MYCT1 controls environmental sensing in human haematopoietic stem cells.

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.

Mast Cells Reveal Their Past Selves.

Mast cells have been thought to derive from bone marrow hematopoietic stem cells. In this issue of Immunity, Gentek et al. (2018) reveal that mast cells have dual developmental origins in primitive and definitive hematopoiesis and that adult mast cell maintenance is largely bone marrow independent.

Granulocyte-Monocyte Progenitors and Monocyte-Dendritic Cell Progenitors Independently Produce Functionally Distinct Monocytes.

Granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs) produce monocytes during homeostasis and in response to increased demand during infection. Both progenitor populations are thought to derive from common myeloid progenitors (CMPs), and a hierarchical relationship (CMP-GMP-MDP-monocyte) is presumed to underlie monocyte differentiation. Here, however, we demonstrate that mouse MDPs arose from CMPs independently of GMPs, and that GMPs and MDPs produced monocytes via similar but distinct monocyte-committed progenitors. GMPs and MDPs yielded classical (Ly6Chi) monocytes with gene expression signatures that were defined by their origins and impacted their function. GMPs produced a subset of "neutrophil-like" monocytes, whereas MDPs gave rise to a subset of monocytes that yielded monocyte-derived dendritic cells. GMPs and MDPs were also independently mobilized to produce specific combinations of myeloid cell types following the injection of microbial components. Thus, the balance of GMP and MDP differentiation shapes the myeloid cell repertoire during homeostasis and following infection.

Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging.

Microglia, the brain's resident macrophages, maintain brain homeostasis and respond to injury and infection. During aging they undergo functional changes, but the underlying mechanisms and their contributions to neuroprotection versus neurodegeneration are unclear. Previous studies suggested that microglia are sex dimorphic, so we compared microglial aging in mice of both sexes. RNA-sequencing of hippocampal microglia revealed more aging-associated changes in female microglia than male microglia, and more sex differences in old microglia than young microglia. Pathway analyses and subsequent validation assays revealed a stronger AKT-mTOR-HIF1α-driven shift to glycolysis among old female microglia and indicated that C3a production and detection was elevated in old microglia, especially in females. Recombinant C3a induced AKT-mTOR-HIF1α signaling and increased the glycolytic and phagocytic activity of young microglia. Single cell analyses attributed the aging-associated sex dimorphism to more abundant disease-associated microglia (DAM) in old female mice than old male mice, and evaluation of an Alzheimer's Disease mouse model revealed that the metabolic and complement changes are also apparent in the context of neurodegenerative disease and are strongest in the neuroprotective DAM2 subset. Collectively, our data implicate autocrine C3a-C3aR signaling in metabolic reprogramming of microglia to neuroprotective DAM during aging, especially in females, and also in Alzheimer's Disease.

Microbial Sensing by Hematopoietic Stem and Progenitor Cells.

Balanced production of immune cells is critical for the maintenance of steady-state immune surveillance, and increased production of myeloid cells is sometimes necessary to eliminate pathogens. Hematopoietic stem and progenitor cell (HSPC) sensing of commensal microbes and invading pathogens has a notable impact on hematopoiesis. In this review, we examine how commensal microbes regulate bone marrow HSPC activity to maintain balanced hematopoiesis in the steady state, and how HSPCs proliferate and differentiate during emergency myelopoiesis in response to infection. HSPCs express a variety of pattern recognition receptors and cytokine receptors that they use to sense the presence of microbes, either directly via detection of microbial components and metabolites, or indirectly by responding to cytokines produced by other host cells. We describe direct and indirect mechanisms of microbial sensing by HSPCs and highlight evidence demonstrating long-term effects of acute and chronic microbial stimuli on HSPCs. We also discuss a possible connection between myeloid-biased hematopoiesis and elevated levels of circulating microbiome-derived components in the context of aging and metabolic stress. Finally, we highlight the prospect of trained immunity-based vaccines that could exploit microbial stimulation of HSPCs.

Heterogeneity and origins of myeloid cells.

