Maternal inflammation regulates fetal emergency myelopoiesis.

Neonates are highly susceptible to inflammation and infection. Here, we investigate how late fetal liver (FL) mouse hematopoietic stem and progenitor cells (HSPCs) respond to inflammation, testing the hypothesis that deficits in the engagement of emergency myelopoiesis (EM) pathways limit neutrophil output and contribute to perinatal neutropenia. We show that fetal HSPCs have limited production of myeloid cells at steady state and fail to activate a classical adult-like EM transcriptional program. Moreover, we find that fetal HSPCs can respond to EM-inducing inflammatory stimuli in vitro but are restricted by maternal anti-inflammatory factors, primarily interleukin-10 (IL-10), from activating EM pathways in utero. Accordingly, we demonstrate that the loss of maternal IL-10 restores EM activation in fetal HSPCs but at the cost of fetal demise. These results reveal the evolutionary trade-off inherent in maternal anti-inflammatory responses that maintain pregnancy but render the fetus unresponsive to EM activation signals and susceptible to infection.

An IL-4 signalling axis in bone marrow drives pro-tumorigenic myelopoiesis.

Myeloid cells are known to suppress antitumour immunity1. However, the molecular drivers of immunosuppressive myeloid cell states are not well defined. Here we used single-cell RNA sequencing of human and mouse non-small cell lung cancer (NSCLC) lesions, and found that in both species the type 2 cytokine interleukin-4 (IL-4) was predicted to be the primary driver of the tumour-infiltrating monocyte-derived macrophage phenotype. Using a panel of conditional knockout mice, we found that only deletion of the IL-4 receptor IL-4Rα in early myeloid progenitors in bone marrow reduced tumour burden, whereas deletion of IL-4Rα in downstream mature myeloid cells had no effect. Mechanistically, IL-4 derived from bone marrow basophils and eosinophils acted on granulocyte-monocyte progenitors to transcriptionally programme the development of immunosuppressive tumour-promoting myeloid cells. Consequentially, depletion of basophils profoundly reduced tumour burden and normalized myelopoiesis. We subsequently initiated a clinical trial of the IL-4Rα blocking antibody dupilumab2-5 given in conjunction with PD-1/PD-L1 checkpoint blockade in patients with relapsed or refractory NSCLC who had progressed on PD-1/PD-L1 blockade alone (ClinicalTrials.gov identifier NCT05013450 ). Dupilumab supplementation reduced circulating monocytes, expanded tumour-infiltrating CD8 T cells, and in one out of six patients, drove a near-complete clinical response two months after treatment. Our study defines a central role for IL-4 in controlling immunosuppressive myelopoiesis in cancer, identifies a novel combination therapy for immune checkpoint blockade in humans, and highlights cancer as a systemic malady that requires therapeutic strategies beyond the primary disease site.

The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells.

Invariant natural killer T cells (iNKT cells) are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about the epigenetic regulation of iNKT cell development. Here we found that the H3K27me3 histone demethylase UTX was an essential cell-intrinsic factor that controlled an iNKT-cell lineage-specific gene-expression program and epigenetic landscape in a demethylase-activity-dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT cell signature genes due to a decrease in activation-associated H3K4me3 marks and an increase in repressive H3K27me3 marks within the promoters occupied by UTX. We found that JunB regulated iNKT cell development and that the expression of genes that were targets of both JunB and the iNKT cell master transcription factor PLZF was UTX dependent. We identified iNKT cell super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for commitment to the iNKT cell lineage. Our findings reveal how UTX regulates the development of iNKT cells through multiple epigenetic mechanisms.

High-Dimensional Single-Cell Multimodal Landscape of Human Carotid Atherosclerosis.

BACKGROUND: Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, our understanding of the comprehensive transcriptional and phenotypic landscape of the cells within these lesions is limited. METHODS: To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. RESULTS: We identified 25 cell populations, each with a unique multiomic signature, including macrophages, T cells, NK (natural killer) cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Among the macrophages, we identified 2 proinflammatory subsets enriched in IL-1B (interleukin-1B) or C1Q expression, 2 TREM2-positive foam cells (1 expressing inflammatory genes), and subpopulations with a proliferative gene signature and SMC-specific gene signature with fibrotic pathways upregulated. Further characterization revealed various subsets of SMCs and fibroblasts, including SMC-derived foam cells. These foamy SMCs were localized in the deep intima of coronary atherosclerotic lesions. Utilizing cellular indexing of transcriptomes and epitopes by sequencing data, we developed a flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Lastly, we observed reduced proportions of efferocytotic macrophages, classically activated endothelial cells, and contractile and modulated SMC-derived cells, while inflammatory SMCs were enriched in plaques of clinically symptomatic versus asymptomatic patients. CONCLUSIONS: Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. These findings facilitate both the mapping of cardiovascular disease susceptibility loci to specific cell types and the identification of novel molecular and cellular therapeutic targets for the treatment of the disease.

