Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

Background: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating the regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is a key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has not yet been elucidated. Methods: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 hours post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. Results: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. Conclusions: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Haematopoietic stem cell numbers are not solely determined by niche availability.

Haematopoietic stem cells (HSCs) reside in specialized microenvironments, also referred to as niches, and it has been widely believed that HSC numbers are determined by the niche size alone 1-5 . However, the vast excess of the number of niche cells over that of HSCs raises questions about this model. We initially established a mathematical model of niche availability and occupancy, which predicted that HSC numbers are restricted at both systemic and local levels. To address this question experimentally, we developed a femoral bone transplantation system, enabling us to increase the number of available HSC niches. We found that the addition of niches does not alter total HSC numbers in the body, regardless of whether the endogenous (host) niche is intact or defective, suggesting that HSC numbers are limited at the systemic level. Additionally, HSC numbers in transplanted wild-type femurs did not increase beyond physiological levels when HSCs were mobilized from defective endogenous niches to the periphery, indicating that HSC numbers are also constrained at the local level. Our study demonstrates that HSC numbers are not solely determined by niche availability, thereby rewriting the long-standing model for the regulation of HSC numbers.

Periosteal skeletal stem cells can migrate into the bone marrow and support hematopoiesis after injury.

Functional stromal cells are known to support bone marrow regeneration after chemotherapy or radiation-induced injury to prevent prolonged myelosuppression. However, it is not known how stromal cells within the bone marrow are regenerated after injury. We have utilized a whole bone transplantation model that mimics the initial bone marrow necrosis and fatty infiltration that is seen after bone marrow injury and subsequent recovery. We demonstrate that periosteal skeletal stem cells (P-SSCs) can migrate into the bone marrow and contribute to stromal regeneration and hematopoietic recovery. Once in the bone marrow, P-SSCs are phenotypically and functionally reprogrammed into bone marrow mesenchymal stem cells (BM-MSCs), expressing high levels of hematopoietic stem cell (HSC) niche factors, such as Cxcl12 and Kitl. Additionally, our results further indicate that P-SSCs are more resistant to acute stress than BM-MSCs. Here, we report a new function of P-SSCs, highlighting their major plasticity and the role of the periosteum as a potential source of BM-MSCs following acute bone marrow injury.

Whole bone subcutaneous transplantation as a strategy to study precisely the bone marrow niche.

Hematopoietic stem cells are maintained in a specialized microenvironment, known as the 'niche', within the bone marrow. Understanding the contribution of cellular and molecular components within the bone marrow niche for the maintenance of hematopoietic stem cells is crucial for the success of therapeutic applications. So far, the roles of crucial mechanisms within the bone marrow niche have been explored in transgenic animals in which genetic modifications are ubiquitously introduced in the whole body. The lack of precise tools to explore genetic alterations exclusively within the bone marrow prevents our determination of whether the observed outcomes result from confounding effects from other organs. Here, we developed a new method - 'whole bone subcutaneous transplantation'- to study the bone marrow niche in transgenic animals precisely. Using immunolabeling of CD45.1 (donor) vs. CD45.2 (recipient) hematopoeitic stem cells, we demonstrated that hematopoeitic stem cells from the host animals colonize the subcutaneously transplanted femurs after transplantation, while the hematopoietic stem cells from the donor disappear. Strikinlgy, the bone marrow niche of these subcutaneously transplanted femurs remain from the donor mice, enabling us to study specifically cells of the bone marrow niche using this model. We also showed that genetic ablation of peri-arteriolar cells specifically in donor femurs reduced the numbers of hematopoietic stem cells in these bones. This supports the use of this strategy as a model, in combination with genetic tools, to evaluate how bone marrow niche specific modifications may impact non-modified hematopoietic stem cells. Thus, this approach can be utilized for genetic manipulation in vivo of specific cell types only within the bone marrow. The combination of whole bone subcutaneous transplantation with rodent transgenic models will facilitate a more precise, complex and comprehensive understanding of existing problems in the study of the hematopoietic stem cell bone marrow niche.

Dietary iron restriction protects against vaso-occlusion and organ damage in murine sickle cell disease.

