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Due to a combination of rapid disease progression and the lack of curative treatment options, hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Infiltrated, monocyte-derived, tumor-associated macrophages are known to play a role in HCC pathogenesis, but the involvement of Kupffer cells (KCs) remains elusive. Here, we used the Clec4F-diphteria toxin receptor transgenic mouse model to specifically investigate the effect of KC depletion on HCC initiation, progression and neoplastic growth following liver resection. For this purpose, several HCC mouse models with varying underlying etiologies were used and partial hepatectomy was performed. Our results show that in HCC, developed on a fibrotic or non-alcoholic steatohepatitis background, depletion of embryonic KCs at the onset of HCC induction and the subsequent replacement by monocyte-derived KCs does not affect the tumor burden, tumor microenvironment or the phenotype of isolated KCs at end-stage disease. In non-chronic liver disease-associated diethylnitrosamine-induced HCC, ablation of Clec4F+ KCs did not alter tumor progression or neoplastic growth following liver resection. Our results show that temporal ablation of resident KCs does not impact HCC pathogenesis, neither in the induction phase nor in advanced disease, and indicate that bone marrow-derived KCs are able to swiftly repopulate the available KC niche and adopt their phenotype.

Primary sclerosing cholangitis (PSC) is an idiopathic chronic immune-mediated cholestatic liver disease characterized by fibro-inflammatory bile duct strictures, progressive hepatobiliary fibrosis, and gut-liver axis disruption. The pathophysiology of PSC remains insufficiently characterized, which hampers the development of effective therapies. Hepatic macrophages (MFs) such as Kupffer cells (KCs) are implicated in PSC pathogenesis, but their exact role is unclear. Using the latest markers to discriminate resident KCs (ResKCs) from their monocyte-derived counterparts (MoKCs), and two models of intrahepatic and extrahepatic cholestasis, respectively, this study showed that CLEC4F+TIM4+ ResKCs were depleted after chronic cholestatic liver injury. The infiltrating CLEC4F+TIM4- MoKCs were already enriched during the acute phase of PSC. Transcriptional profiling of hepatic MF subsets during early cholestatic injury indicated that ResKCs were indeed activated and that MoKCs expressed higher levels of pro-inflammatory and proliferative markers compared with those of ResKCs. As indicated in experiments with Clec4fDTR transgenic mice, conditional depletion of KCs, before and during early cholestasis induction, had no effect on the composition of the hepatic myeloid cell pool following injury progression and did not affect disease outcomes. Taken together, these results provide new insights into the heterogeneity of the MF pool during experimental PSC and evidence that depletion of resident and activated KCs during sclerosing cholangitis does not affect disease outcome in mice.

To improve COVID-19 therapy, it is essential to understand the mechanisms driving critical illness. The complement system is an essential part of innate host defense that can also contribute to injury. All complement pathways have been implicated in COVID-19 pathogenesis, however the upstream drivers and downstream consequences on tissue injury remain ill-defined. Here, we demonstrate that complement activation is mediated by the alternative pathway and we provide a comprehensive atlas of the alterations in complement around the time of respiratory deterioration. Proteome and single-cell sequencing mapping across cell types and tissues reveals a division of labor between lung epithelial, stromal and myeloid cells in the production of complement, in addition to liver-derived factors. Upstream, IL-6 drives complement responses, linking complement dysregulation to approved COVID-19 therapies. In an exploratory proteomic study, C5 inhibition improves epithelial damage and markers of disease severity. Collectively, these results identify complement dysregulation as a key druggable feature of COVID-19.

