Eyal David

Tissue-Resident Macrophage Enhancer Landscapes Are Shaped by the Local Microenvironment

Macrophages are critical for innate immune defense and also control organ homeostasis in a tissue-specific manner. They provide a fitting model to study the impact of ontogeny and microenvironment on chromatin state and whether chromatin modifications contribute to macrophage identity. Here, we profile the dynamics of four histone modifications across seven tissue-resident macrophage populations. We identify 12,743 macrophage-specific enhancers and establish that tissue-resident macrophages have distinct enhancer landscapes beyond what can be explained by developmental origin. Combining our enhancer catalog with gene expression profiles and open chromatin regions, we show that a combination of tissue- and lineage-specific transcription factors form the regulatory networks controlling chromatin specification in tissue-resident macrophages. The environment is capable of shaping the chromatin landscape of transplanted bone marrow precursors, and even differentiated macrophages can be reprogrammed when transferred into a new microenvironment. These results provide a comprehensive view of macrophage regulatory landscape and highlight the importance of the microenvironment, along with pioneer factors in orchestrating identity and plasticity.

Host microbiota constantly control maturation and function of microglia in the CNS

As the tissue macrophages of the CNS, microglia are critically involved in diseases of the CNS. However, it remains unknown what controls their maturation and activation under homeostatic conditions. We observed substantial contributions of the host microbiota to microglia homeostasis, as germ-free (GF) mice displayed global defects in microglia with altered cell proportions and an immature phenotype, leading to impaired innate immune responses. Temporal eradication of host microbiota severely changed microglia properties. Limited microbiota complexity also resulted in defective microglia. In contrast, recolonization with a complex microbiota partially restored microglia features. We determined that short-chain fatty acids (SCFA), microbiota-derived bacterial fermentation products, regulated microglia homeostasis. Accordingly, mice deficient for the SCFA receptor FFAR2 mirrored microglia defects found under GF conditions. These findings suggest that host bacteria vitally regulate microglia maturation and function, whereas microglia impairment can be rectified to some extent by complex microbiota.

Chromatin state dynamics during blood formation

Opening and closing blood enhancers As cells develop and differentiate into different types, the shape and accessibility of their DNA can change. Lara-Astiaso et al. studied this phenomenon in blood. They developed a technique that examines a relatively small number of cells to identify the changes that affect DNA during blood development. They found that the DNA of noncoding regions, called enhancers, is set in an open position when cells are undifferentiated and able to take on a variety of roles and gradually closes as cells mature into their final forms. Science, this issue p. 943 A chromatin precipitation technique identifies changes during the differentiation of blood cells. Chromatin modifications are crucial for development, yet little is known about their dynamics during differentiation. Hematopoiesis provides a well-defined model to study chromatin state dynamics; however, technical limitations impede profiling of homogeneous differentiation intermediates. We developed a high-sensitivity indexing-first chromatin immunoprecipitation approach to profile the dynamics of four chromatin modifications across 16 stages of hematopoietic differentiation. We identify 48,415 enhancer regions and characterize their dynamics. We find that lineage commitment involves de novo establishment of 17,035 lineage-specific enhancers. These enhancer repertoire expansions foreshadow transcriptional programs in differentiated cells. Combining our enhancer catalog with gene expression profiles, we elucidate the transcription factor network controlling chromatin dynamics and lineage specification in hematopoiesis. Together, our results provide a comprehensive model of chromatin dynamics during development.

Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors

Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.

A Unique Microglia Type Associated with Restricting Development of Alzheimer’s Disease

Alzheimers disease (AD) is a detrimental neurodegenerative disease with no effective treatments. Due to cellular heterogeneity, defining the roles of immune cell subsets in AD onset and progression has been challenging. Using transcriptional single-cell sorting, we comprehensively map all immune populations in wild-type and AD-transgenic (Tg-AD) mouse brains. We describe a novel microglia type associated with neurodegenerative diseases (DAM) and identify markers, spatial localization, and pathways associated with these cells. Immunohistochemical staining of mice and human brain slices shows DAM with intracellular/phagocytic Aβ particles. Single-cell analysis of DAM in Tg-AD and triggering receptor expressed on myeloid cells 2 (Trem2)-/- Tg-AD reveals that the DAM program is activated in a two-step process. Activation is initiated in a Trem2-independent manner that involves downregulation of microglia checkpoints, followed by activation of a Trem2-dependent program. This unique microglia-type has the potential to restrict neurodegeneration, which may have important implications for future treatment of AD and other neurodegenerative diseases. VIDEO ABSTRACT.