PURPOSE OF REVIEW: Myeloid cells - granulocytes, monocytes, macrophages and dendritic cells (DCs) - are innate immune cells that play key roles in pathogen defense and inflammation, as well as in tissue homeostasis and repair. Over the past 5 years, in part due to more widespread use of single cell omics technologies, it has become evident that these cell types are significantly more heterogeneous than was previously appreciated. In this review, we consider recent studies that have demonstrated heterogeneity among neutrophils, monocytes, macrophages and DCs in mice and humans. We also discuss studies that have revealed the sources of their heterogeneity. RECENT FINDINGS: Recent studies have confirmed that ontogeny is a key determinant of diversity, with specific subsets of myeloid cells arising from distinct progenitors. However, diverse microenvironmental cues also strongly influence myeloid fate and function. Accumulating evidence therefore suggests that a combination of these mechanisms underlies myeloid cell diversity. SUMMARY: Consideration of the heterogeneity of myeloid cells is critical for understanding their diverse activities, such as the role of macrophages in tissue damage versus repair, or tumor growth versus elimination. Insights into these mechanisms are informing the design of novel therapeutic approaches.

Reticulon-1 and Reduced Migration toward Chemoattractants by Macrophages Differentiated from the Bone Marrow of Ultraviolet-Irradiated and Ultraviolet-Chimeric Mice.

The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV-chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity.

Autocrine Type I IFN Signaling in Dendritic Cells Stimulated with Fungal ß-Glucans or Lipopolysaccharide Promotes CD8 T Cell Activation.

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection, but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal ß-glucans stimulate IFN-ß production by dendritic cells (DCs) following detection by the Dectin-1 receptor, but the effects of ß-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal ß-glucan particle-stimulated DCs. We demonstrate that ß-glucan-stimulated DCs induce CD8 T cell proliferation, activation marker (CD44 and CD69) expression, and production of IFN-γ, IL-2, and granzyme B. Moreover, we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by ß-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically, type I IFNs promote Ag presentation on MHC I molecules, CD86 and CD40 expression, and the production of IL-12 p70, IL-2, IL-6, and TNF-α by ß-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation, although, in the context of LPS stimulation, this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection, which may be useful in the rational design of vaccines directed against pathogens and tumors.

UV Irradiation of Skin Enhances Glycolytic Flux and Reduces Migration Capabilities in Bone Marrow-Differentiated Dendritic Cells.

A systemic immunosuppression follows UV irradiation of the skin of humans and mice. In this study, dendritic cells (DCs) differentiating from the bone marrow (BM) of UV-irradiated mice had a reduced ability to migrate toward the chemokine (C-C motif) ligand 21. Fewer DCs also accumulated in the peritoneal cavity of UV-chimeric mice (ie, mice transplanted with BM from UV-irradiated mice) after injection of an inflammatory stimulus into that site. We hypothesized that different metabolic states underpin altered DC motility. Compared with DCs from the BM of nonirradiated mice, those from UV-irradiated mice produced more lactate, consumed more glucose, and had greater glycolytic flux in a bioenergetics stress test. Greater expression of 3-hydroxyanthranilate 3,4-dioxygenase was identified as a potential contributor to increased glycolysis. Inhibition of 3-hydroxyanthranilate 3,4-dioxygenase by 6-chloro-dl-tryptophan prevented both increased lactate production and reduced migration toward chemokine (C-C motif) ligand 21 by DCs differentiated from BM of UV-irradiated mice. UV-induced prostaglandin E2 has been implicated as an intermediary in the effects of UV radiation on BM cells. DCs differentiating from BM cells pulsed in vitro for 2 hours with dimethyl prostaglandin E2 were functionally similar to those from the BM of UV-irradiated mice. Reduced migration of DCs to lymph nodes associated with increased glycolytic flux may contribute to their reduced ability to initiate new immune responses in UV-irradiated mice.

IRF8 acts in lineage-committed rather than oligopotent progenitors to control neutrophil vs monocyte production.

