Alexandra C. McQuattie-Pimentel

Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span.

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.

Flow Cytometry Reveals Similarities Between Lung Macrophages in Humans and Mice.

Findings in murine models implicate subpopulations of alveolar macrophages in the pathogenesis of lung injury and fibrosis, however, the relevance of these findings for humans with chronic lung disease is unknown in part due to a lack of proper tools to identify macrophage heterogeneity in the human lung. Here we report a flow cytometry protocol that allows unambiguous identification of alveolar macrophages, interstitial macrophages and monocytes in the human lung and in bronchoalveolar lavage fluid. We validated this panel using normal lung tissue and tissue from patients with COPD and lung fibrosis. We found evidence of heterogeneity within human alveolar macrophage populations, which suggest parallels between murine and human macrophage development and differentiation. Abstract word count: 113.

Donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction

Donor nonclassical monocytes mediate primary lung allograft dysfunction by recruiting neutrophils via MyD88-dependent production of CXCL2. Nonclassical monocytes prompt primary graft dysfunction Despite concerted efforts, primary graft dysfunction is a major cause of graft failure after organ transplantation. In lung transplantation, primary graft dysfunction is known to be mediated by early neutrophil infiltration. Zheng et al. used syngeneic and allogeneic mouse models of lung transplantation to show that nonclassical monocytes were the key cell population recruiting these destructive neutrophils. These intravascular cells were donor-derived and were also detectable in human lung grafts being used for transplant. Because depletion of nonclassical monocytes prevented primary graft dysfunction in the mouse models, targeting this cell population during human transplant could lead to improved rates of graft success. 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.

Single-Cell Transcriptomic Analysis of Human Lung Reveals Complex Multicellular Changes During Pulmonary Fibrosis

Pulmonary fibrosis is a devastating disorder that results in the progressive replacement of normal lung tissue with fibrotic scar. Available therapies slow disease progression, but most patients go on to die or require lung transplantation. Single-cell RNA-seq is a powerful tool that can reveal cellular identity via analysis of the transcriptome, but its ability to provide biologically or clinically meaningful insights in a disease context is largely unexplored. Accordingly, we performed single-cell RNA-seq on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and one bronchoscopic cryobiospy sample. Integrated single-cell transcriptomic analysis of donors and patients with pulmonary fibrosis identified the emergence of distinct populations of epithelial cells and macrophages that were common to all patients with lung fibrosis. Analysis of transcripts in the Wnt pathway suggested that within the same cell type, Wnt secretion and response are restricted to distinct non-overlapping cells, which was confirmed using in situ RNA hybridization. Single-cell RNA-seq revealed heterogeneity within alveolar macrophages from individual patients, which was confirmed by immunohistochemistry. These results support the feasibility of discovery-based approaches applying next generation sequencing technologies to clinically obtained samples with a goal of developing personalized therapies. One Sentence Summary Single-cell RNA-seq applied to tissue from diseased and donor lungs and a living patient with pulmonary fibrosis identifies cell type-specific disease-associated molecular pathways.

Metformin Targets Mitochondrial Electron Transport to Reduce Air-Pollution-Induced Thrombosis.

Urban particulate matter air pollution induces the release of pro-inflammatory cytokines including interleukin-6 (IL-6) from alveolar macrophages, resulting in an increase in thrombosis. Here, we report that metformin provides protection in this murine model. Treatment of mice with metformin or exposure of murine or human alveolar macrophages to metformin prevented the particulate matter-induced generation of complex III mitochondrial reactive oxygen species, which were necessary for the opening of calcium release-activated channels (CRAC) and release of IL-6. Targeted genetic deletion of electron transport or CRAC channels in alveolar macrophages in mice prevented particulate matter-induced acceleration of arterial thrombosis. These findings suggest metformin as a potential therapy to prevent some of the premature deaths attributable to air pollution exposure worldwide.