Sickle cell disease (SCD) is an inherited disorder resulting from a ß-globin gene mutation, and SCD patients experience erythrocyte sickling, vaso-occlusive episodes (VOE), and progressive organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron overload, and another subpopulation of SCD patients manifest iron deficiency. To elucidate connections between dietary iron, the microbiome, and SCD pathogenesis, we treated SCD mice with an iron-restricted diet (IRD). IRD treatment reduced iron availability and hemolysis, decreased acute VOE, and ameliorated chronic organ damage in SCD mice. Our results extend previous studies indicating that the gut microbiota regulate disease in SCD mice. IRD alters microbiota load and improves gut integrity, together preventing crosstalk between the gut microbiome and inflammatory factors such as aged neutrophils, dampening VOE, and organ damage. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments, which are under development, focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related- and novel-therapeutic targets.

Tissue-resident glial cells associate with tumoral vasculature and promote cancer progression.

Cancer cells are embedded within the tissue and interact dynamically with its components during cancer progression. Understanding the contribution of cellular components within the tumor microenvironment is crucial for the success of therapeutic applications. Here, we reveal the presence of perivascular GFAP+/Plp1+ cells within the tumor microenvironment. Using in vivo inducible Cre/loxP mediated systems, we demonstrated that these cells derive from tissue-resident Schwann cells. Genetic ablation of endogenous Schwann cells slowed down tumor growth and angiogenesis. Schwann cell-specific depletion also induced a boost in the immune surveillance by increasing tumor-infiltrating anti-tumor lymphocytes, while reducing immune-suppressor cells. In humans, a retrospective in silico analysis of tumor biopsies revealed that increased expression of Schwann cell-related genes within melanoma was associated with improved survival. Collectively, our study suggests that Schwann cells regulate tumor progression, indicating that manipulation of Schwann cells may provide a valuable tool to improve cancer patients' outcomes.

Inflammatory exposure drives long-lived impairment of hematopoietic stem cell self-renewal activity and accelerated aging.

Hematopoietic stem cells (HSCs) mediate regeneration of the hematopoietic system following injury, such as following infection or inflammation. These challenges impair HSC function, but whether this functional impairment extends beyond the duration of inflammatory exposure is unknown. Unexpectedly, we observed an irreversible depletion of functional HSCs following challenge with inflammation or bacterial infection, with no evidence of any recovery up to 1 year afterward. HSCs from challenged mice demonstrated multiple cellular and molecular features of accelerated aging and developed clinically relevant blood and bone marrow phenotypes not normally observed in aged laboratory mice but commonly seen in elderly humans. In vivo HSC self-renewal divisions were absent or extremely rare during both challenge and recovery periods. The progressive, irreversible attrition of HSC function demonstrates that temporally discrete inflammatory events elicit a cumulative inhibitory effect on HSCs. This work positions early/mid-life inflammation as a mediator of lifelong defects in tissue maintenance and regeneration.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis.

Ten-eleven translocation (Tet) enzymes promote DNA demethylation by oxidizing 5-methylcytosine. They are expressed during development and are essential for mouse gastrulation. However, their postgastrulation functions are not well established. We find that global or endothelial-specific loss of all three Tet enzymes immediately after gastrulation leads to reduced number of hematopoietic stem and progenitor cells (HSPCs) and lethality in mid-gestation mouse embryos. This is due to defects in specification of HSPCs from endothelial cells (ECs) that compromise primitive and definitive hematopoiesis. Mechanistically, loss of Tet enzymes in ECs led to hypermethylation and down-regulation of NFκB1 and master hematopoietic transcription factors (Gata1/2, Runx1, and Gfi1b). Restoring Tet catalytic activity or overexpression of these factors in Tet-deficient ECs rescued hematopoiesis defects. This establishes Tet enzymes as activators of hematopoiesis programs in ECs for specification of HSPCs during embryogenesis, which is distinct from their roles in adult hematopoiesis, with implications in deriving HSPCs from pluripotent cells.

VCAM1 confers innate immune tolerance on haematopoietic and leukaemic stem cells.

Haematopoietic stem cells (HSCs) home to the bone marrow via, in part, interactions with vascular cell adhesion molecule-1 (VCAM1)1-3. Once in the bone marrow, HSCs are vetted by perivascular phagocytes to ensure their self-integrity. Here we show that VCAM1 is also expressed on healthy HSCs and upregulated on leukaemic stem cells (LSCs), where it serves as a quality-control checkpoint for entry into bone marrow by providing 'don't-eat-me' stamping in the context of major histocompatibility complex class-I (MHC-I) presentation. Although haplotype-mismatched HSCs can engraft, Vcam1 deletion, in the setting of haplotype mismatch, leads to impaired haematopoietic recovery due to HSC clearance by mononuclear phagocytes. Mechanistically, VCAM1 'don't-eat-me' activity is regulated by ß2-microglobulin MHC presentation on HSCs and paired Ig-like receptor-B (PIR-B) on phagocytes. VCAM1 is also used by cancer cells to escape immune detection as its expression is upregulated in multiple cancers, including acute myeloid leukaemia (AML), where high expression associates with poor prognosis. In AML, VCAM1 promotes disease progression, whereas VCAM1 inhibition or deletion reduces leukaemia burden and extends survival. These results suggest that VCAM1 engagement regulates a critical immune-checkpoint gate in the bone marrow, and offers an alternative strategy to eliminate cancer cells via modulation of the innate immune tolerance.

