The high prevalence of pain and the at times low efficacy of current treatments represent a significant challenge to healthcare systems worldwide. Effective treatment strategies require consideration ...of the diverse pathophysiologies that underlie various pain conditions. Indeed, our understanding of the mechanisms contributing to aberrant sensory neuron function has advanced considerably. However, sensory neurons operate in a complex dynamic microenvironment that is controlled by multidirectional interactions of neurons with non-neuronal cells, including immune cells, neuronal accessory cells, fibroblasts, adipocytes, and keratinocytes. Each of these cells constitute and control the microenvironment in which neurons operate, inevitably influencing sensory function and the pathology of pain. This review highlights the importance of the neuronal microenvironment for sensory function and pain, focusing on cellular interactions in the skin, nerves, dorsal root ganglia, and spinal cord. We discuss the current understanding of the mechanisms by which neurons and non-neuronal cells communicate to promote or resolve pain, and how this knowledge could be used for the development of mechanism-based treatments.
Hematopoietic stem cells (HSC) reside in perivascular regions of the bone marrow (BM) embedded within a complex regulatory unit called the niche. Cellular components of HSC niches include vascular ...endothelial cells, mesenchymal stromal progenitor cells and a variety of mature hematopoietic cells such as macrophages, neutrophils, and megakaryocytes—further regulated by sympathetic nerves and complement components as described in this review. Three decades ago the discovery that cytokines induce a large number of HSC to mobilize from the BM into the blood where they are easily harvested, revolutionised the field of HSC transplantation—curative for immune-deficiencies and some malignancies. However, despite now routine use of granulocyte-colony stimulating factor (G-CSF) to mobilise HSC for transplant, only in last 15 years has research on the mechanisms behind why and how HSC can be induced to move into the blood began. These studies have revealed the complexity of the niche that retains HSC in the BM. This review describes how BM niches and HSC themselves change during administration of G-CSF—or in the recovery phase of chemotherapy—to facilitate movement of HSC into the blood, and research now leading to development of novel therapeutics to further boost HSC mobilization and transplant success.
The endothelial cell adhesion molecule E-selectin is a key component of the bone marrow hematopoietic stem cell (HSC) vascular niche regulating balance between HSC self-renewal and commitment. We now ...report in contrast, E-selectin directly triggers signaling pathways that promote malignant cell survival and regeneration. Using acute myeloid leukemia (AML) mouse models, we show AML blasts release inflammatory mediators that upregulate endothelial niche E-selectin expression. Alterations in cell-surface glycosylation associated with oncogenesis enhances AML blast binding to E-selectin and enable promotion of pro-survival signaling through AKT/NF-κB pathways. In vivo AML blasts with highest E-selectin binding potential are 12-fold more likely to survive chemotherapy and main contributors to disease relapse. Absence (in Sele
hosts) or therapeutic blockade of E-selectin using small molecule mimetic GMI-1271/Uproleselan effectively inhibits this niche-mediated pro-survival signaling, dampens AML blast regeneration, and strongly synergizes with chemotherapy, doubling the duration of mouse survival over chemotherapy alone, whilst protecting endogenous HSC.
In the bone marrow, hematopoietic stem cells (HSCs) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate the regulation of these endosteal niches, we studied the ...mobilization of HSCs into the bloodstream in response to granulocyte colony-stimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts, leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression, and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed, in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the: (1) loss of endosteal osteoblasts and (2) marked reduction of HSC-trophic cytokines at the endosteum, with (3) HSC mobilization into the blood, as observed during G-CSF administration. Together, these results establish that bone marrow macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSCs into the blood.
Distinct subsets of resident tissue macrophages are important in hematopoietic stem cell niche homeostasis and erythropoiesis. We used a myeloid reporter gene (Csf1r-eGFP) to dissect the persistence ...of bone marrow and splenic macrophage subsets following lethal irradiation and autologous hematopoietic stem cell transplantation in a mouse model. Multiple recipient bone marrow and splenic macrophage subsets survived after autologous hematopoietic stem cell transplantation with organ-specific persistence kinetics. Short-term persistence (5 weeks) of recipient resident macrophages in spleen paralleled the duration of extramedullary hematopoiesis. In bone marrow, radiation-resistant recipient CD169+ resident macrophages and erythroid-island macrophages self-repopulated long-term after transplantation via autonomous cell division. Posttransplant peak expansion of recipient CD169+ resident macrophage number in bone marrow aligned with the persistent engraftment of phenotypic long-term reconstituting hematopoietic stem cells within bone marrow. Selective depletion of recipient CD169+ macrophages significantly compromised the engraftment of phenotypic long-term reconstituting hematopoietic stem cells and consequently impaired hematopoietic reconstitution. Recipient bone marrow resident macrophages are essential for optimal hematopoietic stem cell transplantation outcomes and could be an important consideration in the development of pretransplant conditioning therapies and/or chemoresistance approaches.
