Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction

Dysregulation of ribosome biogenesis causes human diseases, such as Diamond-Blackfan anemia, del (5q-) syndrome and bone marrow failure. However, the mechanisms of blood disorders in these diseases remain elusive. Through genetic mapping, molecular cloning and mechanism characterization of the zebrafish mutant cas002, we reveal a novel connection between ribosomal dysfunction and excessive autophagy in the regulation of hematopoietic stem/progenitor cells (HSPCs). cas002 carries a recessive lethal mutation in kri1l gene that encodes an essential component of rRNA small subunit processome. We show that Kri1l is required for normal ribosome biogenesis, expansion of definitive HSPCs and subsequent lineage differentiation. Through live imaging and biochemical studies, we find that loss of Kri1l causes the accumulation of misfolded proteins and excessive PERK activation-dependent autophagy in HSPCs. Blocking autophagy but not inhibiting apoptosis by Bcl2 overexpression can fully rescue hematopoietic defects, but not the lethality of kri1l^cas002 embryos. Treatment with autophagy inhibitors (3-MA and Baf A1) or PERK inhibitor (GSK2656157), or knockdown of beclin1 or perk can markedly restore HSPC proliferation and definitive hematopoietic cell differentiation. These results may provide leads for effective therapeutics that benefit patients with anemia or bone marrow failure caused by ribosome disorders.     

AMD3100 improves ovariectomy-induced osteoporosis in mice by facilitating mobilization of hematopoietic stem/progenitor cells

Inhibition of an increase of osteoclasts has become the most important treatment for osteoporosis. The CXCR4 antagonist, AMD3100, plays an important role in the mobilization of osteoclast precursors within bone marrow (BM). However, the actual therapeutic impact of AMD3100 in osteoporosis has not yet been ascertained. Here we demonstrate the therapeutic effect of AMD3100 in the treatment of ovariectomy-induced osteoporosis in mice. We found that treatment with AMD3100 resulted in direct induction of release of SDF-1 from BM to blood and mobilization of hematopoietic stem/progenitor cells (HSPCs) in an osteoporosis model. AMD3100 prevented bone density loss after ovariectomy by mobilization of HSPCs, suggesting a therapeutic strategy to reduce the number of osteoclasts on bone surfaces. These findings support the hypothesis that treatment with AMD3100 can result in efficient mobilization of HSPCs into blood through direct blockade of the SDF-1/CXCR4 interaction in BM and can be considered as a potential new therapeutic intervention for osteoporosis. [BMB Reports 2014; 47(8): 439-444]     

Rosiglitazone Promotes Bone Marrow Adipogenesis to Impair Myelopoiesis under Stress

Objective

The therapeutic use of thiazolidinediones (TZDs) causes unwanted hematological side effects, although the underlying mechanisms of these effects are poorly understood. This study tests the hypothesis that rosiglitazone impairs the maintenance and differentiation of hematopoietic stem/progenitor cells, which ultimately leads to hematological abnormalities.

Methods

Mice were fed a rosiglitazone-supplemented diet or a normal diet for 6 weeks. To induce hematopoietic stress, all mice were injected once with 250 mg/kg 5-fluorouracil (5-Fu) intraperitoneally. Next, hematopoietic recovery, hematopoietic stem/progenitor cells (HSPCs) subsets, and myeloid differentiation after 5-Fu treatment were evaluated. The adipogenesis induced by rosiglitazone was assessed by histopathology and oil red O staining. The effect of adipocytes on HSPCs was studied with an in vitro co-culture system.

Results

Rosiglitazone significantly enhanced bone marrow adipogenesis and delayed hematopoietic recovery after 5-Fu treatment. Moreover, rosiglitazone inhibited proliferation of a granulocyte/monocyte progenitor (GMP) cell population and granulocyte/macrophage colony-stimulating factor (GM-CSF) colonies, although the proliferation and mobilization of Lin-c-kit+Sca-1+ cells (LSK) was maintained following hematopoietic stress. These effects could be partially reversed by the selective PPARγ antagonist BADGE. Finally, we demonstrated in a co-culture system that differentiated adipocytes actively suppressed the myeloid differentiation of HSPCs.

