A microglia clonal inflammatory disorder in Alzheimer's Disease.

Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however few studies have investigated its role in neurodegenerative processes such as Alzheimer's Disease (AD). Here we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in human, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients.

The nuclear factor ID3 endows macrophages with a potent anti-tumour activity.

Macrophage activation is controlled by a balance between activating and inhibitory receptors1-7, which protect normal tissues from excessive damage during infection8,9 but promote tumour growth and metastasis in cancer7,10. Here we report that the Kupffer cell lineage-determining factor ID3 controls this balance and selectively endows Kupffer cells with the ability to phagocytose live tumour cells and orchestrate the recruitment, proliferation and activation of natural killer and CD8 T lymphoid effector cells in the liver to restrict the growth of a variety of tumours. ID3 shifts the macrophage inhibitory/activating receptor balance to promote the phagocytic and lymphoid response, at least in part by buffering the binding of the transcription factors ELK1 and E2A at the SIRPA locus. Furthermore, loss- and gain-of-function experiments demonstrate that ID3 is sufficient to confer this potent anti-tumour activity to mouse bone-marrow-derived macrophages and human induced pluripotent stem-cell-derived macrophages. Expression of ID3 is therefore necessary and sufficient to endow macrophages with the ability to form an efficient anti-tumour niche, which could be harnessed for cell therapy in cancer.

Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization.

Tumor-infiltrating macrophages support critical steps in tumor progression, and their accumulation in the tumor microenvironment (TME) is associated with adverse outcomes and therapeutic resistance across human cancers. In the TME, macrophages adopt diverse phenotypic alterations, giving rise to heterogeneous immune activation states and induction of cell cycle. While the transcriptional profiles of these activation states are well-annotated across human cancers, the underlying signals that regulate macrophage heterogeneity and accumulation remain incompletely understood. Here, we leveraged a novel ex vivo organotypic TME (oTME) model of breast cancer, in vivo murine models, and human samples to map the determinants of functional heterogeneity of TME macrophages. We identified a subset of F4/80highSca-1+ self-renewing macrophages maintained by type-I interferon (IFN) signaling and requiring physical contact with cancer-associated fibroblasts. We discovered that the contact-dependent self-renewal of TME macrophages is mediated via Notch4, and its inhibition abrogated tumor growth of breast and ovarian carcinomas in vivo, as well as lung dissemination in a PDX model of triple-negative breast cancer (TNBC). Through spatial multi-omic profiling of protein markers and transcriptomes, we found that the localization of macrophages further dictates functionally distinct but reversible phenotypes, regardless of their ontogeny. Whereas immune-stimulatory macrophages (CD11C+CD86+) populated the tumor epithelial nests, the stroma-associated macrophages (SAMs) were proliferative, immunosuppressive (Sca-1+CD206+PD-L1+), resistant to CSF-1R depletion, and associated with worse patient outcomes. Notably, following cessation of CSF-1R depletion, macrophages rebounded primarily to the SAM phenotype, which was associated with accelerated growth of mammary tumors. Our work reveals the spatial determinants of macrophage heterogeneity in breast cancer and highlights the disruption of macrophage self-renewal as a potential new therapeutic strategy.

Plasma membrane abundance dictates phagocytic capacity and functional crosstalk in myeloid cells.

Professional phagocytes like neutrophils and macrophages tightly control what they eat, how much they eat, and when they move after eating. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G-protein subunit Gb4 exhibit profound plasma membrane expansion due to enhanced production of sphingolipids. This increased membrane allocation dramatically enhances phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. Gb4 deficient neutrophils are also defective in the normal inhibition of migration following cargo uptake. In Gb4 knockout mice, myeloid cells exhibit enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. These results reveal an unexpected, biophysical control mechanism lying at the heart of myeloid functional decision-making.

The macrophage genetic cassette inr/dtor/pvf2 is a nutritional status checkpoint for developmental timing.

