CXCL12-abundant Reticular Research Articles

8709 FABP4+ Bone Marrow Endothelial Cells Support Hematopoietic Stem Cell Niches And Osteogenesis In Response To Vascular Insufficiency

Abstract Disclosure: Y. Kim: None. M. Song: None. H. Sun: None. S. Cho: None. Background: Numerous non-communicable and aging-related diseases are linked to systemic vascular insufficiency. The consequences of vascular insufficiency in bone marrow extend to compromising osteogenesis and hematopoiesis, with a more pronounced impact on aging individuals compared to younger counterparts affected by these conditions. Recent advances in scRNA-seq have shed light on the cellular taxonomy in bone marrow; however, a comprehensive understanding of cellular complexity requires further elucidation, and there are limitations in delineating impaired osteogenesis and hematopoiesis resulting from vascular insufficiency. In this study, we employed a combined approach of scRNA-seq and intensive ex vivo investigations to elucidate the regeneration process of bone marrow microenvironment following vascular insufficiency. Method Female C57BL/6 mice at different ages (6-wkks and 14 weeks) were subjected to sham (sham group) or right femoral artery ligation surgery (ischemia group). Reperfusion duration was quantified by color laser doppler. Osteogenesis was assessed by H&E staining, DXA and microCT scan. Flow cytometry was employed to count the number of type H endothelial cell (THEC, Endomucinhi). Bone marrow regeneration capacity was examined in vitro by treating with conditioned medium (CM) of THEC and in vivo via THEC injection. scRNA-seq was performed on the bone marrow of both sham and ischemia group 7 days post-ischemia. Result In young mice, reperfusion occurred within 7 days and osteogenesis was not different between sham and ischemia group. However, in old mice, reperfusion extended to 6 weeks, and accompanied by a reduction in osteogenesis, which did not recover until 12 weeks post-ischemia compared to sham group. The percentage of THEC increased after ischemia in young mice from 1.01% to 3.12 % (n=10, p<0.05), while those were comparable between sham and ischemia group in old mice (from 0.66% to 1.02%, n = 10, p=0.56). in MC3T3-E1 preosteoblast cell line, treatment with THEC-CM upregulated gene expressions associated with bone formation such as Alpl, Bglap, and Col1a1. In vivo THEC injection in old mice following ischemia surgery resulted in a reduction in reperfusion duration and an improvement in osteogenesis. scRNA-seq of bone marrow identified a significant increase of bone marrow stromal cells (BMSCs) and Cxcl12-abundant reticular (CAR) cells 7-days post-ischemia, which provides favorable microenvironment for dormant hematopoietic stem cells (HSCs). The number of FABP4+ THEC synchronized with the increase in BMSCs and CARs. In addition, FABP4+ THEC expressed Col1a1 and Col1a2, associated with osteogenesis, along with Cxcl12 and Foxc1, linked to HSC niches. Conclusion: FABP4+ THECs contribute to favorable microenvironment for HSCs and play a pivotal role in angiogenesis-osteogenesis coupling following vascular insufficiency. Presentation: 6/1/2024

Dietary restriction plus exercise change gene expression of Cxcl12 abundant reticular cells in female mice.

Low energy availability due to excessive exercise lowers bone mass and impairs various physiological functions, including immunity and hematopoiesis. We focused on Cxcl12 abundant reticular (CAR) cells, which are bone marrow mesenchymal stem cells and are essential for the maintenance of hematopoietic and immune cells in bone marrow. We examine the functional changes in CAR cells resulting from dietary restriction combined with exercise. Five-week-old wild-type female mice were divided into an ad libitum group (CON), a 60% dietary restriction group (DR), an ad libitum with exercise group (CON + ex), and a 60% dietary restriction with exercise group (DR + ex). Blood parameters, bone structure parameters, and bone marrow fat volume were evaluated after 5weeks. In addition, bone marrow CAR cells were isolated by cell sorting and analyzed for gene expression by RT-qPCR. Bone mineral density (BMD) was significantly decreased in DR and DR + ex compared to CON and CON + ex. Especially, cortical bone mass and thickness were significantly decreased in DR and DR + ex groups, whereas trabecular bone mass was significantly increased. Bone marrow fat volume was significantly increased in DR and DR + ex groups compared to CON and CON + ex. The number of leukocytes in the blood was significantly decreased in the DR + ex group compared to the other three groups. RT-qPCR showed a significant decrease in gene expression of both Foxc1 and Runx2 in CAR cells of the DR + ex group compared to CON. Dietary restriction combined with exercise promotes CAR cell differentiation into bone marrow adipocyte and suppresses osteoblast differentiation.

Leptin receptor (+) stromal cells respond to periodontitis and attenuate alveolar bone repair via CCRL2-mediated Wnt inhibition.

