Multi-organ single-cell RNA sequencing in mice reveals early hyperglycemia responses that converge on fibroblast dysregulation.

Diabetes causes a range of complications that can affect multiple organs. Hyperglycemia is an important driver of diabetes-associated complications, mediated by biological processes such as dysfunction of endothelial cells, fibrosis, and alterations in leukocyte number and function. Here, we dissected the transcriptional response of key cell types to hyperglycemia across multiple tissues using single-cell RNA sequencing (scRNA-seq) and identified conserved, as well as organ-specific, changes associated with diabetes complications. By studying an early time point of diabetes, we focus on biological processes involved in the initiation of the disease, before the later organ-specific manifestations had supervened. We used a mouse model of type 1 diabetes and performed scRNA-seq on cells isolated from the heart, kidney, liver, and spleen of streptozotocin-treated and control male mice after 8 weeks and assessed differences in cell abundance, gene expression, pathway activation, and cell signaling across organs and within organs. In response to hyperglycemia, endothelial cells, macrophages, and monocytes displayed organ-specific transcriptional responses, whereas fibroblasts showed similar responses across organs, exhibiting altered metabolic gene expression and increased myeloid-like fibroblasts. Furthermore, we found evidence of endothelial dysfunction in the kidney, and of endothelial-to-mesenchymal transition in streptozotocin-treated mouse organs. In summary, our study represents the first single-cell and multi-organ analysis of early dysfunction in type 1 diabetes-associated hyperglycemia, and our large-scale dataset (comprising 67 611 cells) will serve as a starting point, reference atlas, and resource for further investigating the events leading to early diabetic disease.

Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.

AIMS: Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS: Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS: Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis.

BACKGROUND: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS: Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Altered Macrophage Polarization Induces Experimental Pulmonary Hypertension and Is Observed in Patients With Pulmonary Arterial Hypertension.

OBJECTIVE: To determine whether global reduction of CD68 (cluster of differentiation) macrophages impacts the development of experimental pulmonary arterial hypertension (PAH) and whether this reduction affects the balance of pro- and anti-inflammatory macrophages within the lung. Additionally, to determine whether there is evidence of an altered macrophage polarization in patients with PAH. Approach and Results: Macrophage reduction was induced in mice via doxycycline-induced CD68-driven cytotoxic diphtheria toxin A chain expression (macrophage low [MacLow] mice). Chimeric mice were generated using bone marrow transplant. Mice were phenotyped for PAH by echocardiography and closed chest cardiac catheterization. Murine macrophage phenotyping was performed on lungs, bone marrow-derived macrophages, and alveolar macrophages using immunohistochemical and flow cytometry. Monocyte-derived macrophages were isolated from PAH patients and healthy volunteers and polarization capacity assessed morphologically and by flow cytometry. After 6 weeks of macrophage depletion, male but not female MacLow mice developed PAH. Chimeric mice demonstrated a requirement for both MacLow bone marrow and MacLow recipient mice to cause PAH. Immunohistochemical analysis of lung sections demonstrated imbalance in M1/M2 ratio in male MacLow mice only, suggesting that this imbalance may drive the PAH phenotype. M1/M2 imbalance was also seen in male MacLow bone marrow-derived macrophages and PAH patient monocyte-derived macrophages following stimulation with doxycycline and IL (interleukin)-4, respectively. Furthermore, MacLow-derived alveolar macrophages showed characteristic differences in terms of their polarization and expression of diphtheria toxin A chain following stimulation with doxycycline. CONCLUSIONS: These data further highlight a sex imbalance in PAH and further implicate immune cells into this paradigm. Targeting imbalance of macrophage population may offer a future therapeutic option.

A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.

Divergent Roles for TRAIL in Lung Diseases.

The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a widely expressed cytokine that can bind five different receptors. TRAIL has been of particular interest for its proposed ability to selectively induce apoptosis in tumour cells. However, it has also been found to regulate a wide variety of non-canonical cellular effects including survival, migration and proliferation via kinase signalling pathways. Lung diseases represent a wide range of conditions affecting multiple tissues. TRAIL has been implicated in several biological processes underlying lung diseases, including angiogenesis, inflammation, and immune regulation. For example, TRAIL is detrimental in pulmonary arterial hypertension-it is upregulated in patient serum and lungs, and drives the underlying proliferative pulmonary vascular remodelling in rodent models. However, TRAIL protects against pulmonary fibrosis in mice models-by inducing apoptosis of neutrophils-and reduced serum TRAIL is found in patients. Conversely, in the airways TRAIL positively regulates inflammation and immune response. In COPD patients and asthmatic patients challenged with antigen, TRAIL and its death receptors are upregulated in serum and airways. Furthermore, TRAIL-deleted mouse models have reduced airway inflammation and remodelling. In the context of respiratory infections, TRAIL assists in immune response, e.g., via T-cell toxicity in influenza infection, and neutrophil killing in S. pneumoniae infection. In this mini-review, we examine the functions of TRAIL and highlight the diverse roles TRAIL has in diseases affecting the lung. Disentangling the facets of TRAIL signalling in lung diseases could help in understanding their pathogenic processes and targeting novel treatments.

Genomic investigation of alpha-synuclein multiplication and parkinsonism.

OBJECTIVE: Copy number variation is a common polymorphic phenomenon within the human genome. Although the majority of these events are non-deleterious they can also be highly pathogenic. Herein we characterize five families with parkinsonism that have been identified to harbor multiplication of the chromosomal 4q21 locus containing the alpha-synuclein gene (SNCA). METHODS: A methodological approach using fluorescent in situ hybridization and Affymetrix (Santa Clara, CA) 250K SNP microarrays was used to characterize the multiplication in each family and to identify the genes encoded within the region. The telomeric and centromeric breakpoints of each family were further narrowed using semiquantitative polymerase chain reaction with microsatellite markers and then screened for transposable repeat elements. RESULTS: The severity of clinical presentation is correlated with SNCA dosage and does not appear to be overtly affected by the presence of other genes in the multiplicated region. With the exception of the Lister kindred, in each family the multiplication event appears de novo. The type and position of Alu/LINE repeats are also different at each breakpoint. Microsatellite analysis demonstrates two genomic mechanisms are responsible for chromosome 4q21 multiplications, including both SNCA duplication and recombination. INTERPRETATION: SNCA dosage is responsible for parkinsonism, autonomic dysfunction, and dementia observed within each family. We hypothesize dysregulated expression of wild-type alpha-synuclein results in parkinsonism and may explain the recent association of common SNCA variants in sporadic Parkinson's disease. SNCA genomic duplication results from intraallelic (segmental duplication) or interallelic recombination with unequal crossing over, whereas both mechanisms appear to be required for genomic SNCA triplication.