Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

Misharin et al. elucidate the fate and function of monocyte-derived alveolar macrophages during the course of pulmonary fibrosis. These cells persisted throughout the life span, were enriched for the expression of profibrotic genes, and their genetic ablation ameliorated development of pulmonary fibrosis.

Recent epidemiological and laboratory-based studies suggest that the anti-diabetic drug metformin prevents cancer progression. How metformin diminishes tumor growth is not fully understood. In this study, we report that in human cancer cells, metformin inhibits mitochondrial complex I (NADH dehydrogenase) activity and cellular respiration. Metformin inhibited cellular proliferation in the presence of glucose, but induced cell death upon glucose deprivation, indicating that cancer cells rely exclusively on glycolysis for survival in the presence of metformin. Metformin also reduced hypoxic activation of hypoxia-inducible factor 1 (HIF-1). All of these effects of metformin were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed. In vivo, the administration of metformin to mice inhibited the growth of control human cancer cells but not those expressing NDI1. Thus, we have demonstrated that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on its ability to inhibit mitochondrial complex I.DOI: http://dx.doi.org/10.7554/eLife.02242.001

To facilitate the proposed use of graphene and its derivative graphene oxide (GO) in widespread applications, we explored strategies that improve the biocompatibility of graphene nanomaterials in the lung. In particular, solutions of aggregated graphene, Pluronic dispersed graphene, and GO were administered directly into the lungs of mice. The introduction of GO resulted in severe and persistent lung injury. Furthermore, in cells, GO increased the rate of mitochondrial respiration and the generation of reactive oxygen species, activating inflammatory and apoptotic pathways. In contrast, this toxicity was significantly reduced in the case of pristine graphene after liquid phase exfoliation, and was further minimized when the unoxidized graphene was well-dispersed with the block copolymer Pluronic. Our results demonstrate that the covalent oxidation of graphene is a major contributor to its pulmonary toxicity and suggest that dispersion of pristine graphene in Pluronic provides a pathway for the safe handling and potential biomedical application of two-dimensional carbon nanomaterials.

Excitement surrounding the attractive physical and chemical characteristics of single walled carbon nanotubes (SWCNTs) has been tempered by concerns regarding their potential health risks. Here we consider the lung toxicity of nanoscale dispersed SWCNTs (mean diameter ~ 1 nm). Because dispersion of the SWCNTs increases their aspect ratio relative to as-produced aggregates, we directly test the prevailing hypothesis that lung toxicity associated with SWCNTs compared with other carbon structures is attributable to the large aspect ratio of the individual particles. Thirty days after their intratracheal administration to mice, the granuloma-like structures with mild fibrosis in the large airways observed in mice treated with aggregated SWCNTs were absent in mice treated with nanoscale dispersed SWCNTs. Examination of lung sections from mice treated with nanoscale dispersed SWCNTs revealed uptake of the SWCNTs by macrophages and gradual clearance over time. We conclude that the toxicity of SWCNTs in vivo is attributable to aggregation of the nanomaterial rather than the large aspect ratio of the individual nanotubes. Biocompatible nanoscale dispersion provides a scalable method to generate purified preparations of SWCNTs with minimal toxicity, thus allowing them to be used safely in commercial and biomedical applications.