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.

Primary graft dysfunction is the predominant driver of mortality and graft loss after lung transplantation. Recruitment of neutrophils as a result of ischemia-reperfusion injury is thought to cause primary graft dysfunction; however, the mechanisms that regulate neutrophil influx into the injured lung are incompletely understood. We found that donor-derived intravascular nonclassical monocytes (NCMs) are retained in human and murine donor lungs used in transplantation and can be visualized at sites of endothelial injury after reperfusion. When NCMs in the donor lungs were depleted, either pharmacologically or genetically, neutrophil influx and lung graft injury were attenuated in both allogeneic and syngeneic models. Similar protection was observed when the patrolling function of donor NCMs was impaired by deletion of the fractalkine receptor CX3CR1. Unbiased transcriptomic profiling revealed up-regulation of MyD88 pathway genes and a key neutrophil chemoattractant, CXCL2, in donor-derived NCMs after reperfusion. Reconstitution of NCM-depleted donor lungs with wild-type but not MyD88-deficient NCMs rescued neutrophil migration. Donor NCMs, through MyD88 signaling, were responsible for CXCL2 production in the allograft and neutralization of CXCL2 attenuated neutrophil influx. These findings suggest that therapies to deplete or inhibit NCMs in donor lung might ameliorate primary graft dysfunction with minimal toxicity to the recipient.

Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1β production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction-associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic.

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Hyperammonemia is a highly fatal syndrome in lung recipients that is usually refractory to medical therapy. We recently reported that infection by a Mollicute, Ureaplasma, is causative for hyperammonemia and can be successfully treated with antimicrobial agents. However, it remains unknown whether the pathogenic strain of Ureaplasma is donor or recipient derived. Here we provide evidence that donor-derived Ureaplasma infection can be pathogenic. As such, we uncover a previously unknown lethal donor-derived opportunistic infection in lung recipients. Given the high mortality associated with hyperammonemia, strategies for routine donor screening or prophylaxis should be further evaluated in prospective studies.