The microbiota regulates hematopoietic stem cell fate decisions by controlling iron availability in bone marrow.

Host microbiota crosstalk is essential for the production and functional modulation of blood-cell lineages. Whether, and if so how, the microbiota influences hematopoietic stem cells (HSCs) is unclear. Here, we show that the microbiota regulates HSC self-renewal and differentiation under stress conditions by modulating local iron availability in the bone marrow (BM). In microbiota-depleted mice, HSC self-renewal was enhanced during regeneration, while the commitment toward differentiation was dramatically compromised. Mechanistically, microbiota depletion selectively impaired the recycling of red blood cells (RBCs) by BM macrophages, resulting in reduced local iron levels without affecting systemic iron homeostasis. Limiting iron availability in food (in vivo) or in culture (ex vivo), or by CD169+ macrophage depletion, enhanced HSC self-renewal and expansion. These results reveal an intricate interplay between the microbiota, macrophages, and iron, and their essential roles in regulating critical HSC fate decisions under stress.

Bone marrow NG2+/Nestin+ mesenchymal stem cells drive DTC dormancy via TGFß2.

In the bone marrow (BM) microenvironment, where breast cancer (BC) disseminated tumour cells (DTCs) can remain dormant for decades, NG2+/Nestin+ mesenchymal stem cells (MSCs) promote hematopoietic stem cell quiescence. Here, we reveal that periarteriolar BM-resident NG2+/Nestin+ MSCs can also instruct BC DTCs to enter dormancy. NG2+/Nestin+ MSCs produce TGFß2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase result in p27 induction. Genetic depletion of MSCs or conditional knock-out of TGFß2 in MSCs using an NG2-CreER driver led to bone metastatic outgrowth of otherwise dormant p27+/Ki67- DTCs. Also ER+ BC patients without systemic recurrence displayed higher frequency of TGFß2 and BMP7 detection in the BM. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy and suggest that aging or extrinsic factors that affect the NG2+/Nestin+ MSC niche homeostasis may result in a break from dormancy and BC bone relapse.

Future directions in preclinical and translational cancer neuroscience research.

Recent advances in cancer neuroscience necessitate the systematic analysis of neural influences in cancer as potential therapeutic targets in oncology. Here, we outline recommendations for future preclinical and translational research in this field.

MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells.

Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid potential. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. Our results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.

Olinciguat, a stimulator of soluble guanylyl cyclase, attenuates inflammation, vaso-occlusion and nephropathy in mouse models of sickle cell disease.

BACKGROUND AND PURPOSE: Reduced bioavailability of NO, a hallmark of sickle cell disease (SCD), contributes to intravascular inflammation, vasoconstriction, vaso-occlusion and organ damage observed in SCD patients. Soluble guanylyl cyclase (sGC) catalyses synthesis of cGMP in response to NO. cGMP-amplifying agents, including NO donors and phosphodiesterase 9 inhibitors, alleviate TNFα-induced inflammation in wild-type C57BL/6 mice and in 'humanised' mouse models of SCD. EXPERIMENTAL APPROACH: Effects of the sGC stimulator olinciguat on intravascular inflammation and renal injury were studied in acute (C57BL6 and Berkeley mice) and chronic (Townes mice) mouse models of TNFα-induced and systemic inflammation associated with SCD. KEY RESULTS: Acute treatment with olinciguat attenuated increases in plasma biomarkers of endothelial cell activation and leukocyte-endothelial cell interactions in TNFα-challenged mice. Co-treatment with hydroxyurea, an FDA-approved SCD therapeutic agent, further augmented the anti-inflammatory effect of olinciguat. In the Berkeley mouse model of TNFα-induced vaso-occlusive crisis, a single dose of olinciguat attenuated leukocyte-endothelial cell interactions, improved blood flow and prolonged survival time compared to vehicle-treated mice. In Townes SCD mice, plasma biomarkers of inflammation and endothelial cell activation were lower in olinciguat- than in vehicle-treated mice. In addition, kidney mass, water consumption, 24-h urine excretion, plasma levels of cystatin C and urinary excretion of N-acetyl-ß-d-glucosaminidase and neutrophil gelatinase-associated lipocalin were lower in Townes mice treated with olinciguat than in vehicle-treated mice. CONCLUSION AND IMPLICATIONS: Our results suggest that the sGC stimulator olinciguat attenuates inflammation, vaso-occlusion and kidney injury in mouse models of SCD and systemic inflammation.