Conclusion

Taken together, our results demonstrate that rosiglitazone inhibits myeloid differentiation of HSPCs after stress partially by inducing bone marrow adipogenesis. Targeting the bone marrow microenvironment might be one mechanism by which rosiglitazone impairs stress-induced hematopoiesis.     

MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic,but Not Syngeneic Recipient Mice

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.     

Heat-inactivated Escherichia coli promotes hematopoietic regeneration after irradiation with IL-1β

Background aimsHematopoietic stem and progenitor cells (HSPCs) are known to produce short-lived mature blood cells via proliferation and differentiation in a process that depends partially on regulatory cytokines from the bone marrow (BM) microenvironment. Delayed BM recovery after tremendous damage to the hematopoietic system can lead to neutropenia, anemia, thrombopenia and even death. However, efficiently promote BM recovery is still a big problem to be solved. Here, the authors aim to use heat-inactivated Escherichia coli (HIEC) to enhance BM recovery and further to find out the potential mechanism.MethodsX-rad was used to establish HIEC/IL-1β-induced radioprotection model. Single-cell RNA sequencing, RT-PCR, and western blotting were performed to detect the expression of IL-1R1 on HSPCs. Flow cytometry and automated hematology analyzer were used to analyze the percentage and absolute number of different populations of hematopoietic cells. The effects of IL-1β on HSPCs were studied using in vivo and in vitro experiments.ResultsHIEC/IL-1β pre-treatment can significantly increase the survival rate of lethally irradiated mice, and these mice showed better hematopoietic regeneration compared with control group. IL-1R was expressed on HSPCs, and IL-1β could directly function on HSPCs to promote the proliferation and differentiation of HSPCs, and inhibit the apoptosis of HSPCs.ConclusionsHIEC pre-treatment can rescue lethally irradiated mice by promoting hematopoietic recovery via IL-1β/IL-1R1 signaling, which can promote the proliferation of HSPCs by enhancing the cell cycle and attenuating the apoptosis of HSPCs.     

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.     

Arbutin ameliorates glucocorticoid-induced osteoporosis through activating autophagy in osteoblasts

Chronic long-term glucocorticoid use causes osteoporosis partly by interrupting osteoblast homeostasis and exacerbating bone loss. Arbutin, a natural hydroquinone glycoside, has been reported to have biological activities related to the differentiation of osteoblasts and osteoclasts. However, the role and underlying mechanism of arbutin in glucocorticoid-induced osteoporosis are elusive. In this study, we demonstrated that arbutin administration ameliorated osteoporotic disorders in glucocorticoid dexamethasone (Dex)-induced mouse model, including attenuating the loss of bone mass and trabecular microstructure, promoting bone formation, suppressing bone resorption, and activating autophagy in bone tissues. Furthermore, Dex-stimulated mouse osteoblastic MC3T3-E1 cells were treated with arbutin. Arbutin treatment rescued Dex-induced repression of osteoblast differentiation and mineralization, the downregulation of osteogenic gene expression, reduced autophagic marker expression, and decreased autophagic puncta formation. The application of autophagy inhibitor 3-MA decreased autophagy, differentiation, and mineralization of MC3T3-E1 cells triggered by arbutin. Taken together, our findings suggest that arbutin treatment fends off glucocorticoid-induced osteoporosis, partly through promoting differentiation and mineralization of osteoblasts by autophagy activation.     