A small number of signaling molecules, used reiteratively, control differentiation programs, but the mechanisms that adapt developmental timing to environmental cues are less understood. We report here that a macrophage inr/dtor/pvf2 genetic cassette is a developmental timing checkpoint in Drosophila, which either licenses or delays biosynthesis of the steroid hormone in the endocrine gland and metamorphosis according to the larval nutritional status. Insulin receptor/dTor signaling in macrophages is required and sufficient for production of the PDGF/VEGF family growth factor Pvf2, which turns on transcription of the sterol biosynthesis Halloween genes in the prothoracic gland via its receptor Pvr. In response to a starvation event or genetic manipulation, low Pvf2 signal delays steroid biosynthesis until it becomes Pvr-independent, thereby prolonging larval growth before pupariation. The significance of this developmental timing checkpoint for host fitness is illustrated by the observation that it regulates the size of the pupae and adult flies.

Early life high fructose exposure disrupts microglia function and impedes neurodevelopment.

Despite the success of fructose as a low-cost food additive, recent epidemiological evidence suggests that high fructose consumption by pregnant mothers or during adolescence is associated with disrupted neurodevelopment 1-7 . An essential step in appropriate mammalian neurodevelopment is the synaptic pruning and elimination of newly-formed neurons by microglia, the central nervous system's (CNS) resident professional phagocyte 8-10 . Whether early life high fructose consumption affects microglia function and if this directly impacts neurodevelopment remains unknown. Here, we show that both offspring born to dams fed a high fructose diet and neonates exposed to high fructose exhibit decreased microglial density, increased uncleared apoptotic cells, and decreased synaptic pruning in vivo . Importantly, deletion of the high affinity fructose transporter SLC2A5 (GLUT5) in neonates completely reversed microglia dysfunction, suggesting that high fructose directly affects neonatal development. Mechanistically, we found that high fructose treatment of both mouse and human microglia suppresses synaptic pruning and phagocytosis capacity which is fully reversed in GLUT5-deficient microglia. Using a combination of in vivo and in vitro nuclear magnetic resonance- and mass spectrometry-based fructose tracing, we found that high fructose drives significant GLUT5-dependent fructose uptake and catabolism, rewiring microglia metabolism towards a hypo-phagocytic state. Importantly, mice exposed to high fructose as neonates exhibited cognitive defects and developed anxiety-like behavior which were rescued in GLUT5-deficient animals. Our findings provide a mechanistic explanation for the epidemiological observation that early life high fructose exposure is associated with increased prevalence of adolescent anxiety disorders.

Physiology and diseases of tissue-resident macrophages.

Embryo-derived tissue-resident macrophages are the first representatives of the haematopoietic lineage to emerge in metazoans. In mammals, resident macrophages originate from early yolk sac progenitors and are specified into tissue-specific subsets during organogenesis-establishing stable spatial and functional relationships with specialized tissue cells-and persist in adults. Resident macrophages are an integral part of tissues together with specialized cells: for instance, microglia reside with neurons in brain, osteoclasts reside with osteoblasts in bone, and fat-associated macrophages reside with white adipocytes in adipose tissue. This ancillary cell type, which is developmentally and functionally distinct from haematopoietic stem cell and monocyte-derived macrophages, senses and integrates local and systemic information to provide specialized tissue cells with the growth factors, nutrient recycling and waste removal that are critical for tissue growth, homeostasis and repair. Resident macrophages contribute to organogenesis, promote tissue regeneration following damage and contribute to tissue metabolism and defence against infectious disease. A correlate is that genetic or environment-driven resident macrophage dysfunction is a cause of degenerative, metabolic and possibly inflammatory and tumoural diseases. In this Review, we aim to provide a conceptual outline of our current understanding of macrophage physiology and its importance in human diseases, which may inform and serve the design of future studies.

Hematopoietic Somatic Mosaicism Is Associated With an Increased Risk of Postoperative Atrial Fibrillation.

