Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

Slow cycling and durable Flt3+ progenitors contribute to hematopoiesis under native conditions.

The dynamics of the hematopoietic flux responsible for blood cell production in native conditions remains a matter of debate. Using CITE-seq analyses, we uncovered a distinct progenitor population that displays a cell cycle gene signature similar to the one found in quiescent hematopoietic stem cells. We further determined that the CD62L marker can be used to phenotypically enrich this population in the Flt3+ multipotent progenitor (MPP4) compartment. Functional in vitro and in vivo analyses validated the heterogeneity of the MPP4 compartment and established the quiescent/slow-cycling properties of the CD62L- MPP4 cells. Furthermore, studies under native conditions revealed a novel hierarchical organization of the MPP compartments in which quiescent/slow-cycling MPP4 cells sustain a prolonged hematopoietic activity at steady-state while giving rise to other lineage-biased MPP populations. Altogether, our data characterize a durable and productive quiescent/slow-cycling hematopoietic intermediary within the MPP4 compartment and highlight early paths of progenitor differentiation during unperturbed hematopoiesis.

The multifaceted role of mitochondria in HSC fate decisions: energy and beyond.

Hematopoietic stem cells (HSCs) have the properties to self-renew and/or differentiate into all-mature blood cell lineages. The fate decisions to generate progeny that retain stemness properties or that commit to differentiation is a fundamental process to maintain tissue homeostasis and must be tightly regulated to prevent HSC overgrowth or exhaustion. HSC fate decisions are inherently coupled to cell division. The transition from quiescence to activation is accompanied by major metabolic and mitochondrial changes that are important for cell cycle entry and for balanced decisions between self-renewal and differentiation. In this review, we discuss the current understanding of the role of mitochondrial metabolism in HSC transition from quiescence to activation and fate decisions.

Linking cell cycle to hematopoietic stem cell fate decisions.

Hematopoietic stem cells (HSCs) have the properties to self-renew and/or differentiate into any blood cell lineages. In order to balance the maintenance of the stem cell pool with supporting mature blood cell production, the fate decisions to self-renew or to commit to differentiation must be tightly controlled, as dysregulation of this process can lead to bone marrow failure or leukemogenesis. The contribution of the cell cycle to cell fate decisions has been well established in numerous types of stem cells, including pluripotent stem cells. Cell cycle length is an integral component of hematopoietic stem cell fate. Hematopoietic stem cells must remain quiescent to prevent premature replicative exhaustion. Yet, hematopoietic stem cells must be activated into cycle in order to produce daughter cells that will either retain stem cell properties or commit to differentiation. How the cell cycle contributes to hematopoietic stem cell fate decisions is emerging from recent studies. Hematopoietic stem cell functions can be stratified based on cell cycle kinetics and divisional history, suggesting a link between Hematopoietic stem cells activity and cell cycle length. Hematopoietic stem cell fate decisions are also regulated by asymmetric cell divisions and recent studies have implicated metabolic and organelle activity in regulating hematopoietic stem cell fate. In this review, we discuss the current understanding of the mechanisms underlying hematopoietic stem cell fate decisions and how they are linked to the cell cycle.

Rap1b-loss increases neutrophil lactate dehydrogenase activity to enhance neutrophil migration and acute inflammation in vivo.

