Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1 + SiglecF + transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo . Human orthologues of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.

Tissues are complex milieus consisting of numerous cell types. Several recent methods have attempted to enumerate cell subsets from transcriptomes. However, the available methods have used limited sources for training and give only a partial portrayal of the full cellular landscape. Here we present xCell, a novel gene signature-based method, and use it to infer 64 immune and stromal cell types. We harmonized 1822 pure human cell type transcriptomes from various sources and employed a curve fitting approach for linear comparison of cell types and introduced a novel spillover compensation technique for separating them. Using extensive in silico analyses and comparison to cytometry immunophenotyping, we show that xCell outperforms other methods. xCell is available at http://xCell.ucsf.edu/.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1349-1) contains supplementary material, which is available to authorized users.

Type 2 diabetes mellitus (DM) is characterized by insulin resistance and pancreatic ␤ cell dysfunction. In high-risk subjects, the earliest detectable abnormality is insulin resistance in skeletal muscle. Impaired insulin-mediated signaling, gene expression, glycogen synthesis, and accumulation of intramyocellular triglycerides have all been linked with insulin resistance, but no specific defect responsible for insulin resistance and DM has been identified in humans. To identify genes potentially important in the pathogenesis of DM, we analyzed gene expression in skeletal muscle from healthy metabolically characterized nondiabetic (family history negative and positive for DM) and diabetic Mexican-American subjects. We demonstrate that insulin resistance and DM associate with reduced expression of multiple nuclear respiratory factor-1 (NRF-1)-dependent genes encoding key enzymes in oxidative metabolism and mitochondrial function. Although NRF-1 expression is decreased only in diabetic subjects, expression of both PPAR␥ coactivator 1-␣ and-␤ (PGC1-␣͞PPARGC1 and PGC1-␤͞PERC), coactivators of NRF-1 and PPAR␥-dependent transcription, is decreased in both diabetic subjects and family history-positive nondiabetic subjects. Decreased PGC1 expression may be responsible for decreased expression of NRF-dependent genes, leading to the metabolic disturbances characteristic of insulin resistance and DM.I nsulin resistance precedes and predicts the development of type 2 diabetes mellitus (DM) (1, 2). Defects in insulin signal transduction, gene expression, and muscle glycogen synthesis, and accumulation of intramyocellular triglycerides have all been identified as potential mediators of insulin resistance in high-risk individuals (1, 3-7). However, the molecular pathogenesis of DM remains unknown. Mouse data highlight the importance of glucose uptake into muscle but suggest a role for novel mechanisms, distinct from insulin signaling pathways (8). The importance of genetic risk factors is exemplified by the high concordance of DM in identical twins, the strong influence of family history and ethnicity on risk, and the identification of DNA sequence alterations in both rare and common forms of DM (9). Environmental factors, including obesity, inactivity, and aging, also play critical roles in DM risk. Because both genotype and environment converge to influence cellular function via gene and protein expression, we hypothesize that alterations in expression define a phenotype that parallels the metabolic evolution of DM and provides potential clues to pathogenesis. We used high-density oligonucleotide arrays to identify genes differentially expressed in skeletal muscle from nondiabetic and type 2 diabetic subjects. Because hyperglycemia per se can modulate expression, we also evaluated gene expression in insulin-resistant subjects at high risk for DM (''prediabetes'') on the basis of family history of DM and Mexican-American ethnicity (10). We demonstrate that prediabetic and diabetic muscle is characterized by decreased expressi...