BACKGROUND: On-pump cardiac surgery triggers sterile inflammation and postoperative complications such as postoperative atrial fibrillation (POAF). Hematopoietic somatic mosaicism (HSM) is a recently identified risk factor for cardiovascular diseases and results in a shift toward a chronic proinflammatory monocyte transcriptome and phenotype. OBJECTIVES: The aim of this study was to assess the prevalence, characteristics, and impact of HSM on preoperative blood and myocardial myeloid cells as well as on outcomes after cardiac surgery. METHODS: Blood DNA from 104 patients referred for surgical aortic valve replacement (AVR) was genotyped using the HemePACT panel (576 genes). Four screening methods were applied to assess HSM, and postoperative outcomes were explored. In-depth blood and myocardial leukocyte phenotyping was performed in selected patients using mass cytometry and preoperative and postoperative RNA sequencing analysis of classical monocytes. RESULTS: The prevalence of HSM in the patient cohort ranged from 29%, when considering the conventional HSM panel (97 genes) with variant allelic frequencies ≥2%, to 60% when considering the full HemePACT panel and variant allelic frequencies ≥1%. Three of 4 explored HSM definitions were significantly associated with higher risk for POAF. On the basis of the most inclusive definition, HSM carriers exhibited a 3.5-fold higher risk for POAF (age-adjusted OR: 3.5; 95% CI: 1.52-8.03; P = 0.003) and an exaggerated inflammatory response following AVR. HSM carriers presented higher levels of activated CD64+CD14+CD16- circulating monocytes and inflammatory monocyte-derived macrophages in presurgery myocardium. CONCLUSIONS: HSM is frequent in candidates for AVR, is associated with an enrichment of proinflammatory cardiac monocyte-derived macrophages, and predisposes to a higher incidence of POAF. HSM assessment may be useful in the personalized management of patients in the perioperative period. (Post-Operative Myocardial Incident & Atrial Fibrillation [POMI-AF]; NCT03376165).

Inherited human c-Rel deficiency disrupts myeloid and lymphoid immunity to multiple infectious agents.

We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8+ T cells, memory CD4+ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4+ T cells. In naive CD4+ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient's naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.

Diet-regulated production of PDGFcc by macrophages controls energy storage.

The mechanisms by which macrophages regulate energy storage remain poorly understood. We identify in a genetic screen a platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF)-family ortholog, Pvf3, that is produced by macrophages and is required for lipid storage in fat-body cells of Drosophila larvae. Genetic and pharmacological experiments indicate that the mouse Pvf3 ortholog PDGFcc, produced by adipose tissue-resident macrophages, controls lipid storage in adipocytes in a leptin receptor- and C-C chemokine receptor type 2-independent manner. PDGFcc production is regulated by diet and acts in a paracrine manner to control lipid storage in adipose tissues of newborn and adult mice. At the organismal level upon PDGFcc blockade, excess lipids are redirected toward thermogenesis in brown fat. These data identify a macrophage-dependent mechanism, conducive to the design of pharmacological interventions, that controls energy storage in metazoans.

Origins, Biology, and Diseases of Tissue Macrophages.

Tissue-resident macrophages are present in most tissues with developmental, self-renewal, or functional attributes that do not easily fit into a textbook picture of a plastic and multifunctional macrophage originating from hematopoietic stem cells; nor does it fit a pro- versus anti-inflammatory paradigm. This review presents and discusses current knowledge on the developmental biology of macrophages from an evolutionary perspective focused on the function of macrophages, which may aid in study of developmental, inflammatory, tumoral, and degenerative diseases. We also propose a framework to investigate the functions of macrophages in vivo and discuss how inherited germline and somatic mutations may contribute to the roles of macrophages in diseases.

NR4A1 Deletion in Marginal Zone B Cells Exacerbates Atherosclerosis in Mice-Brief Report.

OBJECTIVE: NR4A orphan receptors have been well studied in vascular and myeloid cells where they play important roles in the regulation of inflammation in atherosclerosis. NR4A1 (nerve growth factor IB) is among the most highly induced transcription factors in B cells following BCR (B-cell receptor) stimulation. Given that B cells substantially contribute to the development of atherosclerosis, we examined whether NR4A1 regulates B-cell function during atherogenesis. Approach and Results: We found that feeding Ldlr-/- mice a Western diet substantially increased Nr4a1 expression in marginal zone B (MZB) cells compared with follicular B cells. We then generated Ldlr-/- mice with complete B- or specific MZB-cell deletion of Nr4a1. Complete B-cell deletion of Nr4a1 led to increased atherosclerosis, which was accompanied by increased T follicular helper cell-germinal center axis response, as well as increased serum total cholesterol and triglycerides levels. Interestingly, specific MZB-cell deletion of Nr4a1 increased atherosclerosis in association with an increased T follicular helper-germinal center response but without any impact on serum cholesterol or triglyceride levels. Nr4a1-/- MZB cells showed decreased PDL1 (programmed death ligand-1) expression, which may have contributed to the enhanced T follicular helper response. CONCLUSIONS: Our findings reveal a previously unsuspected role for NR4A1 in the atheroprotective role of MZB cells.

