Alexander Mildner

Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions

Microglia are crucially important myeloid cells in the CNS and constitute the first immunological barrier against pathogens and environmental insults. The factors controlling microglia recruitment from the blood remain elusive and the direct circulating microglia precursor has not yet been identified in vivo. Using a panel of bone marrow chimeric and adoptive transfer experiments, we found that circulating Ly-6ChiCCR2+ monocytes were preferentially recruited to the lesioned brain and differentiated into microglia. Notably, microglia engraftment in CNS pathologies, which are not associated with overt blood-brain barrier disruption, required previous conditioning of brain (for example, by direct tissue irradiation). Our results identify Ly-6ChiCCR2+ monocytes as direct precursors of microglia in the adult brain and establish the importance of local factors in the adult CNS for microglia engraftment.

Massively parallel single cell RNA-Seq for marker-free decomposition of tissues into cell types

Sequencing of RNA from thousands of individual immune cells allows unbiased identification of cellular subtypes. In multicellular organisms, biological function emerges when heterogeneous cell types form complex organs. Nevertheless, dissection of tissues into mixtures of cellular subpopulations is currently challenging. We introduce an automated massively parallel single-cell RNA sequencing (RNA-seq) approach for analyzing in vivo transcriptional states in thousands of single cells. Combined with unsupervised classification algorithms, this facilitates ab initio cell-type characterization of splenic tissues. Modeling single-cell transcriptional states in dendritic cells and additional hematopoietic cell types uncovers rich cell-type heterogeneity and gene-modules activity in steady state and after pathogen activation. Cellular diversity is thereby approached through inference of variable and dynamic pathway activity rather than a fixed preprogrammed cell-type hierarchy. These data demonstrate single-cell RNA-seq as an effective tool for comprehensive cellular decomposition of complex tissues. Introducing MARS-Seq Immune cells are typically differentiated by surface markers; however, this designation is somewhat crude and does not allow for fine distinctions that might be characterized by their RNA transcripts. Jaitin et al. (p. 776) used massively parallel single-cell RNA-sequencing (MARS-Seq) analysis to explore cellular heterogeneity within the immune system by assembling an automated experimental platform that enables RNA profiling of cells sorted from tissues using flow cytometry. More than 1000 cells could be sequenced, and unsupervised clustering analysis of the RNA profiles revealed distinct cellular groupings that corresponded to B cells, macrophages, and dendritic cells. This approach provides the ability to perform a bottom-up characterization of in vivo cell-type landscapes independent of cell markers or prior knowledge.

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.

Distinct and nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the central nervous system

The action of type I interferons in the central nervous system (CNS) during autoimmunity is largely unknown. Here, we demonstrate elevated interferon beta concentrations in the CNS, but not blood, of mice with experimental autoimmune encephalomyelitis (EAE), a model for CNS autoimmunity. Furthermore, mice devoid of the broadly expressed type I IFN receptor (IFNAR) developed exacerbated clinical disease accompanied by a markedly higher inflammation, demyelination, and lethality without shifting the T helper 17 (Th17) or Th1 cell immune response. Whereas adoptive transfer of encephalitogenic T cells led to enhanced disease in Ifnar1(-/-) mice, newly created conditional mice with B or T lymphocyte-specific IFNAR ablation showed normal EAE. The engagement of IFNAR on neuroectodermal CNS cells had no protective effect. In contrast, absence of IFNAR on myeloid cells led to severe disease with an enhanced effector phase and increased lethality, indicating a distinct protective function of type I IFNs during autoimmune inflammation of the CNS.

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.

Development and function of dendritic cell subsets.

Classical dendritic cells (cDCs) form a critical interface between innate and adaptive immunity. As myeloid immune cell sentinels, cDCs are specialized in the sensing of pathogen challenges and cancer. They translate the latter for T cells into peptide form. Moreover, cDCs provide additional critical information on the original antigen context to trigger a diverse spectrum of appropriate protective responses. Here we review recent progress in our understanding of cDC subsets in mice. We will discuss cDC subset ontogeny and transcription factor dependencies, as well as emerging functional specializations within the cDC compartment in lymphoid and nonlymphoid tissues.

