A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

Dynamic control of gene expression by ISGF3 and IRF1 during IFNß and IFNγ signaling.

Type I interferons (IFN-I, including IFNß) and IFNγ produce overlapping, yet clearly distinct immunological activities. Recent data show that the distinctness of global transcriptional responses to the two IFN types is not apparent when comparing their immediate effects. By analyzing nascent transcripts induced by IFN-I or IFNγ over a period of 48 h, we now show that the distinctiveness of the transcriptomes emerges over time and is based on differential employment of the ISGF3 complex as well as of the second-tier transcription factor IRF1. The distinct transcriptional properties of ISGF3 and IRF1 correspond with a largely diverse nuclear protein interactome. Mechanistically, we describe the specific input of ISGF3 and IRF1 into enhancer activation and the regulation of chromatin accessibility at interferon-stimulated genes (ISG). We further report differences between the IFN types in altering RNA polymerase II pausing at ISG 5' ends. Our data provide insight how transcriptional regulators create immunological identities of IFN-I and IFNγ.

Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin dependent-kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. We here describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, proposing a concept where MAZ and NFY-A are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.

JAK-STAT signaling maintains homeostasis in T cells and macrophages.

Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

Coordinated ARP2/3 and glycolytic activities regulate the morphological and functional fitness of human CD8+ T cells.

Actin cytoskeleton remodeling sustains the ability of cytotoxic T cells to search for target cells and eliminate them. We here investigated the relationship between energetic status, actin remodeling, and functional fitness in human CD8+ effector T cells. Cell spreading during migration or immunological synapse assembly mirrored cytotoxic activity. Morphological and functional fitness were boosted by interleukin-2 (IL-2), which also stimulated the transcription of glycolytic enzymes, actin isoforms, and actin-related protein (ARP)2/3 complex subunits. This molecular program scaled with F-actin content and cell spreading. Inhibiting glycolysis impaired F-actin remodeling at the lamellipodium, chemokine-driven motility, and adhesion, while mitochondrial oxidative phosphorylation blockade impacted cell elongation during confined migration. The severe morphological and functional defects of ARPC1B-deficient T cells were only partially corrected by IL-2, emphasizing ARP2/3-mediated actin polymerization as a crucial energy state integrator. The study therefore underscores the tight coordination between metabolic and actin remodeling programs to sustain the cytotoxic activity of CD8+ T cells.

Non-IgE-reactive allergen peptides deteriorate the skin barrier in house dust mite-sensitized atopic dermatitis patients.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by type 2 cytokine-driven skin inflammation and epithelial barrier dysfunction. The latter is believed to allow the increased penetration of chemicals, toxins, and allergens into the skin. House dust mite allergens, particularly Der p 2, are important triggers in sensitized individuals with AD; the precise actions of these allergens in epithelial biology remain, however, incompletely understood. In this study, we compared the effects of the protein allergen Der p 2 and a mix of non-IgE-reactive Der p 2 peptides on skin cells using patch tests in AD patients and healthy participants. We then analyzed mRNA expression profiles of keratinocytes by single-cell RNA-sequencing. We report that existing barrier deficiencies in the non-lesional skin of AD patients allow deep penetration of Der p 2 and its peptides, leading to local microinflammation. Der p 2 protein specifically upregulated genes involved in the innate immune system, stress, and danger signals in suprabasal KC. Der p 2 peptides further downregulated skin barrier genes, in particular the expression of genes involved in cell-matrix and cell-cell adhesion. Peptides also induced genes involved in hyperproliferation and caused disturbances in keratinocyte differentiation. Furthermore, inflammasome-relevant genes and IL18 were overexpressed, while KRT1 was downregulated. Our data suggest that Der p 2 peptides contribute to AD initiation and exacerbation by augmenting hallmark features of AD, such as skin inflammation, barrier disruption, and hyperplasia of keratinocytes.

Single-cell transcriptomics and epigenomics unravel the role of monocytes in neuroblastoma bone marrow metastasis.

