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Granulomas are lumps of immune cells that can form in various organs. Most granulomas appear unstructured, yet they have some resemblance to lymphoid organs. To better understand granuloma formation, we performed single-cell sequencing and spatial transcriptomics on granulomas from patients with sarcoidosis and bioinformatically reconstructed the underlying gene regulatory networks. We discovered an immune stimulatory environment in granulomas that repurposes transcriptional programs associated with lymphoid organ development. Granuloma formation followed characteristic spatial patterns and involved genes linked to immunometabolism, cytokine and chemokine signaling, and extracellular matrix remodeling. Three cell types emerged as key players in granuloma formation: metabolically reprogrammed macrophages, cytokine-producing Th17.1 cells, and fibroblasts with inflammatory and tissue-remodeling phenotypes. Pharmacological inhibition of one of the identified processes attenuated granuloma formation in a sarcoidosis mouse model. We show that human granulomas adopt characteristic aspects of normal lymphoid organ development in aberrant combinations, indicating that granulomas constitute aberrant lymphoid organs. [Display omitted] • Performed single-cell sequencing and spatial transcriptomics on sarcoidosis granulomas • Reconstructed the architecture of granulomas and their gene regulatory networks • Identified a network of pathogenic macrophages, T cells, and fibroblasts in granulomas • Tested mechanisms shared with lymphoid organ development as drug targets in sarcoidosis Granulomas are accumulations of immune cells that help contain infections but can also give rise to diseases. To better understand granuloma formation, Krausgruber et al. perform single-cell and spatial profiling of sarcoidosis-associated skin granulomas. They find that granulomas exploit molecular mechanisms underlying the formation of tertiary lymphoid structures but lack their overall control, indicating that granulomas constitute aberrant lymphoid organs. [ABSTRACT FROM AUTHOR]

FOXP3+ regulatory T cells (Treg cells) are abundant in the intestine, where they prevent dysregulated inflammatory responses to self and environmental stimuli. It is now appreciated that Treg cells acquire tissue-specific adaptations that facilitate their survival and function; however, key host factors controlling the Treg response in the intestine are poorly understood. The interleukin (IL)-1 family member IL-33 is constitutively expressed in epithelial cells at barrier sites, where it functions as an endogenous danger signal, or alarmin, in response to tissue damage. Recent studies in humans have described high levels of IL-33 in inflamed lesions of inflammatory bowel disease patients, suggesting a role for this cytokine in disease pathogenesis. In the intestine, both protective and pathological roles for IL-33 have been described in murine models of acute colitis, but its contribution to chronic inflammation remains ill defined. Here we show in mice that the IL-33 receptor ST2 is preferentially expressed on colonic Treg cells, where it promotes Treg function and adaptation to the inflammatory environment. IL-33 signalling in T cells stimulates Treg responses in several ways. First, it enhances transforming growth factor (TGF)-β1-mediated differentiation of Treg cells and, second, it provides a necessary signal for Treg-cell accumulation and maintenance in inflamed tissues. Strikingly, IL-23, a key pro-inflammatory cytokine in the pathogenesis of inflammatory bowel disease, restrained Treg responses through inhibition of IL-33 responsiveness. These results demonstrate a hitherto unrecognized link between an endogenous mediator of tissue damage and a major anti-inflammatory pathway, and suggest that the balance between IL-33 and IL-23 may be a key controller of intestinal immune responses. [ABSTRACT FROM AUTHOR]

Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (TH1)-TH17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Autoinflammatory diseases, while having a variety of underlying causes, are mediated by dysfunctional innate immune responses. Therefore, standard treatments target innate cytokines or block their receptors. Despite excellent responses in some patients, first-line treatments fail in others, for reasons which remain to be understood. We studied the effects of IL-37, an anti-inflammatory cytokine, on immune cells using multi-omics profiling of 325 healthy adults. Our findings show that IL-37 is associated with inflammation control and generally reduced immune cell activity. Further, genetic variants in IL37 are associated with impaired trained immunity, a memory phenotype of innate immune cells contributing to autoinflammation. To underpin the medical potential of IL-37, an explorative cohort of seven autoinflammatory disorders was built. In vitro stimulation experiments argue for recombinant IL-37 as a potential therapy in IL-6-, and IL-22-driven conditions. Concluding, IL-37 is highlighted as a cytokine with broad anti-inflammatory functions, implicating its potential as therapeutic intervention. [Display omitted] • The induction of trained immunity in humans can be regulated by IL-37 in vivo. • IL-37 is a potential therapeutic in inflammatory conditions. • IL-37 regulates cellular activity, effector functions, and inflammation in humans. • IL-37 controls lipid, fatty acid, and amino acid metabolism. [ABSTRACT FROM AUTHOR]

