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.

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The mechanisms by which exposure to particulate matter increases the risk of cardiovascular events are not known. Recent human and animal data suggest that particulate matter may induce alterations in hemostatic factors. In this study we determined the mechanisms by which particulate matter might accelerate thrombosis. We found that mice treated with a dose of well characterized particulate matter of less than 10 μM in diameter exhibited a shortened bleeding time, decreased prothrombin and partial thromboplastin times (decreased plasma clotting times), increased levels of fibrinogen, and increased activity of factor II, VIII, and X. This prothrombotic tendency was associated with increased generation of intravascular thrombin, an acceleration of arterial thrombosis, and an increase in bronchoalveolar fluid concentration of the prothrombotic cytokine IL-6. Knockout mice lacking IL-6 were protected against particulate matter-induced intravascular thrombin formation and the acceleration of arterial thrombosis. Depletion of macrophages by the intratracheal administration of liposomal clodronate attenuated particulate matter-induced IL-6 production and the resultant prothrombotic tendency. Our findings suggest that exposure to particulate matter triggers IL-6 production by alveolar macrophages, resulting in reduced clotting times, intravascular thrombin formation, and accelerated arterial thrombosis. These results provide a potential mechanism linking ambient particulate matter exposure and thrombotic events.

Hypercapnia (elevated CO 2 levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO 2 -induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO 2 levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO 2 -triggered increase in intracellular Ca 2+ concentration and Ca 2+ /calmodulin-dependent kinase kinase-β (CaMKK-β). Chelating intracellular Ca 2+ or abrogating CaMKK-β function by gene silencing or chemical inhibition prevented the CO 2 -induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-ζ and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-α 1 prevented CO 2 -induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a β-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO 2 levels are sensed by AECs and that AMPK mediates CO 2 -induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with β-adrenergic agonists and cAMP.

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-Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of ␣-smooth muscle actin (␣-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, suggesting that hypoxia induces EMT in AEC. Both severe hypoxia and moderate hypoxia induced EMT. The reactive oxygen species (ROS) scavenger Euk-134 prevented hypoxia-induced EMT. Moreover, hypoxia-induced expression of ␣-SMA and vimentin was prevented in mitochondria-deficient 0 cells, which are incapable of ROS production during hypoxia. CoCl2 and dimethyloxaloylglycine, two compounds that stabilize hypoxiainducible factor (HIF)-␣ under normoxia, failed to induce ␣-SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1␣ did not induce ␣-SMA. However, loss of HIF-1␣ or HIF-2␣ abolished induction of ␣-SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-␤1, and preincubation of AEC with SB431542, an inhibitor of the TGF-␤1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-␤1 dependent. Furthermore, both ROS and HIF-␣ were necessary for hypoxia-induced TGF-␤1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-␤1 signaling. alveolar epithelial cells; pulmonary fibrosis; transforming growth factor-␤1 EPITHELIAL-MESENCHYMAL TRANSITION (EMT) is a cellular process during which epithelial cells acquire mesenchymal properties while losing cell-cell interactions and apicobasal polarity (33,44). EMT is characterized by changes in cell morphology and acquisition of mesenchymal markers such as ␣-smooth muscle actin (␣-SMA) and vimentin as well as loss of epithelial makers, including E-cadherin (53). Transforming growth factor (TGF)-␤1 is considered to be the prototypical cytokine for the induction of EMT (53). Active TGF-␤1 binds to the transmembrane serine-threonine kinase receptor II and receptor I and activates Smad-mediated transcription of target genes, including ␣-SMA and vimentin, which leads to EMT (33,53,54). TGF-␤1 is reported to induce EMT in renal proximal tubular epithelial cells, lens epithelial cells, and, most recently, alveolar epithelial cells (AEC) (19,23,40,48,55).AEC perform many tasks necessary for normal alveolus functioning, including surfactant protein production and fluid and ion transport (17, 57). Recent evidence suggests that AEC may undergo EMT, contributing to the pathogenesis of pulmonary fibrosis (26,49).AEC are exposed to hypoxia in human lung diseases, including acute lung injury and pulmonary fibrosis (20, 41, 57). It has been described that during hypoxia, mitochondria increase the production of reactive oxy...

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