The impaired bone healing in tooth extraction sockets due to periodontitis presents a major obstacle to restoring oral health. The mechanisms regulating the osteogenic capacity of jawbone-derived stromal cells in the periodontitis microenvironment remain elusive. Leptin receptor (LepR) expressing stromal cells, which largely overlap with Cxcl12-abundant reticular (CAR) cells in bone tissue, rapidly proliferate and differentiate into bone-forming cells during extraction socket healing to support alveolar bone repair. In this study, we identify that CCRL2 is significantly expressed and inhibits osteogenesis in LepR+/CAR cells of alveolar bones with periodontitis. The Ccrl2-KO mice exhibit significant improvements in bone healing in extraction sockets with periodontitis. Specifically, the binding of CCRL2 to SFRP1 on the surface of LepR+/CAR cells can amplify the suppressive effect of SFRP1 on Wnt signaling under inflammation, thus hindering the osteogenic differentiation of LepR+/CAR cells and resulting in poor bone healing in extraction sockets with periodontitis. Together, we clarify that the CCRL2 receptor of LepR+/CAR cells can respond to periodontitis and crosstalk with Wnt signaling to deteriorate extraction socket healing.

Acute malaria suppresses the B lymphocytic niche in the bone marrow through the alteration of CXCL12-abundant reticular cells.

Bone marrow is a dynamic organ composed of stem cells that constantly receive signals from stromal cells and other hematopoietic cells in the niches of the bone marrow to maintain hematopoiesis and generate immune cells. Perturbation of the bone marrow microenvironment by infection and inflammation affects hematopoiesis and may affect immune cell development. Little is known about the effect of malaria on the bone marrow stromal cells that govern the hematopoietic stem cell (HSC) niche. In this study, we demonstrate that the mesenchymal stromal CXCL12-abundant reticular (CAR) cell population is reduced during acute malaria infection. The reduction of CXCL12 and IL-7 signals in the bone marrow impairs the lymphopoietic niche, leading to the depletion of common lymphoid progenitors, B cell progenitors and mature B cells, including plasma cells in the bone marrow. We found that IFNγ is responsible for the upregulation of Sca1 on CAR cells, yet the decline in CAR cell and B cell populations in the bone marrow is IFNγ-independent. In contrast to the decline in B cell populations, HSCs and multipotent progenitors increased with expansion of myelopoiesis and erythropoiesis, indicating a bias in the differentiation of multipotent progenitors during malaria infection. These findings suggest that malaria may affect host immunity by modulating the bone marrow niche.

CD_99 G1 neutrophils modulate osteogenic differentiation of mesenchymal stem cells in the pathological process of ankylosing spondylitis

ObjectivesThis study aimed to identify the types and heterogeneity of cells within the spinal enthesis and investigate the underlying mechanisms of osteogenesis.MethodsSingle-cell RNA sequencing was used to identify cell populations...

Microenvironmental CXCL12 deletion enhances Flt3-ITD acute myeloid leukemia stem cell response to therapy by reducing p38 MAPK signaling.

Fms-like tyrosine kinase 3 (Flt3) tyrosine kinase inhibitors (Flt3-TKI) have improved outcomes for patients with Flt3-mutated acute myeloid leukemia (AML) but are limited by resistance and relapse, indicating persistence of leukemia stem cells (LSC). Here utilizing a Flt3-internal tandem duplication (Flt3-ITD) and Tet2-deleted AML genetic mouse model we determined that FLT3-ITD AML LSC were enriched within the primitive ST-HSC population. FLT3-ITD LSC showed increased expression of the CXCL12 receptor CXCR4. CXCL12-abundant reticular (CAR) cells were increased in Flt3-ITD AML marrow. CXCL12 deletion from the microenvironment enhanced targeting of AML cells by Flt3-TKI plus chemotherapy treatment, including enhanced LSC targeting. Both treatment and CXCL12 deletion partially reduced p38 mitogen-activated protein kinase (p38) signaling in AML cells and further reduction was seen after treatment in CXCL12 deleted mice. p38 inhibition reduced CXCL12-dependent and -independent maintenance of both murine and human Flt3-ITD AML LSC by MSC and enhanced their sensitivity to treatment. p38 inhibition in combination with chemotherapy plus TKI treatment leads to greater depletion of Flt3-ITD AML LSC compared with CXCL12 deletion. Our studies support roles for CXCL12 and p38 signaling in microenvironmental protection of AML LSC and provide a rationale for inhibiting p38 signaling to enhance Flt3-ITD AML targeting.

