Morphometric Analysis of the Thymic Epithelial Cell (TEC) Network Using Integrated and Orthogonal Digital Pathology Approaches.

The thymus, a central primary lymphoid organ of the immune system, plays a key role in T cell development. Surprisingly, the thymus is quite neglected with regards to standardized pathology approaches and practices for assessing structure and function. Most studies use multispectral flow cytometry to define the dynamic composition of the thymus at the cell population level, but they are limited by lack of contextual insight. This knowledge gap hinders our understanding of various thymic conditions and pathologies, particularly how they affect thymic architecture, and subsequently, immune competence. Here, we introduce a digital pathology pipeline to address these challenges. Our approach can be coupled to analytical algorithms and utilizes rationalized morphometric assessments of thymic tissue, ranging from tissue-wide down to microanatomical and ultrastructural levels. This pipeline enables the quantitative assessment of putative changes and adaptations of thymic structure to stimuli, offering valuable insights into the pathophysiology of thymic disorders. This versatile pipeline can be applied to a wide range of conditions that may directly or indirectly affect thymic structure, ranging from various cytotoxic stimuli inducing acute thymic involution to autoimmune diseases, such as myasthenia gravis. Here, we demonstrate applicability of the method in a mouse model of age-dependent thymic involution, both by confirming established knowledge, and by providing novel insights on intrathymic remodeling in the aged thymus. Our orthogonal pipeline, with its high versatility and depth of analysis, promises to be a valuable and practical toolset for both basic and translational immunology laboratories investigating thymic function and disease.

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.

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.

CD36-Mediated Fatty Acid Oxidation in Hematopoietic Stem Cells Is a Novel Mechanism of Emergency Hematopoiesis in Response to Infection.

Hematopoietic homeostasis depends on the close regulation of hematopoietic stem cell (HSC) activity in the bone marrow. Quiescence and activation in response to stress, among other changes in state, are mediated by shifts in HSC metabolic activity. Although HSC steady-state metabolism is well established, the mechanisms driving HSC activation, proliferation, and differentiation in response to stress remain poorly understood. Here we discuss a study by Mistry et al. that describes a novel metabolic mechanism that fuels HSC activation and expansion. The authors show that to meet their metabolic needs in response to infection, hematopoietic stem and progenitor cells uptake free fatty acids from their microenvironment via CD36 to fuel fatty acid oxidation. These exciting findings suggest that in the context of infection, HSCs undergo a metabolic shift toward fatty acid metabolism that drives emergency hematopoiesis and raise questions about the role of the microenvironment in this process.

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.

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.

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.

Clinically Actionable Strategies for Studying Neural Influences in Cancer.

Neuro-glial activation is a recently identified hallmark of growing cancers. Targeting tumor hyperinnervation in preclinical and small clinical trials has yielded promising antitumor effects, highlighting the need of systematic analysis of neural influences in cancer (NIC). Here, we outline the strategies translating these findings from bench to the clinic.

Nestin+NG2+ Cells Form a Reserve Stem Cell Population in the Mouse Prostate.

In the prostate, stem and progenitor cell regenerative capacities have been ascribed to both basal and luminal epithelial cells. Here, we show that a rare subset of mesenchymal cells in the prostate are epithelial-primed Nestin-expressing cells (EPNECs) that can generate self-renewing prostate organoids with bipotential capacity. Upon transplantation, these EPNECs can form prostate gland tissue grafts at the clonal level. Lineage-tracing analyses show that cells marked by Nestin or NG2 transgenic mice contribute to prostate epithelium during organogenesis. In the adult, modest contributions in repeated rounds of regression and regeneration are observed, whereas prostate epithelial cells derived from Nestin/NG2-marked cells are dramatically increased after severe irradiation-induced organ damage. These results indicate that Nestin/NG2 expression marks a novel radioresistant prostate stem cell that is active during development and displays reserve stem cell activity for tissue maintenance.

Engineering a haematopoietic stem cell niche by revitalizing mesenchymal stromal cells.

Haematopoietic stem cells (HSCs) are maintained by bone marrow niches in vivo1,2, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP+ mesenchymal-derived stromal cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified five genes encoding transcription factors (Klf7, Ostf1, Xbp1, Irf3 and Irf7) that restored HSC niche function in cultured bone marrow-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed an approximately sevenfold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. Analysis of the reprogramming mechanisms uncovered a role for myocyte enhancer factor 2c (Mef2c) in the revitalization of MSCs. These results provide insight into the transcriptional regulation of the niche with implications for stem cell-based therapies.

Author Correction: Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche.

In the version of this article originally published, the key for Fig. 4c was incorrect. The symbols for 'Sham' and 'Den' were reversed. The error has been corrected in the PDF and HTML versions of the manuscript.

Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche.