Introduction: Neutrophils are critical for host immune defense; yet, aberrant neutrophil tissue infiltration triggers tissue damage. Neutrophils are heterogeneous functionally, and adopt 'normal' or 'pathogenic' effector function responses. Understanding neutrophil heterogeneity could provide specificity in targeting inflammation. We previously identified a signaling pathway that suppresses neutrophilmediated inflammation via integrin-mediated Rap1b signaling pathway. Methods: Here, we used Rap1-deficient neutrophils and proteomics to identify pathways that specifically control pathogenic neutrophil effector function. Results: We show neutrophil acidity is normally prevented by Rap1b during normal immune response with loss of Rap1b resulting in increased neutrophil acidity via enhanced Ldha activity and abnormal neutrophil behavior. Acidity drives the formation of abnormal invasive-like protrusions in neutrophils, causing a shift to transcellular migration through endothelial cells. Acidity increases neutrophil extracellular matrix degradation activity and increases vascular leakage in vivo. Pathogenic inflammatory condition of ischemia/reperfusion injury is associated with increased neutrophil transcellular migration and vascular leakage. Reducing acidity with lactate dehydrogenase inhibition in vivo limits tissue infiltration of pathogenic neutrophils but less so of normal neutrophils, and reduces vascular leakage. Discussion: Acidic milieu renders neutrophils more dependent on Ldha activity such that their effector functions are more readily inhibited by small molecule inhibitor of Ldha activity, which offers a therapeutic window for antilactate dehydrogenase treatment in specific targeting of pathogenic neutrophils in vivo.

Acquired and hereditary bone marrow failure: A mitochondrial perspective.

The disorders known as bone marrow failure syndromes (BMFS) are life-threatening disorders characterized by absence of one or more hematopoietic lineages in the peripheral blood. Myelodysplastic syndromes (MDS) are now considered BMF disorders with associated cellular dysplasia. BMFs and MDS are caused by decreased fitness of hematopoietic stem cells (HSC) and poor hematopoiesis. BMF and MDS can occur de novo or secondary to hematopoietic stress, including following bone marrow transplantation or myeloablative therapy. De novo BMF and MDS are usually associated with specific genetic mutations. Genes that are commonly mutated in BMF/MDS are in DNA repair pathways, epigenetic regulators, heme synthesis. Despite known and common gene mutations, BMF and MDS are very heterogenous in nature and non-genetic factors contribute to disease phenotype. Inflammation is commonly found in BMF and MDS, and contribute to ineffective hematopoiesis. Another common feature of BMF and MDS, albeit less known, is abnormal mitochondrial functions. Mitochondria are the power house of the cells. Beyond energy producing machinery, mitochondrial communicate with the rest of the cells via triggering stress signaling pathways and by releasing numerous metabolite intermediates. As a result, mitochondria play significant roles in chromatin regulation and innate immune signaling pathways. The main goal of this review is to investigate BMF processes, with a focus mitochondria-mediated signaling in acquired and inherited BMF.

The new metabolic needs of hematopoietic stem cells.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) are endowed with high regenerative potential to supply mature blood cells throughout life, under steady state or stress conditions. HSCs are thought to rely on glycolysis when in a quiescent state and to switch to oxidative phosphorylation to meet their metabolic needs during activation. Recently, a series of important studies reveals a higher degree of complexity that goes well beyond the dichotomy between glycolysis and oxidative phosphorylation. The purpose of this review is to summarize the recent findings highlighting the multifaceted metabolic requirements of HSC homeostasis. RECENT FINDINGS: Emerging evidence points to the importance of lysosomal catabolic activity and noncanonical retinoic acid pathway in maintaining HSC quiescence and stemness. HSC activation into cycle seems to be accompanied by a switch to glycolysis-mitochondrial coupling and to anabolic pathways, including Myc, aspartate-mediated purine synthesis. SUMMARY: Knowledge of metabolism of HSCs has dramatically increased in the past 2 years and reveals unexpected needs of HSCs during both their quiescent and activated state. Understanding how HSCs use metabolism for their functions will offer new opportunity for HSC-based therapies.

Hungry Hematopoietic Stem Cells during Bacterial Infection: Fatty Acid for Food.

Hematopoietic stem cells (HSC) directly initiate a response to bacterial infections by rapidly entering the cell cycle in order to produce mature blood cells. An important issue in the field of HSC biology is to understand how metabolic activities of HSC are fueled during specific condition that require HSC activation. In their paper, Mistry et al. provide evidence that bacterial infections trigger an increased in free fatty acid uptake by HSC that fuel fatty acid oxidation and mitochondrial respiration activities. This increased fatty acid uptake is exclusively dependent on the upregulation of the fatty acid transporter CD36. This study shed important light into the metabolic needs of HSC during septic conditions.

