Efferocytosis by bone marrow mesenchymal stromal cells disrupts osteoblastic differentiation via mitochondrial remodeling.

The efficient clearance of dead and dying cells, efferocytosis, is critical to maintain tissue homeostasis. In the bone marrow microenvironment (BMME), this role is primarily fulfilled by professional bone marrow macrophages, but recent work has shown that mesenchymal stromal cells (MSCs) act as a non-professional phagocyte within the BMME. However, little is known about the mechanism and impact of efferocytosis on MSCs and on their function. To investigate, we performed flow cytometric analysis of neutrophil uptake by ST2 cells, a murine bone marrow-derived stromal cell line, and in murine primary bone marrow-derived stromal cells. Transcriptional analysis showed that MSCs possess the necessary receptors and internal processing machinery to conduct efferocytosis, with Axl and Tyro3 serving as the main receptors, while MerTK was not expressed. Moreover, the expression of these receptors was modulated by efferocytic behavior, regardless of apoptotic target. MSCs derived from human bone marrow also demonstrated efferocytic behavior, showing that MSC efferocytosis is conserved. In all MSCs, efferocytosis impaired osteoblastic differentiation. Transcriptional analysis and functional assays identified downregulation in MSC mitochondrial function upon efferocytosis. Experimentally, efferocytosis induced mitochondrial fission in MSCs. Pharmacologic inhibition of mitochondrial fission in MSCs not only decreased efferocytic activity but also rescued osteoblastic differentiation, demonstrating that efferocytosis-mediated mitochondrial remodeling plays a critical role in regulating MSC differentiation. This work describes a novel function of MSCs as non-professional phagocytes within the BMME and demonstrates that efferocytosis by MSCs plays a key role in directing mitochondrial remodeling and MSC differentiation. Efferocytosis by MSCs may therefore be a novel mechanism of dysfunction and senescence. Since our data in human MSCs show that MSC efferocytosis is conserved, the consequences of MSC efferocytosis may impact the behavior of these cells in the human skeleton, including bone marrow remodeling and bone loss in the setting of aging, cancer and other diseases.

What is the role of the microenvironment in MDS?

Treating myelodysplastic syndromes (MDS) remains challenging. Hematopoiesis occurs within a heterogeneous, complex and dynamic microenvironment, and a multiplicity of mutations in hematopoietic stem and progenitor cells (HSPCs) lead to MDS. But is there a role for the microenvironment? Here we review experimental and conceptual arguments that support a role for the microenvironment, provide evidence for the disruption of the microenvironment in MDS, and explore microenvironmental signals that may provide a targetable and conserved vulnerability in MDS that transcend genetic heterogeneity.

Aged marrow macrophages expand platelet-biased hematopoietic stem cells via Interleukin1B.

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mφs) directs HSC platelet-bias. Mφs from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals. Aged marrow Mφs also displayed decreased phagocytic function. Senescent neutrophils, typically cleared by marrow Mφs, were markedly increased in aged mice, consistent with functional defects in Mφ phagocytosis and efferocytosis. In aged mice, Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity, which can process pro-IL1B, was increased in marrow Mφs and neutrophils. Mechanistically, IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice, depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mφs induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mφs and IL1B in the age-associated lineage-skewing of HSCs, and reveals the therapeutic potential of their manipulation as antigeronic targets.

EVI1 overexpression reprograms hematopoiesis via upregulation of Spi1 transcription.

Inv(3q26) and t(3:3)(q21;q26) are specific to poor-prognosis myeloid malignancies, and result in marked overexpression of EVI1, a zinc-finger transcription factor and myeloid-specific oncoprotein. Despite extensive study, the mechanism by which EVI1 contributes to myeloid malignancy remains unclear. Here we describe a new mouse model that mimics the transcriptional effects of 3q26 rearrangement. We show that EVI1 overexpression causes global distortion of hematopoiesis, with suppression of erythropoiesis and lymphopoiesis, and marked premalignant expansion of myelopoiesis that eventually results in leukemic transformation. We show that myeloid skewing is dependent on DNA binding by EVI1, which upregulates Spi1, encoding master myeloid regulator PU.1. We show that EVI1 binds to the -14 kb upstream regulatory element (-14kbURE) at Spi1; knockdown of Spi1 dampens the myeloid skewing. Furthermore, deletion of the -14kbURE at Spi1 abrogates the effects of EVI1 on hematopoietic stem cells. These findings support a novel mechanism of leukemogenesis through EVI1 overexpression.