Microglia development follows a stepwise program to regulate brain homeostasis.

Microglia development follows a stepwise program Microglia are cells that defend the central nervous system. However, because they migrate into the brain during development, the changes that they undergo, including those that affect gene expression, have been difficult to document. Matcovitch-Natan et al. transcriptionally profiled gene expression and analyzed epigenetic signatures of microglia at the single-cell level in the early postnatal life of mice. They identified three stages of microglia development, which are characterized by gene expression and linked with chromatin changes, occurring in sync with the developing brain. Furthermore, they showed that the proper development of microglia is affected by the microbiome. Science, this issue p. 789 The microbiota help regulate the development of active immune defense in the central nervous system of mice. INTRODUCTION Microglia, as the resident myeloid cells of the central nervous system, play an important role in life-long brain maintenance and in pathology. Microglia are derived from erythromyeloid progenitors that migrate to the brain starting at embryonic day 8.5 and continuing until the blood-brain barrier is formed; after this, self-renewal is the only source of new microglia in the healthy brain. As the brain develops, microglia must perform different functions to accommodate temporally changing needs: first, actively engaging in synapse pruning and neurogenesis, and later, maintaining homeostasis. Although the interactions of microglia with the brain environment at steady state and in response to immune challenges have been well studied, their dynamics during development have not been fully elucidated. RATIONALE We systematically studied the transcriptional and epigenomic regulation of microglia throughout brain development to decipher the dynamics of the chromatin state and gene networks governing the transformation from yolk sac progenitor to adult microglia. We used environmental and genetic perturbation models to investigate how timed disruptions to microglia impact their natural development. RESULTS Global profiles of transcriptional states indicated that microglia development proceeds through three distinct temporal stages, which we define as early microglia (until embryonic day 14), pre-microglia (from embryonic day 14 to a few weeks after birth), and adult microglia (from a few weeks after birth onward). ATAC-seq (assay for transposase-accessible chromatin followed by sequencing) for chromatin accessibility and ChIP-seq (chromatin immunoprecipitation followed by sequencing) for histone modifications further characterized the differential regulatory elements in each developmental phase. Single-cell transcriptome analysis revealed minor mixing of the gene expression programs across phases, suggesting that individual cells shift their regulatory networks during development in a coordinated manner. Specific markers and regulatory factors distinguish each phase: For example, we identified MAFB as an important transcription factor of the adult microglia program. Microglia-specific knockout of MafB led to disruption of homeostasis in adulthood and increased expression of interferon and inflammation pathways. We found that microglia from germ-free mice exhibited dysregulation of dozens of genes associated with the adult phase and immune response. In addition, maternal immune activation, which has been linked to behavioral disorders in adult offspring, had the greatest impact on pre-microglia, resulting in a transcriptional shift toward the more advanced developmental stage. CONCLUSION Our work identifies a stepwise developmental program of microglia in synchrony with the developing brain. Each stage of microglia development has evolved distinct pathways for processing the relevant signals from the environment to balance their time-dependent role in neurogenesis with regulation of immune responses that may cause collateral damage. Genetic or environmental perturbations of these pathways can disrupt stage-specific functions of microglia and lead to loss of brain homeostasis, which may be associated with neurodevelopmental disorders. Microglia development proceeds in a stepwise manner. Microglia were isolated from mice throughout development from embryo to adult. Data from population-level RNA-seq, ChIP-seq, and ATAC-seq, as well as single-cell RNA-seq, show that microglia development proceeds through three distinct stages—early, pre-, and adult— with characteristic gene expression and functional states. Perturbations of this developmental process, such as from MafB knockout, lead to disrupted brain homeostasis by the dysregulation of adult and inflammatory genes. Tn5, transposase 5. Microglia, the resident myeloid cells of the central nervous system, play important roles in life-long brain maintenance and in pathology. Despite their importance, their regulatory dynamics during brain development have not been fully elucidated. Using genome-wide chromatin and expression profiling coupled with single-cell transcriptomic analysis throughout development, we found that microglia undergo three temporal stages of development in synchrony with the brain—early, pre-, and adult microglia—which are under distinct regulatory circuits. Knockout of the gene encoding the adult microglia transcription factor MAFB and environmental perturbations, such as those affecting the microbiome or prenatal immune activation, led to disruption of developmental genes and immune response pathways. Together, our work identifies a stepwise microglia developmental program integrating immune response pathways that may be associated with several neurodevelopmental disorders.