Transforming growth factor-ß signaling modifies the hematopoietic acute inflammatory response to drive bone marrow failure.

Bone marrow failure syndromes are characterized by ineffective hematopoiesis due to impaired fitness of hematopoietic stem cells. They can be acquired during bone marrow stress or innate and are associated with driver genetic mutations. Patients with a bone marrow failure syndrome are at higher risk of developing secondary neoplasms, including myelodysplastic syndromes and leukemia. Despite the identification of genetic driver mutations, the hematopoietic presentation of the disease is quite heterogeneous, raising the possibility that non-genetic factors contribute to the pathogenesis of the disease. The role of inflammation has emerged as an important contributing factor, but remains to be understood in detail. In this study, we examined the effect of increased transforming growth factor-b (TGFb) signaling, in combination or not with an acute innate immune challenge using polyinosinc:polycytidilic acid (pIC), on the hematopoietic system without genetic mutations. We show that acute rounds of pIC alone drive a benign age-related myeloid cell expansion and increased TGFb signaling alone causes a modest anemia in old mice. In sharp contrast, increased TGFb signaling plus acute pIC challenge result in chronic pancytopenia, expanded hematopoietic stem and progenitor cell pools, and increased bone marrow dysplasia 3-4 months after stress, which are phenotypes similar to human bone marrow failure syndromes. Mechanistically, this disease phenotype is uniquely associated with increased mitochondrial content, increased reactive oxygen species and enhanced caspase-1 activity. Our results suggest that chronic increased TGFb signaling modifies the memory of an acute immune response to drive bone marrow failure without the need for a preexisting genetic insult. Hence, non-genetic factors in combination are sufficient to drive bone marrow failure.

The deubiquitinase USP15 modulates cellular redox and is a therapeutic target in acute myeloid leukemia.

Ubiquitin-specific peptidase 15 (USP15) is a deubiquitinating enzyme implicated in critical cellular and oncogenic processes. We report that USP15 mRNA and protein are overexpressed in human acute myeloid leukemia (AML) as compared to normal hematopoietic progenitor cells. This high expression of USP15 in AML correlates with KEAP1 protein and suppression of NRF2. Knockdown or deletion of USP15 in human and mouse AML models significantly impairs leukemic progenitor function and viability and de-represses an antioxidant response through the KEAP1-NRF2 axis. Inhibition of USP15 and subsequent activation of NRF2 leads to redox perturbations in AML cells, coincident with impaired leukemic cell function. In contrast, USP15 is dispensable for human and mouse normal hematopoietic cells in vitro and in vivo. A preclinical small-molecule inhibitor of USP15 induced the KEAP1-NRF2 axis and impaired AML cell function, suggesting that targeting USP15 catalytic function can suppress AML. Based on these findings, we report that USP15 drives AML cell function, in part, by suppressing a critical oxidative stress sensor mechanism and permitting an aberrant redox state. Furthermore, we postulate that inhibition of USP15 activity with small molecule inhibitors will selectively impair leukemic progenitor cells by re-engaging homeostatic redox responses while sparing normal hematopoiesis.

Decline in IGF1 in the bone marrow microenvironment initiates hematopoietic stem cell aging.

Decline in hematopoietic stem cell (HSC) function with age underlies limited health span of our blood and immune systems. In order to preserve health into older age, it is necessary to understand the nature and timing of initiating events that cause HSC aging. By performing a cross-sectional study in mice, we discover that hallmarks of aging in HSCs and hematopoiesis begin to accumulate by middle age and that the bone marrow (BM) microenvironment at middle age induces and is indispensable for hematopoietic aging. Using unbiased approaches, we find that decreased levels of the longevity-associated molecule IGF1 in the local middle-aged BM microenvironment are a factor causing HSC aging. Direct stimulation of middle-aged HSCs with IGF1 rescues molecular and functional hallmarks of aging, including restored mitochondrial activity. Thus, although decline in IGF1 supports longevity, our work indicates that this also compromises HSC function and limits hematopoietic health span.