Metaphyseal Stromal Progenitor Cells Are Skewed Via Wnt-Activation Towards Osteogenesis in Progressed Myelofibrosis

Bone remodeling occurs actively alongside bone marrow (BM) fibrosis in the context of myeloproliferative neoplasms (MPN). Osteosclerosis in MPN is characterized by para-trabecular apposition of new bone and increase in bone density. Yet, the pathogenesis of osteosclerosis is largely unknown and it remains an unanswered question how the 1) peri-vascular, and 2) endosteal stromal niche contribute to the formation of new bone. Here, we employed genetic fate tracing and high-resolution imaging combined with single-cell RNA sequencing (scRNAseq) to map out the BM stromal niche in steady-state, after transplant of control or thrombopoietin (ThPO)-overexpressing hematopoietic stem and progenitor cells (HSPCs) which robustly and with defined kinetics results in BM fibrosis/osteosclerosis. High-resolution confocal imaging revealed that PdgfrbCreERt2-derived tdTomato+ cells are abundant throughout the whole marrow, growth plate and bone. Gli1CreERt2;tdTomato+ have a distinct localization specifically at the growth plate, in the trabecular region of the metaphysis and around the central artery in the diaphysis. Grem1CreERt2;tdTomato+ cells are the least abundant, specifically located at the growth plate. Integrated data sets provided a high resolution of the BM stroma representing Cxcl12-abundant reticular (CAR) cells, fibroblasts (FBs), osteo-lineage cells (OLCs), osteoblasts (OBs), (pre)chondrocytes, Schwann cells and endothelial subpopulations. We next asked how these different subsets contribute to BM fibrosis and osteosclerosis. CAR cells were functionally reprogrammed in fibrosis: they lost their hematopoiesis-supporting capacity, gained a pro-fibrotic phenotype, produced extra-cellular matrix (ECM) and were less frequent compared to control conditions. BM-resident FBs rather gained a pro-inflammatory phenotype, comparable to inflammatory fibroblasts described in solid organ fibrosis, and were enriched in fibrosis but did not show a "pro-fibrotic" expression profile. Strikingly, the major expansion of tdTomato+ stromal cells occurred at the metaphysis, overlapping with increased reticulin deposition in this region. Deconvolution revealed that mainly OLCs, pre-chondrocytes and chondrocytes but also FBs reside in the metaphysis. Cell trajectory analysis suggested that a subset of OLCs acts as a mesenchymal precursor reservoir for OBs, chondrocytes and FBs. In line with our hypothesis, only metaphyseal, but not diaphyseal, PdgfrbCreERt2;tdTomato+ cells from steady-state mice harbored CFU-F capacity and gave rise to colonies in vitro. Receptor-ligand interaction demonstrated that OLCs function as important information-hubs to CAR cells after BM transplantation compared to steady-state bone. This pro-regenerative phase is characterized by upregulation of adipogenesis-related genes, potentially prompting the BM niche to recover hematopoiesis. In response to a fibrotic cue, OLCs expand, lose cellular crosstalk reflected by decreased receptor-ligand pairs, downregulate adipogenic signatures but upregulate osteogenic signatures, thus skewing the BM stroma towards osteogenesis. Time-course imaging of the stepwise invasion of Gli1CreERt2;tdTomato+ cells into the BM from the growth plate additionally highlighted the active bone remodeling occurring at the chondrocyte-OLC border during fibrosis. µCT imaging revealed increased ossification specifically at the metaphyseal region. Pathways analysis demonstrated that metaphyseal cells remain active even in progressed fibrosis and show enriched Wnt, PI3K and ECM receptor signaling. Interestingly, Wnt pathway inhibitors were downregulated in pro-fibrotic-CAR cells, whereas Wnt signaling was increased in OLCs as mesenchymal progenitor cells upon fibrotic transformation. In summary, we provide evidence that active bone remodeling is co-occurring with the fibrotic transformation with a skewing of stromal-cell fate towards osteogenesis rather than adipogenesis. Our analysis highlights the functional differences of metaphyseal and diaphyseal macro-niches within bone and postulates that a metaphyseal stromal progenitor is activated in an injury-specific manner, being an attractive cellular target.

