The use of cultured human alveolar basal cells to mimic honeycomb formation in idiopathic pulmonary fibrosis.

BACKGROUND: Honeycomb cysts (HC) within the alveolar region are distinct histopathological features in the lungs of idiopathic pulmonary fibrosis (IPF) patients. HC are lined with a single-or stratified layer of basal cells (BC), or with a bronchiolar-like epithelium composed of basal-, ciliated- and secretory epithelial cells. By using cultured IPF patient-derived alveolar BC, we aimed to establish an in vitro- and in vivo model to mimic HC formation in IPF. We (1) optimized conditions to culture and propagate IPF patient-derived alveolar BC, (2) cultured the cells on an air liquid interface (ALI) or in a three dimensional (3D) organoid model, and (3) investigated the cells` behavior after instillation into bleomycin-challenged mice. METHODS: Alveolar BC were cultured from peripheral IPF lung tissue and grown on tissue-culture treated plastic, an ALI, or in a 3D organoid model. Furthermore, cells were instilled into bleomycin-challenged NRG mice. Samples were analyzed by TaqMan RT-PCR, immunoblotting, immunocytochemistry/immunofluorescence (ICC/IF), or immunohistochemistry (IHC)/IF. Mann-Whitney tests were performed using GraphPad Prism software. RESULTS: Cultured alveolar BC showed high expression of canonical basal cell markers (TP63, keratin (KRT)5, KRT14, KRT17), robust proliferation, and wound closure capacity. The cells could be cryopreserved and propagated for up to four passages without a significant loss of basal cell markers. When cultured on an ALI or in a 3D organoid model, alveolar BC differentiated to ciliated- and secretory epithelial cells. When instilled into bleomycin-challenged mice, human alveolar BC cells formed HC-like structures composed of human basal-, and secretory epithelial cells within the mouse parenchyma. CONCLUSION: IPF patient-derived alveolar BC on an ALI, in 3D organoids or after instillation into bleomycin-challenged mice form HC-like structures that closely resemble HC within the IPF lung. These models therefore represent powerful tools to study honeycomb formation, and its potential therapeutic inhibition in IPF.

Multilamellated Basement Membranes in the Capillary Network of Alveolar Capillary Dysplasia.

A minimal diffusion barrier is key to the pulmonary gas exchange. In alveolar capillary dysplasia (ACD), a rare genetically driven disease of early infancy, this crucial fibrovascular interface is compromised while the underlying pathophysiology is insufficiently understood. Recent in-depth analyses of vascular alterations in adult lung disease encouraged researchers to extend these studies to ACD and compare the changes of the microvasculature. Lung tissue samples of children with ACD (n = 12), adults with non-specific interstitial pneumonia (n = 12), and controls (n = 20) were studied using transmission electron microscopy, single-gene sequencing, immunostaining, exome sequencing, and broad transcriptome profiling. In ACD, pulmonary capillary basement membranes were hypertrophied, thickened, and multilamellated. Transcriptome profiling revealed increased CDH5, COL4A1, COL15A1, PTK2B, and FN1 and decreased VIT expression, confirmed by immunohistochemistry. In contrast, non-specific interstitial pneumonia samples showed a regular basement membrane architecture with preserved VIT expression but also increased COL15A1+ vessels. This study provides insight into the ultrastructure and pathophysiology of ACD. The lack of normally developed lung capillaries appeared to cause a replacement by COL15A1+ vessels, a mechanism recently described in interstitial lung disease. The VIT loss and FN1 overexpression might contribute to the unique appearance of basement membranes in ACD. Future studies are needed to explore the therapeutic potential of down-regulating the expression of FN1 and balancing VIT deficiency.

CAR-NK Cells Targeting HER1 (EGFR) Show Efficient Anti-Tumor Activity against Head and Neck Squamous Cell Carcinoma (HNSCC).

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.

Fibrosis-Related Gene Profiling in Liver Biopsies of PiZZ α1-Antitrypsin Children with Different Clinical Courses.