Aging-induced type I interferon response at the choroid plexus negatively affects brain function

Excess signaling is bad for the aging brain Preventing antiviral-like responses may protect function in the aging brain. Baruch et al. monitored messenger RNA production in the choroid plexus, the interface between the blood and cerebrospinal fluid, in young and old mice (see the Perspective by Ransohoff). They detected an inflammatory response in older mice not present in the brain of young mice that was also seen in old aged human samples postmortem. Preventing signaling by the cytokine interferon-I, which normally helps in the antiviral response of the immune system, helped prevent the decrease in cognitive function seen in aged mice. Science, this issue p. 89; see also p. 36 Excess signaling by type I interferon contributes to cognitive decline in aged mice. [Also see Perspective by Ransohoff] Aging-associated cognitive decline is affected by factors produced inside and outside the brain. By using multiorgan genome-wide analysis of aged mice, we found that the choroid plexus, an interface between the brain and the circulation, shows a type I interferon (IFN-I)–dependent gene expression profile that was also found in aged human brains. In aged mice, this response was induced by brain-derived signals, present in the cerebrospinal fluid. Blocking IFN-I signaling within the aged brain partially restored cognitive function and hippocampal neurogenesis and reestablished IFN-II–dependent choroid plexus activity, which is lost in aging. Our data identify a chronic aging-induced IFN-I signature, often associated with antiviral response, at the brain’s choroid plexus and demonstrate its negative influence on brain function, thereby suggesting a target for ameliorating cognitive decline in aging.

Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations

The intestinal microbiota undergoes diurnal compositional and functional oscillations that affect metabolic homeostasis, but the mechanisms by which the rhythmic microbiota influences host circadian activity remain elusive. Using integrated multi-omics and imaging approaches, we demonstrate that the gut microbiota features oscillating biogeographical localization and metabolome patterns that determine the rhythmic exposure of the intestinal epithelium to different bacterial species and their metabolites over the course of a day. This diurnal microbial behavior drives, in turn, the global programming of the host circadian transcriptional, epigenetic, and metabolite oscillations. Surprisingly, disruption of homeostatic microbiome rhythmicity not only abrogates normal chromatin and transcriptional oscillations of the host, but also incites genome-wide de novo oscillations in both intestine and liver, thereby impacting diurnal fluctuations of host physiology and disease susceptibility. As such, the rhythmic biogeography and metabolome of the intestinal microbiota regulates the temporal organization and functional outcome of host transcriptional and epigenetic programs.

PD-1 immune checkpoint blockade reduces pathology and improves memory in mouse models of Alzheimer's disease

Systemic immune suppression may curtail the ability to mount the protective, cell-mediated immune responses that are needed for brain repair. By using mouse models of Alzheimers disease (AD), we show that immune checkpoint blockade directed against the programmed death-1 (PD-1) pathway evokes an interferon (IFN)-γ–dependent systemic immune response, which is followed by the recruitment of monocyte-derived macrophages to the brain. When induced in mice with established pathology, this immunological response leads to clearance of cerebral amyloid-β (Aβ) plaques and improved cognitive performance. Repeated treatment sessions were required to maintain a long-lasting beneficial effect on disease pathology. These findings suggest that immune checkpoints may be targeted therapeutically in AD.

Single-cell spatial reconstruction reveals global division of labour in the mammalian liver

The mammalian liver consists of hexagon-shaped lobules that are radially polarized by blood flow and morphogens. Key liver genes have been shown to be differentially expressed along the lobule axis, a phenomenon termed zonation, but a detailed genome-wide reconstruction of this spatial division of labour has not been achieved. Here we measure the entire transcriptome of thousands of mouse liver cells and infer their lobule coordinates on the basis of a panel of zonated landmark genes, characterized with single-molecule fluorescence in situ hybridization. Using this approach, we obtain the zonation profiles of all liver genes with high spatial resolution. We find that around 50% of liver genes are significantly zonated and uncover abundant non-monotonic profiles that peak at the mid-lobule layers. These include a spatial order of bile acid biosynthesis enzymes that matches their position in the enzymatic cascade. Our approach can facilitate the reconstruction of similar spatial genomic blueprints for other mammalian organs.