of modification states. Based on the specificity of the enzymatic cross-linking reaction we utilized follow-up variable modification searches to facilitate identification with a wider range of analytical workflows. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl-hydroxylase and lysyl-oxidase enzymes.

Trimannose-coupled antimiR-21 for macrophage-targeted inhalation treatment of acute inflammatory lung damage.

Recent studies of severe acute inflammatory lung disease including COVID-19 identify macrophages to drive pulmonary hyperinflammation and long-term damage such as fibrosis. Here, we report on the development of a first-in-class, carbohydrate-coupled inhibitor of microRNA-21 (RCS-21), as a therapeutic means against pulmonary hyperinflammation and fibrosis. MicroRNA-21 is among the strongest upregulated microRNAs in human COVID-19 and in mice with acute inflammatory lung damage, and it is the strongest expressed microRNA in pulmonary macrophages. Chemical linkage of a microRNA-21 inhibitor to trimannose achieves rapid and specific delivery to macrophages upon inhalation in mice. RCS-21 reverses pathological activation of macrophages and prevents pulmonary dysfunction and fibrosis after acute lung damage in mice. In human lung tissue infected with SARS-CoV-2 ex vivo, RCS-21 effectively prevents the exaggerated inflammatory response. Our data imply trimannose-coupling for effective and selective delivery of inhaled oligonucleotides to pulmonary macrophages and report on a first mannose-coupled candidate therapeutic for COVID-19.

Emerging Roles of Airway Epithelial Cells in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with incompletely understood aetiology and limited treatment options. Traditionally, IPF was believed to be mainly caused by repetitive injuries to the alveolar epithelium. Several recent lines of evidence, however, suggest that IPF equally involves an aberrant airway epithelial response, which contributes significantly to disease development and progression. In this review, based on recent clinical, high-resolution imaging, genetic, and single-cell RNA sequencing data, we summarize alterations in airway structure, function, and cell type composition in IPF. We furthermore give a comprehensive overview on the genetic and mechanistic evidence pointing towards an essential role of airway epithelial cells in IPF pathogenesis and describe potentially implicated aberrant epithelial signalling pathways and regulation mechanisms in this context. The collected evidence argues for the investigation of possible therapeutic avenues targeting these processes, which thus represent important future directions of research.

Quantitative proteomics of differentiated primary bronchial epithelial cells from chronic obstructive pulmonary disease and control identifies potential novel host factors post-influenza A virus infection.

Background: Chronic obstructive pulmonary disease (COPD) collectively refers to chronic and progressive lung diseases that cause irreversible limitations in airflow. Patients with COPD are at high risk for severe respiratory symptoms upon influenza virus infection. Airway epithelial cells provide the first-line antiviral defense, but whether or not their susceptibility and response to influenza virus infection changes in COPD have not been elucidated. Therefore, this study aimed to compare the susceptibility of COPD- and control-derived airway epithelium to the influenza virus and assess protein changes during influenza virus infection by quantitative proteomics. Materials and methods: The presence of human- and avian-type influenza A virus receptor was assessed in control and COPD lung sections as well as in fully differentiated primary human bronchial epithelial cells (phBECs) by lectin- or antibody-based histochemical staining. PhBECs were from COPD lungs, including cells from moderate- and severe-stage diseases, and from age-, sex-, smoking, and history-matched control lung specimens. Protein profiles pre- and post-influenza virus infection in vitro were directly compared using quantitative proteomics, and selected findings were validated by qRT-PCR and immunoblotting. Results: The human-type influenza receptor was more abundant in human airways than the avian-type influenza receptor, a property that was retained in vitro when differentiating phBECs at the air-liquid interface. Proteomics of phBECs pre- and post-influenza A virus infection with A/Puerto Rico/8/34 (PR8) revealed no significant differences between COPD and control phBECs in terms of flu receptor expression, cell type composition, virus replication, or protein profile pre- and post-infection. Independent of health state, a robust antiviral response to influenza virus infection was observed, as well as upregulation of several novel influenza virus-regulated proteins, including PLSCR1, HLA-F, CMTR1, DTX3L, and SHFL. Conclusion: COPD- and control-derived phBECs did not differ in cell type composition, susceptibility to influenza virus infection, and proteomes pre- and post-infection. Finally, we identified novel influenza A virus-regulated proteins in bronchial epithelial cells that might serve as potential targets to modulate the pathogenicity of infection and acute exacerbations.

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models.

Rationale: Human coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively. Methods: Primary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication. Results: Both CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model. Conclusion: The immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.