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.

A major complication of factor replacement therapy for haemophilia is the development of anti-factor neutralizing antibodies (inhibitors). Here we show that liver gene therapy by lentiviral vectors (LVs) expressing factor IX (FIX) strongly reduces pre-existing anti-FIX antibodies and eradicates FIX inhibitors in haemophilia B mice. Concomitantly, plasma FIX levels and clotting activity rose to 50–100% of normal. The treatment was effective in 75% of treated mice. FIX-specific plasma cells (PCs) and memory B cells were reduced, likely because of memory B-cell depletion in response to constant exposure to high doses of FIX. Regulatory T cells displaying FIX-specific suppressive capacity were induced in gene therapy treated mice and controlled FIX-specific T helper cells. Gene therapy proved safer than a regimen mimicking immune tolerance induction (ITI) by repeated high-dose FIX protein administration, which induced severe anaphylactoid reactions in inhibitors-positive haemophilia B mice. Liver gene therapy can thus reverse pre-existing immunity, induce active tolerance to FIX and establish sustained FIX activity at therapeutic levels. These data position gene therapy as an attractive treatment option for inhibitors-positive haemophilic patients.

Since the first publications coining the term RNA-seq (RNA sequencing) appeared in 2008, the number of publications containing RNA-seq data has grown exponentially, hitting an all-time high of 2,808 publications in 2016 (PubMed). With this wealth of RNA-seq data being generated, it is a challenge to extract maximal meaning from these datasets, and without the appropriate skills and background, there is risk of misinterpretation of these data. However, a general understanding of the principles underlying each step of RNA-seq data analysis allows investigators without a background in programming and bioinformatics to critically analyze their own datasets as well as published data. Our goals in the present review are to break down the steps of a typical RNA-seq analysis and to highlight the pitfalls and checkpoints along the way that are vital for bench scientists and biomedical researchers performing experiments that use RNA-seq.

Prolonged exposures to IH during the sleep period induce pronounced vWAT inflammation and insulin resistance despite concomitant vWAT mass reductions. These changes are only partially reversible after 3 months of normoxic recovery. Thus, long-lasting OSA may preclude complete reversibility of metabolic changes.

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Antigen (Ag)-specific tolerance in type 1 diabetes (T1D) in human has not been achieved yet. Targeting lentiviral vector (LV)-mediated gene expression to hepatocytes induces active tolerance toward the encoded Ag. The insulin B chain 9-23 (InsB9-23) is an immunodominant T cell epitope in nonobese diabetic (NOD) mice. To determine whether auto-Ag gene transfer to hepatocytes induces tolerance and control of T1D, NOD mice were treated with integrase-competent LVs (ICLVs) that selectively target the expression of InsB9-23 to hepatocytes. ICLV treatment induced InsB9-23-specific effector T cells but also FoxP3(+) regulatory T cells (Tregs), which halted islet immune cell infiltration, and protected from T1D. Moreover, ICLV treatment combined with a single suboptimal dose of anti-CD3 monoclonal antibody (mAb) is effective in T1D reversal. Splenocytes from LV.InsB9-23-treated mice, but not from LV.OVA (ovalbumin)-treated control mice, stopped diabetes development, demonstrating that protection is Ag-specific. Depletion of CD4(+)CD25(+)FoxP3(+) T cells led to diabetes progression, indicating that Ag-specific FoxP3(+) Tregs mediate protection. Integrase-defective LVs (IDLVs).InsB9-23, which alleviate the concerns for insertional mutagenesis and support transient transgene expression in hepatocytes, were also efficient in protecting from T1D. These data demonstrate that hepatocyte-targeted auto-Ag gene expression prevents and resolves T1D and that stable integration of the transgene is not required for this protection. Gene transfer to hepatocytes can be used to induce Ag-specific tolerance in autoimmune diseases.