PiZZ (Glu342Lys) α1-antitrypsin deficiency (AATD) is characterized by intrahepatic AAT polymerization and is a risk factor for liver disease development in children. The majority of PiZZ children are disease free, hence this mutation alone is not sufficient to cause the disease. We investigated Z-AAT polymers and the expression of fibrosis-related genes in liver tissues of PiZZ children with different clinical courses. Liver biopsies obtained during 1979-2010 at the Department of Paediatrics, Karolinska University Hospital, Sweden, were subjected to histological re-evaluation, immunohistochemistry and NanoString-based transcriptome profiling using a panel of 760 fibrosis plus 8 bile acid-related genes. Subjects were divided into three groups based on clinical outcomes: NCH (neonatal cholestasis, favourable outcome, n = 5), NCC (neonatal cholestasis, early cirrhosis and liver transplantation, n = 4), and NNCH (no neonatal cholestasis, favourable outcome, n = 5, six biopsies). Hepatocytes containing Z-AAT polymers were abundant in all groups whereas NCC showed higher expression of genes related to liver fibrosis/cirrhosis and lower expression of genes related to lipid, aldehyde/ketone, and bile acid metabolism. Z-AAT accumulation per se cannot explain the clinical outcomes of PiZZ children; however, changes in the expression of specific genes and pathways involved in lipid, fatty acid, and steroid metabolism appear to reflect the degree of liver injury.

A Morphomolecular Approach to Alveolar Capillary Dysplasia.

Alveolar capillary dysplasia (ACD) is a rare lung developmental disorder leading to persistent pulmonary arterial hypertension and fatal outcomes in newborns. The current study analyzed the microvascular morphology and the underlying molecular background of ACD. One ACD group (n = 7), one pulmonary arterial hypertension group (n = 20), and one healthy con1trol group (n = 16) were generated. Samples of histologically confirmed ACD were examined by exome sequencing and array-based comparative genomic hybridization. Vascular morphology was analyzed using scanning electron microscopy of microvascular corrosion casts. Gene expression and biological pathways were analyzed using two panels on inflammation/kinase-specific genes and a comparison analysis tool. Compartment-specific protein expression was analyzed using immunostaining. In ACD, there was an altered capillary network, a high prevalence of intussusceptive angiogenesis, and increased activity of C-X-C motif chemokine receptor 4 (CXCR4), hypoxia-inducible factor 1α (HIF1A), and angiopoietin signaling pathways compared with pulmonary arterial hypertension/healthy controls. Histologically, there was a markedly increased prevalence of endothelial tyrosine kinase receptor (TEK/TIE2)+ macrophages in ACD, compared with the other groups, whereas the CXCR4 ligand CXCL12 and HIF1A showed high expression in all groups. ACD is characterized by dysfunctional capillaries and a high prevalence of intussusceptive angiogenesis. The results indicate that endothelial CXCR4, HIF1A, and angiopoietin signaling as well as TIE2+ macrophages are crucial for the induction of intussusceptive angiogenesis and vascular remodeling. Future studies should address the use of anti-angiogenic agents in ACD, where TIE2 appears as a promising target.

Alveolar Basal Cells Differentiate towards Secretory Epithelial- and Aberrant Basaloid-like Cells In Vitro.

In idiopathic pulmonary fibrosis (IPF), keratin (KRT)17+/KRT5+ basal and KRT17+/KRT5- aberrant basaloid cells are atypically present within the alveolar space. We previously described the fibrosis-enriched outgrowth of alveolar basal cells from peripheral fibrotic lung tissue. Using single cell RNA sequencing (scRNA-seq), we here characterize the transcriptome of these cultured alveolar basal cells under different culture conditions. METHODS: Fibrotic peripheral lung tissue pieces were placed in DMEM growth medium. Outgrown cells were analysed by scRNA-seq, TaqMan-PCR or immunofluorescence (IF) either directly or after medium change to an epithelial cell specific medium (Cnt-PR-A). RESULTS: A fraction of alveolar basal cells cultured in DMEM growth medium showed close transcriptomic similarities to IPF basal cells. However, although they expressed KRT5, the transcriptome of the majority of cells matched best to the transcriptome of recently described KRT17+/KRT5- aberrant basaloid cells, co-expressing the canonical basal cell marker KRT17 and mesenchymal cell marker (VIM, FN1). A smaller fraction of cells matched best to secretory epithelial cells. Two differentiation gradients from basal to aberrant basaloid-like cells and basal to secretory epithelial-like cells were apparent. Interestingly, these differentiation paths seemed reversed when the cell culture medium was changed to Cnt-PR-A. CONCLUSIONS: Our results suggest that cultured alveolar basal cells have the capacity to differentiate towards secretory epithelial-like cells and to aberrant basaloid-like cells. However, due to the persistent expression of KRT5, a complete differentiation towards aberrant basaloid cells did not seem to be achieved in our culture conditions. Importantly, differentiation seemed reversible by changing the cells microenvironment. Determining specific factors influencing these differentiation paths may help to define novel drug targets for IPF therapy.