The Small GTPase Cdc42 Negatively Regulates the Formation of Neutrophil Extracellular Traps by Engaging Mitochondria.

Neutrophil granulocytes represent the first line of defense against invading pathogens. In addition to the production of Reactive Oxygen Species, degranulation, and phagocytosis, these specialized cells are able to extrude Neutrophil Extracellular Traps. Extensive work was done to elucidate the mechanism of this special form of cell death. However, the exact mechanisms are still not fully uncovered. Here we demonstrate that the small GTPase Cdc42 is a negative regulator of NET formation in primary human and murine neutrophils. We present a functional role for Cdc42 activity in NET formation that differs from the already described NETosis pathways. We show that Cdc42 deficiency induces NETs independent of the NADPH-oxidase but dependent on protein kinase C. Furthermore, we demonstrate that Cdc42 deficiency induces NETosis through activation of SK-channels and that mitochondria play a crucial role in this process. Our data therefore suggests a mechanistic role for Cdc42 activity in primary human neutrophils, and identify Cdc42 activity as a target to modulate the formation of Neutrophil Extracellular Traps.

Hematopoietic stem cell (HSC) divisional memory: The journey of mitochondrial metabolism through HSC division.

Hematopoietic stem cells (HSCs) are characterized by their ability to produce all cells of the blood and immune system and have been used for transplantation for decades. Although the regenerative potential of HSCs is high, their self-renewal potential is limited. HSC functions are inversely correlated with their divisional history. Recent advances in our understanding of the regulation of HSCs through cell division suggest that HSCs may never replicate into identical self, but rather replicate into progeny that gradually lose functionality at each round of division. HSC division is accompanied by major transcriptional and metabolic changes. In this perspective, the possibility that mitochondrial metabolism confers HSC division memory and programs HSCs for extinction is discussed.

The Small GTPase Cdc42 Is a Major Regulator of Neutrophil Effector Functions.

Neutrophil granulocytes are key components of the innate immune system. As the first responders to inflammatory cues, they rapidly migrate toward the site of infection or inflammation and fulfill diverse effector functions. Since these effector functions can be both beneficial and harmful to the host and surrounding tissue, they require a strict control. The small GTPase Cdc42 is known to regulate neutrophil locomotion by controlling cytoskeleton rearrangement in murine neutrophils. However, the role of Cdc42 in other neutrophil functions in human neutrophils is still poorly understood. Here we demonstrate that in primary human neutrophils, Cdc42 controls directed and random migration, activation, and degranulation as well as the formation of reactive oxygen species, in a stimulus dependent manner. In addition, we show that Cdc42 regulates pathogen killing efficiency, both in murine and human neutrophils. Cdc42 regulation of neutrophil functions is linked to differential regulation of Akt, p38, and p42/44. Our data, therefore, suggests a mechanistic role for Cdc42 activity in primary human neutrophil biology, and identify Cdc42 activity as a target to modulate neutrophil effector mechanisms and killing efficacy.

Yap1-Scribble polarization is required for hematopoietic stem cell division and fate.

Yap1 and its paralogue Taz largely control epithelial tissue growth. We have identified that hematopoietic stem cell (HSC) fitness response to stress depends on Yap1 and Taz. Deletion of Yap1 and Taz induces a loss of HSC quiescence, symmetric self-renewal ability, and renders HSC more vulnerable to serial myeloablative 5-fluorouracil treatment. This effect depends on the predominant cytosolic polarization of Yap1 through a PDZ domain-mediated interaction with the scaffold Scribble. Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity and HSC fate determination in vivo. Deletion of Scribble disrupts Yap1 copolarization with Cdc42 and decreases Cdc42 activity, resulting in increased self-renewing HSC with competitive reconstitution advantages. These data suggest that Scribble/Yap1 copolarization is indispensable for Cdc42-dependent activity on HSC asymmetric division and fate. The combined loss of Scribble, Yap1, and Taz results in transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, Rac activation, and HSC fitness restoration. Scribble links Cdc42 and the cytosolic functions of the Hippo signaling cascade in HSC fate determination.

