Addressing Technical Pitfalls in Pursuit of Molecular Factors That Mediate Immunoglobulin Gene Regulation.

The expressed Ab repertoire is a critical determinant of immune-related phenotypes. Ab-encoding transcripts are distinct from other expressed genes because they are transcribed from somatically rearranged gene segments. Human Abs are composed of two identical H and L chain polypeptides derived from genes in IGH locus and one of two L chain loci. The combinatorial diversity that results from Ab gene rearrangement and the pairing of different H and L chains contributes to the immense diversity of the baseline Ab repertoire. During rearrangement, Ab gene selection is mediated by factors that influence chromatin architecture, promoter/enhancer activity, and V(D)J recombination. Interindividual variation in the composition of the Ab repertoire associates with germline variation in IGH, implicating polymorphism in Ab gene regulation. Determining how IGH variants directly mediate gene regulation will require integration of these variants with other functional genomic datasets. In this study, we argue that standard approaches using short reads have limited utility for characterizing regulatory regions in IGH at haplotype resolution. Using simulated and chromatin immunoprecipitation sequencing reads, we define features of IGH that limit use of short reads and a single reference genome, namely 1) the highly duplicated nature of the DNA sequence in IGH and 2) structural polymorphisms that are frequent in the population. We demonstrate that personalized diploid references enhance performance of short-read data for characterizing mappable portions of the locus, while also showing that long-read profiling tools will ultimately be needed to fully resolve functional impacts of IGH germline variation on expressed Ab repertoires.

Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa chain locus in individuals of diverse ancestry.

Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of the IG loci has hindered use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we use long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n = 36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and novel structural variants harboring functional IGKV genes. Among 47 functional IGKV genes, we identify 145 alleles, 67 of which were not previously curated. We report inter-population differences in allele frequencies for 10 IGKV genes, including alleles unique to specific populations within this dataset. We identify haplotypes carrying signatures of gene conversion that associate with SNV enrichment in the IGK distal region, and a haplotype with an inversion spanning the proximal and distal regions. These data provide a critical resource of curated genomic reference information from diverse ancestries, laying a foundation for advancing our understanding of population-level genetic variation in the IGK locus.

Endothelial-Specific Loss of Sphingosine-1-Phosphate Receptor 1 Increases Vascular Permeability and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell-(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1-/-) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1-/- mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1-/- mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.

Retinoid signaling regulates angiogenesis and blood-retinal barrier integrity in neonatal mouse retina.

OBJECTIVE: The neonatal mouse retina is a well-characterized experimental model for investigating factors impacting retinal angiogenesis and inner blood-retinal barrier (BRB) integrity. Retinoic acid (RA) is an essential signaling molecule. RA is needed for vasculogenic development in embryos and endothelial barrier integrity in zebrafish retina and adult mouse brain; however, the function of this signaling molecule in developing mammalian retinal vasculature remains unknown. This study aims to investigate the role of RA signaling in angiogenesis and inner BRB integrity in mouse neonatal retina. METHODS: RA distribution in the developing neurovascular retina was assessed in mice carrying an RA-responsive transgene. RA function in retinal angiogenesis was determined by treating C57BL/6 neonatal pups with a pharmacological inhibitor of RA signaling BMS493 or control vehicle. BRB integrity assessed by monitoring leakage of injected tracer into extravascular retinal tissue. RESULTS: RA signaling activity is present in peripheral astrocytes in domains corresponding to RA activity of the underlying neural retina. RA inhibition impaired retinal angiogenesis and reduced endothelial cell proliferation. RA inhibition also compromised BRB integrity. Vascular leakage was not associated with altered expression of CLDN5, PLVAP, LEF1, or VEcad. CONCLUSIONS: RA signaling is needed for angiogenesis and integrity of the BRB in the neonatal mouse retina.

Lysolipids in Vascular Development, Biology, and Disease.

Membrane phospholipid metabolism forms lysophospholipids, which possess unique biochemical and biophysical properties that influence membrane structure and dynamics. However, lysophospholipids also function as ligands for G-protein-coupled receptors that influence embryonic development, postnatal physiology, and disease. The 2 most well-studied species-lysophosphatidic acid and S1P (sphingosine 1-phosphate)-are particularly relevant to vascular development, physiology, and cardiovascular diseases. This review summarizes the role of lysophosphatidic acid and S1P in vascular developmental processes, endothelial cell biology, and their roles in cardiovascular disease processes. In addition, we also point out the apparent connections between lysophospholipid biology and the Wnt (int/wingless family) pathway, an evolutionarily conserved fundamental developmental signaling system. The discovery that components of the lysophospholipid signaling system are key genetic determinants of cardiovascular disease has warranted current and future research in this field. As pharmacological approaches to modulate lysophospholipid signaling have entered the clinical sphere, new findings in this field promise to influence novel therapeutic strategies in cardiovascular diseases.