GPX4 and vitamin E cooperatively protect hematopoietic stem and progenitor cells from lipid peroxidation and ferroptosis

Ferroptosis, a newly defined mode of regulated cell death caused by unbalanced lipid redox metabolism, is implicated in various tissue injuries and tumorigenesis. However, the role of ferroptosis in stem cells has not yet been investigated. Glutathione peroxidase 4 (GPX4) is a critical suppressor of lipid peroxidation and ferroptosis. Here, we study the function of GPX4 and ferroptosis in hematopoietic stem and progenitor cells (HSPCs) in mice with Gpx4 deficiency in the hematopoietic system. We find that Gpx4 deletion solely in the hematopoietic system has no significant effect on the number and function of HSPCs in mice. Notably, hematopoietic stem cells (HSCs) and hematopoietic progenitor cells lacking Gpx4 accumulated lipid peroxidation and underwent ferroptosis in vitro. α-Tocopherol, the main component of vitamin E, was shown to rescue the Gpx4-deficient HSPCs from ferroptosis in vitro. When Gpx4 knockout mice were fed a vitamin E-depleted diet, a reduced number of HSPCs and impaired function of HSCs were found. Furthermore, increased levels of lipid peroxidation and cell death indicated that HSPCs undergo ferroptosis. Collectively, we demonstrate that GPX4 and vitamin E cooperatively maintain lipid redox balance and prevent ferroptosis in HSPCs.Subject terms: Cell biology, Physiology, Stem-cell research     

Rumba and Haus3 are essential factors for the maintenance of hematopoietic stem/progenitor cells during zebrafish hematopoiesis

The hallmark of vertebrate definitive hematopoiesis is the establishment of the hematopoietic stem/progenitor cell (HSPC) pool during embryogenesis. This process involves a defined ontogenic switching of HSPCs in successive hematopoietic compartments and is evolutionarily conserved from teleost fish to human. In zebrafish, HSPCs originate from the ventral wall of the dorsal aorta (VDA), from which they subsequently mobilize to an intermediate hematopoietic site known as the caudal hematopoietic tissue (CHT) and finally colonize the kidney for adult hematopoiesis. Despite substantial understanding of the ontogeny of HSPCs, the molecular basis governing migration, colonization and maintenance of HSPCs remains to be explored fully. Here, we report the isolation and characterization of two zebrafish mutants, rumba(hkz1) and samba(hkz2), that are defective in generating definitive hematopoiesis. We find that HSPC initiation in the VDA and subsequent homing to the CHT are not affected in these two mutants. However, the further development of HSPCs in the CHT is compromised in both mutants. Positional cloning reveals that Rumba is a novel nuclear C2H2 zinc-finger factor with unknown function and samba encodes an evolutionarily conserved protein that is homologous to human augmin complex subunit 3 (HAUS3). Furthermore, we show that these two factors independently regulate cell cycle progression of HSPCs and are cell autonomously required for HPSC development in the CHT. Our study identifies Rumba and Haus3 as two essential regulators of HSPC maintenance during zebrafish fetal hematopoiesis.     

Immune Humanization of Immunodeficient Mice Using Diagnostic Bone Marrow Aspirates from Carcinoma Patients

Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses.     

The miRNA-15b/USP7/KDM6B axis engages in the initiation of osteoporosis by modulating osteoblast differentiation and autophagy

Osteoporosis is a metabolic disease that results from oxidative stress or inflammation in renal disorders. microRNAs (miRNAs) are recently implicated to participate in osteoporosis, but the mechanism remains largely unexplored. Herein, we aimed to explore the potential role of miR-15b in osteoblast differentiation and autophagy in osteoporosis. We established osteoporosis models through ovariectomy and determined that miR-15b was highly expressed whereas USP7 and KDM6B were poorly expressed in tissue of osteoporosis mice. Treatment of silenced miR-15b resulted in the elevation of decreased bone mineral density (BMD), the maximum elastic stress and the maximum load of osteoporosis mice. In osteoblasts, miR-15 overexpression decreased proliferation but suppressed the cell differentiation and autophagy, accompanied with decreased expression of USP7. Mechanistically, miR-15 bound and inhibited USP7 expression, while overexpression of USP7 promoted autophagy of osteoblasts. USP7, importantly, strengthened the stability of KDM6B and promoted KDM6B expression. MG132 protease inhibitor increased KDM6B and USP7 expression in osteoblasts. Silencing of KDM6B reversed the promoting effect on autophagy and proliferation induced by overexpression of USP7. Taken altogether, miR-15b inhibits osteoblast differentiation and autophagy to aggravate osteoporosis by targeting USP7 to regulate KDM6B expression.     