Brain-immune cross-talk and neuroinflammation critically shape brain physiology in health and disease. A detailed understanding of the brain immune landscape is essential for developing new treatments for neurological disorders. Single-cell technologies offer an unbiased assessment of the heterogeneity, dynamics and functions of immune cells. Here we provide a protocol that outlines all the steps involved in performing single-cell multi-omic analysis of the brain immune compartment. This includes a step-by-step description on how to microdissect the border regions of the mouse brain, together with dissociation protocols tailored to each of these tissues. These combine a high yield with minimal dissociation-induced gene expression changes. Next, we outline the steps involved for high-dimensional flow cytometry and droplet-based single-cell RNA sequencing via the 10x Genomics platform, which can be combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and offers a higher throughput than plate-based methods. Importantly, we detail how to implement CITE-seq with large antibody panels to obtain unbiased protein-expression screening coupled to transcriptome analysis. Finally, we describe the main steps involved in the analysis and interpretation of the data. This optimized workflow allows for a detailed assessment of immune cell heterogeneity and activation in the whole brain or specific border regions, at RNA and protein level. The wet lab workflow can be completed by properly trained researchers (with basic proficiency in cell and molecular biology) and takes between 6 and 11 h, depending on the chosen procedures. The computational analysis requires a background in bioinformatics and programming in R.

Single-cell and spatial transcriptomic technologies have revealed an underappreciated heterogeneity of liver macrophages. This has led us to rethink the involvement of macrophages in liver homeostasis and disease. Identification of conserved gene signatures within these cells across species and diseases is enabling the correct identification of specific macrophage subsets and the generation of more specific tools to track and study the functions of these cells. Here, we discuss what is currently known about the definitions of these different macrophage populations, the markers that can be used to identify them, how they are wired within the liver, and their functional specializations in health and disease.

Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume-EM with high-resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high-resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. LAY DESCRIPTION: As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10-9 m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high-resolution contextual information provided by the electron microscope. In this study we present a workflow to guide a tissue sample from the light microscope to the electron microscope and image the ultra-structure of a specific cell type in the liver. In particular we focus on the incorporation of fiducial markers during the sample preparation to help navigate through the tissue in 3D in both microscopes. One sample is followed throughout the workflow to visualize the important steps in the process, showing the final result; a dataset combining fluorescent labels with ultra-structural detail.

Many encapsulated bacteria use capsules to cause invasive diseases. However, it remains largely unknown how the capsules enhance bacterial virulence under in vivo infection conditions. Here we show that the capsules primarily target the liver to enhance bacterial survival at the onset of blood-borne infections. In a mouse sepsis model, the capsules enabled human pathogens Streptococcus pneumoniae and Escherichia coli to circumvent the recognition of liver-resident macrophage Kupffer cells (KCs) in a capsular serotype-dependent manner. In contrast to effective capture of acapsular bacteria by KCs, the encapsulated bacteria are partially (low-virulence types) or completely (high-virulence types) “untouchable” for KCs. We finally identified the asialoglycoprotein receptor (ASGR) as the first known capsule receptor on KCs to recognize the low-virulence serotype-7F and -14 pneumococcal capsules. Our data identify the molecular interplay between the capsules and KCs as a master controller of the fate and virulence of encapsulated bacteria, and suggest that the interplay is targetable for therapeutic control of septic infections.

Summary: Liver-resident macrophages Kupffer cells (KCs) and infiltrating Ly6Chi monocytes both contribute to liver tissue regeneration in various pathologies but also to disease progression upon disruption of orderly consecutive regeneration cascades. Little is known about molecular pathways that regulate their differentiation, maintenance, or inflammatory behavior during injury. Here, we show that copper metabolism MURR1 domain (COMMD)10-deficient KCs adopt liver-specific identity. Strikingly, COMMD10 deficiency in KCs and in other tissue-resident macrophages impedes their homeostatic survival, leading to their continuous replacement by Ly6Chi monocytes. While COMMD10 deficiency in KCs mildly worsens acetaminophen-induced liver injury (AILI), its deficiency in Ly6Chi monocytes results in exacerbated and sustained hepatic damage. Monocytes display unleashed inflammasome activation and a reduced type I interferon response and acquire “neutrophil-like” and lipid-associated macrophage differentiation fates. Collectively, COMMD10 appears indispensable for KC and other tissue-resident macrophage survival and is an important regulator of Ly6Chi monocyte fate decisions and reparative behavior in the diseased liver.