HDL-bound sphingosine-1-phosphate restrains lymphopoiesis and neuroinflammation.

Lipid mediators influence immunity in myriad ways. For example, circulating sphingosine-1-phosphate (S1P) is a key regulator of lymphocyte egress. Although the majority of plasma S1P is bound to apolipoprotein M (ApoM) in the high-density lipoprotein (HDL) particle, the immunological functions of the ApoM-S1P complex are unknown. Here we show that ApoM-S1P is dispensable for lymphocyte trafficking yet restrains lymphopoiesis by activating the S1P1 receptor on bone marrow lymphocyte progenitors. Mice that lacked ApoM (Apom(-/-)) had increased proliferation of Lin(-) Sca-1(+) cKit(+) haematopoietic progenitor cells (LSKs) and common lymphoid progenitors (CLPs) in bone marrow. Pharmacological activation or genetic overexpression of S1P1 suppressed LSK and CLP cell proliferation in vivo. ApoM was stably associated with bone marrow CLPs, which showed active S1P1 signalling in vivo. Moreover, ApoM-bound S1P, but not albumin-bound S1P, inhibited lymphopoiesis in vitro. Upon immune stimulation, Apom(-/-) mice developed more severe experimental autoimmune encephalomyelitis, characterized by increased lymphocytes in the central nervous system and breakdown of the blood-brain barrier. Thus, the ApoM-S1P-S1P1 signalling axis restrains the lymphocyte compartment and, subsequently, adaptive immune responses. Unique biological functions imparted by specific S1P chaperones could be exploited for novel therapeutic opportunities.

Complete sequencing of ape genomes.

We present haplotype-resolved reference genomes and comparative analyses of six ape species, namely: chimpanzee, bonobo, gorilla, Bornean orangutan, Sumatran orangutan, and siamang. We achieve chromosome-level contiguity with unparalleled sequence accuracy (<1 error in 500,000 base pairs), completely sequencing 215 gapless chromosomes telomere-to-telomere. We resolve challenging regions, such as the major histocompatibility complex and immunoglobulin loci, providing more in-depth evolutionary insights. Comparative analyses, including human, allow us to investigate the evolution and diversity of regions previously uncharacterized or incompletely studied without bias from mapping to the human reference. This includes newly minted gene families within lineage-specific segmental duplications, centromeric DNA, acrocentric chromosomes, and subterminal heterochromatin. This resource should serve as a definitive baseline for all future evolutionary studies of humans and our closest living ape relatives.

The Vasculature in Pulmonary Fibrosis.

Purpose of Review: The current paradigm of idiopathic pulmonary fibrosis (IPF) pathogenesis involves recurrent injury to a sensitive alveolar epithelium followed by impaired repair responses marked by fibroblast activation and deposition of extracellular matrix. Multiple cell types are involved in this response with potential roles suggested by advances in single-cell RNA sequencing and lung developmental biology. Notably, recent work has better characterized the cell types present in the pulmonary endothelium and identified vascular changes in patients with IPF. Recent Findings: Lung tissue from patients with IPF has been examined at single-cell resolution, revealing reductions in lung capillary cells and expansion of a population of vascular cells expressing markers associated with bronchial endothelium. In addition, pre-clinical models have demonstrated a fundamental role for aging and vascular permeability in the development of pulmonary fibrosis. Summary: Mounting evidence suggests that the endothelium undergoes changes in the context of fibrosis, and these changes may contribute to the development and/or progression of pulmonary fibrosis. Additional studies will be needed to further define the functional role of these vascular changes.

Sphingosine 1-Phosphate Receptor Signaling Establishes AP-1 Gradients to Allow for Retinal Endothelial Cell Specialization.

Transcriptional mechanisms that drive angiogenesis and organotypic vascular endothelial cell specialization are poorly understood. Here, we show that retinal endothelial sphingosine 1-phosphate receptors (S1PRs), which restrain vascular endothelial growth factor (VEGF)-induced angiogenesis, spatially restrict expression of JunB, a member of the activator protein 1 (AP-1) family of transcription factors (TFs). Mechanistically, VEGF induces JunB expression at the sprouting vascular front while S1PR-dependent vascular endothelial (VE)-cadherin assembly suppresses JunB expression in the nascent vascular network, thus creating a gradient of this TF. Endothelial-specific JunB knockout mice showed diminished expression of neurovascular guidance genes and attenuated retinal vascular network progression. In addition, endothelial S1PR signaling is required for normal expression of ß-catenin-dependent genes such as TCF/LEF1 and ZIC3 TFs, transporters, and junctional proteins. These results show that S1PR signaling restricts JunB function to the expanding vascular front, thus creating an AP-1 gradient and enabling organotypic endothelial cell specialization of the vascular network.

Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta.