P1399: EXTRAMEDULLARY HEMATOPOIESIS IN THE ADULT ADRENAL GLAND AS A MODEL OF THE MINIMAL HEMATOPOIETIC NICHE

Background: Hematopoietic Stem and Progenitor Cells (HSPCs) reside in the hematopoietic niche, a structure that regulates the balance of cellular quiescence, self-renewal and commitment towards progenitor cells in a demand-adapted manner. The bone marrow (BM) hematopoietic niche is formed by several cellular players, mainly endothelial cells, osteoblasts, adipocytes, and a variety of stromal cells. While the BM niche contains a complex stromal population, evidence exists for simpler, albeit functional, extramedullary hematopoietic (EMH) niches. EMH can be found associated to adipose tissue. An example of adipose-associated EMH is myelolipoma, an adrenal tumor composed of myeloid and adipose tissue. Myelolipoma is frequent in patients with congenital adrenal hyperplasia, an endocrine disorder that associates an increase in ACTH. Aims: We aimed to produce an EMH animal model that would recapitulate myelolipoma to study a minimalistic, boneless hematopoietic niche. Methods: We designed a strategy to induce EMH in the adrenal gland. For this, we injected hematopoietic cytokines (Granulocyte colony-stimulating factor (G-CSF)) and pituitary axis hormones (ACTH) as well as androgens (testosterone) in splenectomized C57BL/6J mice and studied the hematopoietic-supportive capacity of the induced adrenal glands. Results: Here, we show that the adult adrenal gland can be transformed into a hematopoietic supportive environment. Upon hormonal stimulation, the adult adrenal gland can be induced to recruit and host hematopoietic cells, including serially transplantable HSCPs. The adrenal stroma supports HSPC proliferation and function; furthermore, the adrenal stroma contains a CXCL12+ population, reminiscent of BM CXCL12-Abundant Reticular (CAR). These findings are mirrored by the presence of CXCL12+ cells in human myelolipoma samples, a benign adrenal tumor composed of adipose and hematopoietic tissue. Summary/Conclusion: We present our model as a novel tool to increase our understanding of the physiology of hematopoietic support and to facilitate the development of a boneless niche model.

ERα Signaling in a Subset of CXCL12-Abundant Reticular Cells Regulates Trabecular Bone in Mice.

ABSTRACTEstrogen has pronounced effects on the immune system, which also influences bone homeostasis. In recent years, stromal cells in lymphoid organs have gained increasing attention as they not only support the regulation of immune responses but also affect bone remodeling. A conditional knockout mouse model where estrogen receptor alpha (ERα) is deleted in CCL19‐expressing stromal cells (Ccl19‐Cre ERα fl/fl mice) was generated and bone densitometry was performed to analyze the importance of stromal cell–specific ERα signaling on the skeleton. Results showed that female Ccl19‐Cre ERα fl/fl mice display reduced total bone mineral density and detailed X‐ray analyses revealed that ERα expression in CCL19‐expressing stromal cells is important for trabecular but not cortical bone homeostasis. Further analysis showed that the trabecular bone loss is caused by increased osteoclastogenesis. Additionally, the bone formation rate was reduced; however, the expression of osteoprogenitor genes was not altered. Analysis of the bone marrow stromal cell compartment revealed a deletion of ERα in a subgroup of CXCL12‐abundant reticular (CAR) cells resulting in increased secretion of the pro‐osteoclastogenic chemokine CXCL12. In conclusion, this study reveals the importance of ERα signaling in CAR cells for bone health. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development.

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow-associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; Rosa26fs-tdTomato and Col10a1Cre; Rosa26fs-tdTomato) in combination with a PDGFRaH2B-GFP transgenic line, single-cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow-associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte-derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

A Subset of Osteosarcoma Bears Markers of CXCL12-Abundant Reticular Cells.

ABSTRACTCurrently, the cell of origin for osteosarcoma or other primary skeletal tumors is largely unknown. Recent reports identifying specific cell types comprising bone now newly enable investigation of this topic. Specifically, CXC motif chemokine 12 (CXCL12)‐abundant reticular (CAR) cells are a specific skeletal stromal cell type that orchestrate the bone marrow microenvironment through cross‐talk with hematopoietic and endothelial cells and a likely candidate cell of origin for at least a subset of primary skeletal tumors. Here, we analyze osteosarcomas via immunohistochemistry for known markers of CAR cells such as leptin receptor (LEPR), B‐cell factor 3 (EBF3), CXCL12, and platelet‐derived growth factor receptor alpha (PDGFRA). A large proportion of high‐grade tumors expressed LEPR, PDGFRA, and EBF3 but not CXCL12. These data raise the hypothesis that CAR cells are the cell of origin of this osteoblastic osteosarcoma subset, a finding with implications for the cellular oncogenesis of primary osteosarcoma and the development of effective targeted therapies. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Bone Marrow Stromal Antigen 2 Facilitates Activation of Hematopoietic Stem Cell through the Induction of ERK Signaling