Basal-Like Cell-Conditioned Medium Exerts Anti-Fibrotic Effects In Vitro and In Vivo.

In idiopathic pulmonary fibrosis (IPF), basal-like cells are atypically present in the alveolar region, where they may affect adjacent stromal cells by paracrine mechanisms. We here aimed to confirm the presence of basal-like cells in peripheral IPF lung tissue in vivo, to culture and characterize the cells in vitro, and to investigate their paracrine effects on IPF fibroblasts in vitro and in bleomycin-injured rats in vivo. Basal-like cells are mainly localized in areas of pathological bronchiolization or honeycomb cysts in peripheral IPF lung tissue. Single-cell RNA sequencing (scRNA-seq) demonstrated an overall homogeneity, the expression of the basal cell markers cytokeratin KRT5 and KRT17, and close transcriptomic similarities to basal cells in the majority of cells cultured in vitro. Basal-like cells secreted significant levels of prostaglandin E2 (PGE2), and their conditioned medium (CM) inhibited alpha-smooth muscle actin (α-SMA) and collagen 1A1 (Col1A1) and upregulated matrix metalloproteinase-1 (MMP-1) and hepatocyte growth factor (HGF) by IPF fibroblasts in vitro. The instillation of CM in bleomycin-injured rat lungs resulted in reduced collagen content, improved lung architecture, and reduced α-SMA-positive cells. Our data suggested that basal-like cells may limit aberrant fibroblast activation and differentiation in IPF through paracrine mechanisms.

Comparative Analysis of Gene Expression in Fibroblastic Foci in Patients with Idiopathic Pulmonary Fibrosis and Pulmonary Sarcoidosis.

BACKGROUND: Fibroblastic foci (FF) are characteristic features of usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) and one cardinal feature thought to represent a key mechanism of pathogenesis. Hence, FF have a high impact on UIP/IPF diagnosis in current guidelines. However, although less frequent, these histomorphological hallmarks also occur in other fibrotic pulmonary diseases. Currently, there is therefore a gap in knowledge regarding the underlying molecular similarities and differences of FF in different disease entities. METHODS: In this work, we analyzed the compartment-specific gene expression profiles of FF in IPF and sarcoidosis in order to elucidate similarities and differences as well as shared pathomechanisms. For this purpose, we used laser capture microdissection, mRNA and protein expression analysis. Biological pathway analysis was performed using two different gene expression databases. As control samples, we used healthy lung tissue that was donated but not used for lung transplantation. RESULTS: Based on Holm Bonferroni corrected expression data, mRNA expression analysis revealed a significantly altered expression signature for 136 out of 760 genes compared to healthy controls while half of these showed a similar regulation in both groups. Immunostaining of selected markers from each group corroborated these results. However, when comparing all differentially expressed genes with the fdr-based expression data, only 2 of these genes were differentially expressed between sarcoidosis and IPF compared to controls, i.e., calcium transport protein 1 (CAT1) and SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), both in the sarcoidosis group. Direct comparison of sarcoidosis and IPF did not show any differentially regulated genes independent from the statistical methodology. Biological pathway analysis revealed a number of fibrosis-related pathways pronounced in IPF without differences in the regulatory direction. CONCLUSIONS: These results demonstrate that FF of end-stage IPF and sarcoidosis lungs, although different in initiation, are similar in gene and protein expression, encouraging further studies on the use of antifibrotic agents in sarcoidosis.

PACS2-TRPV1 axis is required for ER-mitochondrial tethering during ER stress and lung fibrosis.