Aging of hematopoietic stem cells (HSCs) is associated with a decline in their regenerative capacity and multilineage differentiation potential, contributing to the development of blood disorders. The bone marrow microenvironment has recently been suggested to influence HSC aging, but the underlying mechanisms remain largely unknown. Here we show that HSC aging critically depends on bone marrow innervation by the sympathetic nervous system (SNS), as loss of SNS nerves or adrenoreceptor ß3 signaling in the bone marrow microenvironment of young mice led to premature HSC aging, as evidenced by appearance of HSC phenotypes reminiscent of physiological aging. Strikingly, supplementation of a sympathomimetic acting selectively on adrenoreceptor ß3 to old mice significantly rejuvenated the in vivo function of aged HSCs, suggesting that the preservation or restitution of bone marrow SNS innervation during aging may hold the potential for new HSC rejuvenation strategies.

Neural Regulation of Bone and Bone Marrow.

Bones provide both skeletal scaffolding and space for hematopoiesis in its marrow. Previous work has shown that these functions were tightly regulated by the nervous system. The central and peripheral nervous systems tightly regulate compact bone remodeling, its metabolism, and hematopoietic homeostasis in the bone marrow (BM). Accumulating evidence indicates that the nervous system, which fine-tunes inflammatory responses and alterations in neural functions, may regulate autoimmune diseases. Neural signals also influence the progression of hematological malignancies such as acute and chronic myeloid leukemias. Here, we review the interplay of the nervous system with bone, BM, and immunity, and discuss future challenges to target hematological diseases through modulation of activity of the nervous system.

Adrenergic nerves activate an angio-metabolic switch in prostate cancer.

Nerves closely associate with blood vessels and help to pattern the vasculature during development. Recent work suggests that newly formed nerve fibers may regulate the tumor microenvironment, but their exact functions are unclear. Studying mouse models of prostate cancer, we show that endothelial ß-adrenergic receptor signaling via adrenergic nerve-derived noradrenaline in the prostate stroma is critical for activation of an angiogenic switch that fuels exponential tumor growth. Mechanistically, this occurs through alteration of endothelial cell metabolism. Endothelial cells typically rely on aerobic glycolysis for angiogenesis. We found that the loss of endothelial Adrb2, the gene encoding the ß2-adrenergic receptor, leads to inhibition of angiogenesis through enhancement of endothelial oxidative phosphorylation. Codeletion of Adrb2 and Cox10, a gene encoding a cytochrome IV oxidase assembly factor, prevented the metabolic shift induced by Adrb2 deletion and rescued prostate cancer progression. This cross-talk between nerves and endothelial metabolism could potentially be targeted as an anticancer therapy.

T-Regulating Hair Follicle Stem Cells.

Regulatory T (Treg) cells are well known to modulate inflammatory responses. In a recent issue of Cell, Ali et al. (2017) reveal a function for Treg cells in stem cell maintenance by showing that skin-resident Foxp3+ Treg cells preferentially localize to the hair follicle stem cell (HFSC) niche to control HFSC-mediated hair regeneration.

A Time Bomb for Leukemia.

Alterations of the circadian clock have been linked to cancer development. Puram et al. (in this issue) now uncover differential requirements between healthy hematopoietic and diseased leukemic stem cells for core circadian transcription factors, wherein leukemic cells depend on the clock machinery for survival and growth.

An MTCH2 pathway repressing mitochondria metabolism regulates haematopoietic stem cell fate.

The metabolic state of stem cells is emerging as an important determinant of their fate. In the bone marrow, haematopoietic stem cell (HSC) entry into cycle, triggered by an increase in intracellular reactive oxygen species (ROS), corresponds to a critical metabolic switch from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Here we show that loss of mitochondrial carrier homologue 2 (MTCH2) increases mitochondrial OXPHOS, triggering HSC and progenitor entry into cycle. Elevated OXPHOS is accompanied by an increase in mitochondrial size, increase in ATP and ROS levels, and protection from irradiation-induced apoptosis. In contrast, a phosphorylation-deficient mutant of BID, MTCH2's ligand, induces a similar increase in OXPHOS, but with higher ROS and reduced ATP levels, and is associated with hypersensitivity to irradiation. Thus, our results demonstrate that MTCH2 is a negative regulator of mitochondrial OXPHOS downstream of BID, indispensible in maintaining HSC homeostasis.

Neural regulation of hematopoiesis, inflammation, and cancer.

Although the function of the autonomic nervous system (ANS) in mediating the flight-or-fight response was recognized decades ago, the crucial role of peripheral innervation in regulating cell behavior and response to the microenvironment has only recently emerged. In the hematopoietic system, the ANS regulates stem cell niche homeostasis and regeneration and fine-tunes the inflammatory response. Additionally, emerging data suggest that cancer cells take advantage of innervating neural circuitry to promote their progression. These new discoveries outline the need to redesign therapeutic strategies to target this underappreciated stromal constituent. Here, we review the importance of neural signaling in hematopoietic homeostasis, inflammation, and cancer.