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here, we present a spatial proteogenomic atlas of the healthy human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and bile-duct macrophages. We also uncover the respective spatially-resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that bile-duct macrophages are induced by local lipid exposure, while Kupffer cells crucially depend on their crosstalk with hepatic stellate cells via the evolutionarily-conserved ALK1-BMP9/10 axis.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) instructs monocytes to differentiate into alveolar macrophages (AM) that preserve lung homeostasis. By comparing AM development in mouse and human, we discovered that COVID-19 patients showed marked defects in GM-CSF-dependent AM instruction. The multi-center, open-label, randomized, controlled SARPAC-trial evaluated the efficacy and safety of 5 days of inhalation of rhu-GM-CSF (sargramostim, Leukine®) in 81 non-ventilated patients with COVID-19 and hypoxemic respiratory failure identified by PaO2/FiO2 ratio < 350mmHg. At day 6, more patients in the sargramostim group experienced at least 25% improvement in oxygenation compared with the standard of care group. Higher numbers of circulating class-switched B cells and effector virus-specific CD8 lymphocytes were found in the sargramostim group. Treatment adverse events, including signs of cytokine storm, were not different between active and control group. This proof-of-concept study demonstrates the feasibility and safety of inhaled GM-CSF in restoring alveolar gas exchange, while simultaneously boosting anti-COVID-19 immunity. ClinicalTrials.gov (NCT04326920).

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer’s disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2(−/−) mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1(+) and CX3CR1(+) macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.

The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400 basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400bp region (Zeb2(Δ−165) mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC) derived lineages. Zeb2(Δ−165) mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2(Δ−165) mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at +164-kb that was selectively active in embryonically derived lineages, but not hematopoietic stem cells (HSCs) derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the −165kb Zeb2 enhancer.

Liver resident macrophages Kupffer cells (KCs) and infiltrating Ly6C(hi) monocytes both contribute to liver tissue regeneration in various pathologies, but also to disease progression upon disruption of orderly consecutive regeneration cascades. Little is known about molecular pathways that regulate their differentiation, maintenance, or their inflammatory behavior during injury. Here we show that COMMD10-deficient KCs adopt liver-specific identity. Strikingly, COMMD10-deficiency in KCs and in other tissue resident macrophages impedes their homeostatic survival, leading to their continuous replacement by Ly6C(hi) monocytes. While COMMD10-deficiency in KCs mildly worsens acetaminophen-induced liver injury (AILI), its deficiency in Ly6C(hi) monocytes results in exacerbated and sustained hepatic damage. Monocytes display unleashed inflammasome activation and reduced type I interferon response and acquire ‘neutrophil-like’ and lipid associated macrophage differentiation fates. Collectively, COMMD10 appears indispensable for KC and other tissue resident macrophage survival and is an important regulator of Ly6C(hi) monocyte fate decisions and reparative behavior in the diseased liver.

We dissect metabolic variability of mononuclear phagocyte (MNP) subpopulations across different tissues through integrative analysis of three large scale datasets. Specifically, we introduce ImmGen MNP Open Source dataset that profiled 337 samples and extended previous ImmGen effort which included 202 samples of mononuclear phagocytes and their progenitors. Next, we analysed Tabula Muris Senis dataset to extract data for 51,364 myeloid cells from 18 tissues. Taken together, a compendium of data assembled in this work covers phagocytic populations found across 38 different tissues. To analyse common metabolic features, we developed novel network-based computational approach for unbiased identification of key metabolic subnetworks based on cellular transcriptional profiles in large-scale datasets. Using ImmGen MNP Open Source dataset as baseline, we define 9 metabolic subnetworks that encapsulate the metabolic differences within mononuclear phagocytes, and demonstrate that these features are robustly found across all three datasets, including lipid metabolism, cholesterol biosynthesis, glycolysis, and a set of fatty acid related metabolic pathways, as well as nucleotide and folate metabolism. We systematically define major features specific to macrophage and dendritic cell subpopulations. Among other things, we find that cholesterol synthesis appears particularly active within the migratory dendritic cells. We demonstrate that interference with this pathway through statins administration diminishes migratory capacity of the dendritic cells in vivo. This result demonstrates the power of our approach and highlights importance of metabolic diversity among mononuclear phagocytes.

During postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone marrow derived hematopoietic progenitors that migrate through the bloodstream. In human, the nature of these thymus immigrants has remained unclear. Here, we employ single-cell RNA sequencing on approximately 70.000 CD34+ thymocytes to unravel the heterogeneity of the human immature postnatal thymocytes. Integration of bone marrow and peripheral blood precursors datasets identifies several putative thymus seeding precursors that display heterogeneity for currently used surface markers as revealed by CITEseq. Besides T cell precursors, we discover branches of intrathymic developing dendritic cells with predominantly plasmacytoid DCs. Trough trajectory inference, we delineate the transcriptional dynamics underlying early human T-lineage development from which we predict transcription factor modules that drive stage-specific steps of human T cell development. Thus, our work resolves the heterogeneity of thymus seeding precursors in human and reveals the molecular mechanisms that drive their in vivo cell fate.

Glioblastomas are aggressive primary brain cancers that recur as therapy-resistant tumors. Myeloid cells control glioblastoma malignancy, but their dynamics during disease progression remain poorly understood. Here, we employed single-cell RNA sequencing and CITE-seq to map the glioblastoma immune landscape in mouse tumors and in patients with newly diagnosed disease or recurrence. This revealed a large and diverse myeloid compartment, with dendritic cell and macrophage populations that were conserved across species and dynamic across disease stages. Tumor-associated macrophages (TAMs) consisted of microglia- or monocyte-derived populations, with both exhibiting additional heterogeneity, including subsets with conserved lipid and hypoxic signatures. Microglia- and monocyte-derived TAMs were self-renewing populations that competed for space and could be depleted via CSF1R blockade. Microglia-derived TAMs were predominant in newly diagnosed tumors, but were outnumbered by monocyte-derived TAMs following recurrence, especially in hypoxic tumor environments. Our results unravel the glioblastoma myeloid landscape and provide a framework for future therapeutic interventions. ispartof: NATURE NEUROSCIENCE vol:24 issue:4 pages:595-610 ispartof: location:United States status: published

Summary Self-maintaining resident macrophages populate all mammalian organs. In addition to their role as immune sentinels, macrophages perform day-to-day functions essential to tissue homeostasis. The homeostatic functions of macrophages are regulated by so-called tissular “niches” that control the size of the macrophage population and imprint their tissue-specific identity. Here, we review the mechanisms underlying self-maintenance of distinct macrophage populations and outline the organizing principles of the macrophage niche. We examine recent studies that uncovered mutually beneficial cell-cell circuits established between macrophages and their niche and propose a modular view of tissues that integrates the resident macrophage as an essential component of each individual module. Manipulating macrophage niche cells to control the function of resident macrophages in vivo might have therapeutic value in various disease settings.

The NFIL3–ZEB2–ID2 transcription-factor regulatory circuit switches the E protein–dependent +41 kb Irf8 enhancer in DC progenitors to the BATF3-dependent +32 kb Irf8 enhancer in mature cDC1s. Deletion of the cryptic +41 kb Irf8 enhancer impedes cDC1 development.

In this issue of Blood , Kurotaki et al demonstrate that IRF8 expression delineates a subset of lymphoid-primed multipotent progenitors (LMPPs) biased toward the generation of conventional type 1 dendritic cells (cDC1s) by epigenetically priming the precursors for the expression of dendritic cell (DC) lineage genes. 1