Despite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. We report the comprehensive characterization of transcriptomes (bulk and single-cell) and chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse aortic endothelial cells. First, S1PR1 regulates NFκB and nuclear glucocorticoid receptor pathways to suppress inflammation-related mRNAs. Second, S1PR1 signaling in the heterogenous endothelial cell (EC) subtypes occurs at spatially-distinct areas of the aorta. For example, a transcriptomically distinct arterial EC population at vascular branch points (aEC1) exhibits ligand-independent S1PR1/ß-arrestin coupling. In contrast, circulatory S1P-dependent S1PR1/ß-arrestin coupling was observed in non-branch point aEC2 cells that exhibit an inflammatory gene expression signature. Moreover, S1P/S1PR1 signaling regulates the expression of lymphangiogenic and inflammation-related transcripts in an adventitial lymphatic EC (LEC) population in a ligand-dependent manner. These insights add resolution to existing concepts of endothelial heterogeneity, GPCR signaling and S1P biology.

Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes.

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) activate G protein-coupled receptors (GPCRs) to regulate biological processes. Using a genome-wide CRISPR/dCas9-based GPCR signaling screen, LPAR1 was identified as an inducer of S1PR1/ß-arrestin coupling while suppressing Gαi signaling. S1pr1 and Lpar1-positive lymphatic endothelial cells (LECs) of lymph nodes exhibit constitutive S1PR1/ß-arrestin signaling, which was suppressed by LPAR1 antagonism. Pharmacological inhibition or genetic loss of function of Lpar1 reduced the frequency of punctate junctions at sinus-lining LECs. Ligand activation of transfected LPAR1 in endothelial cells remodeled junctions from continuous to punctate structures and increased transendothelial permeability. In addition, LPAR1 antagonism in mice increased lymph node retention of adoptively transferred lymphocytes. These data suggest that cross-talk between LPAR1 and S1PR1 promotes the porous junctional architecture of sinus-lining LECs, which enables efficient lymphocyte trafficking. Heterotypic inter-GPCR coupling may regulate complex cellular phenotypes in physiological milieu containing many GPCR ligands.

Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis: Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type.

Gene duplication confers genetic redundancy that can facilitate subfunctionalization, the partitioning of ancestral functions between paralogs. We capitalize on a recent genome duplication in Xenopus laevis (African clawed frog) to interrogate possible functional differentiation between alloalleles of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates toxicity of dioxin-like compounds and plays a role in the physiology and development of the cardiovascular, hepatic, and immune systems in vertebrates. X. laevis has 2 AHR genes, AHR1α and AHR1ß To test the hypothesis that the encoded proteins exhibit different molecular functions, we used TALENs in XLK-WG cells, generating mutant lines lacking functional versions of each AHR and measuring the transcriptional responsiveness of several target genes to the toxic xenobiotic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the candidate endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ). Mutation of either AHR1α or AHR1ß reduced TCDD induction of the canonical AHR target, Cytochrome P4501A6, by 75%, despite the much lower abundance of AHR1ß in wild-type cells. More modestly induced target genes, encoding aryl hydrocarbon receptor repressor (AHRR), spectrin repeat-containing nuclear envelope protein 1 (SYNE-1), and gap junction protein gamma 1 (GJC1), were regulated solely by AHR1α. AHR1ß was responsible for CYP1A6 induction by FICZ, while AHR1α mediated FICZ induction of AHRR We conclude that AHR1α and AHR1ß have distinct transcriptional functions in response to specific agonists, even within a single cell type. Functional analysis of frog AHR paralogs advances the understanding of AHR evolution and as well as the use of frog models of developmental toxicology such as FETAX.

An engineered S1P chaperone attenuates hypertension and ischemic injury.

Endothelial dysfunction, a hallmark of vascular disease, is restored by plasma high-density lipoprotein (HDL). However, a generalized increase in HDL abundance is not beneficial, suggesting that specific HDL species mediate protective effects. Apolipoprotein M-containing HDL (ApoM+HDL), which carries the bioactive lipid sphingosine 1-phosphate (S1P), promotes endothelial function by activating G protein-coupled S1P receptors. Moreover, HDL-bound S1P is limiting in several inflammatory, metabolic, and vascular diseases. We report the development of a soluble carrier for S1P, ApoM-Fc, which activated S1P receptors in a sustained manner and promoted endothelial function. In contrast, ApoM-Fc did not modulate circulating lymphocyte numbers, suggesting that it specifically activated endothelial S1P receptors. ApoM-Fc administration reduced blood pressure in hypertensive mice, attenuated myocardial damage after ischemia/reperfusion injury, and reduced brain infarct volume in the middle cerebral artery occlusion model of stroke. Our proof-of-concept study suggests that selective and sustained targeting of endothelial S1P receptors by ApoM-Fc could be a viable therapeutic strategy in vascular diseases.