Using CT-guided stereotactic prostate radiation therapy (CT-SPRT) to assess sustained murine prostate ablation.

The prostate is a hormone-responsive organ where testicular androgens drive the proliferation and survival of prostatic cells, ensuring the development and functioning of this gland throughout life. Androgen deprivation therapy leads to apoptosis of prostatic cells and organ regression, and is a cornerstone of prostate cancer and benign prostatic hypertrophy treatment. For several decades, androgen deprivation has been used as an adjuvant to external beam radiotherapy, however, emerging data suggests that the low rates of epithelial proliferation in the castrated prostate imparts radio-resistance. As proliferating cells exhibit increased sensitivity to radiation, we hypothesized that short bursts of synchronized epithelial proliferation, which can be achieved by exogeneous testosterone supplementation prior to targeted high-dose radiation, would maximize sustained prostate ablation, while minimizing damage to surrounding tissues. To test this hypothesis, we designed a novel computed-tomography (CT)-guided stereotactic prostate radiation therapy (CT-SPRT) technique to deliver a single high-dose 25 Gy fraction of X-ray radiation. Sustained prostatic cell ablation was assessed post CT-SPRT by measuring prostate weight, epithelial cell number, and relative contributions of luminal and basal epithelial populations in control and testosterone-pretreated glands. CT-SPRT was safely delivered with no observed damage to surrounding rectal and bladder tissues. Importantly, castrated mice that received a pulse of testosterone to induce synchronous cell proliferation prior to CT-SPRT exhibited significant sustained gland ablation compared to control mice. These results provide new insights in stereotactic radiotherapy sensitivity to maximize prostatic cell ablation and improve our understanding of prostate gland regeneration that can potentially lead to improved non-invasive therapies for benign prostatic hypertrophy and prostate cancer.

Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1.

Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal.

Nociceptive nerves regulate haematopoietic stem cell mobilization.

Haematopoietic stem cells (HSCs) reside in specialized microenvironments in the bone marrow-often referred to as 'niches'-that represent complex regulatory milieux influenced by multiple cellular constituents, including nerves1,2. Although sympathetic nerves are known to regulate the HSC niche3-6, the contribution of nociceptive neurons in the bone marrow remains unclear. Here we show that nociceptive nerves are required for enforced HSC mobilization and that they collaborate with sympathetic nerves to maintain HSCs in the bone marrow. Nociceptor neurons drive granulocyte colony-stimulating factor (G-CSF)-induced HSC mobilization via the secretion of calcitonin gene-related peptide (CGRP). Unlike sympathetic nerves, which regulate HSCs indirectly via the niche3,4,6, CGRP acts directly on HSCs via receptor activity modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CALCRL) to promote egress by activating the Gαs/adenylyl cyclase/cAMP pathway. The ingestion of food containing capsaicin-a natural component of chili peppers that can trigger the activation of nociceptive neurons-significantly enhanced HSC mobilization in mice. Targeting the nociceptive nervous system could therefore represent a strategy to improve the yield of HSCs for stem cell-based therapeutic agents.

Nociceptors protect sickle cell disease mice from vaso-occlusive episodes and chronic organ damage.

Sickle cell disease (SCD) is a common hereditary hematologic disorder. SCD patients suffer from acute vaso-occlusive episodes (VOEs), chronic organ damage, and premature death, with few therapeutic options. Although severe pain is a major clinical manifestation of SCD, it remains unknown whether nociception plays a role in SCD pathogenesis. To address this question, we generated nociceptor-deficient SCD mice and found, unexpectedly, that the absence of nociception led to more severe and more lethal VOE, indicating that somatosensory nerves protect SCD mice from VOE. Mechanistically, the beneficial effects of sensory nerves were induced by the neuropeptide calcitonin gene-related peptide (CGRP), which acted on hematopoietic cells. Additionally, oral capsaicin consumption, which can activate somatosensory nerves by binding to TRPV1, dramatically alleviated acute VOE and significantly prevented chronic liver and kidney damage in SCD mice. Thus, the manipulation of nociception may provide a promising approach to treat SCD.