The adult human skin contains a vast number of T cells that are essential for skin homeostasis and pathogen defense. T cells are first observed in the skin at the early stages of gestation; however, our understanding of their contribution to early immunity has been limited by their low abundance and lack of comprehensive methodologies for their assessment. Here, we describe a new workflow for isolating and expanding significant amounts of T cells from fetal human skin. Using multiparametric flow cytometry and in situ immunofluorescence, we found a large population with a naive phenotype and small populations with a memory and regulatory phenotype. Their molecular state was characterized using single-cell transcriptomics and TCR repertoire profiling. Importantly, culture of total fetal skin biopsies facilitated T cell expansion without a substantial impact on their phenotype, a major prerequisite for subsequent functional assays. Collectively, our experimental approaches and data advance the understanding of fetal skin immunity and potential use in future therapeutic interventions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BACKGROUNDThe antituberculosis vaccine bacillus Calmette-Guérin (BCG) reduces overall infant mortality. Induction of innate immune memory, also termed trained immunity, contributes toward protection against heterologous infections. Since immune cells display oscillations in numbers and function throughout the day, we investigated the effect of BCG administration time on the induction of trained immunity.METHODSEighteen volunteers were vaccinated with BCG at 6 pm and compared with 36 age- and sex-matched volunteers vaccinated between 8 am and 9 am. Peripheral blood mononuclear cells were stimulated with Staphylococcus aureus and Mycobacterium tuberculosis before, as well as 2 weeks and 3 months after, BCG vaccination. Cytokine production was measured to assess the induction of trained immunity and adaptive responses, respectively. Additionally, the influence of vaccination time on induction of trained immunity was studied in an independent cohort of 302 individuals vaccinated between 8 am and 12 pm with BCG.RESULTSCompared with evening vaccination, morning vaccination elicited both a stronger trained immunity and adaptive immune phenotype. In a large cohort of 302 volunteers, early morning vaccination resulted in a superior cytokine production capacity compared with later morning. A cellular, rather than soluble, substrate of the circadian effect of BCG vaccination was demonstrated by the enhanced capacity to induce trained immunity in vitro in morning- compared with evening-isolated monocytes.CONCLUSIONSBCG vaccination in the morning induces stronger trained immunity and adaptive responses compared with evening vaccination. Future studies should take vaccine administration time into account when studying specific and nonspecific effects of vaccines; early morning should be the preferred moment of BCG administration.FUNDINGThe Netherlands Organization for Scientific Research, the European Research Council, and the Danish National Research Foundation. [ABSTRACT FROM AUTHOR]

The superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration and is composed of a rich diversity of excitatory and inhibitory neurons and glia. However, the developmental principles directing the generation of SC cell-type diversity are not understood. Here, we pursued systematic cell lineage tracing in silico and in vivo , preserving full spatial information, using genetic mosaic analysis with double markers (MADM)-based clonal analysis with single-cell sequencing (MADM-CloneSeq). The analysis of clonally related cell lineages revealed that radial glial progenitors (RGPs) in SC are exceptionally multipotent. Individual resident RGPs have the capacity to produce all excitatory and inhibitory SC neuron types, even at the stage of terminal division. While individual clonal units show no pre-defined cellular composition, the establishment of appropriate relative proportions of distinct neuronal types occurs in a PTEN-dependent manner. Collectively, our findings provide an inaugural framework at the single-RGP/-cell level of the mammalian SC ontogeny. [Display omitted] • SC cell-type diversity originates from a single common pool of progenitors • Rapid RGP lineage progression without temporal order of layer production in SC • Individual RGPs are multipotent with the capacity to generate all SC neuronal types • Cell-autonomous requirement of Pten for the establishment of SC cell-type diversity Cheung et al. combine MADM-based lineage tracing with single-cell RNA sequencing to uncover the ontogenetic framework of the mouse superior colliculus. Individual progenitors show an exceptional capacity to produce all known excitatory and inhibitory neuron types and thus overall neuronal cell-type diversity in superior colliculus in a PTEN-dependent manner. [ABSTRACT FROM AUTHOR]