•Recipient macrophages persist in hematopoietic tissues and self-repopulate via in situ proliferation after syngeneic transplantation.•Targeted depletion of recipient CD169+ macrophages after transplant impaired long-term bone marrow engraftment of hematopoietic stem cells.
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Hematopoietic stem cell (HSC) niches have been reported at the endosteum or adjacent to bone marrow (BM) vasculature. To investigate functional attributes of these niches, mice were perfused with ...Hoechst 33342 (Ho) in vivo before BM cell collection in presence of pump inhibitors and antibody stained. We report that the position of phenotypic HSCs, multipotent and myeloid progenitors relative to blood flow, follows a hierarchy reflecting differentiation stage, whereas mesenchymal stromal cells are perivascular. Furthermore, during granulocyte colony-stimulating factor–induced mobilization, HSCs migrated closer to blood flow, whereas stromal cells did not. Interestingly, phenotypic Lin−Sca1+KIT+CD41−CD48−CD150+ HSCs segregated into 2 groups (Honeg or Homed), based on degree of blood/Ho perfusion of their niche. HSCs capable of serial transplantation and long-term bromodeoxyuridine label retention were enriched in Honeg HSCs, whereas Homed HSCs cycled more frequently and only reconstituted a single host. This suggests that the most potent HSC niches are enriched in locally secreted factors and low oxygen tension due to negligible blood flow. Importantly, blood perfusion of niches correlates better with HSC function than absolute distance from vasculature. This technique enables prospective isolation of serially reconstituting HSCs distinct from other less potent HSCs of the same phenotype, based on the in vivo niche in which they reside.
The erythroblastic island (EBI) is a multicellular functional erythropoietic unit comprising a central macrophage nurturing a rosette of maturing erythroblasts. Since the discovery of EBIs more than ...half a century ago, EBIs are still studied by traditional microscopy methods after enrichment by sedimentation. These isolation methods are not quantitative and do not enable precise quantification of EBI numbers or frequency in the bone marrow or spleen tissues. Conventional flow cytometric methods have enabled quantification of cell aggregates co-expressing macrophage and erythroblast markers; however, it is unknown whether these aggregates contain EBIs as these aggregates cannot be visually assessed for EBI content. Combining the strengths of both microscopy and flow cytometry methods, in this chapter we describe an imaging flow cytometry method to analyze and quantitatively measure EBIs from the mouse bone marrow. This method is adaptable to other tissues such as the spleen or to other species provided that fluorescent antibodies specific to macrophages and erythroblasts are available.
The microenvironment, or niche, surrounding a stem cell largely governs its cellular fate. Two anatomical niches for hematopoietic stem cells (HSCs) have been reported in the bone marrow, but a ...distinct function for each of these niches remains unclear. Here we report a new role for the adhesion molecule E-selectin expressed exclusively by bone marrow endothelial cells in the vascular HSC niche. HSC quiescence was enhanced and self-renewal potential was increased in E-selectin knockout (Sele(-/-)) mice or after administration of an E-selectin antagonist, demonstrating that E-selectin promotes HSC proliferation and is a crucial component of the vascular niche. These effects are not mediated by canonical E-selectin ligands. Deletion or blockade of E-selectin enhances HSC survival threefold to sixfold after treatment of mice with chemotherapeutic agents or irradiation and accelerates blood neutrophil recovery. As bone marrow suppression is a severe side effect of high-dose chemotherapy, transient blockade of E-selectin is potentially a promising treatment for the protection of HSCs during chemotherapy or irradiation.
•Widely used “humanized” chimeric mice aid basic research.•NSG-SGM3 mice support robust human myeloid and T-cell development.•Optimized protocol achieves efficient human CD45+ ...reconstitution.•Engrafting neonatal NSG-SGM3 without irradiation delays anemia and HLH.
Immunodeficient mice bearing human immune systems, or “humanized” chimeric mice, are widely used in basic research, along with the preclinical stages of drug development. Nonobese diabetic-severe combined immunodeficiency (NOD-SCID) IL2Rγnull (NSG) mice expressing human stem cell factor, granulocyte-macrophage colony stimulating factor, and interleukin-3 (NSG-SGM3) support robust development of human myeloid cells and T cells but have reduced longevity due to the development of fatal hemophagocytic lymphohistiocytosis (HLH). Here, we describe an optimized protocol for development of human immune chimerism in NSG-SGM3 mice. We demonstrate that efficient human CD45+ reconstitution can be achieved and HLH delayed by engraftment of neonatal NSG-SGM3 with low numbers of human umbilical cord-derived CD34+ hematopoietic stem cells in the absence of preconditioning irradiation.
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