Chronic infections are prevalent worldwide and contribute to bone marrow failure syndromes. Long-term homeostatic production of all blood cells is dependent on the ability of hematopoietic stem cells (HSCs) to remain mostly quiescent and, upon activation, must carefully balance between their ability to self-renew and differentiate. However, infections trigger inflammatory cytokines, such as interferon-gamma (IFNy), that facilitate HSC proliferative exhaustion by disrupting both quiescence and self-renewal, thereby contributing to cytopenias and bone marrow failure syndromes. Our previously published results indicate that the IFN-induced surface protein, Bone Marrow Stromal Antigen 2 (BST2), facilitates HSC activation, and the loss of BST2 protected against the depletion of HSCs during chronic infection. However, the mechanism by which BST2 impacts HSCs activation is unknown. BST2 is known to promote activation of intracellular signaling in both dendritic cells and cancer cell types. In particular BST2 is a known activator of the NFkB signaling pathway. We hypothesized that BST2 could promote activation of HSCs through the induction of intracellular signaling pathways.Intracellular signaling pathways have been shown to be important for the activation of not only HSCs but also downstream immune cells. Indeed, the loss of BST2 has been shown to impair the function of T cells, NK cells, and antigen presenting cells in the presence of viral infections via impaired induction of NFkB signaling. However, there have been no studies on the role of BST2 during bacterial infections. To evaluate the role of BST2 in hematopoiesis during a bacterial infection, we obtained BST2KO mice and characterized hematopoietic populations in the presence or absence of Mycobacterium avium infection, a model bacterial infection that generates high levels of IFNy. We observed no changes in activation or production of cytokines from macrophage, NK, CD8, or neutrophil populations. Surprisingly we observed enhanced dendritic cell activation at day 15, 30, and 42 as measured by co-stimulatory molecules and MHC II expression. Additionally, we observed increased tetramer-specific T cell numbers early on during the infection. Interestingly, these results pair with decreased bacterial load at day 30 and day 42 despite having the similar spleen sizes. These results suggest that BST2 regulates the activation of dendritic cells, which may trigger more robust earlier T cell responses early in the course of infection.Our previous work showed that the loss of BST2 protects against HSC activation by displacement of HSCs from CXCL12-abundant reticular (CAR) cells in the niche. To determine whether HSC activation is related to distancing from the vascular niche or increased proximity to an activated osteoblastic niche, we performed intravital imaging using osteocalcin-GFP reporter mice. We observed no change in proximity to the osteoblastic niche after administration of recombinant IFNy using either WT or BST2KO HSCs. These results suggest that the effects of BST2 may be more related to distancing from CAR cells than migration towards other niche locations.To evaluate the mechanisms underlying BST2-dependent HSC activation, we performed RNA sequencing on WT versus BST2KO HSCs in the presence of IFNy. GSEA-pathway analysis of differentially expressed genes revealed that BST2KO HSCs have a decreased response to IFNy and diminished activation of the P13K-AKT-MTOR signaling pathways. Western blot analysis on hematopoietic stem cell progenitor cells (HSPCs) indicate a decrease in phosphorylation of the ERK signaling pathway in BST2KO HSPCs. These results were analogous to work performed in 32D cells in vitro, in which ERK-phosphorylation has been shown to be important for both HSC maintenance and proliferation. Future studies are aimed at assessing the impact of ERK inhibition on HSC activation during various inflammatory stresses.Collectively our results show that the loss of BST2 in downstream immune cells surprisingly enhances dendritic cell and T cell activation in response to bacterial infection. BST2 enhances HSC activation through activation of downstream ERK signaling. Future targeting of BST2 and/or ERK signaling could have important consequences for patients that suffer from cytopenias, bone marrow failure, and engraftment failure due to high levels of inflammatory stimuli. DisclosuresNo relevant conflicts of interest to declare.

Patient-Derived Bone Marrow Spheroids Reveal Leukemia-Initiating Cells Supported by Mesenchymal Hypoxic Niches in Pediatric B-ALL.

B-cell acute lymphoblastic leukemia (B-ALL) results from the expansion of malignant lymphoid precursors within the bone marrow (BM), where hematopoietic niches and microenvironmental signals provide leukemia-initiating cells (LICs) the conditions to survive, proliferate, initiate disease, and relapse. Normal and malignant lymphopoiesis are highly dependent on the BM microenvironment, particularly on CXCL12-abundant Reticular (CAR) cells, which provide a niche for maintenance of primitive cells. During B-ALL, leukemic cells hijack BM niches, creating a proinflammatory milieu incompetent to support normal hematopoiesis but favoring leukemic proliferation. Although the lack of a phenotypic stem cell hierarchy is apparent in B-ALL, LICs are a rare and quiescent population potentially responsible for chemoresistance and relapse. Here, we developed novel patient-derived leukemia spheroids (PDLS), an ex vivo avatar model, from mesenchymal stromal cells (MSCs) and primary B-ALL cells, to mimic specialized niche structures and cell-to-cell intercommunication promoting normal and malignant hematopoiesis in pediatric B-ALL. 3D MSC spheroids can recapitulate CAR niche-like hypoxic structures that produce high levels of CXCL10 and CXCL11. We found that PDLS were preferentially enriched with leukemia cells displaying functional properties of LICs, such as quiescence, low reactive oxygen species, drug resistance, high engraftment in immunodeficient mice, and long-term leukemogenesis. Moreover, the combination of PDLS and patient-derived xenografts confirmed a microenvironment-driven hierarchy in their leukemic potential. Importantly, transcriptional profiles of MSC derived from primary patient samples revealed two unique signatures (1), a CXCL12low inflammatory and leukemia expansion (ILE)-like niche, that likely supports leukemic burden, and (2) a CXCL11hi immune-suppressive and leukemia-initiating cell (SLIC)-like niche, where LICs are likely sustained. Interestingly, the CXCL11+ hypoxic zones were recapitulated within the PDLS that are capable of supporting LIC functions. Taken together, we have implemented a novel PDLS system that enriches and supports leukemia cells with stem cell features driven by CXCL11+ MSCs within hypoxic microenvironments capable of recapitulating key features, such as tumor reemergence after exposure to chemotherapy and tumor initiation. This system represents a unique opportunity for designing ex vivo personalized avatars for B-ALL patients to evaluate their own LIC pathobiology and drug sensitivity in the context of the tumor microenvironment.

