Mechanisms of myeloid cell entry to the healthy and diseased central nervous system.

Myeloid cells in the central nervous system (CNS), such as microglia, CNS-associated macrophages (CAMs), dendritic cells and monocytes, are vital for steady-state immune homeostasis as well as the resolution of tissue damage during brain development or disease-related pathology. The complementary usage of multimodal high-throughput and high-dimensional single-cell technologies along with recent advances in cell-fate mapping has revealed remarkable myeloid cell heterogeneity in the CNS. Despite the establishment of extensive expression profiles revealing myeloid cell multiplicity, the local anatomical conditions for the temporal- and spatial-dependent cellular engraftment are poorly understood. Here we highlight recent discoveries of the context-dependent mechanisms of myeloid cell migration and settlement into distinct subtissular structures in the CNS. These insights offer better understanding of the factors needed for compartment-specific myeloid cell recruitment, integration and residence during development and perturbation, which may lead to better treatment of CNS diseases.

Author Correction: Novel Hexb-based tools for studying microglia in the CNS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Novel Hexb-based tools for studying microglia in the CNS.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

Microglia regulate central nervous system myelin growth and integrity.

Myelin is required for the function of neuronal axons in the central nervous system, but the mechanisms that support myelin health are unclear. Although macrophages in the central nervous system have been implicated in myelin health1, it is unknown which macrophage populations are involved and which aspects they influence. Here we show that resident microglia are crucial for the maintenance of myelin health in adulthood in both mice and humans. We demonstrate that microglia are dispensable for developmental myelin ensheathment. However, they are required for subsequent regulation of myelin growth and associated cognitive function, and for preservation of myelin integrity by preventing its degeneration. We show that loss of myelin health due to the absence of microglia is associated with the appearance of a myelinating oligodendrocyte state with altered lipid metabolism. Moreover, this mechanism is regulated through disruption of the TGFß1-TGFßR1 axis. Our findings highlight microglia as promising therapeutic targets for conditions in which myelin growth and integrity are dysregulated, such as in ageing and neurodegenerative disease2,3.

Origin, fate and dynamics of macrophages at central nervous system interfaces.

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

A Subset of Skin Macrophages Contributes to the Surveillance and Regeneration of Local Nerves.

The skin comprises tissue macrophages as the most abundant resident immune cell type. Their diverse tasks including resistance against invading pathogens, attraction of bypassing immune cells from vessels, and tissue repair require dynamic specification. Here, we delineated the postnatal development of dermal macrophages and their differentiation into subsets by adapting single-cell transcriptomics, fate mapping, and imaging. Thereby we identified a phenotypically and transcriptionally distinct subset of prenatally seeded dermal macrophages that self-maintained with very low postnatal exchange by hematopoietic stem cells. These macrophages specifically interacted with sensory nerves and surveilled and trimmed the myelin sheath. Overall, resident dermal macrophages contributed to axon sprouting after mechanical injury. In summary, our data show long-lasting functional specification of macrophages in the dermis that is driven by stepwise adaptation to guiding structures and ensures codevelopment of ontogenetically distinct cells within the same compartment.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single-cell profiling.

Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases.

Interleukin-22 protects intestinal stem cells against genotoxic stress.

Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development1-3. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis4,5. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and γδ T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR)6, and AhR-mediated signalling in ILC3 and γδ T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated 'sensing' of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells.

Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

Microglia have critical roles not only in neural development and homeostasis, but also in neurodegenerative and neuroinflammatory diseases of the central nervous system1-4. These highly diverse and specialized functions may be executed by subsets of microglia that already exist in situ, or by specific subsets of microglia that develop from a homogeneous pool of cells on demand. However, little is known about the presence of spatially and temporally restricted subclasses of microglia in the central nervous system during development or disease. Here we combine massively parallel single-cell analysis, single-molecule fluorescence in situ hybridization, advanced immunohistochemistry and computational modelling to comprehensively characterize subclasses of microglia in multiple regions of the central nervous system during development and disease. Single-cell analysis of tissues of the central nervous system during homeostasis in mice revealed specific time- and region-dependent subtypes of microglia. Demyelinating and neurodegenerative diseases evoked context-dependent subtypes of microglia with distinct molecular hallmarks and diverse cellular kinetics. Corresponding clusters of microglia were also identified in healthy human brains, and the brains of patients with multiple sclerosis. Our data provide insights into the endogenous immune system of the central nervous system during development, homeostasis and disease, and may also provide new targets for the treatment of neurodegenerative and neuroinflammatory pathologies.

