The cell type specific sequences of transcriptional programs during lung regeneration have remained elusive. Using time-series single cell RNA-seq of the bleomycin lung injury model, we resolved transcriptional dynamics for 28 cell types. Trajectory modeling together with lineage tracing revealed that airway and alveolar stem cells converge on a unique Krt8 + transitional stem cell state during alveolar regeneration. These cells have squamous morphology, feature p53 and NFkB activation and display transcriptional features of cellular senescence. The Krt8+ state appears in several independent models of lung injury and persists in human lung fibrosis, creating a distinct cell–cell communication network with mesenchyme and macrophages during repair. We generated a model of gene regulatory programs leading to Krt8+ transitional cells and their terminal differentiation to alveolar type-1 cells. We propose that in lung fibrosis, perturbed molecular checkpoints on the way to terminal differentiation can cause aberrant persistence of regenerative intermediate stem cell states.

Inhaled nanoparticles constitute a potential health hazard due to their size‐dependent lung deposition and large surface to mass ratio. Exposure to high levels contributes to the risk of developing respiratory and cardiovascular diseases, as well as of lung cancer. Particle‐induced acute phase response may be an important mechanism of action of particle‐induced cardiovascular disease. Here, the authors review new important scientific evidence showing causal relationships between inhalation of particle and nanomaterials, induction of acute phase response, and risk of cardiovascular disease. Particle‐induced acute phase response provides a means for risk assessment of particle‐induced cardiovascular disease and underscores cardiovascular disease as an occupational disease.

Translation of mRNA into protein is an evolutionarily conserved, fundamental process of life. A prerequisite for translation is the accurate charging of tRNAs with their cognate amino acids, a reaction catalyzed by specific aminoacyl-tRNA synthetases. One of these enzymes is the aspartyl-tRNA synthetase DARS, which pairs aspartate with its corresponding tRNA. Missense mutations of the gene encoding DARS result in the leukodystrophy hypomyelination with brainstem and spinal cord involvement and leg spasticity (HBSL) with a distinct pattern of hypomyelination, motor abnormalities, and cognitive impairment. A thorough understanding of the DARS expression domains in the central nervous system is essential for the development of targeted therapies to treat HBSL. Here, we analyzed endogenous DARS expression on the mRNA and protein level in different brain regions and cell types of human post mortem brain tissue as well as in human stem cell derived neurons, oligodendrocytes, and astrocytes. DARS expression is significantly enriched in the cerebellum, a region affected in HBSL patients and important for motor control. Although obligatorily expressed in all cells, DARS shows a distinct expression pattern with enrichment in neurons but only low abundance in oligodendrocytes, astrocytes, and microglia. Our results reveal little homogeneity across the different cell types, largely matching previously published data in the murine brain. This human gene expression study will significantly contribute to the understanding of DARS gene function and HBSL pathology and will be instrumental for future development of animal models and targeted therapies. In particular, we anticipate high benefit from a gene replacement approach in neurons of HBSL mouse models, given the abundant endogenous DARS expression in this lineage cell.

Introduction: Interstitial lung disease (ILD) is a heterogenous group of lung disorders where destruction and incomplete regeneration of the lung parenchyma often results in persistent architectural distortion of the pulmonary scaffold. Continuous mesenchyme-centered, disease-relevant signaling likely initiates and perpetuates the fibrotic remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area.Methods: With the aim of identifying functional mediators of the lung mesenchymal-epithelial crosstalk with potential as new targets for therapeutic strategies, we developed a 3D organoid co-culture model based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells that form alveolar organoids in presence of lung fibroblasts from fibrotic-ILD patients, in our study referring to cases of pulmonary fibrosis, as well as control cell line (IMR-90).Results: While organoid formation capacity and size was comparable in the presence of fibrotic-ILD or control lung fibroblasts, metabolic activity was significantly increased in fibrotic-ILD co-cultures. Alveolar organoids cultured with fibrotic-ILD fibroblasts further demonstrated reduced stem cell function as reflected by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. To screen for key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used mass spectrometry and characterized the secretome of end stage fibrotic-ILD lung fibroblasts in comparison to non-chronic lung disease (CLD) patient fibroblasts. Out of the over 2000 proteins detected by this experimental approach, 47 proteins were differentially abundant comparing fibrotic-ILD and non-CLD fibroblast secretome. The fibrotic-ILD secretome profile was dominated by chemokines, including CXCL1, CXCL3, and CXCL8, interfering with growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating alveolar organoids with IL11, we recapitulated the co-culture results obtained with primary fibrotic-ILD fibroblasts including changes in metabolic activity.Conclusion: We identified mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD. In our alveolar organoid co-cultures, we were able to highlight the importance of fibroblast-initiated aberrant epithelial differentiation and confirmed IL11 as a key player in fibrotic-ILD pathogenesis by unbiased fibroblast secretome analysis.

Lung epithelial organoids for the hazard assessment of inhaled nanomaterials offer a promising improvement to in vitro culture systems used so far. Organoids grow in three-dimensional (3D) spheres and can be derived from either induced pluripotent stem cells (iPSC) or primary lung tissue stem cells from either human or mouse. In this perspective we will highlight advantages and disadvantages of traditional culture systems frequently used for testing nanomaterials and compare them to lung epithelial organoids. We also discuss the differences between tissue and iPSC-derived organoids and give an outlook in which direction the whole field could possibly go with these versatile tools.

Positive controls are an important feature in experimental studies as they show the responsiveness of the model under investigation. An often applied reagent for a pro-inflammatory stimulus is the endotoxin lipopolysaccharide (LPS), which has been shown to induce a cytokine release by various cell cultures. The effect of LPS in monocultures of 16HBE14o-, a bronchial cell line, and of A549, an alveolar cell line, were compared in submerged and air-liquid interface cultures, as well as in co-cultures of the two epithelial cells with monocyte-derived macrophages and dendritic cells. The protein and mRNA levels of the two most relevant pro-inflammatory mediators, Tumor necrosis factor alpha (TNF) and Interleukin 8 (CXCL8), were measured after 4 h and 24 h exposure. 16HBE14o- cells alone as well as in co-cultures are non-responsive to an LPS stimulus, but an already increased basal expression of both pro-inflammatory mediators after prolonged time in culture was observed. In contrary, A549 in monocultures showed increased CXCL8 production at the gene and protein level after LPS exposure, while TNF-levels were below detection limit. In A549 co-cultured with immune cells both mediators were upregulated. This study shows the importance of a careful evaluation of the culture system used, including the application of positive controls. In addition, the use of co-cultures with immune cells more adequately reflects the inflammatory response upon exposure to toxicants.