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.

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.

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.

Exploring the heterogeneous transcriptional response of the CNS to systemic LPS and Poly(I:C).

Peripheral contact to pathogen-associated molecular patterns (PAMPs) evokes a systemic innate immune response which is rapidly relayed to the central nervous system (CNS). The remarkable cellular heterogeneity of the CNS poses a significant challenge to the study of cell type and stimulus dependent responses of neural cells during acute inflammation. Here we utilized single nuclei RNA sequencing (snRNAseq), serum proteome profiling and primary cell culture methods to systematically compare the acute response of the mammalian brain to the bacterial PAMP lipopolysaccharide (LPS) and the viral PAMP polyinosinic:polycytidylic acid (Poly(I:C)), at single cell resolution. Our study unveiled convergent transcriptional cytokine and cellular stress responses in brain vascular and ependymal cells and a downregulation of several key mediators of directed blood brain barrier (BBB) transport. In contrast the neuronal response to PAMPs was limited in acute neuroinflammation. Moreover, our study highlighted the dominant role of IFN signalling upon Poly(I:C) challenge, particularly in cells of the oligodendrocyte lineage. Collectively our study unveils heterogeneous, shared and distinct cell type and stimulus dependent acute responses of the CNS to bacterial and viral PAMP challenges. Our findings highlight inflammation induced dysregulations of BBB-transporter gene expression, suggesting potential translational implications on drug pharmacokinetics variability during acute neuroinflammation. The pronounced dependency of oligodendrocytes on IFN stimulation during viral PAMP challenges, emphasizes their limited molecular viral response repertoire.

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.

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.

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.

Single-cell RNA sequencing reveals markers of disease progression in primary cutaneous T-cell lymphoma.

BACKGROUND: In early-stage mycosis fungoides (MF), the most common primary cutaneous T-cell lymphoma, limited skin involvement with patches and plaques is associated with a favorable prognosis. Nevertheless, approximately 20-30% of cases progress to tumors or erythroderma, resulting in poor outcome. At present, factors contributing to this switch from indolent to aggressive disease are only insufficiently understood. METHODS: In patients with advanced-stage MF, we compared patches with longstanding history to newly developed plaques and tumors by using single-cell RNA sequencing, and compared results with early-stage MF as well as nonlesional MF and healthy control skin. RESULTS: Despite considerable inter-individual variability, lesion progression was uniformly associated with downregulation of the tissue residency markers CXCR4 and CD69, the heat shock protein HSPA1A, the tumor suppressors and immunoregulatory mediators ZFP36 and TXNIP, and the interleukin 7 receptor (IL7R) within the malignant clone, but not in benign T cells. This phenomenon was not only found in conventional TCR-αß MF, but also in a case of TCR-γδ MF, suggesting a common mechanism across MF subtypes. Conversely, malignant cells in clinically unaffected skin from MF patients showed upregulation of these markers. CONCLUSIONS: Our data reveal a specific panel of biomarkers that might be used for monitoring MF disease progression. Altered expression of these genes may underlie the switch in clinical phenotype observed in advanced-stage MF.

The Extracellular Matrix in Skin Inflammation and Infection.

The extracellular matrix (ECM) is an integral component of all organs and plays a pivotal role in tissue homeostasis and repair. While the ECM was long thought to mostly have passive functions by providing physical stability to tissues, detailed characterization of its physical structure and biochemical properties have uncovered an unprecedented broad spectrum of functions. It is now clear that the ECM not only comprises the essential building block of tissues but also actively supports and maintains the dynamic interplay between tissue compartments as well as embedded resident and recruited inflammatory cells in response to pathologic stimuli. On the other hand, certain pathogens such as bacteria and viruses have evolved strategies that exploit ECM structures for infection of cells and tissues, and mutations in ECM proteins can give rise to a variety of genetic conditions. Here, we review the composition, structure and function of the ECM in cutaneous homeostasis, inflammatory skin diseases such as psoriasis and atopic dermatitis as well as infections as a paradigm for understanding its wider role in human health.

Single-Cell RNA Sequencing Reveals Tissue Compartment-Specific Plasticity of Mycosis Fungoides Tumor Cells.

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (TRM, CD69+CD27-NR4A1+RGS1+AHR+ ). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype (KLF2+TCF7+S1PR1+SELL+CCR7+ ), while retaining tissue-homing receptors (CLA, CCR10). The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory (IDO1) and Th2-associated mediators (CCL13, CCL17, CCL22). Given their expression of PVR, TNFRSF14 and CD80/CD86, they might be under direct control by TIGIT+CTLA4+CSF2+TNFSF14+ tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the TRM-like phenotype enables long-term skin residence of MF cells. Their switch to a TCM-like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF.

The skin immune atlas: three-dimensional analysis of cutaneous leukocyte subsets by multiphoton microscopy.

Site-specific differences in skin response to pathogens and in the course of cutaneous inflammatory diseases are well appreciated. The composition and localization of cutaneous leukocytes has been studied extensively using histology and flow cytometry. However, the precise three-dimensional (3D) distribution of distinct immune cell subsets within skin at different body sites requires visualization of intact living skin. We used intravital multiphoton microscopy in transgenic reporter mice in combination with quantitative flow cytometry to generate a 3D immune cell atlas of mouse skin. The 3D location of innate and adaptive immune cells and site-specific differences in the densities of macrophages, T cells, and mast cells at four defined sites (ear, back, footpad, and tail) is presented. The combinatorial approach further demonstrates an as yet unreported age-dependent expansion of dermal gamma-delta T cells. Localization of dermal immune cells relative to anatomical structures was also determined. Although dendritic cells were dispersed homogeneously within the dermis, mast cells preferentially localized to the perivascular space. Finally, we show the functional relevance of site-specific mast cell disparities using the passive cutaneous anaphylaxis model. These approaches are applicable to assessing immune cell variations and potential functional consequences in the setting of infection, as well as the pathogenesis of inflammatory skin conditions.