Author Correction: Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

In this Letter, Dominic Grün and Sagar have been added to the author list (affiliated with Max-Planck-Institute of Immunology and Epigenetics (MPI-IE), Freiburg, Germany). The author list, 'Author contribution' and 'Acknowledgements' sections have been corrected online. See accompanying Amendment.

Task-relevant visual feedback uncertainty attenuates visuomotor adaptation.

Motor adaptation is attenuated when sensory feedback about the movement is uncertain. Although this was initially shown for small visual errors, attenuation seems not to hold when visual errors are larger and the contributions of implicit adaptation are isolated with the error-clamp method, which makes visual feedback task-irrelevant. Here we ask whether adaptation to a similarly large perturbation is attenuated when task-relevant visual feedback is uncertain. In a first experiment, we tested participants on a 30° movement-contingent visuomotor rotation under both low (cursor) and high (cloud of moving dots) visual feedback uncertainty. In line with optimal integration, we found that the early increase in adaptation and final extent of adaptation were reduced with high feedback uncertainty. In a second experiment, we included several blocks of no-feedback trials during the perturbation block to quantify the contribution of implicit adaptation. Results showed that implicit adaptation was smaller with high compared to low feedback uncertainty throughout the perturbation block. The estimated contribution of explicit adaptation was overall small, particularly for high feedback uncertainty. Our results demonstrate an influence of task-relevant visual feedback, and the resulting target errors, on implicit adaptation. We show that our motor system is sensitive to the feedback it receives even for larger error sizes and accordingly adjusts its learning properties when our ability to achieve the task goal is affected.NEW & NOTEWORTHY Motor adaptation is linked to the estimation of our actions. Whereas uncertainty of task-irrelevant visual feedback appears not to influence implicit adaptation for errors beyond a certain size, here we tested whether this is still the case for task-relevant feedback. We show that implicit adaptation is attenuated when task-relevant visual feedback is uncertain, suggesting a dependency on the assessment of not just sensory prediction errors but also target errors.

Optimality of multisensory integration while compensating for uncertain visual target information with artificial vibrotactile cues during reach planning.

BACKGROUND: Planning and executing movements requires the integration of different sensory modalities, such as vision and proprioception. However, neurological diseases like stroke can lead to full or partial loss of proprioception, resulting in impaired movements. Recent advances focused on providing additional sensory feedback to patients to compensate for the sensory loss, proving vibrotactile stimulation to be a viable option as it is inexpensive and easy to implement. Here, we test how such vibrotactile information can be integrated with visual signals to estimate the spatial location of a reach target. METHODS: We used a center-out reach paradigm with 31 healthy human participants to investigate how artificial vibrotactile stimulation can be integrated with visual-spatial cues indicating target location. Specifically, we provided multisite vibrotactile stimulation to the moving dominant arm using eccentric rotating mass (ERM) motors. As the integration of inputs across multiple sensory modalities becomes especially relevant when one of them is uncertain, we additionally modulated the reliability of visual cues. We then compared the weighing of vibrotactile and visual inputs as a function of visual uncertainty to predictions from the maximum likelihood estimation (MLE) framework to decide if participants achieve quasi-optimal integration. RESULTS: Our results show that participants could estimate target locations based on vibrotactile instructions. After short training, combined visual and vibrotactile cues led to higher hit rates and reduced reach errors when visual cues were uncertain. Additionally, we observed lower reaction times in trials with low visual uncertainty when vibrotactile stimulation was present. Using MLE predictions, we found that integration of vibrotactile and visual cues followed optimal integration when vibrotactile cues required the detection of one or two active motors. However, if estimating the location of a target required discriminating the intensities of two cues, integration violated MLE predictions. CONCLUSION: We conclude that participants can quickly learn to integrate visual and artificial vibrotactile information. Therefore, using additional vibrotactile stimulation may serve as a promising way to improve rehabilitation or the control of prosthetic devices by patients suffering loss of proprioception.