CXCL12-abundant reticular cells are the major source of IL-6 upon LPS stimulation and thereby regulate hematopoiesis

AbstractHematopoiesis is maintained by hematopoietic stem and progenitor cells that are located in the bone marrow (BM) where they are embedded within a complex supportive microenvironment consisting of a multitude of various non-hematopoietic and hematopoietic cell types. The BM microenvironment not only regulates steady-state hematopoiesis by provision of growth factors, cytokines, and cell–cell contact but is also an emerging key player during the adaptation to infectious and inflammatory insults (emergency hematopoiesis). Through a combination of gene expression analyses in prospectively isolated non-hematopoietic BM cell populations and various mouse models, we found that BM CXCL12-abundant reticular (CAR) cells are a major source of systemic and local BM interleukin-6 (IL-6) levels during emergency hematopoiesis after lipopolysaccharide (LPS) stimulation. Importantly, although IL-6 is dispensable during the initial phase of LPS-induced emergency hematopoiesis, it is required to sustain an adequate hematopoietic output during chronic repetitive inflammation. Our data highlight the essential role of the non-hematopoietic BM microenvironment for the sensing and integration of pathogen-derived signals into sustained demand-adapted hematopoietic responses.

IFNg Increases HSC Homing and Induces Relocalization within the Bone Marrow Niche

Infections affect hematopoietic stem cell (HSC) biology, with significant implications in ageing, cancer development, and bone marrow failure syndromes. We previously showed that some infections induce HSCs to proliferate through a cell autonomous response to interferon gamma (IFNg). Given that interactions of HSCs with their bone marrow niche are a critical factor in maintaining quiescence, we considered how IFNg affects the interaction between HSCs and the niche. We hypothesized that infection would displace HSCs from the niche and reduce homing in an IFNg-dependent manner, thereby contributing to activation.We used a Mycobacterium avium model of infection to characterize the effects of infection on HSC homing. In this model HSCs are activated to divide and differentiate via an IFNg-dependent mechanism with depletion of HSCs after several months of infection. We compared the homing of hematopoietic stem and progenitor cells (HSPCs) from M. avium infected versus non-infected animals by IV administration of CSFE-labeled cKit+ cells into lethally irradiated recipient mice. Homing was determined by the frequency with which labeled cells could be found in the bone marrow 17 hours later, relative to a DDAO-stained internal control. Surprisingly, HSPCs from M. avium -infected animals displayed improved homing compared to control HSPCs. A similar phenotype was seen for cKit+ cells isolated from mice treated with recombinant IFNg, whereas cKit+ cells from Ifngr1-/- mice showed decreased homing compared to wildtype. Using transplantation assays, we found decreased engraftment of Ifngr1-deficient whole bone marrow when cells were administered intravenously; but engraftment was normal when cells were infused intrafemorally. Altogether, these findings show that basal IFNg signaling contributes to normal HSPC homing and that IFNg induced during M. avium infection increases homing to the bone marrow.The above studies reflect homing of HSPCs to the bone marrow, not cellular interactions within the niche. Since proximity to Cxcl12-expressing cells in the perivascular niche can affect HSC activation status, we used 3-D intravital imaging to study the effects of IFNg on HSC-niche interactions. We injected CMTMR-stained HSCs into Cxcl12-GFP transgenic mice and measured the distance of the HSCs from quiescence-associated Cxcl12-abundant reticular (CAR) cells. CMTMR-stained HSCs were seen moving upon IFNg treatment, and 24hrs post-IFNg HSCs were located significantly further away from CAR cells compared to untreated or TGF-b treated controls. These results suggest that IFNg can induce movement away from CAR cells, thereby allowing HSC proliferation during inflammatory conditions.To define the mechanism by which IFNg causes HSC relocalization, we performed gene expression profiling of HSCs from control or IFNg-treated mice. Gene set enrichment analysis of differentially regulated genes showed modulation of the expected immune and cytokine response pathways, but also genes related to adhesion and extracellular matrix remodeling. Of note, the glycoprotein Bst2 (Tetherin) was upregulated in HSCs following IFNg treatment. Bst2 has been shown to bind to E-selectin, and localization to E-selectin+ endothelial cells is associated with increased HSC proliferation. Thus we hypothesized that IFNg-induced upregulation of surface Bst2 may facilitate proliferation by promoting HSC binding to E-selectin. We confirmed induction of Bst2 mRNA and protein by qPCR and flow cytometry, and we found that Bst2 was upregulated following M. avium infection via an IFNg-dependent mechanism. In vitro binding assays showed increased HSPC binding to recombinant E-selectin following IFNg treatment. Thus, IFNg-dependent cell surface expression of Bst2 may facilitate the displacement of HSCs from the quiescence-enforcing CAR niche to an active E-selectin-positive niche. Because E-selectin expression along blood vessel walls is also important for adhesion and transendothelial migration of HSCs, increased Bst2 on HSCs may also contribute to more efficient homing. In summary, IFNg improves HSPC homing but displaces HSCs from quiescence-enforcing CAR cells in the niche, potentially by inducing Bst2-mediated movement to the E-selectin rich proliferative niche. Future studies will ascertain whether blocking Bst2 is a feasible method to protect HSCs from the deleterious effects of chronic inflammation. DisclosuresNo relevant conflicts of interest to declare.

