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.

Peroxisome proliferator activated-receptor (PPAR) isoforms, ␣ and ␥, function as important coregulators of energy (lipid) homeostasis. PPAR␣ regulates fatty acid oxidation primarily in liver and to a lesser extent in adipose tissue, whereas PPAR␥ serves as a key regulator of adipocyte differentiation and lipid storage. Of the two PPAR␥ isoforms, PPAR␥1 and PPAR␥2 generated by alternative splicing, PPAR␥1 isoform is expressed in liver and other tissues, whereas PPAR␥2 isoform is expressed exclusively in adipose tissue where it regulates adipogenesis and lipogenesis. Since the function of PPAR␥1 in liver is not clear, we have, in this study, investigated the biological impact of overexpression of PPAR␥1 in mouse liver. Adenovirus-PPAR␥1 injected into the tail vein induced hepatic steatosis in PPAR␣ ؊/؊ mice. Northern blotting and gene expression profiling results showed that adipocyte-specific genes and lipogenesis-related genes are highly induced in PPAR␣ ؊/؊ livers with PPAR␥1 overexpression. These include adipsin, adiponectin, aP2, caveolin-1, fasting-induced adipose factor, fat-specific gene 27 (FSP27), CD36, ⌬ 9 desaturase, and malic enzyme among others, implying adipogenic transformation of hepatocytes. Of interest is that hepatic steatosis per se, induced either by feeding a diet deficient in choline or developing in fasted PPAR␣ ؊/؊ mice, failed to induce the expression of these PPAR␥-regulated adipogenesis-related genes in steatotic liver. These results suggest that a high level of PPAR␥ in mouse liver is sufficient for the induction of adipogenic transformation of hepatocytes with adipose tissue-specific gene expression and lipid accumulation. We conclude that excess PPAR␥ activity can lead to the development of a novel type of adipogenic hepatic steatosis.

Fasting causes lipolysis in adipose tissue leading to the release of large quantities of free fatty acids into circulation that reach the liver where they are metabolized to generate ketone bodies to serve as fuels for other tissues. Since fatty acid-metabolizing enzymes in the liver are transcriptionally regulated by peroxisome proliferator-activated receptor ␣ (PPAR␣), we investigated the role of PPAR␣ in the induction of these enzymes in response to fasting and their relationship to the development of hepatic steatosis in mice deficient in PPAR␣ (PPAR␣ ؊/؊ ), peroxisomal fatty acyl-CoA oxidase (AOX ؊/؊ ), and in both PPAR␣ and AOX (double knockout (DKO)). Fasting for 48 -72 h caused profound impairment of fatty acid oxidation in both PPAR␣؊/؊ and DKO mice, and DKO mice revealed a greater degree of hepatic steatosis when compared with PPAR␣ ؊/؊ mice. The absence of PPAR␣ in both PPAR␣ ؊/؊ and DKO mice impairs the induction of mitochondrial ␤-oxidation in liver following fasting which contributes to hypoketonemia and hepatic steatosis. Pronounced steatosis in DKO mouse livers is due to the added deficiency of peroxisomal ␤-oxidation system in these animals due to the absence of AOX. In mice deficient in AOX alone, the sustained hyperactivation of PPAR␣ and up-regulation of mitochondrial ␤-oxidation and microsomal -oxidation systems as well as the regenerative nature of a majority of hepatocytes containing numerous spontaneously proliferated peroxisomes, which appear refractory to store triglycerides, blunt the steatotic response to fasting. Starvation for 72 h caused a decrease in PPAR␣ hepatic mRNA levels in wild type mice, with no perceptible compensatory increases in PPAR␥ and PPAR␦ mRNA levels. PPAR␥ and PPAR␦ hepatic mRNA levels were lower in fed PPAR␣ ؊/؊ and DKO mice when compared with wild type mice, and fasting caused a slight increase only in PPAR␥ levels and a decrease in PPAR␦ levels. Fasting did not change the PPAR isoform levels in AOX ؊/؊ mouse liver. These observations point to the critical importance of PPAR␣ in the transcriptional regulatory responses to fasting and in determining the severity of hepatic steatosis.

Peroxisomal ␤-oxidation system consists of four consecutive reactions to preferentially metabolize very long chain fatty acids. The first step of this system, catalyzed by acyl-CoA oxidase (AOX), converts fatty acylCoA to 2-trans-enoyl-CoA. Herein, we show that mice deficient in AOX exhibit steatohepatitis, increased hepatic H 2 O 2 levels, and hepatocellular regeneration, leading to a complete reversal of fatty change by 6 to 8 months of age. The liver of AOX؊/؊ mice with regenerated hepatocytes displays profound generalized spontaneous peroxisome proliferation and increased mRNA levels of genes that are regulated by peroxisome proliferator-activated receptor ␣ (PPAR␣). Hepatic adenomas and carcinomas develop in AOX؊/؊ mice by 15 months of age due to sustained activation of PPAR␣. These observations implicate acyl-CoA and other putative substrates for AOX, as biological ligands for PPAR␣; thus, a normal AOX gene is indispensable for the physiological regulation of PPAR␣.

Lung transplantation can potentially be a life-saving treatment for patients with non-resolving COVID-19-associated respiratory failure. Concerns limiting lung transplantation include recurrence of SARS-CoV-2 infection in the allograft, technical challenges imposed by viral-mediated injury to the native lung, and the potential risk for allograft infection by pathogens causing ventilator-associated pneumonia in the native lung. Importantly, the native lung might recover, resulting in long-term outcomes preferable to those of transplant. Here, we report the results of lung transplantation in three patients with non-resolving COVID-19-associated respiratory failure. We performed single molecule fluorescent in situ hybridization (smFISH) to detect both positive and negative strands of SARS-CoV-2 RNA in explanted lung tissue from the three patients and in additional control lung tissue samples. We conducted extracellular matrix imaging and single cell RNA sequencing on explanted lung tissue from the three patients who underwent transplantation and on warm post-mortem lung biopsies from two patients who had died from COVID-19-associated pneumonia. Lungs from these five patients with prolonged COVID-19 disease were free of SARS-CoV-2 as detected by smFISH, but pathology showed extensive evidence of injury and fibrosis that resembled end-stage pulmonary fibrosis. Using machine learning, we compared single cell RNA sequencing data from the lungs of patients with late stage COVID-19 to that from the lungs of patients with pulmonary fibrosis and identified similarities in gene expression across cell lineages. Our findings suggest that some patients with severe COVID-19 develop fibrotic lung disease for which lung transplantation is their only option for survival.