Immune responses are tightly regulated yet highly variable between individuals. To investigate human population variation of trained immunity, we immunized healthy individuals with Bacillus Calmette-Guérin (BCG). This live-attenuated vaccine induces not only an adaptive immune response against tuberculosis but also triggers innate immune activation and memory that are indicative of trained immunity. We established personal immune profiles and chromatin accessibility maps over a 90-day time course of BCG vaccination in 323 individuals. Our analysis uncovered genetic and epigenetic predictors of baseline immunity and immune response. BCG vaccination enhanced the innate immune response specifically in individuals with a dormant immune state at baseline, rather than providing a general boost of innate immunity. This study advances our understanding of BCG's heterologous immune-stimulatory effects and trained immunity in humans. Furthermore, it highlights the value of epigenetic cell states for connecting immune function with genotype and the environment. [Display omitted] • Clinical study with 323 healthy individuals over a 90-day time course of BCG vaccination • Time-resolved personal immune profiles, chromatin accessibility maps, and genotypes • Baseline immunity, seasonality, genotype, and chromatin predict trained immunity responses • Trained immunity is specific to individuals with a dormant innate immune state at baseline The response to vaccination and immune stimuli varies widely between individuals. To dissect population variation in immune function, Moorlag, Folkman, ter Horst, Krausgruber, et al. perform multi-omics analysis before and after BCG vaccination. They find that trained immunity induction is most effective in individuals with a dormant immune state at baseline. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem-cell transplantation (HSCT) seeks to reconstitute the host's immune system from donor stem cells. The success of HSCT is threatened by complications including leukemia relapse or graft-versus-host-disease (GvHD). To investigate the underlying regulatory processes in central and peripheral T cell recovery, we performed sequential multi-omics analysis of T cells of the skin and blood during HSCT. We detected rapid effector T cell reconstitution, while emergence of regulatory T cells was delayed. Epigenetic and gene-regulatory programs were associated with recovering T cells and diverged greatly between skin and blood T cells. The BRG1/BRM-associated factor chromatin remodeling complex and histone deacetylases (HDACs) were epigenetic regulators involved in restoration of T cell homeostasis after transplantation. In isolated T cells of patients after HSCT, we observed class I HDAC-inhibitors to modulate their dysbalance. The present study highlights the importance of epigenetic regulation in the recovery of T cells following HSCT. • Epigenetic and transcriptional profiling shows imbalanced reconstitution of T cells after HSCT. • Skin and blood T cells display different transcription factor activities post-transplantation. • Expression of histone deacetylases is altered in recovering T cells. [ABSTRACT FROM AUTHOR]

Summary The role of intestinal eosinophils in immune homeostasis is enigmatic and the molecular signals that drive them from protective to tissue damaging are unknown. Most commonly associated with Th2 cell-mediated diseases, we describe a role for eosinophils as crucial effectors of the interleukin-23 (IL-23)-granulocyte macrophage colony-stimulating factor (GM-CSF) axis in colitis. Chronic intestinal inflammation was characterized by increased bone marrow eosinopoiesis and accumulation of activated intestinal eosinophils. IL-5 blockade or eosinophil depletion ameliorated colitis, implicating eosinophils in disease pathogenesis. GM-CSF was a potent activator of eosinophil effector functions and intestinal accumulation, and GM-CSF blockade inhibited chronic colitis. By contrast neutrophil accumulation was GM-CSF independent and dispensable for colitis. In addition to TNF secretion, release of eosinophil peroxidase promoted colitis identifying direct tissue-toxic mechanisms. Thus, eosinophils are key perpetrators of chronic inflammation and tissue damage in IL-23-mediated immune diseases and it suggests the GM-CSF-eosinophil axis as an attractive therapeutic target. [ABSTRACT FROM AUTHOR]

IFNs λ1, λ2, and λ3, or type Ill IFNs, are recently identified cytokines distantly related to type I IFN5. Despite an early evolutionary divergence, the 2 types of IFN5 display similar antiviral activities, and both are produced primarily in dendritic cells. Although virus induction of the type I IFN-β gene had served as a paradigm of gene regulation, relatively little is known about the regulation of IFN-λ gene expression. Studies of virus induction of IFN-λ1 identified an essential role of IFN regulatory factors (IRF) 3 and 7, which bind to a regulatory DNA sequence near the start site of transcription. Here, we report that the proximal promoter region of the IFN-λ1 regulatory region is not sufficient for maximal gene induction in response to bacterial IPS. and we identify an essential cluster of homotypic NF-κB binding sites. Remarkably, these sites, which bind efficiently to NF-κB and function independently of the IRF3/7 binding sites, originate as transposable elements of the Alu and LTR families. We also show that depletion of the NF-κB RelA protein significantly reduces the level of the IFN-λ1 gene expression. We conclude that IFN-λ1 gene expression requires NF-κB, and we propose a model for IFN-λ1 gene regulation, in which IRF and NF-κB activate gene expression independently via spatially separated promoter elements. These observations provide insights into the independent evolution of the IFN-λ1 and IFN-β promoters and directly implicate transposable elements in the regulation of the IFN-λV;1 gene by NF-κB. [ABSTRACT FROM AUTHOR]

Summary Non-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4+ regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation. Graphical Abstract Highlights • scRNA-seq reveals Treg cell tissue-specific signatures • The phenotype of Treg cells shows progressive adaptation to barrier tissues • Treg cells share transcriptional dynamics of adaptation to colon and skin • Modules of peripheral Treg cell identity are conserved between mouse and human Treg cells are known to display tissue-specific heterogeneity. Miragaia and colleagues probe Treg cells in lymphoid and barrier tissues by single-cell transcriptomics. They show that Treg cells segregate into subpopulations along a continuum of tissue adaptation and present conserved expression programs between homeostasis and disease and mouse and human. [ABSTRACT FROM AUTHOR]