CXCL12-Abundant Reticular (CAR) Cells Direct Megakaryocyte Protrusions across the Bone Marrow Sinusoid Wall.

Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation.

Phosphate restriction impairs mTORC1 signaling leading to increased bone marrow adipose tissue and decreased bone in growing mice.

Bone marrow stromal cells (BMSCs) are multipotent cells that differentiate into cells of the osteogenic and adipogenic lineage. A striking inverse relationship between bone marrow adipose tissue (BMAT) and bone volume is seen in several conditions, suggesting that differentiation of BMSCs into bone marrow adipocytes diverts cells from the osteogenic lineage, thereby compromising the structural and mechanical properties of bone. Phosphate restriction of growing mice acutely decreases bone formation, blocks osteoblast differentiation and increases BMAT. Studies performed to evaluate the cellular and molecular basis for the effects of acute phosphate restriction demonstrate that it acutely increases 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling in osteoblasts. This is accompanied by decreased expression of Wnt10b in BMSCs. Phosphate restriction also promotes expression of the key adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer binding protein α (CEBPα), in CXCL12 abundant reticular (CAR) cells, which represent undifferentiated BMSCs and are the main source of BMAT and osteoblasts in the adult murine skeleton. Consistent with this, lineage tracing studies reveal that the BMAT observed in phosphate-restricted mice is of CAR cell origin. To determine whether circumventing the decrease in mTORC1 signaling in maturing osteoblasts attenuates the osteoblast and BMAT phenotype, phosphate-restricted mice with OSX-CreERT2 -mediated haploinsufficiency of the mTORC1 inhibitor, TSC2, were generated. TSC2 haploinsufficiency in preosteoblasts/osteoblasts normalized bone volume and osteoblast number in phosphate-restricted mice and attenuated the increase in BMAT observed. Thus, acute phosphate restriction leads to decreased bone and increases BMAT by impairing mTORC1 signaling in osterix-expressing cells. © 2021 American Society for Bone and Mineral Research (ASBMR).

Interferon Gamma Mediates Hematopoietic Stem Cell Activation and Niche Relocalization through BST2

During chronic infection, the inflammatory cytokine interferon gamma (IFNγ) damages hematopoietic stem cells (HSCs) by disrupting quiescence and promoting excessive terminal differentiation. However, the mechanism by which IFNγ hinders HSC quiescence remains undefined. Using intravital 3-dimensional microscopy, we find that IFNγ disrupts the normally close interaction between HSCs and CXCL12-abundant reticular (CAR) cells in the HSC niche. IFNγ stimulation increases expression of the cell surface protein BST2, which we find is required for IFNγ-dependent HSC relocalization and activation. IFNγ stimulation of HSCs increases their E-selectin binding by BST2 and homing to the bone marrow, which depends on E-selectin binding. Upon chronic infection, HSCs from mice lacking BST2 are more quiescent and more resistant to depletion than HSCs from wild-type mice. Overall, this study defines a critical mechanism by which IFNγ promotes niche relocalization and activation in response to inflammatory stimulation and identifies BST2 as a key regulator of HSC quiescence. VIDEO ABSTRACT.

Intercellular Interactions of an Adipogenic CXCL12-Expressing Stromal Cell Subset in Murine Bone Marrow.