Asymmetrically Segregated Mitochondria Provide Cellular Memory of Hematopoietic Stem Cell Replicative History and Drive HSC Attrition.

The metabolic requirements of hematopoietic stem cells (HSCs) change with their cell cycle activity. However, the underlying role of mitochondria remains ill-defined. Here we found that, after mitochondrial activation with replication, HSCs irreversibly remodel the mitochondrial network and that this network is not repaired after HSC re-entry into quiescence, contrary to hematopoietic progenitors. HSCs keep and accumulate dysfunctional mitochondria through asymmetric segregation during active division. Mechanistically, mitochondria aggregate and depolarize after stress because of loss of activity of the mitochondrial fission regulator Drp1 onto mitochondria. Genetic and pharmacological studies indicate that inactivation of Drp1 causes loss of HSC regenerative potential while maintaining HSC quiescence. Molecularly, HSCs carrying dysfunctional mitochondria can re-enter quiescence but fail to synchronize the transcriptional control of core cell cycle and metabolic components in subsequent division. Thus, loss of fidelity of mitochondrial morphology and segregation is one type of HSC divisional memory and drives HSC attrition.

Single-Cell Assays Using Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem cells (HSCs) undergo division, making two daughter cells with unique fate decision choices, that is, whether to self-renew to maintain stemness or differentiate to committed progenitors. Since HSCs are heterogeneous in nature understanding this phenomenon at the single cell level is important. In vitro single-cell assays like the paired-daughter cell and myeloid multilineage differentiation are useful to understand this unique stem cell process. Both assays are performed using cytokine combination which allows four-lineage myeloid differentiation-neutrophil, erythroid, macrophage/monocyte, and megakaryocyte. Paired-daughter cell assay examines symmetric or asymmetric retention of four myeloid lineages after first cell division in the paired-daughter cells. Thus, it defines asymmetric versus symmetric division patterns in the paired daughter cells. Thus, this assay may provide HSC fate decision cues. Myeloid multilineage differentiation assay examines the ability of a single cell to form multipotent clones containing four or less myeloid lineages. Here, we discuss in detail methodology of these assays.

Neutrophil transendothelial migration: updates and new perspectives.

Neutrophils represent the first line of cellular defense against invading microorganism by rapidly moving across the blood-endothelial cell (EC) barrier and exerting effector cell functions. The neutrophil recruitment cascade to inflamed tissues involves elements of neutrophil rolling, firm adhesion, and crawling onto the EC surface before extravasating by breaching the EC barrier. The interaction between neutrophils and ECs occurs via various adhesive modules and is a critical event determining the mode of neutrophil transmigration, either at the EC junction (paracellular) or directly through the EC body (transcellular). Once thought to be a homogenous entity, new evidence clearly points to the plasticity of neutrophil functions. This review will focus on recent advances in our understanding of the mechanism of the neutrophil transmigration process. It will discuss how neutrophil-EC interactions and the subsequent mode of diapedesis, junctional or nonjunctional, can be context dependent and how this plasticity may be exploited clinically.

Mitochondria in the maintenance of hematopoietic stem cells: new perspectives and opportunities.

The hematopoietic system produces new blood cells throughout life. Mature blood cells all derived from a pool of rare long-lived hematopoietic stem cells (HSCs) that are mostly quiescent but occasionally divide and self-renew to maintain the stem cell pool and to insure the continuous replenishment of blood cells. Mitochondria have recently emerged as critical not only for HSC differentiation and commitment but also for HSC homeostasis. Mitochondria are dynamic organelles that orchestrate a number of fundamental metabolic and signaling processes, producing most of the cellular energy via oxidative phosphorylation. HSCs have a relatively high amount of mitochondria that are mostly inactive. Here, we review recent advances in our understanding of the role of mitochondria in HSC homeostasis and discuss, among other topics, how mitochondrial dynamism and quality control might be implicated in HSC fate, self-renewal, and regenerative potential.