Abstract
While footprinting analysis of ATAC-seq data can theoretically enable investigation of transcription factor (TF) binding, the lack of a computational tool able to conduct different levels of ...footprinting analysis has so-far hindered the widespread application of this method. Here we present TOBIAS, a comprehensive, accurate, and fast footprinting framework enabling genome-wide investigation of TF binding dynamics for hundreds of TFs simultaneously. We validate TOBIAS using paired ATAC-seq and ChIP-seq data, and find that TOBIAS outperforms existing methods for bias correction and footprinting. As a proof-of-concept, we illustrate how TOBIAS can unveil complex TF dynamics during zygotic genome activation in both humans and mice, and propose how zygotic Dux activates cascades of TFs, binds to repeat elements and induces expression of novel genetic elements.
Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition ...to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.
Monocyte-derived and tissue-resident macrophages are ontogenetically distinct components of the innate immune system. Assessment of their respective functions in pathology is complicated by changes ...to the macrophage phenotype during inflammation. Here we find that Cxcr4-CreER enables permanent genetic labeling of hematopoietic stem cells (HSCs) and distinguishes HSC-derived monocytes from microglia and other tissue-resident macrophages. By combining Cxcr4-CreER-mediated lineage tracing with Cxcr4 inhibition or conditional Cxcr4 ablation in photothrombotic stroke, we find that Cxcr4 promotes initial monocyte infiltration and subsequent territorial restriction of monocyte-derived macrophages to infarct tissue. After transient focal ischemia, Cxcr4 deficiency reduces monocyte infiltration and blunts the expression of pattern recognition and defense response genes in monocyte-derived macrophages. This is associated with an altered microglial response and deteriorated outcomes. Thus, Cxcr4 is essential for an innate-immune-system-mediated defense response after cerebral ischemia. We further propose Cxcr4-CreER as a universal tool to study functions of HSC-derived cells.
Induction of trained immunity by Bacille-Calmette-Guérin (BCG) vaccination mediates beneficial heterologous effects, but the mechanisms underlying its persistence and magnitude remain elusive. In ...this study, we show that BCG vaccination in healthy human volunteers induces a persistent transcriptional program connected to myeloid cell development and function within the hematopoietic stem and progenitor cell (HSPC) compartment in the bone marrow. We identify hepatic nuclear factor (HNF) family members 1a and b as crucial regulators of this transcriptional shift. These findings are corroborated by higher granulocyte numbers in BCG-vaccinated infants, HNF1 SNP variants that correlate with trained immunity, and elevated serum concentrations of the HNF1 target alpha-1 antitrypsin. Additionally, transcriptomic HSPC remodeling was epigenetically conveyed to peripheral CD14+ monocytes, displaying an activated transcriptional signature three months after BCG vaccination. Taken together, transcriptomic, epigenomic, and functional reprogramming of HSPCs and peripheral monocytes is a hallmark of BCG-induced trained immunity in humans.
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•Human BCG vaccination induces a persistent innate immune training of CD14+ monocytes•BCG vaccination imprints a persistent transcriptomic myeloid bias on human HSPCs•Hepatic nuclear factors are regulators of BCG-induced trained immunity in HSPCs•BCG induces persistent epigenetic changes in peripheral CD14+ monocytes
Cirovic and de Bree et al. investigate the effects of BCG vaccination in humans and reveal the induction of a transcriptomic rewiring of human stem and progenitor cells toward the myeloid cell lineage, instructed by hepatic nuclear factors, resulting in epigenetic and functional changes within CD14+ peripheral monocytes.
Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive ...mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.
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•HPGD is specifically upregulated in Treg cells and confers suppressive capabilities•HPGD-induced Tconv cell suppression is in part mediated by PPARγ signaling•HPGD expression in Treg cells is vital for VAT homeostasis and metabolic regulation•T2D patients have a reduced Treg cell fraction expressing lower amounts of HPGD
Regulatory T (Treg) cells are important for preventing autoimmunity and maintaining tissue homeostasis. Schmidleithner et al. report a hydroxyprostaglandin dehydrogenase (HPGD)-mediated tissue- and context-dependent suppressor mechanism used by Treg cells to maintain adipose tissue homeostasis through the metabolism of PGE2 into the PPARγ ligand 15-keto-PGE2, which is conserved in human and mouse.
Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, ...transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.
•In vitro monocyte cultures model in vivo inflammatory dendritic cells and macrophages•Monocyte-derived dendritic cells integrate interleukin-4 signaling time dependently•NCOR2 controls differentiation of in vitro generated monocyte-derived dendritic cells•In vitro generated monocyte-derived cells are phenotypically heterogeneous
Monocyte-derived cellular derivatives are used clinically and are a crucial tool in basic research. Sander and colleagues now show that they transcriptionally relate to in vivo inflammatory monocytes, that they integrate differentiation cues time dependently, and that in vitro differentiated monocytes are phenotypically heterogeneous.