Endoplasmic reticulum (ER) and mitochondria (mito) play a vital role in alveolar type II cell (AEC2) homeostasis and are both stressed in patients with idiopathic pulmonary fibrosis (IPF). Up to now, no data are available with regard to ER-mito cross talk and tethering under conditions of IPF. We here demonstrate that ER-mitochondrial tethering is reduced upon experimental ER stress in vitro and in the IPF AECII ex vivo, and this is-at least in part-due to decreased phosphofurin acidic cluster sorting protein 2 (PACS-2, also called PACS2) protein levels. PACS2 levels are influenced by its interaction with the transient receptor potential cation channel subfamily V member 1 (TRPV1) and can be experimentally modified by the TRPV1-modulating drug capsaicin (CPS). Employing alveolar epithelial cells with overexpression of the terminal ER stress signaling factor Chop or the IPF-associated surfactant protein C mutation (SPCΔexon4) in vitro, we observed a restoration of PACS2 levels upon treatment with CPS. Similarly, treatment of precision cut lung slices from IPF patients with CPS ex vivo forwarded similar effects. Importantly, in all models such kind of intervention also greatly reduced the extent of alveolar epithelial apoptosis. We therefore conclude that therapeutic targeting of the PACS2-TRPV1 axis represents an interesting novel, epithelial-protective approach in IPF.

Time-Dependent Molecular Motifs of Pulmonary Fibrogenesis in COVID-19.

(1) Background: In COVID-19 survivors there is an increased prevalence of pulmonary fibrosis of which the underlying molecular mechanisms are poorly understood; (2) Methods: In this multicentric study, n = 12 patients who succumbed to COVID-19 due to progressive respiratory failure were assigned to an early and late group (death within ≤7 and >7 days of hospitalization, respectively) and compared to n = 11 healthy controls; mRNA and protein expression as well as biological pathway analysis were performed to gain insights into the evolution of pulmonary fibrogenesis in COVID-19; (3) Results: Median duration of hospitalization until death was 3 (IQR25-75, 3-3.75) and 14 (12.5-14) days in the early and late group, respectively. Fifty-eight out of 770 analyzed genes showed a significantly altered expression signature in COVID-19 compared to controls in a time-dependent manner. The entire study group showed an increased expression of BST2 and IL1R1, independent of hospitalization time. In the early group there was increased activity of inflammation-related genes and pathways, while fibrosis-related genes (particularly PDGFRB) and pathways dominated in the late group; (4) Conclusions: After the first week of hospitalization, there is a shift from pro-inflammatory to fibrogenic activity in severe COVID-19. IL1R1 and PDGFRB may serve as potential therapeutic targets in future studies.

3D virtual histopathology of cardiac tissue from Covid-19 patients based on phase-contrast X-ray tomography.

For the first time, we have used phase-contrast X-ray tomography to characterize the three-dimensional (3d) structure of cardiac tissue from patients who succumbed to Covid-19. By extending conventional histopathological examination by a third dimension, the delicate pathological changes of the vascular system of severe Covid-19 progressions can be analyzed, fully quantified and compared to other types of viral myocarditis and controls. To this end, cardiac samples with a cross-section of 3.5mm were scanned at a laboratory setup as well as at a parallel beam setup at a synchrotron radiation facility the synchrotron in a parallel beam configuration. The vascular network was segmented by a deep learning architecture suitable for 3d datasets (V-net), trained by sparse manual annotations. Pathological alterations of vessels, concerning the variation of diameters and the amount of small holes, were observed, indicative of elevated occurrence of intussusceptive angiogenesis, also confirmed by high-resolution cone beam X-ray tomography and scanning electron microscopy. Furthermore, we implemented a fully automated analysis of the tissue structure in the form of shape measures based on the structure tensor. The corresponding distributions show that the histopathology of Covid-19 differs from both influenza and typical coxsackie virus myocarditis.

Method for 3D airway topology extraction.

