Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible interstitial pneumonia caused by the excessive production and deposition of extracellular matrix components, including type I collagen. Activated fibroblasts, called α-SMA (α-smooth muscle actin)-expressing myofibroblasts, are the major source of type I collagen in pulmonary fibrosis (PF), but the mechanisms underlying disease progression have not been fully elucidated. Here, we obtained lung fibroblasts from patients with IPF from both nonfibrotic and fibrotic areas as determined by a lung computed tomography scan and compared gene expression between these areas by DNA microarray. We found that ANGPTL4 (angiopoietin-like 4) was highly expressed only in fibroblasts from the fibrotic area. ANGPTL4 was selectively expressed in the fibroblastic area of IPF lungs, where the myofibroblast marker α-SMA was also expressed. ANGPTL4 also regulates the gene expression of fibrosis-related markers, cell migration, and proliferation. In addition, ANGPTL4 expression in a murine model of PF induced by treatment with bleomycin was significantly induced in the lungs from the acute to the chronic phase. Single-cell transcriptome analysis during the course of bleomycin-induced PF revealed that Angptl4 was predominantly expressed in the activated fibroblasts and myofibroblasts. Moreover, the administration of recombinant ANGPTL4 to the bleomycin-induced fibrosis model significantly increased collagen deposition and exacerbated the PF. In contrast, the pathogenesis of PF in Angptl4-deficient mice was improved. These results indicate that ANGPTL4 is critical for the progression of PF and might be an early diagnostic marker and therapeutic target for IPF.

Profibrotic properties of C1q+ interstitial macrophages in silica-induced pulmonary fibrosis in mice.

Pulmonary fibrosis (PF) is a progressive fibrotic disease with poor prognosis and suboptimal therapeutic options. Although macrophages have been implicated in PF, the role of macrophage subsets, particularly interstitial macrophages (IMs), remains unknown. We performed a time-series single-cell RNA sequencing analysis of the silica-induced mouse PF model. Among the macrophage subsets in fibrotic lungs, Lyve1lo MHC IIhi IMs increased with fibrosis, and highly expressed profibrotic genes. Additionally, we identified C1q as an IM-specific marker. Experiments with C1q-diphtheria toxin receptor-GFP knock-in (C1qKI) mice revealed that IMs are distributed around fibrotic nodules. Depletion of C1q+ IMs in C1qKI mice decreased activated fibroblasts and epithelial cells; however, bodyweight loss and neutrophil infiltration were exacerbated in silica-induced PF. Collectively, these results suggest that IMs have profibrotic and anti-inflammatory properties and that the selective inhibition of the profibrotic function of IMs without compromising their anti-inflammatory effects is a potential novel therapeutic strategy for PF.

Complement protein C1q activates lung fibroblasts and exacerbates silica-induced pulmonary fibrosis in mice.

Pulmonary fibrosis is a progressive fibrotic disease with a poor prognosis and has suboptimal therapeutic options. The complement protein, C1q, which has various functions, such as promoting phagocytosis and signal transduction, has been shown to exacerbate several fibrosis-related diseases such as myofibrosis. In this study, we examined the role and cellular targets of C1q in pulmonary fibrosis. Silica-induced pulmonary fibrotic C1q-deficient mice showed improvement in fibrosis, and intratracheal administration of C1q to normal mice led to the induction of fibrotic changes. Single-cell RNA sequencing analysis revealed the early activation of fibroblasts and type 2 alveolar epithelial cells after intratracheal administration of C1q, and treatment of primary lung fibroblasts with C1q induced the expression of profibrotic genes. Thus, the inhibition of C1q may be regarded as a therapeutic target for pulmonary fibrosis.

Macrophages in lung fibrosis.

Pulmonary fibrosis (PF) is a disease in which excessive extracellular matrix (ECM) accumulation occurs in the lungs, which induces thickening of the alveolar walls, ultimately leading to the destruction of alveolar structures and respiratory failure. Idiopathic PF, the cause of which is unknown, has a poor prognosis with a median survival of 2-4 years after diagnosis. There is currently no known curative treatment. The mechanism underlying PF is thought to be initiated by the dysfunction of type II alveolar epithelial cells, which leads to ECM overproduction through the activation of fibroblasts. In addition, it has been suggested that a variety of cells contribute to fibrotic processes. In particular, clinical and basic research findings examining the roles of macrophages suggest that they may be pivotal regulators of PF. In this review, we discuss the characteristics, functions and origins of subsets of macrophages involved in PF, including resident alveolar, interstitial and monocyte-derived macrophages.

Single-Cell Transcriptome Profiling of Pancreatic Islets From Early Diabetic Mice Identifies Anxa10 for Ca2+ Allostasis Toward ß-Cell Failure.