The origin, fate and function of macrophages in the peripheral nervous system-an update.

The field of macrophage biology has made enormous progress over recent years. This was triggered by the advent of several new techniques such as the establishment of Cre/loxP-based transgenic mouse models that allowed for the first time delineation of the ontogeny and function of specific macrophage populations across many tissues. In addition, the introduction of new high-throughput technologies like bulk RNA sequencing and later single-cell RNA sequencing as well as advances in epigenetic analysis have helped to establish gene expression profiles, enhancer landscapes and local signaling cues that define and shape the identity of diverse macrophage populations. Nonetheless, some macrophage populations, like the ones residing in the peripheral nervous system (PNS), have not been studied in such detail yet. Here, we discuss recent studies that shed new light on the ontogeny, heterogeneity and gene expression profiles of resident macrophages in peripheral nerves and described differential activation of macrophage subsets during and after acute sciatic nerve injury.

Central nervous system-associated macrophages modulate the immune response following stroke in aged mice.

Age is a major nonmodifiable risk factor for ischemic stroke. Central nervous system-associated macrophages (CAMs) are resident immune cells located along the brain vasculature at the interface between the blood circulation and the parenchyma. By using a clinically relevant thromboembolic stroke model in young and aged male mice and corresponding human tissue samples, we show that during aging, CAMs acquire a central role in orchestrating immune cell trafficking after stroke through the specific modulation of adhesion molecules by endothelial cells. The absence of CAMs provokes increased leukocyte infiltration (neutrophils and CD4+ and CD8+ T lymphocytes) and neurological dysfunction after stroke exclusively in aged mice. Major histocompatibility complex class II, overexpressed by CAMs during aging, plays a significant role in the modulation of immune responses to stroke. We demonstrate that during aging, CAMs become central coordinators of the neuroimmune response that ensure a long-term fine-tuning of the immune responses triggered by stroke.

Interaction between subventricular zone microglia and neural stem cells impacts the neurogenic response in a mouse model of cortical ischemic stroke.

After a stroke, the neurogenic response from the subventricular zone (SVZ) to repair the brain is limited. Microglia, as an integral part of the distinctive SVZ microenvironment, control neural stem / precursor cell (NSPC) behavior. Here, we show that discrete stroke-associated SVZ microglial clusters negatively impact the innate neurogenic response, and we propose a repository of relevant microglia-NSPC ligand-receptor pairs. After photothrombosis, a mouse model of ischemic stroke, the altered SVZ niche environment leads to immediate activation of microglia in the niche and an abnormal neurogenic response, with cell-cycle arrest of neural stem cells and neuroblast cell death. Pharmacological restoration of the niche environment increases the SVZ-derived neurogenic repair and microglial depletion increases the formation and survival of newborn neuroblasts in the SVZ. Therefore, we propose that altered cross-communication between microglial subclusters and NSPCs regulates the extent of the innate neurogenic repair response in the SVZ after stroke.

A comparative analysis of microglial inducible Cre lines.

Cre/LoxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. The field of microglial biology has particularly benefited from this technology as microglia have historically been difficult to transduce with virus or electroporation methods for gene delivery. Here, we interrogate four of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency and spontaneous recombination, depending on the Cre line and loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency in microglia, which could be extended to other cell types. There is increasing evidence that microglia are key regulators of neural circuit structure and function. Microglia are also major drivers of a broad range of neurological diseases. Thus, reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field of microglial biology and the development of microglia-based therapeutics.

A comparative analysis of microglial inducible Cre lines.

Cre/loxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. Microglial biology has particularly benefited because microglia historically have been difficult to transduce with virus or electroporation methods for gene delivery. Here, we investigate five of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency, cell-type specificity, and spontaneous recombination, depending on the Cre line and inter-loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency, particularly in microglia. There is increasing evidence that microglia are key regulators of neural circuits and major drivers of a broad range of neurological diseases. Reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field and the development of microglia-based therapeutics.