The microenvironment in myelodysplastic syndromes: Niche-mediated disease initiation and progression.

Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoietic stem and progenitor cells and represent the most common cause of acquired marrow failure. Hallmarked by ineffective hematopoiesis, dysplastic marrow, and risk of transformation to acute leukemia, MDS remains a poorly treated disease. Although identification of hematopoietic aberrations in human MDS has contributed significantly to our understanding of MDS pathogenesis, evidence now identify the bone marrow microenvironment (BMME) as another key contributor to disease initiation and progression. With improved understanding of the BMME, we are beginning to refine the role of the hematopoietic niche in MDS. Despite genetic diversity in MDS, interaction between MDS and the BMME appears to be a common disease feature and therefore represents an appealing therapeutic target. Further understanding of the interdependent relationship between MDS and its niche is needed to delineate the mechanisms underlying hematopoietic failure and how the microenvironment can be targeted clinically. This review provides an overview of data from human MDS and murine models supporting a role for BMME dysfunction at several steps of disease pathogenesis. Although no models or human studies so far have combined all of these findings, we review current data identifying BMME involvement in each step of MDS pathogenesis organized to reflect the chronology of BMME contribution as the normal hematopoietic system becomes myelodysplastic and MDS progresses to marrow failure and transformation. Although microenvironmental heterogeneity and dysfunction certainly add complexity to this syndrome, data are already demonstrating that targeting microenvironmental signals may represent novel therapeutic strategies for MDS treatment.

Targeting of the bone marrow microenvironment improves outcome in a murine model of myelodysplastic syndrome.

In vitro evidence suggests that the bone marrow microenvironment (BMME) is altered in myelodysplastic syndromes (MDSs). Here, we study the BMME in MDS in vivo using a transgenic murine model of MDS with hematopoietic expression of the translocation product NUP98-HOXD13 (NHD13). This model exhibits a prolonged period of cytopenias prior to transformation to leukemia and is therefore ideal to interrogate the role of the BMME in MDS. In this model, hematopoietic stem and progenitor cells (HSPCs) were decreased in NHD13 mice by flow cytometric analysis. The reduction in the total phenotypic HSPC pool in NHD13 mice was confirmed functionally with transplantation assays. Marrow microenvironmental cellular components of the NHD13 BMME were found to be abnormal, including increases in endothelial cells and in dysfunctional mesenchymal and osteoblastic populations, whereas megakaryocytes were decreased. Both CC chemokine ligand 3 and vascular endothelial growth factor, previously shown to be increased in human MDS, were increased in NHD13 mice. To assess whether the BMME contributes to disease progression in NHD13 mice, we performed transplantation of NHD13 marrow into NHD13 mice or their wild-type (WT) littermates. WT recipients as compared with NHD13 recipients of NHD13 marrow had a lower rate of the combined outcome of progression to leukemia and death. Moreover, hematopoietic function was superior in a WT BMME as compared with an NHD13 BMME. Our data therefore demonstrate a contributory role of the BMME to disease progression in MDS and support a therapeutic strategy whereby manipulation of the MDS microenvironment may improve hematopoietic function and overall survival.

Rapamycin nanoparticles target defective autophagy in muscular dystrophy to enhance both strength and cardiac function.

Duchenne muscular dystrophy in boys progresses rapidly to severe impairment of muscle function and death in the second or third decade of life. Current supportive therapy with corticosteroids results in a modest increase in strength as a consequence of a general reduction in inflammation, albeit with potential untoward long-term side effects and ultimate failure of the agent to maintain strength. Here, we demonstrate that alternative approaches that rescue defective autophagy in mdx mice, a model of Duchenne muscular dystrophy, with the use of rapamycin-loaded nanoparticles induce a reproducible increase in both skeletal muscle strength and cardiac contractile performance that is not achievable with conventional oral rapamycin, even in pharmacological doses. This increase in physical performance occurs in both young and adult mice, and, surprisingly, even in aged wild-type mice, which sets the stage for consideration of systemic therapies to facilitate improved cell function by autophagic disposal of toxic byproducts of cell death and regeneration.