Metastasis is the major cause of cancer-related deaths. Neuroblastoma (NB), a childhood tumor has been molecularly defined at the primary cancer site, however, the bone marrow (BM) as the metastatic niche of NB is poorly characterized. Here we perform single-cell transcriptomic and epigenomic profiling of BM aspirates from 11 subjects spanning three major NB subtypes and compare these to five age-matched and metastasis-free BM, followed by in-depth single cell analyses of tissue diversity and cell-cell interactions, as well as functional validation. We show that cellular plasticity of NB tumor cells is conserved upon metastasis and tumor cell type composition is NB subtype-dependent. NB cells signal to the BM microenvironment, rewiring via macrophage mgration inhibitory factor and midkine signaling specifically monocytes, which exhibit M1 and M2 features, are marked by activation of pro- and anti-inflammatory programs, and express tumor-promoting factors, reminiscent of tumor-associated macrophages. The interactions and pathways characterized in our study provide the basis for therapeutic approaches that target tumor-to-microenvironment interactions.

Cytokine-directed cellular cross-talk imprints synovial pathotypes in rheumatoid arthritis.

INTRODUCTION: Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. METHODS: Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4+ T cells from patients with RA was established to quantify cell-cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell-cell interaction. RESULTS: Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4+ T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. CONCLUSION: Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.

Biallelic NFATC1 mutations cause an inborn error of immunity with impaired CD8+ T-cell function and perturbed glycolysis.

The nuclear factor of activated T cells (NFAT) family of transcription factors plays central roles in adaptive immunity in murine models; however, their contribution to human immune homeostasis remains poorly defined. In a multigenerational pedigree, we identified 3 patients who carry germ line biallelic missense variants in NFATC1, presenting with recurrent infections, hypogammaglobulinemia, and decreased antibody responses. The compound heterozygous NFATC1 variants identified in these patients caused decreased stability and reduced the binding of DNA and interacting proteins. We observed defects in early activation and proliferation of T and B cells from these patients, amenable to rescue upon genetic reconstitution. Stimulation induced early T-cell activation and proliferation responses were delayed but not lost, reaching that of healthy controls at day 7, indicative of an adaptive capacity of the cells. Assessment of the metabolic capacity of patient T cells revealed that NFATc1 dysfunction rendered T cells unable to engage in glycolysis after stimulation, although oxidative metabolic processes were intact. We hypothesized that NFATc1-mutant T cells could compensate for the energy deficit due to defective glycolysis by using enhanced lipid metabolism as an adaptation, leading to a delayed, but not lost, activation responses. Indeed, we observed increased 13C-labeled palmitate incorporation into citrate, indicating higher fatty acid oxidation, and we demonstrated that metformin and rosiglitazone improved patient T-cell effector functions. Collectively, enabled by our molecular dissection of the consequences of loss-of-function NFATC1 mutations and extending the role of NFATc1 in human immunity beyond receptor signaling, we provide evidence of metabolic plasticity in the context of impaired glycolysis observed in patient T cells, alleviating delayed effector responses.

Single-cell RNA sequencing defines disease-specific differences between chronic nodular prurigo and atopic dermatitis.

BACKGROUND: Chronic nodular prurigo (CNPG) is an inflammatory skin disease that is maintained by a chronic itch-scratch cycle likely rooted in neuroimmunological dysregulation. This condition may be associated with atopy in some patients, and there are now promising therapeutic results from blocking type 2 cytokines such as IL-4, IL-13, and IL-31. OBJECTIVES: This study aimed to improve the understanding of pathomechanisms underlying CNPG as well as molecular relationships between CNPG and atopic dermatitis (AD). METHODS: We profiled skin lesions from patients with CNPG in comparison with AD and healthy control individuals using single-cell RNA sequencing combined with T-cell receptor sequencing. RESULTS: We found type 2 immune skewing in both CNPG and AD, as evidenced by CD4+ helper T cells expressing IL13. However, only AD harbored an additional, oligoclonally expanded CD8A+IL9R+IL13+ cytotoxic T-cell population, and immune activation pathways were highly upregulated in AD, but less so in CNPG. Conversely, CNPG showed signatures of extracellular matrix organization, collagen synthesis, and fibrosis, including a unique population of CXCL14-IL24+ secretory papillary fibroblasts. Besides known itch mediators such as IL31 and oncostatin M, we also detected increased levels of neuromedin B in fibroblasts of CNPG lesions compared with AD and HC, with neuromedin B receptors detectable on some nerve endings. CONCLUSIONS: These data show that CNPG does not harbor the strong disease-specific immune activation pathways that are typically found in AD but is rather characterized by upregulated stromal remodeling mechanisms that might have a direct impact on itch fibers.