Interferon regulatory factor 8 (IRF8) is a key regulator of myelopoiesis in mice and humans. IRF8-deficient mice exhibit increased neutrophil numbers but defective monocyte and dendritic cell (DC) production. It has therefore been hypothesized that IRF8 regulates granulocyte vs monocyte/DC lineage commitment by oligopotent progenitors. Alternatively, IRF8 could control the differentiation of lineage-committed progenitors. In this study, we defined the role of IRF8 in lineage commitment and neutrophil vs monocyte differentiation using a novel sorting strategy that for the first time allows us to separate oligopotent granulocyte-monocyte progenitors (GMPs) and their lineage-committed progeny: granulocyte progenitors (GPs) and monocyte progenitors (MPs). We show that IRF8 is highly expressed by both GPs and MPs, but not GMPs, and is not required for GP or MP production by GMPs. In fact, IRF8-deficient mice have more GPs and MPs. This is not due to IRF8-mediated suppression of GP and MP production by GMPs, but rather to selective effects in GPs and MPs. We identify roles for IRF8 in regulating progenitor survival and differentiation and preventing leukemic cell accumulation. Thus, IRF8 does not regulate granulocytic vs monocytic fate in GMPs, but instead acts downstream of lineage commitment to selectively control neutrophil and monocyte production.

Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'.

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects ß-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate ß-glucan polymers, Dectin-1 signalling is only activated by particulate ß-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.

Interferon regulatory factor 8 and the regulation of neutrophil, monocyte, and dendritic cell production.

PURPOSE OF REVIEW: Interferon regulatory factor 8 (IRF8) is a transcription factor that plays central roles in the regulation of myeloid cell fate. In both mice and humans, IRF8 is required for the differentiation of most monocyte and dendritic cell subsets, but suppresses neutrophil production. IRF8 mutations can cause immunodeficiency, and the dysregulated differentiation that underlies myeloid leukemia has been attributed in part to reduced IRF8 expression. In this review we discuss recent studies that have revealed molecular mechanisms underlying the regulation of myelopoiesis by IRF8, which cooperates with other transcription factors to control the initiation of gene expression programs that define the development of specific myeloid cell subsets. RECENT FINDINGS: It is now clear that IRF8 regulates cell fate choice by both promoting monocyte/dendritic cell differentiation and suppressing neutrophil differentiation. Recent studies have shown that it collaborates with PU.1 to promote monocyte gene expression (in part via induction of Krüppel-like factor-4), associates with Batf3 to induce CD8α conventional dendritic cell differentiation via autoregulation of its own expression, and restricts neutrophil gene expression by disrupting the binding of c/EBPα to target genes. SUMMARY: These studies have emphasized the importance of IRF8 in the regulation of myelopoiesis and are revealing novel therapeutic targets.

Mechanisms of Fc receptor and dectin-1 activation for phagocytosis.

Phagocytosis is a key cellular process, both during homeostasis and upon infection or tissue damage. Receptors on the surface of professional phagocytic cells bind to target particles either directly or through opsonizing ligands, and trigger actin-mediated ingestion of the particles. The process must be carefully controlled to ensure that phagocytosis is triggered efficiently and specifically, and that the antimicrobial cytotoxic responses that often accompany it are initiated only when required. In this review, we will describe and compare the molecular mechanisms that regulate phagocytosis triggered by Fcγ receptors, which mediate the uptake of immunoglobulin G-opsonized targets, and Dectin-1, which is responsible for internalization of fungi with exposed cell wall ß-glucan. We will examine how these receptors detect their ligands, how signal transduction is initiated and regulated, and how internalization is instructed to achieve rapid and yet controlled uptake of their targets.

TLRs control hematopoiesis during infection.

Recent research has shown that (i) Toll-like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to proliferate and differentiate along the myeloid lineage in vitro, and (ii) direct TLR-mediated stimulation of HSPCs also promotes macrophage differentiation in vivo following infection. These new insights demonstrate that TLR signaling in HSPCs, in addition to other TLR-dependent mechanisms, can contribute to HSPC expansion and myeloid differentiation after infection. Evidence is, therefore, mounting that direct TLR-induced programming of hematopoiesis plays a key role in host defense by rapidly replenishing the innate immune system with the cells needed to deal with pathogens.

Detection of a TLR2 agonist by hematopoietic stem and progenitor cells impacts the function of the macrophages they produce.