c-Kit-mediated functional positioning of stem cells to their niches is essential for maintenance and regeneration of adult hematopoiesis

The mechanism by which hematopoietic stem and progenitor cells (HSPCs) through interaction with their niches maintain and reconstitute adult hematopoietic cells is unknown. To functionally and genetically track localization of HSPCs with their niches, we employed novel mutant loxPs, lox66 and lox71 and Cre-recombinase technology to conditionally delete c-Kit in adult mice, while simultaneously enabling GFP expression in the c-Kit-deficient cells. Conditional deletion of c-Kit resulted in hematopoietic failure and splenic atrophy both at steady state and after marrow ablation leading to the demise of the treated adult mice. Within the marrow, the c-Kit-expressing GFP(+) cells were positioned to Kit ligand (KL)-expressing niche cells. This c-Kit-mediated cellular adhesion was essential for long-term maintenance and expansion of HSPCs. These results lay the foundation for delivering KL within specific niches to maintain and restore hematopoiesis.     

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L ‐Leucyl‐L‐leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy‐dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.     

稳态及衰老情况下骨髓微环境对造血干细胞的调控作用

稳态下,骨髓微环境(bone marrow microenvironment)被证实能通过多种信号通路和细胞因子调控造血干细胞(hematopoietic stem cells,HSCs)的自我更新、增殖、分化和迁移能力以维持造血系统的稳定.在衰老过程中,HSCs功能受损会导致造血系统功能的退化以及年龄相关的免疫应答的...     

Pannexin-1 channel “fuels” by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells

An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1⁺ and CD11b⁺ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

     

Adipose tissue as a potential source of hematopoietic stem/progenitor cells

It has been more than 30 years since adipose tissue (AT) has been recognized as a central modulator orchestrating sophisticated process termed "immunometabolism". Nonetheless, despite its unique involvement in the regulation of immune and endocrine homeostasis, recent studies demonstrated that AT also contains significant number of hematopoietic stem/progenitor cells (HSPCs) that may be there "settling down" throughout life. In this article we will focus on presenting the current concepts regarding endocrine, immunological, and molecular mechanisms that may contribute to and regulate bone marrow (BM)-derived HSPCs homing into AT environment, as well as, highlight various structural and morphological similarities between BM and AT that might be involved in creating appropriate tissue niches for BM-derived HSPCs in AT. Finally, we will discuss how development of obesity or type 2 diabetes may influence balance of homing signals for HSPCs in AT environment.     

Endocannabinoids Are Expressed in Bone Marrow Stromal Niches and Play a Role in Interactions of Hematopoietic Stem and Progenitor Cells with the Bone Marrow Microenvironment

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB₁ and CB₂. The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB₁ receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10⁷ cells), and 2-AG (75.2 ng/10⁷ cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10⁷ cells) and 2-AG (98.8 ng/10⁷ cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB₁ agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1^−/− showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)^−/− mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1^−/−, as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.     

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood^[1-2].We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.     

自噬与骨质疏松症的相关性

骨质疏松症是一种全身性骨骼疾病,其特征为低骨量和骨组织微结构退化。自噬是一个动态的、高度规律的自我消化过程,负责细胞存活和氧化应激反应,可以控制人体老化和骨质疏松症。尽管目前自噬对骨质疏松症的调控机制并没有完全解释清楚,但是随着对自噬研究的不断深入,其与骨质疏松症之间可能的联系和相互影响机制不断被揭示。本文回顾了近年来自噬与骨质疏松症的相关研究文献,探寻自噬与骨质疏松症相关的科学依据,发现对自噬与骨质疏松症共同影响的机制包括衰老、基因调控等。同时,自噬对糖皮质激素诱导的骨质疏松发病及药物治疗均有一定的影响。为进一步利用与自噬相关的调控体系来防治骨质疏松症提供证据。