Summary Peripheral tolerance is crucial for avoiding activation of self-reactive T cells to tissue-restricted antigens. Sterile tissue injury can break peripheral tolerance, but it is unclear how autoreactive T cells get activated in response to self. An example of a sterile injury is myocardial infarction (MI). We hypothesized that tissue necrosis is an activator of dendritic cells (DCs), which control tolerance to self-antigens. DC subsets of a murine healthy heart consisted of IRF8-dependent conventional (c)DC1, IRF4-dependent cDC2, and monocyte-derived DCs. In steady state, cardiac self-antigen α-myosin was presented in the heart-draining mediastinal lymph node (mLN) by cDC1s, driving the proliferation of antigen-specific CD4+ TCR-M T cells and their differentiation into regulatory cells (Tregs). Following MI, all DC subsets infiltrated the heart, whereas only cDCs migrated to the mLN. Here, cDC2s induced TCR-M proliferation and differentiation into interleukin-(IL)-17/interferon-(IFN)γ-producing effector cells. Thus, cardiac-specific autoreactive T cells get activated by mature DCs following myocardial infarction., Graphical Abstract, Highlights • IRF8+ cDC1, IRF4+ cDC2, moDCs, and macrophages are the APCs of the murine heart • Self-antigen presentation in the steady state drives Treg development via cDC1s • Myocardial infarction promotes infiltration, activation, and maturation of all DCs • Myocardial infarction promotes priming of Th1/Th17 autoreactive T cells via cDC2s, Van der Borght et al. demonstrate that myocardial infarction induces the priming of autoreactive CD4+ T cells specific for cardiac self-antigen α-myosin in the heart-draining lymph node through the maturation and migration of conventional dendritic cells. Using ex vivo co-culture systems, cDC2s are shown to be superior in presenting α-myosin.

The authors identify two subsets of peripheral nerve macrophages residing in the endoneurium and the epineurium and displaying a distinct transcriptome and response to injury. These cells lack the main microglia identity and have a distinct origin. While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: Relm alpha(+)Mgl1(+) snMacs in the epineurium and Relm alpha(-)Mgl1(-) snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages.

Using single-cell RNA sequencing of both immune and non-immune cells in the developing lung, Cohen et al. map candidate cell-cell interactions during alveolar macrophage development. This revealed potential cross-talk between epithelial cells, ILC2s, basophils, and the developing macrophages, which were validated both in vitro and in vivo.

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Two studies show that adding NOTCH ligands to Flt3L cultures increases the yield of cDC1s from mouse and human progenitors. Moreover, these NOTCH-DCs more closely resemble in vivo DCs transcriptionally and functionally. This will be extremely valuable for studying DCs in vitro and for further developing autologous DC-based antitumor vaccinations.

Summary During postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone-marrow-derived hematopoietic progenitors that migrate through the bloodstream. The current understanding of the nature of thymic immigrants is largely based on data from pre-clinical models. Here, we employed single-cell RNA sequencing (scRNA-seq) to examine the immature postnatal thymocyte population in humans. Integration of bone marrow and peripheral blood precursor datasets identified two putative thymus seeding progenitors that varied in expression of CD7; CD10; and the homing receptors CCR7, CCR9, and ITGB7. Whereas both precursors supported T cell development, only one contributed to intrathymic dendritic cell (DC) differentiation, predominantly of plasmacytoid dendritic cells. Trajectory inference delineated the transcriptional dynamics underlying early human T lineage development, enabling prediction of transcription factor (TF) modules that drive stage-specific steps of human T cell development. This comprehensive dataset defines the expression signature of immature human thymocytes and provides a resource for the further study of human thymopoiesis.

While the roles of parenchymal microglia in brain homeostasis and disease are fairly clear, other brain-resident myeloid cells remain less well understood. By dissecting border regions and combining single-cell RNA-sequencing with high-dimensional cytometry, bulk RNA-sequencing, fate-mapping and microscopy, we reveal the diversity of non-parenchymal brain macrophages. Border-associated macrophages (BAMs) residing in the dura mater, subdural meninges and choroid plexus consisted of distinct subsets with tissue-specific transcriptional signatures, and their cellular composition changed during postnatal development. BAMs exhibited a mixed ontogeny, and subsets displayed distinct self-renewal capacity following depletion and repopulation. Single-cell and fate-mapping analysis both suggested that there is a unique microglial subset residing on the apical surface of the choroid plexus epithelium. Finally, gene network analysis and conditional deletion revealed IRF8 as a master regulator that drives the maturation and diversity of brain macrophages. Our results provide a framework for understanding host-macrophage interactions in both the healthy and diseased brain. ispartof: NATURE NEUROSCIENCE vol:22 issue:6 pages:1021-1035 ispartof: location:United States status: published

The hygiene hypothesis postulates that the recent increase in allergic diseases such as asthma and hay fever observed in Western countries is linked to reduced exposure to childhood infections. Here we investigated how infection with a gammaherpesvirus affected the subsequent development of allergic asthma. We found that murid herpesvirus 4 (MuHV-4) inhibited the development of house dust mite (HDM)-induced experimental asthma by modulating lung innate immune cells. Specifically, infection with MuHV-4 caused the replacement of resident alveolar macrophages (AMs) by monocytes with regulatory functions. Monocyte-derived AMs blocked the ability of dendritic cells to trigger a HDM-specific response by the TH2 subset of helper T cells. Our results indicate that replacement of embryonic AMs by regulatory monocytes is a major mechanism underlying the long-term training of lung immunity after infection.