Several groups have shown that detection of microbial components by TLRs on hematopoietic stem and progenitor cells (HSPCs) instructs myeloid cell generation, raising interest in the possibility of targeting TLRs on HSPCs to boost myelopoiesis. However, although "TLR-derived" cells exhibit myeloid cell characteristics (phagocytosis, cytokine production, antigen presentation), it is not clear whether they are functionally equivalent to macrophages derived in the absence of TLR activation. Our in vitro and in vivo studies show that macrophages derived from mouse and human HSPC subsets (including stem cells) exposed to a TLR2 agonist prior to or during macrophage differentiation produce lower levels of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and reactive oxygen species. This is in contrast to prior exposure of differentiated macrophages to the TLR2 agonist ("tolerance"), which suppresses inflammatory cytokine production, but elevates reactive oxygen species. Soluble factors produced following exposure of HSPCs to a TLR2 agonist can also act in a paracrine manner to influence the function of macrophages derived from unexposed HSPCs. Our data demonstrate that macrophage function can be influenced by TLR signaling in the HSPCs from which they are derived, and that this may impact the clinical utility of targeting TLRs on HSPCs to boost myelopoiesis.

NLRP3, NLRP6, and NLRP12 are inflammasomes with distinct expression patterns.

Inflammasomes are sensors that detect cytosolic microbial molecules or cellular damage, and in response they initiate a form of lytic regulated cell death called pyroptosis. Inflammasomes signal via homotypic protein-protein interactions where CARD or PYD domains are crucial for recruiting downstream partners. Here, we screened these domains from NLR family proteins, and found that the PYD domain of NLRP6 and NLRP12 could activate caspase-1 to induce cleavage of IL-1ß and GSDMD. Inflammasome reconstitution verified that full length NLRP6 and NLRP12 formed inflammasomes in vitro, and NLRP6 was more prone to auto-activation. NLRP6 was highly expressed in intestinal epithelial cells (IEC), but not in immune cells. Molecular phylogeny analysis found that NLRP12 was closely related to NLRP3, but the activation mechanisms are different. NLRP3 was highly expressed in monocytes and macrophages, and was modestly but appreciably expressed in neutrophils. In contrast, NLRP12 was specifically expressed in neutrophils and eosinophils, but was not detectable in macrophages. NLRP12 mutations cause a periodic fever syndrome called NLRP12 autoinflammatory disease. We found that several of these patient mutations caused spontaneous activation of caspase-1 in vitro, which likely causes their autoinflammatory disease. Different cell types have unique cellular physiology and structures which could be perturbed by a pathogen, necessitating expression of distinct inflammasome sensors to monitor for signs of infection.

Beta-glucan recognition by the innate immune system.

Beta-glucans are recognized by the innate immune system. This recognition plays important roles in host defense and presents specific opportunities for clinical modulation of the host immune response. Neutrophils, macrophages, and dendritic cells among others express several receptors capable of recognizing beta-glucan in its various forms. This review explores what is currently known about beta-glucan recognition and how this recognition stimulates immune responses. Special emphasis is placed on Dectin-1, as we know the most about how this key beta-glucan receptor translates recognition into intracellular signaling, stimulates cellular responses, and participates in orchestrating the adaptive immune response.

Single-cell transcriptomic analysis of endometriosis.

Endometriosis is a common condition in women that causes chronic pain and infertility and is associated with an elevated risk of ovarian cancer. We profiled transcriptomes of >370,000 individual cells from endometriomas (n = 8), endometriosis (n = 28), eutopic endometrium (n = 10), unaffected ovary (n = 4) and endometriosis-free peritoneum (n = 4), generating a cellular atlas of endometrial-type epithelial cells, stromal cells and microenvironmental cell populations across tissue sites. Cellular and molecular signatures of endometrial-type epithelium and stroma differed across tissue types, suggesting a role for cellular restructuring and transcriptional reprogramming in the disease. Epithelium, stroma and proximal mesothelial cells of endometriomas showed dysregulation of pro-inflammatory pathways and upregulation of complement proteins. Somatic ARID1A mutation in epithelial cells was associated with upregulation of pro-angiogenic and pro-lymphangiogenic factors and remodeling of the endothelial cell compartment, with enrichment of lymphatic endothelial cells. Finally, signatures of ciliated epithelial cells were enriched in ovarian cancers, reinforcing epidemiologic associations between these two diseases.