Stem cells assessed.

The increasing momentum of stem cell research continues, with the better characterization of induced pluripotent stem (iPS) cells, the conversion of differentiated cells into different cell types and the use of pluripotent stem cells to generate whole tissues, among other advances. Here, six experts in the field of stem cell research compare different stem cell models and highlight the importance of pursuing complementary experimental approaches for a better understanding of pluripotency and differentiation and an informed approach to medical applications.

Autophagy maintains the metabolism and function of young and old stem cells.

With age, haematopoietic stem cells lose their ability to regenerate the blood system, and promote disease development. Autophagy is associated with health and longevity, and is critical for protecting haematopoietic stem cells from metabolic stress. Here we show that loss of autophagy in haematopoietic stem cells causes accumulation of mitochondria and an activated metabolic state, which drives accelerated myeloid differentiation mainly through epigenetic deregulations, and impairs haematopoietic stem-cell self-renewal activity and regenerative potential. Strikingly, most haematopoietic stem cells in aged mice share these altered metabolic and functional features. However, approximately one-third of aged haematopoietic stem cells exhibit high autophagy levels and maintain a low metabolic state with robust long-term regeneration potential similar to healthy young haematopoietic stem cells. Our results demonstrate that autophagy actively suppresses haematopoietic stem-cell metabolism by clearing active, healthy mitochondria to maintain quiescence and stemness, and becomes increasingly necessary with age to preserve the regenerative capacity of old haematopoietic stem cells.

Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis.

Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1ß, G-CSF, TGFß and CXCL4) and activation of an inducible Irf8 and ß-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.

The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells.

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.

FOXO3A directs a protective autophagy program in haematopoietic stem cells.

Blood production is ensured by rare, self-renewing haematopoietic stem cells (HSCs). How HSCs accommodate the diverse cellular stresses associated with their life-long activity remains elusive. Here we identify autophagy as an essential mechanism protecting HSCs from metabolic stress. We show that mouse HSCs, in contrast to their short-lived myeloid progeny, robustly induce autophagy after ex vivo cytokine withdrawal and in vivo calorie restriction. We demonstrate that FOXO3A is critical to maintain a gene expression program that poises HSCs for rapid induction of autophagy upon starvation. Notably, we find that old HSCs retain an intact FOXO3A-driven pro-autophagy gene program, and that ongoing autophagy is needed to mitigate an energy crisis and allow their survival. Our results demonstrate that autophagy is essential for the life-long maintenance of the HSC compartment and for supporting an old, failing blood system.

Validation of MdmX as a therapeutic target for reactivating p53 in tumors.

MdmX, also known as Mdm4, is a critical negative regulator of p53, and its overexpression serves to block p53 tumor suppressor function in many cancers. Consequently, inhibiting MdmX has emerged as an attractive approach to restoring p53 function in those cancers that retain functional p53. However, the consequences of acute systemic MdmX inhibition in normal adult tissues remain unknown. To determine directly the effects of systemic MdmX inhibition in normal tissues and in tumors, we crossed mdmX(-/-) mice into the p53ER(TAM) knockin background. In place of wild-type p53, p53ER(TAM) knockin mice express a variant of p53, p53ER(TAM), that is completely dependent on 4-hydroxy-tamoxifen for its activity. MdmX inhibition was then modeled by restoring p53 function in these MdmX-deficient mice. We show that MdmX is continuously required to buffer p53 activity in adult normal tissues and their stem cells. Importantly, the effects of transient p53 restoration in the absence of MdmX are nonlethal and reversible, unlike transient p53 restoration in the absence of Mdm2, which is ineluctably lethal. We also show that the therapeutic impact of restoring p53 in a tumor model is enhanced in the absence of MdmX, affording a significant extension of life span over p53 restoration in the presence of MdmX. Hence, systemic inhibition of MdmX is both a feasible therapeutic strategy for restoring p53 function in tumors that retain wild-type p53 and likely to be significantly safer than inhibition of Mdm2.

Invasive breast cancer reprograms early myeloid differentiation in the bone marrow to generate immunosuppressive neutrophils.