Bone marrow houses a multifunctional stromal cell population expressing C-X-C motif chemokine ligand 12 (CXCL12), termed CXCL12-abundant reticular (CAR) cells, that regulates osteogenesis and adipogenesis. The quiescent pre-adipocyte-like subset of CXCL12+ stromal cells ("Adipo-CAR" cells) is localized to sinusoidal surfaces and particularly enriched for hematopoiesis-supporting cytokines. However, detailed characteristics of these CXCL12+ pre-adipocyte-like stromal cells and how they contribute to marrow adipogenesis remain largely unknown. Here we highlight CXCL12-dependent physical coupling with hematopoietic cells as a potential mechanism regulating the adipogenic potential of CXCL12+ stromal cells. Single-cell computational analyses of RNA velocity and cell signaling reveal that Adipo-CAR cells exuberantly communicate with hematopoietic cells through CXCL12-CXCR4 ligand-receptor interactions but do not interconvert with Osteo-CAR cells. Consistent with this computational prediction, a substantial fraction of Cxcl12-creER+ pre-adipocyte-like cells intertwines with hematopoietic cells in vivo and in single-cell preparation in a protease-sensitive manner. Deletion of CXCL12 in these cells using Col2a1-cre leads to a reduction of stromal-hematopoietic coupling and extensive marrow adipogenesis in adult bone marrow, which appears to involve direct conversion of CXCL12+ cells to lipid-laden marrow adipocytes without altering mesenchymal progenitor cell fates. Therefore, these findings suggest that CXCL12+ pre-adipocyte-like marrow stromal cells prevent their premature differentiation by maintaining physical coupling with hematopoietic cells in a CXCL12-dependent manner, highlighting a possible cell-non-autonomous mechanism that regulates marrow adipogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Bone Marrow Stromal Antigen 2 Is Critical for IFNy-Dependent Hematopoietic Stem Cell Activation

Bone marrow failure is a significant complication of many chronic infections, which affect a third of the world's population. The inflammatory cytokine interferon-gamma (IFNy) contributes to bone marrow failure syndromes by activating hematopoietic stem cells (HSCs) and impairing their self-renewal. IFNy upregulation during many chronic infections such as tuberculosis, HIV and hepatitis B directly depletes hematopoietic stem cells (HSCs). The mechanisms by which IFNy drives the loss of quiescence and ultimate exhaustion of HSCs remain poorly understood, but may be related to changes in the interaction between HSCs and the bone marrow (BM) microenvironment, or BM niche. Current evidence suggests that quiescent HSCs reside predominantly in the vascular niche, where the production of stem cell factor (SCF) from endothelial cells and CXCL12 from perivascular CXCL12 abundant reticular (CAR) stromal cells are critical for maintaining their quiescence. The goal of our work was to determine whether IFNy signaling alters HSC interactions within the niche. We performed transcriptomic analysis of IFNy-stimulated HSCs and focused on changes in cell-surface expressed genes that may influence HSC-niche interactions. Our analysis revealed Bone Marrow Stromal Antigen 2 (BST2) as the only surface protein upregulated on HSCs upon 24-hour IFNy stimulation. To study the effects of BST2 on HSC-niche interactions, we performed intravital imaging using two reporter mouse models: CXCL12-GFP reporter mice in which CAR cells are labeled with GFP, and CXCL12-GFP Krt18-Cre LSL-tdTomato dual reporter mice in which CAR cells are labelled with GFP and HSCs are labeled with tdTomato. After exogenously labeling and transferring HSCs into CXCL12-GFP mice or endogenously labeling HSCs in the CXCL12-GFP Krt18-Cre LSL-tdTomato mice, we observed that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. These findings are consistent with other reports that chemotherapeutic and inflammatory stress disrupts HSC interactions with the niche and promotes HSC migration. Conversely, we observed no change in HSC distancing from CAR cells after IFNy stimulation of IFNy-receptor deficient HSCs, suggesting that the observed HSC displacement was due to a cell autonomous mechanism. These changes were not due to a loss of CXCL12 receptor (CXCR4) expression or disrupted capacity of HSCs to migrate towards CXCL12. Interestingly, Intravital imaging using BST2-deficient HSCs revealed that BST2 KO HSCs do not re-localize from CAR cells during IFNy stimulation. Increased BST2 expression has been linked to the migration, adhesion and metastasis of various cancer cells and we explored whether it could serve a similar role in protein binding in HSCs. Using in vitro plate binding assays, we found that IFNy-treatment promoted increased HSC binding to E-selectin via BST2, as well as increased HSC homing to the bone marrow, a property that is dependent on E-selectin binding. Finally, to determine whether BST2 affects IFNy-dependent HSC activation we performed cell cycle analysis of WT and BST2 KO HSCs. We discovered that the loss of BST2 protects against HSC activation during Mycobacterium avium infection. Furthermore, HSC depletion during chronic infection was mitigated in BST2 KO mice. Our data identifies BST2 as a key protein that influences niche relocalization and activation in response to inflammatory stimulation. This study expands our understanding of factors that contribute to HSC activation and loss of quiescence. These findings could shed light on novel therapeutic interventions for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.