Type 2 diabetes is a progressive disorder denoted by hyperglycemia and impaired insulin secretion. Although a decrease in ß-cell function and mass is a well-known trigger for diabetes, the comprehensive mechanism is still unidentified. Here, we performed single-cell RNA sequencing of pancreatic islets from prediabetic and diabetic db/db mice, an animal model of type 2 diabetes. We discovered a diabetes-specific transcriptome landscape of endocrine and nonendocrine cell types with subpopulations of ß- and α-cells. We recognized a new prediabetic gene, Anxa10, that was induced by and regulated Ca2+ influx from metabolic stresses. Anxa10-overexpressed ß-cells displayed suppression of glucose-stimulated intracellular Ca2+ elevation and potassium-induced insulin secretion. Pseudotime analysis of ß-cells predicted that this Ca2+-surge responder cluster would proceed to mitochondria dysfunction and endoplasmic reticulum stress. Other trajectories comprised dedifferentiation and transdifferentiation, emphasizing acinar-like cells in diabetic islets. Altogether, our data provide a new insight into Ca2+ allostasis and ß-cell failure processes. ARTICLE HIGHLIGHTS: The transcriptome of single-islet cells from healthy, prediabetic, and diabetic mice was studied. Distinct ß-cell heterogeneity and islet cell-cell network in prediabetes and diabetes were found. A new prediabetic ß-cell marker, Anxa10, regulates intracellular Ca2+ and insulin secretion. Diabetes triggers ß-cell to acinar cell transdifferentiation.

Clonal Spreading of Tumor-Infiltrating T Cells Underlies the Robust Antitumor Immune Responses.

The repertoire of tumor-infiltrating T cells is an emerging method for characterizing effective antitumor T-cell responses. Oligoclonal expansion of the tumor T-cell repertoire has been evaluated; however, their association with antitumor effects is unclear. We demonstrate here that the polyclonal fraction of the tumor-reactive T-cell repertoire, consisting of relatively minor clones, increased in tumor-bearing mice treated with monoclonal anti-programmed death-ligand 1 (PD-L1) or anti-CD4, which correlated with antitumor effects. Meanwhile, the size of the oligoclonal fraction consisting of major clones remained unchanged. Moreover, the polyclonal fraction was enriched in progenitor exhausted T cells, which are essential for a durable antitumor response, and was more dependent on CCR7+ migratory dendritic cells, which are responsible for priming tumor-reactive T cells in the tumor-draining lymph nodes. These results suggest that the expansion of diverse tumor-reactive clones ("clonal spreading") represents characteristics of antitumor T-cell responses induced by anti-CD4 and anti-PD-L1 treatment.

TAS-Seq is a robust and sensitive amplification method for bead-based scRNA-seq.

Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data. However, droplet- and plate-based scRNA-seq techniques have cell sampling bias that could affect the cell composition of scRNA-seq datasets. Here we developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq) for scRNA-seq based on a terminator, terminal transferase, and nanowell/bead-based scRNA-seq platform. TAS-Seq showed high tolerance to variations in the terminal transferase reaction, which complicate the handling of existing terminal transferase-based scRNA-seq methods. In murine and human lung samples, TAS-Seq yielded scRNA-seq data that were highly correlated with flow-cytometric data, showing higher gene-detection sensitivity and more robust detection of important cell-cell interactions and expression of growth factors/interleukins in cell subsets than 10X Chromium v2 and Smart-seq2. Expanding TAS-Seq application will improve understanding and atlas construction of lung biology at the single-cell level.

Combining an Alarmin HMGN1 Peptide with PD-L1 Blockade Results in Robust Antitumor Effects with a Concomitant Increase of Stem-Like/Progenitor Exhausted CD8+ T Cells.

The expansion of intratumoral stem-like/progenitor exhausted CD8+ T (Tstem/Tpex) cells provides a potential approach to improve the therapeutic efficacy of immune checkpoint blockade (ICB). Thus, here we demonstrate a strategy to facilitate Tstem/Tpex cell expansion by combining an alarmin high-mobility group nucleosome binding domain 1 (HMGN1) peptide with programmed death-ligand 1 (PD-L1) blockade. The antitumor effects of HMGN1, anti-PD-L1, and their combined treatment were monitored in the B16F10, LLC, Colon26, or EO771 tumor-bearing mice. The comprehensive immunologic analyses, such as high-dimensional flow cytometry, transcriptome analysis, and single-cell RNA-sequencing (scRNA-seq), were used to investigate the cellular and molecular mechanisms of antitumor immune responses after treatments. We identified the immunostimulatory domain (EPKRR SARLS AKPPA KVEAK PKK) on HMGN1 and synthesized this domain as a therapeutic peptide (minP1). Combined treatment with minP1 and PD-L1 blockade induced durable tumor regression in tumor-bearing mice. minP1 increased the number of intratumoral mature DCs enriched in immunoregulatory molecules (mregDC) and enhanced their MHC class I antigen-presenting program. minP1 also synergized with PD-L1 blockade in augmenting intratumoral Tstem/Tpex cell number. Analysis of our scRNA-seq dataset by CellPhonDB suggested potential interactions between mregDCs and Tstem/Tpex cells in tumors. Our results indicate that HMGN1 peptide (minP1) serves as an immunoadjuvant to promote effective anti-PD-L1 immunotherapy with increased Tstem/Tpex cells in tumors.