Expansion of myeloid cells associated with solid tumor development is a key contributor to neoplastic progression. Despite their clinical relevance, the mechanisms controlling myeloid cell production and activity in cancer remains poorly understood. Using a multistage mouse model of breast cancer, we show that production of atypical T cell-suppressive neutrophils occurs during early tumor progression, at the onset of malignant conversion, and that these cells preferentially accumulate in peripheral tissues but not in the primary tumor. Production of these cells results from activation of a myeloid differentiation program in bone marrow (BM) by a novel mechanism in which tumor-derived granulocyte-colony stimulating factor (G-CSF) directs expansion and differentiation of hematopoietic stem cells to skew hematopoiesis toward the myeloid lineage. Chronic skewing of myeloid production occurred in parallel to a decrease in erythropoiesis in BM in mice with progressive disease. Significantly, we reveal that prolonged G-CSF stimulation is both necessary and sufficient for the distinguishing characteristics of tumor-induced immunosuppressive neutrophils. These results demonstrate that prolonged G-CSF may be responsible for both the development and activity of immunosuppressive neutrophils in cancer.

Transcription and methylation analyses of preleukemic promyelocytes indicate a dual role for PML/RARA in leukemia initiation.

Acute promyelocytic leukemia is an aggressive malignancy characterized by the accumulation of promyelocytes in the bone marrow. PML/RARA is the primary abnormality implicated in this pathology, but the mechanisms by which this chimeric fusion protein initiates disease are incompletely understood. Identifying PML/RARA targets in vivo is critical for comprehending the road to pathogenesis. Utilizing a novel sorting strategy, we isolated highly purified promyelocyte populations from normal and young preleukemic animals, carried out microarray and methylation profiling analyses, and compared the results from the two groups of animals. Surprisingly, in the absence of secondary lesions, PML/RARA had an overall limited impact on both the transcriptome and methylome. Of interest, we did identify down-regulation of secondary and tertiary granule genes as the first step engaging the myeloid maturation block. Although initially not sufficient to arrest terminal granulopoiesis in vivo, such alterations set the stage for the later, complete differentiation block seen in leukemia. Further, gene set enrichment analysis revealed that PML/RARA promyelocytes exhibit a subtle increase in expression of cell cycle genes, and we show that this leads to both increased proliferation of these cells and expansion of the promyelocyte compartment. Importantly, this proliferation signature was absent from the poorly leukemogenic p50/RARA fusion model, implying a critical role for PML in the altered cell-cycle kinetics and ability to initiate leukemia. Thus, our findings challenge the predominant model in the field and we propose that PML/RARA initiates leukemia by subtly shifting cell fate decisions within the promyelocyte compartment.

Pro-inflammatory cytokines: emerging players regulating HSC function in normal and diseased hematopoiesis.

Hematopoiesis is the hierarchical process in which all lineages of blood cells are produced by self-renewing hematopoietic stem cells (HSCs) in the bone marrow (BM). While the regulatory factors that maintain proper HSC function and lineage output under normal conditions are well understood, significantly less is known about how HSC fate is regulated in response to inflammation or disease. As many blood disorders are associated with overproduction of pro-inflammatory cytokines, significant interest has emerged in understanding the impact of these factors on HSC function. In this review we highlight key advances demonstrating the impact of pro-inflammatory cytokines on the biology of HSCs and the BM niche, and address ongoing questions regarding their role in normal and pathogenic hematopoiesis.

Essential role of Jun family transcription factors in PU.1 knockdown-induced leukemic stem cells.

Knockdown of the transcription factor PU.1 (encoded by Sfpi1) leads to acute myeloid leukemia (AML) in mice. We examined the transcriptome of preleukemic hematopoietic stem cells (HSCs) in which PU.1 was knocked down (referred to as 'PU.1-knockdown HSCs') to identify transcriptional changes preceding malignant transformation. Transcription factors c-Jun and JunB were among the top-downregulated targets. Restoration of c-Jun expression in preleukemic cells rescued the PU.1 knockdown-initiated myelomonocytic differentiation block. Lentiviral restoration of JunB at the leukemic stage led to loss of leukemic self-renewal capacity and prevented leukemia in NOD-SCID mice into which leukemic PU.1-knockdown cells were transplanted. Examination of human individuals with AML confirmed the correlation between PU.1 and JunB downregulation. These results delineate a transcriptional pattern that precedes leukemic transformation in PU.1-knockdown HSCs and demonstrate that decreased levels of c-Jun and JunB contribute to the development of PU.1 knockdown-induced AML by blocking differentiation and increasing self-renewal. Therefore, examination of disturbed gene expression in HSCs can identify genes whose dysregulation is essential for leukemic stem cell function and that are targets for therapeutic interventions.

Activated Gs signaling in osteoblastic cells alters the hematopoietic stem cell niche in mice.