Histone deacetylase 3 controls lung alveolar macrophage development and homeostasis.

Alveolar macrophages (AMs) derived from embryonic precursors seed the lung before birth and self-maintain locally throughout adulthood, but are regenerated by bone marrow (BM) under stress conditions. However, the regulation of AM development and maintenance remains poorly understood. Here, we show that histone deacetylase 3 (HDAC3) is a key epigenetic factor required for AM embryonic development, postnatal homeostasis, maturation, and regeneration from BM. Loss of HDAC3 in early embryonic development affects AM development starting at E14.5, while loss of HDAC3 after birth affects AM homeostasis and maturation. Single-cell RNA sequencing analyses reveal four distinct AM sub-clusters and a dysregulated cluster-specific pathway in the HDAC3-deficient AMs. Moreover, HDAC3-deficient AMs exhibit severe mitochondrial oxidative dysfunction and deteriorative cell death. Mechanistically, HDAC3 directly binds to Pparg enhancers, and HDAC3 deficiency impairs Pparg expression and its signaling pathway. Our findings identify HDAC3 as a key epigenetic regulator of lung AM development and homeostasis.

Cxcr4 distinguishes HSC-derived monocytes from microglia and reveals monocyte immune responses to experimental stroke.

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.

Macrophage ontogeny in the control of adipose tissue biology.

Macrophages are found in large numbers in the adipose tissue where they closely associate with the adipocytes and the vasculature. Adipose tissue macrophages are a heterogenous population of cells with 'hard wired' diversity brought upon by distinct developmental lineages. The purpose of this review is to provide a brief history of macrophages in control of adipose tissue metabolism with the emphasis on the importance of macrophage ontogeny.

Adult Drosophila Lack Hematopoiesis but Rely on a Blood Cell Reservoir at the Respiratory Epithelia to Relay Infection Signals to Surrounding Tissues.

The use of adult Drosophila melanogaster as a model for hematopoiesis or organismal immunity has been debated. Addressing this question, we identify an extensive reservoir of blood cells (hemocytes) at the respiratory epithelia (tracheal air sacs) of the thorax and head. Lineage tracing and functional analyses demonstrate that the majority of adult hemocytes are phagocytic macrophages (plasmatocytes) from the embryonic lineage that parallels vertebrate tissue macrophages. Surprisingly, we find no sign of adult hemocyte expansion. Instead, hemocytes play a role in relaying an innate immune response to the blood cell reservoir: through Imd signaling and the Jak/Stat pathway ligand Upd3, hemocytes act as sentinels of bacterial infection, inducing expression of the antimicrobial peptide Drosocin in respiratory epithelia and colocalizing fat body domains. Drosocin expression in turn promotes animal survival after infection. Our work identifies a multi-signal relay of organismal humoral immunity, establishing adult Drosophila as model for inter-organ immunity.

Activating mutations in CSF1R and additional receptor tyrosine kinases in histiocytic neoplasms.

Histiocytoses are clonal hematopoietic disorders frequently driven by mutations mapping to the BRAF and MEK1 and MEK2 kinases. Currently, however, the developmental origins of histiocytoses in patients are not well understood, and clinically meaningful therapeutic targets outside of BRAF and MEK are undefined. In this study, we uncovered activating mutations in CSF1R and rearrangements in RET and ALK that conferred dramatic responses to selective inhibition of RET (selpercatinib) and crizotinib, respectively, in patients with histiocytosis.

Liver-Derived Signals Sequentially Reprogram Myeloid Enhancers to Initiate and Maintain Kupffer Cell Identity.

Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-ß) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.