Our understanding of conventional dendritic cell (cDC) development and the functional specializations of distinct subsets in the peripheral tissues has increased greatly in recent years. Here, we review cDC development from the distinct progenitors in the bone marrow through to the distinct cDC subsets found in barrier tissues, providing an overview of the different subsets described in each location. In addition, we detail the transcription factors and local signals that have been proposed to control this developmental process. Importantly, despite these significant advances, numerous questions remain to be answered regarding cDC development. For example, it remains unclear whether the different subsets described, such as the CD103+CD11b+ and CD103-CD11b+ cDCs in the intestines, truly represent different populations or rather distinct developmental or activation stages. Furthermore, whether distinct progenitors exist for these cDC subsets remains to be determined. Thus in the last part of this review we discuss what we believe will be the main questions facing the field for the coming years.

Tissue-resident macrophages can derive from yolk sac macrophages (YS-Macs), fetal liver monocytes (FL-MOs), or adult bone-marrow monocytes (BM-MOs). The relative capacity of these precursors to colonize a niche, self-maintain, and perform tissue-specific functions is unknown. We simultaneously transferred traceable YS-Macs, FL-MOs, and BM-MOs into the empty alveolar macrophage (AM) niche of neonatal Csf2rb(-/-) mice. All subsets produced AMs, but in competition preferential outgrowth of FL-MOs was observed, correlating with their superior granulocyte macrophage-colony stimulating factor (GM-CSF) reactivity and proliferation capacity. When transferred separately, however, all precursors efficiently colonized the alveolar niche and generated AMs that were transcriptionally almost identical, self-maintained, and durably prevented alveolar proteinosis. Mature liver, peritoneal, or colon macrophages could not efficiently colonize the empty AM niche, whereas mature AMs could. Thus, precursor origin does not affect the development of functional self-maintaining tissue-resident macrophages and the plasticity of the mononuclear phagocyte system is largest at the precursor stage.

Defining the origins and developmental pathways of tissue-resident macrophages should help refine our understanding of the role of these cells in various disease settings and enable the design of novel macrophage-targeted therapies. In recent years the long-held belief that macrophage populations in the adult are continuously replenished by monocytes from the bone marrow (BM) has been overturned with the advent of new techniques to dissect cellular ontogeny. The new paradigm suggests that several tissue-resident macrophage populations are seeded during waves of embryonic hematopoiesis and self-maintain independently of BM contribution during adulthood. However, the exact nature of the embryonic progenitors that give rise to adult tissue-resident macrophages is still debated, and the mechanisms enabling macrophage population maintenance in the adult are undefined. Here, we review the emergence of these concepts and discuss current controversies and future directions in macrophage biology.

Summary The phenotypic and functional dichotomy between IRF8+ type 1 and IRF4+ type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4+ T helper (Th) cell polarization while simultaneously presenting antigen to CD8+ T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs., Graphical Abstract, Highlights • Type I interferon drives differentiation of inf-cDC2s that closely resemble MCs • Inf-cDC2s prime CD4+ and CD8+ T cells, whereas MCs lack APC function • Inf-cDC2s internalize antibody-complexed antigen via Fc receptors • IRF8 controls maturation gene module in inf-cDC2s, The dichotomy between type 1 and 2 conventional DCs under steady-state conditions is well defined. Bosteels et al. demonstrate that, upon inflammation, cDC2s acquire a hybrid inf-cDC2 phenotype, sharing phenotype, gene expression, and function with cDC1s and monocyte-derived cells, to optimally boost CD4 and CD8 immunity via Fc receptors.