Adult hematopoiesis occurs primarily in the BM space where hematopoietic cells interact with stromal niche cells. Despite this close association, little is known about the specific roles of osteoblastic lineage cells (OBCs) in maintaining hematopoietic stem cells (HSCs), and how conditions affecting bone formation influence HSC function. Here we use a transgenic mouse model with the ColI(2.3) promoter driving a ligand-independent, constitutively active 5HT4 serotonin receptor (Rs1) to address how the massive increase in trabecular bone formation resulting from increased G(s) signaling in OBCs impacts HSC function and blood production. Rs1 mice display fibrous dysplasia, BM aplasia, progressive loss of HSC numbers, and impaired megakaryocyte/erythrocyte development with defective recovery after hematopoietic injury. These hematopoietic defects develop without compensatory extramedullary hematopoiesis, and the loss of HSCs occurs despite a paradoxical expansion of stromal niche cells with putative HSC-supportive activity (ie, endothelial, mesenchymal, and osteoblastic cells). However, Rs1-expressing OBCs show decreased expression of key HSC-supportive factors and impaired ability to maintain HSCs. Our findings indicate that long-term activation of G(s) signaling in OBCs leads to contextual changes in the BM niche that adversely affect HSC maintenance and blood homeostasis.

Made to order: emergency myelopoiesis and demand-adapted innate immune cell production.

Definitive haematopoiesis is the process by which haematopoietic stem cells, located in the bone marrow, generate all haematopoietic cell lineages in healthy adults. Although highly regulated to maintain a stable output of blood cells in health, the haematopoietic system is capable of extensive remodelling in response to external challenges, prioritizing the production of certain cell types at the expense of others. In this Review, we consider how acute insults, such as infections and cytotoxic drug-induced myeloablation, cause molecular, cellular and metabolic changes in haematopoietic stem and progenitor cells at multiple levels of the haematopoietic hierarchy to drive accelerated production of the mature myeloid cells needed to resolve the initiating insult. Moreover, we discuss how dysregulation or subversion of these emergency myelopoiesis mechanisms contributes to the progression of chronic inflammatory diseases and cancer.

Decoding functional hematopoietic progenitor cells in the adult human lung.

The bone marrow is the main site of blood cell production in adults, however, rare pools of hematopoietic stem and progenitor cells with self-renewal and differentiation potential have been found in extramedullary organs. The lung is primarily known for its role in gas exchange but has recently been described as a site of blood production in mice. Here, we show that functional hematopoietic precursors reside in the extravascular spaces of the human lung, at a frequency similar to the bone marrow, and are capable of proliferation and engraftment. The organ-specific gene signature of pulmonary and medullary CD34+ hematopoietic progenitors indicates greater baseline activation of immune, megakaryocyte/platelet and erythroid-related pathways in lung progenitors. Spatial transcriptomics mapped blood progenitors in the lung to a vascular-rich alveolar interstitium niche. These results identify the lung as a pool for uniquely programmed blood stem and progenitor cells with the potential to support hematopoiesis in humans.

Autophagy counters inflammation-driven glycolytic impairment in aging hematopoietic stem cells.

Autophagy is central to the benefits of longevity signaling programs and to hematopoietic stem cell (HSC) response to nutrient stress. With age, a subset of HSCs increases autophagy flux and preserves regenerative capacity, but the signals triggering autophagy and maintaining the functionality of autophagy-activated old HSCs (oHSCs) remain unknown. Here, we demonstrate that autophagy is an adaptive cytoprotective response to chronic inflammation in the aging murine bone marrow (BM) niche. We find that inflammation impairs glucose uptake and suppresses glycolysis in oHSCs through Socs3-mediated inhibition of AKT/FoxO-dependent signaling, with inflammation-mediated autophagy engagement preserving functional quiescence by enabling metabolic adaptation to glycolytic impairment. Moreover, we show that transient autophagy induction via a short-term fasting/refeeding paradigm normalizes glycolytic flux and significantly boosts oHSC regenerative potential. Our results identify inflammation-driven glucose hypometabolism as a key driver of HSC dysfunction with age and establish autophagy as a targetable node to reset oHSC regenerative capacity.

JunB can substitute for Jun in mouse development and cell proliferation.

The Jun and JunB components of the AP-1 transcription factor are known to have antagonistic functions. Here we show, by a knock-in strategy and a transgenic complementation approach, that Junb can substitute for absence of Jun during mouse development. Junb can rescue both liver and cardiac defects in Jun-null mice in a manner dependent on gene dosage. JunB restores the expression of genes regulated by Jun/Fos, but not those regulated by Jun/ATF, thereby rescuing Jun-dependent defects in vivo as well as in primary fibroblasts and fetal hepatoblasts in vitro. Thus, the transcriptionally less active JunB has the potential to substitute for Jun, indicating that the spatial and temporal regulation of expression of the transcription factor AP-1 may be more important than the coding sequence of its components.