CCR2+Ly-6Chi monocytes are crucial for the effector phase of autoimmunity in the central nervous system

The chemokine receptor CCR2 plays a vital role for the induction of autoimmunity in the central nervous system. However, it remains unclear how the pathogenic response is mediated by CCR2-bearing cells. By combining bone marrow chimerism with gene targeting we detected a mild disease-modulating role of CCR2 during experimental autoimmune encephalomyelitis, a model for central nervous system autoimmunity, on radio-resistant cells that was independent from targeted CCR2 expression on endothelia. Interestingly, absence of CCR2 on lymphocytes did not influence autoimmune demyelination. In contrast, engagement of CCR2 on accessory cells was required for experimental autoimmune encephalomyelitis induction. CCR2+Ly-6Chi monocytes were rapidly recruited to the inflamed central nervous system and were crucial for the effector phase of disease. Selective depletion of this specific monocyte subpopulation through engagement of CCR2 strongly reduced central nervous system autoimmunity. Collectively, these data indicate a disease-promoting role of CCR2+Ly-6Chi monocytes during autoimmune inflammation of the central nervous system.

Axonal loss and neuroinflammation caused by peroxisome-deficient oligodendrocytes.

Oligodendrocytes myelinate axons for rapid impulse conduction and contribute to normal axonal functions in the central nervous system. In multiple sclerosis, demyelination is caused by autoimmune attacks, but the role of oligodendroglial cells in disease progression and axon degeneration is unclear. Here we show that oligodendrocytes harbor peroxisomes whose function is essential for maintaining white matter tracts throughout adult life. By selectively inactivating the import factor PEX5 in myelinating glia, we generated mutant mice that developed normally, but within several months showed ataxia, tremor and premature death. Absence of functional peroxisomes from oligodendrocytes caused widespread axonal degeneration and progressive subcortical demyelination, but did not interfere with glial survival. Moreover, it caused a strong proinflammatory milieu and, unexpectedly, the infiltration of B and activated CD8+ T cells into brain lesions. We conclude that peroxisomes provide oligodendrocytes with an essential neuroprotective function against axon degeneration and neuroinflammation, which is relevant for human demyelinating diseases.

Distinct and Non-Redundant Roles of Microglia and Myeloid Subsets in Mouse Models of Alzheimer's Disease

Mononuclear phagocytes are important modulators of Alzheimers disease (AD), but the specific functions of resident microglia, bone marrow-derived mononuclear cells, and perivascular macrophages have not been resolved. To elucidate the spatiotemporal roles of mononuclear phagocytes during disease, we targeted myeloid cell subsets from different compartments and examined disease pathogenesis in three different mouse models of AD (APPswe/PS1, APPswe, and APP23 mice). We identified chemokine receptor 2 (CCR2)-expressing myeloid cells as the population that was preferentially recruited to β-amyloid (Aβ) deposits. Unexpectedly, AD brains with dysfunctional microglia and devoid of parenchymal bone marrow-derived phagocytes did not show overt changes in plaque pathology and Aβ load. In contrast, restriction of CCR2 deficiency to perivascular myeloid cells drastically impaired β-amyloid clearance and amplified vascular Aβ deposition, while parenchymal plaque deposition remained unaffected. Together, our data advocate selective functions of CCR2-expressing myeloid subsets, which could be targeted specifically to modify disease burden in AD.

Macrophages: Development and Tissue Specialization

Macrophages are myeloid immune cells that are strategically positioned throughout the body tissues, where they ingest and degrade dead cells, debris, and foreign material and orchestrate inflammatory processes. Here we review two major recent paradigm shifts in our understanding of tissue macrophage biology. The first is the realization that most tissue-resident macrophages are established prenatally and maintained through adulthood by longevity and self-renewal. Their generation and maintenance are thus independent from ongoing hematopoiesis, although the cells can be complemented by adult monocyte-derived macrophages. Second, aside from being immune sentinels, tissue macrophages form integral components of their host tissue. This entails their specialization in response to local environmental cues to contribute to the development and specific function of their tissue of residence. Factors that govern tissue macrophage specialization are emerging. Moreover, tissue specialization is reflected in discrete gene expression profiles of macrophages, as well as epigenetic signatures reporting actual and potential enhancer usage.