Single-cell RNA sequencing analysis of a COVID-19-associated maculopapular rash in a patient with psoriasis treated with ustekinumab.

Coronavirus disease 2019 (COVID-19) primarily affects the respiratory system but extrapulmonary manifestations, including the skin, have been well documented. However, transcriptomic profiles of skin lesions have not been performed thus far. Here, we present a single-cell RNA sequencing analysis in a patient with COVID-19 infection with a maculopapular skin rash while on treatment with the interleukin (IL)-12/IL-23 blocker ustekinumab for his underlying psoriasis. Results were compared with healthy controls and untreated psoriasis lesions. We found the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry receptors ACE2 and TMPRSS2 in keratinocytes of the patient with COVID-19, while ACE2 expression was low to undetectable in psoriasis lesions and healthy skin. Among all cell types, ACE2+ keratinocyte clusters showed the highest levels of transcriptomic dysregulation in COVID-19, expressing type 1-associated immune markers such as CXCL9 and CXCL10. In line with a generally type 1-skewed immune microenvironment, cytotoxic lymphocytes showed increased expression of the IFNG gene and other T-cell effector genes, while type 2, type 17, or type 22 T-cell activation was largely absent. Conversely, downregulation of several anti-inflammatory mediators was observed. This first transcriptomic description of a COVID-19-associated rash identifies ACE2+ keratinocytes displaying profound transcriptional changes, and inflammatory immune cells that might help to improve the understanding of SARS-CoV-2-associated skin conditions.

Comparative analysis of genome-scale, base-resolution DNA methylation profiles across 580 animal species.

Methylation of cytosines is a prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms across the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale DNA methylation profiles of multiple organs. Bioinformatic analysis of this large dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions-once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution.

Stress signaling boosts interferon-induced gene transcription in macrophages.

Promoters of antimicrobial genes function as logic boards, integrating signals of innate immune responses. One such set of genes is stimulated by interferon (IFN) signaling, and the expression of these genes [IFN-stimulated genes (ISGs)] can be further modulated by cell stress-induced pathways. Here, we investigated the global effect of stress-induced p38 mitogen-activated protein kinase (MAPK) signaling on the response of macrophages to IFN. In response to cell stress that coincided with IFN exposure, the p38 MAPK-activated transcription factors CREB and c-Jun, in addition to the IFN-activated STAT family of transcription factors, bound to ISGs. In addition, p38 MAPK signaling induced activating histone modifications at the loci of ISGs and stimulated nuclear translocation of the CREB coactivator CRTC3. These actions synergistically enhanced ISG expression. Disrupting this synergy with p38 MAPK inhibitors improved the viability of macrophages infected with Listeria monocytogenes. Our findings uncover a mechanism of transcriptional synergism and highlight the biological consequences of coincident stress-induced p38 MAPK and IFN-stimulated signal transduction.

Processing human skin samples for single-cell assays.

Characterizing resident immune cells in human skin using single-cell assays provides insight into their role in infections, inflammation, and cancer. We describe an optimized protocol to rapidly isolate viable cells from 6-mm skin punch-biopsies. We provide an example in which we coupled single-cell RNA sequencing (scRNA-seq) with single-cell T-cell receptor sequencing (scTCR-seq) of skin and blood cells to study transcriptional profiles and clonotypes of skin resident and peripheral circulating, memory, and effector T cells. This is an improved protocol based on Saluzzo et al. (2021). For complete details on the use and execution of this protocol, please refer to Saluzzo et al. (2021).

Soluble TREM2 levels reflect the recruitment and expansion of TREM2+ macrophages that localize to fibrotic areas and limit NASH.