Macrophages are the principal immune cells of the epididymis and testis, but their origins, heterogeneity, development, and maintenance are not well understood. Here, we describe distinct populations ...of epididymal and testicular macrophages that display an organ-specific cellular identity. Combining in vivo fate-mapping, chimeric and parabiotic mouse models with in-depth cellular analyses, we found that CD64
MHCII
and CD64
MHCII
macrophage populations of epididymis and testis arise sequentially from yolk sac erythro-myeloid progenitors, embryonic hematopoiesis, and nascent neonatal monocytes. While monocytes were the major developmental source of both epididymal and testicular macrophages, both populations self-maintain in the steady-state independent of bone marrow hematopoietic precursors. However, after radiation-induced macrophage ablation or during infection, bone marrow-derived circulating monocytes are recruited to the epididymis and testis, giving rise to inflammatory macrophages that promote tissue damage. These results define the layered ontogeny, maintenance and inflammatory response of macrophage populations in the male reproductive organs.
Cytokines and IFNs downstream of innate immune pathways are critical for mounting an appropriate immune response to microbial infection. However, the expression of these inflammatory mediators is ...tightly regulated, as uncontrolled production can result in tissue damage and lead to chronic inflammatory conditions and autoimmune diseases. Activating transcription factor 3 (ATF3) is an important transcriptional modulator that limits the inflammatory response by controlling the expression of a number of cytokines and chemokines. However, its role in modulating IFN responses remains poorly defined. In this study, we demonstrate that ATF3 expression in macrophages is necessary for governing basal IFN-β expression, as well as the magnitude of IFN-β cytokine production following activation of innate immune receptors. We found that ATF3 acted as a transcriptional repressor and regulated IFN-β via direct binding to a previously unidentified specific regulatory site distal to the Ifnb1 promoter. Additionally, we observed that ATF3 itself is a type I IFN-inducible gene, and that ATF3 further modulates the expression of a subset of inflammatory genes downstream of IFN signaling, suggesting it constitutes a key component of an IFN negative feedback loop. Consistent with this, macrophages deficient in Atf3 showed enhanced viral clearance in lymphocytic choriomeningitis virus and vesicular stomatitis virus infection models. Our study therefore demonstrates an important role for ATF3 in modulating IFN responses in macrophages by controlling basal and inducible levels of IFNβ, as well as the expression of genes downstream of IFN signaling.
Differentiation of inflammatory macrophages from monocytes is characterized by an orderly integration of epi- genetic and transcriptional regulatory mechanisms guided by lineage-determining ...transcription factors such as PU.1. Further activation of macrophages leads to a stimulus- or microenvironment-specific signal integration with subse- quent transcriptional control established by the action of tissue- or signal-associated transcription factors. Here, we assess four histone modifications during human macrophage activation and integrate this information with the gene expression data from 28 different macrophage activation conditions in combination with GM-CSF. Bioinformatically, for inflammatory macrophages we define a unique network of transcriptional and epigenetic regulators （TRs）, which was characterized by accessible promoters independent of the activation signal. In contrast to the general accessibil- ity of promoters of TRs, mRNA expression of central TRs belonging to the TR network displayed stimulus-specific expression patterns, indicating a second level of transcriptional regulation beyond epigenetic chromatin changes. In contrast, stringent integration of epigenetic and transcriptional regulation was observed in networks of TRs estab- lished from somatic tissues and tissue macrophages. In these networks, clusters of TRs with permissive bistone marks were associated with high gene expression whereas clusters with repressive chromatin marks were associated with absent gene expression. Collectively, these results support that macrophage activation during inflammation in con- trast to lineage determination is mainly regulated transcriptionally by a pre-defined TR network.
Tumor-associated macrophages (TAMs) are frequently the most abundant immune cells in cancers and are associated with poor survival. Here, we generated TAM molecular signatures from ...K14cre;Cdh1flox/flox;Trp53flox/flox (KEP) and MMTV-NeuT (NeuT) transgenic mice that resemble human invasive lobular carcinoma (ILC) and HER2+ tumors, respectively. Determination of TAM-specific signatures requires comparison with healthy mammary tissue macrophages to avoid overestimation of gene expression differences. TAMs from the two models feature a distinct transcriptomic profile, suggesting that the cancer subtype dictates their phenotype. The KEP-derived signature reliably correlates with poor overall survival in ILC but not in triple-negative breast cancer patients, indicating that translation of murine TAM signatures to patients is cancer subtype dependent. Collectively, we show that a transgenic mouse tumor model can yield a TAM signature relevant for human breast cancer outcome prognosis and provide a generalizable strategy for determining and applying immune cell signatures provided the murine model reflects the human disease.
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•Murine TAM signatures prognosticate outcomes in corresponding cancer patients•TAM signatures are robust when they are compared with healthy tissue macrophages•TAM transcriptome is dictated by tissue and tumor subtype-related signals•Murine TAM signatures can be translated only when a suitable model is chosen
Tuit et al. show that TAM transcriptomes in murine models of breast cancer are governed mainly by tissue and tumor subtype-specific signals. Clinical translation of murine signatures can be achieved when human and mouse breast tumor subtypes are matched and only upon proper comparison of TAMs with healthy tissue macrophages.
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