In lungs the number of conducting airway generations as well as bifurcation patterns varies across species and shows specific characteristics relating to illnesses or gene variations. A method to characterize the topology of the mouse airway tree using scanning laser optical tomography (SLOT) tomograms is presented in this paper. It is used to test discrimination between two types of mice based on detected differences in their conducting airway pattern. Based on segmentations of the airways in these tomograms, the main spanning tree of the volume skeleton is computed. The resulting graph structure is used to distinguish between wild type and surfactant protein (SP-D) deficient knock-out mice.

Sphingosine-1-phosphate receptors stimulate macrophage plasma-membrane actin assembly via ADP release, ATP synthesis and P2X7R activation.

Eukaryotic plasma membranes assemble actin filaments within seconds of activation of many receptors, especially during chemotaxis. Here, serum or sphingosine-1-phosphate stimulation of J774 and RAW macrophages released ADP within seconds into the extracellular medium, along with an adenylate kinase activity that converted ADP to ATP. ATP then activated the P2X7 receptor (P2X7R) that was necessary for a peak of plasma-membrane actin assembly within 5 to 10 seconds in P2X7R-expressing J774, RAW and primary macrophages. Neither actin assembly nor characteristic P2X7R channel activity was seen in response to ATP in P2X7R-knockout macrophages, as detected by patch-clamp analysis. Since P2X7R has been shown previously to form a macromolecular complex with actin we propose that it is involved in the membrane assembly of actin. Our data reveal a surprisingly rapid and complex relay of signaling and externalization events that precede and control actin assembly induced by sphingosine-1-phosphate. The overall model we present is strongly supported by the data presented in the accompanying paper that focuses on latex bead phagosomes.

Lipids regulate P2X7-receptor-dependent actin assembly by phagosomes via ADP translocation and ATP synthesis in the phagosome lumen.

Latex bead phagosomes isolated from J774 macrophages polymerize actin. We show here that five lipids--phosphatidylinositol-4-phosphate, phosphatidylinositol-(4,5)-bisphosphate, sphingosine-1-phosphate (S1P), ceramide-1-phosphate and phosphatidic acid--stimulate both actin assembly and transport of ADP across the phagosomal membrane into the lumen. Once there, this ADP is converted to ATP by adenylate kinase activity. High luminal ATP concentrations correlated well with phagosome actin assembly under different conditions. The ATP-binding P2X7 receptor (P2X7R) was detected in phagosomes. Although S1P stimulated actin assembly by phagosomes from P2X7R-containing bone marrow macrophages, S1P-stimulated actin assembly was inhibited in phagosomes from cells lacking P2X7R. We propose that luminal ATP accumulates in response to selected lipids and activates the P2X7R that signals across the phagosomal membrane to trigger actin assembly on the cytoplasmic membrane surface. In the accompanying paper by Kuehnel et al. (doi:10.1242/jcs.034207), more evidence is provided in support of this model from the analysis of actin assembly at the plasma membrane of intact macrophages.

cAMP synthesis and degradation by phagosomes regulate actin assembly and fusion events: consequences for mycobacteria.

We showed recently that actin assembly by phagosomal membranes facilitates fusion with late endocytic organelles in macrophages. Moreover, lipids that induced phagosomal actin also stimulated this fusion process. In macrophages infected with pathogenic mycobacteria actin-stimulatory lipids led to an increase in pathogen destruction, whereas inhibitors facilitated their growth. A model was proposed whereby phagosomal membrane actin assembly provides tracks for lysosomes to move towards phagosomes, thereby facilitating fusion. Here, we investigated how cAMP affected phagosomal actin assembly in vitro, and phagosomal actin, acidification and late fusion events in J774 macrophages. Latex bead phagosomes are shown to possess adenylyl cyclase activity, which synthesizes cAMP, and phosphodiesterase activity, which degrades cAMP. The system is regulated by protein kinase A (PKA). Increasing cAMP levels inhibited, whereas decreasing cAMP levels stimulated, actin assembly in vitro and within cells. Increasing cAMP levels also inhibited phagosome-lysosome fusion and acidification in cells, whereas reducing cAMP had the opposite effect. High cAMP levels induced an increase in intraphagosomal growth in macrophages of both the non-pathogenic Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis, whereas low cAMP levels or inhibition of PKA correlated with increased bacterial destruction. We argue that the phagosome cAMP-PKA system behaves as a molecular switch that regulates phagosome actin and maturation in macrophages.