BACKGROUND & AIMS: Previous single-cell RNA-sequencing analyses have shown that Trem2-expressing macrophages are present in the liver during obesity, non-alcoholic steatohepatitis (NASH) and cirrhosis. Herein, we aimed to functionally characterize the role of bone marrow-derived TREM2-expressing macrophage populations in NASH. METHODS: We used bulk RNA sequencing to assess the hepatic molecular response to lipid-dependent dietary intervention in mice. Spatial mapping, bone marrow transplantation in two complementary murine models and single-cell sequencing were applied to functionally characterize the role of TREM2+ macrophage populations in NASH. RESULTS: We found that the hepatic transcriptomic profile during steatohepatitis mirrors the dynamics of recruited bone marrow-derived monocytes that already acquire increased expression of Trem2 in the circulation. Increased Trem2 expression was reflected by elevated levels of systemic soluble TREM2 in mice and humans with NASH. In addition, soluble TREM2 levels were superior to traditionally used laboratory parameters for distinguishing between different fatty liver disease stages in two separate clinical cohorts. Spatial transcriptomics revealed that TREM2+ macrophages localize to sites of hepatocellular damage, inflammation and fibrosis in the steatotic liver. Finally, using multiple murine models and in vitro experiments, we demonstrate that hematopoietic Trem2 deficiency causes defective lipid handling and extracellular matrix remodeling, resulting in exacerbated steatohepatitis, cell death and fibrosis. CONCLUSIONS: Our study highlights the functional properties of bone marrow-derived TREM2+ macrophages and implies the clinical relevance of systemic soluble TREM2 levels in the context of NASH. LAY SUMMARY: Our study defines the origin and function of macrophages (a type of immune cell) that are present in the liver and express a specific protein called TREM2. We find that these cells have an important role in protecting against non-alcoholic steatohepatitis (a progressive form of fatty liver disease). We also show that the levels of soluble TREM2 in the blood could serve as a circulating marker of non-alcoholic fatty liver disease.

DNA methylation signature of chronic low-grade inflammation and its role in cardio-respiratory diseases.

We performed a multi-ethnic Epigenome Wide Association study on 22,774 individuals to describe the DNA methylation signature of chronic low-grade inflammation as measured by C-Reactive protein (CRP). We find 1,511 independent differentially methylated loci associated with CRP. These CpG sites show correlation structures across chromosomes, and are primarily situated in euchromatin, depleted in CpG islands. These genomic loci are predominantly situated in transcription factor binding sites and genomic enhancer regions. Mendelian randomization analysis suggests altered CpG methylation is a consequence of increased blood CRP levels. Mediation analysis reveals obesity and smoking as important underlying driving factors for changed CpG methylation. Finally, we find that an activated CpG signature significantly increases the risk for cardiometabolic diseases and COPD.

Schwann cells contribute to keloid formation.

Keloids are disfiguring, hypertrophic scars with yet poorly understood pathomechanisms, which could lead to severe functional impairments. Here we analyzed the characteristics of keloidal cells by single cell sequencing and discovered the presence of an abundant population of Schwann cells that persisted in the hypertrophic scar tissue after wound healing. In contrast to normal skin, keloidal Schwann cells show a unique, pro-fibrotic phenotype. Our data support the hypothesis that keloidal Schwann cells contribute to the formation of the extracellular matrix and are able to affect M2 polarization of macrophages. Indeed, we show that macrophages in keloids predominantly display a M2 polarization and produce factors that inhibit Schwann cell differentiation. This study suggests the contribution of a Schwann cell - macrophage cross-talk to the continuous expansion of keloids, and that targeting Schwann cells might represent an interesting novel treatment option for keloids.

Single-cell transcriptomics defines keratinocyte differentiation in avian scutate scales.

The growth of skin appendages, such as hair, feathers and scales, depends on terminal differentiation of epidermal keratinocytes. Here, we investigated keratinocyte differentiation in avian scutate scales. Cells were isolated from the skin on the legs of 1-day old chicks and subjected to single-cell transcriptomics. We identified two distinct populations of differentiated keratinocytes. The first population was characterized by mRNAs encoding cysteine-rich keratins and corneous beta-proteins (CBPs), also known as beta-keratins, of the scale type, indicating that these cells form hard scales. The second population of differentiated keratinocytes contained mRNAs encoding cysteine-poor keratins and keratinocyte-type CBPs, suggesting that these cells form the soft interscale epidermis. We raised an antibody against keratin 9-like cysteine-rich 2 (KRT9LC2), which is encoded by an mRNA enriched in the first keratinocyte population. Immunostaining confirmed expression of KRT9LC2 in the suprabasal epidermal layers of scutate scales but not in interscale epidermis. Keratinocyte differentiation in chicken leg skin resembled that in human skin with regard to the transcriptional upregulation of epidermal differentiation complex genes and genes involved in lipid metabolism and transport. In conclusion, this study defines gene expression programs that build scutate scales and interscale epidermis of birds and reveals evolutionarily conserved keratinocyte differentiation genes.