Histone demethylase LSD1 regulates hematopoietic stem cells homeostasis and protects from death by endotoxic shock

Hematopoietic stem cells (HSCs) maintain a quiescent state during homeostasis, but with acute infection, they exit the quiescent state to increase the output of immune cells, the so-called “emergency hematopoiesis.” However, HSCs’ response to severe infection during septic shock and the pathological impact remain poorly elucidated. Here, we report that the histone demethylase KDM1A/LSD1, serving as a critical regulator of mammalian hematopoiesis, is a negative regulator of the response to inflammation in HSCs during endotoxic shock typically observed during acute bacterial or viral infection. Inflammation-induced LSD1 deficiency results in an acute expansion of a pathological population of hyperproliferative and hyperinflammatory myeloid progenitors, resulting in a septic shock phenotype and acute death. Unexpectedly, in vivo administration of bacterial lipopolysaccharide (LPS) to wild-type mice results in acute suppression of LSD1 in HSCs with a septic shock phenotype that resembles that observed following induced deletion of LSD1. The suppression of LSD1 in HSCs is caused, at least in large part, by a cohort of inflammation-induced microRNAs. Significantly, reconstitution of mice with bone marrow progenitor cells expressing inhibitors of these inflammation-induced microRNAs blocked the suppression of LSD1 in vivo following acute LPS administration and prevented mortality from endotoxic shock. Our results indicate that LSD1 activators or miRNA antagonists could serve as a therapeutic approach for life-threatening septic shock characterized by dysfunction of HSCs.

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Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽

10.1182/blood.v124.21.350.350 ◽

2014 ◽

Vol 124 (21) ◽

pp. 350-350

Author(s):

Kyung-Hee Chang ◽

Amitava Sengupta ◽

Ramesh C Nayak ◽

Angeles Duran ◽

Sang Jun Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Negative Regulator ◽

Wild Type ◽

Hematopoietic Stem ◽

P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

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Stress-Mediated Activation of Dormant Hematopoietic Stem Cells In Vivo

Blood ◽

10.1182/blood.v120.21.sci-41.sci-41 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-41-SCI-41

Author(s):

Andreas Trumpp ◽

Marieke Essers

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Strong Increase ◽

Interferon Α ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract SCI-41 Maintenance of the blood system is dependent on dormant hematopoietic stem cells (HSCs), which are characterized by pluripotency and lifelong self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain deeply quiescent during homeostasis, and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines and cytokines, as well as by interactions of HSCs with their niche environment. The transcriptome of dormant CD34-CD150+CD48-LSK- HSCs significantly differs from that of active HSCs with the same phenotype, while the latter are highly similar to MPP1 progenitors which express CD34. One of the genes differentially expressed is the cylindromatosis (CYLD) gene, which encodes a negative regulator of the NF-κB signaling pathway. HSCs failing to express functional CYLD show various defects associated with a disturbed balance between dormant and active HSCs, suggesting a role for NF-κB signaling in establishing dormancy in HSCs. In addition, our studies have recently shown that the cytokine interferon-α (IFNα) very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNα, HSCs exit G0 and enter an active cell cycle. In general, IFNα is produced in response to viral infections by cells of the immune system, and plays an important role in the antiviral host defense. We now questioned whether endogenous IFNα is also produced in response to other types of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNα production in the bone marrow in vivo, we have generated MxCre ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNα in the vicinity of HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in HSC cycling. Surprisingly, since mice lacking the IFNα receptor (Ifnar−/−) still respond to LPS, this effect is independent of IFNAR signaling. Strikingly, LPS-induced HSC activation correlated with increased expression of Sca-1, similar to what occurs upon IFNα treatment. Moreover, as for IFNα, the upregulation of SCA-1 is required for LPS-induced proliferation, since Sca-1−/− mice fail to respond to LPS stimulation. In summary, these data suggest that not only virus-inducible IFNα, but also infections by gram-negative-bacteria-produced LPS induce cycling of progenitors and otherwise dormant HSCs. Disclosures: No relevant conflicts of interest to declare.

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Foxo3a Is Essential for the Quiescence and Self-Renewal of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.442.442 ◽

2006 ◽

Vol 108 (11) ◽

pp. 442-442

Author(s):

Kana Miyamoto ◽

Atsushi Hirao ◽

Kiyomi Y. Araki ◽

Fumio Arai ◽

Kazuhito Naka ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Functions

Abstract Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state in bone marrow (BM). Quiescent stem cells show resistance to various stresses, suggesting that mechanisms for protection of HSC life from stress contribute to maintenance of self-renewal capacity through a whole life in animals. We hypothesized that a signaling pathway for regulating aging might be involved in stem cell functions. FOXO transcription factors belong to the forkhead family of transcriptional regulators characterized by a conserved DNA-binding domain termed “forkhead box”. In C.elegans, genetic analyses have revealed the existence of a conserved insulin-like signaling involved in longevity. Conservation of this pathways lead to speculation that forkhead transcriptional factor are involved in life span in mammals. It was known that active-state Foxo3a is localized in nucleus, and we found HSC-specific nuclear localization of Foxo3a by immunocytochemistric study, therefore we generated gene-targeted Foxo3a−/− mice to analyze roles of Foxo in HSC regulation. Peripheral blood count showed decreased number of red blood cells in Foxo3a−/− mice, but numbers of white blood cells and platelets were normal. In colony-forming assay, we detected the numbers and sizes of myeloid, erythroid and mixed colonies derived from Foxo3a−/− BM mononuclear cells were all normal. These results suggest that the proliferation and differentiation of Foxo3a−/− progenitors were normal. However, the number of colony-forming cells present in long-term culture of Foxo3a−/− c-kit+Sca-1+Lin− (KSL) cells with stroma was significantly reduced. The ability of Foxo3a−/− HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired, indicating that self-renewal capacity of HSCs was defective in Foxo3a−/− mice. To understand the mechanisms of this phenotypes, we evaluated the cell cycle status using BrdU (5-bromodeoxyuridine) incorporation but found no difference in Foxo3a+/+ and Foxo3a−/− progenitor cells. To directly evaluate HSC quiescence in Foxo3a−/− mice, we stained CD34−KSL cells with Pyronin Y. Although most Foxo3a+/+ CD34−KSL cells stained negatively for Pyronin Y, a sizable Pyronin Y+ population was detected among Foxo3a−/− CD34−KSL cells, demonstrating that loss of Foxo3a leads to a defect in the maintenance of HSCs quiescence. Since p38MAPK is selectively activated by environmental stress, we evaluated the activation status of p38MAPK in Foxo3a+/+ and Foxo3a−/− HSCs. Frequency of phosphorylated p38MAPK+ cells in Foxo3a−/−CD34−KSL cells was significantly increased than that of Foxo3a+/+CD34−KSL cells. Our results suggest that Foxo3a−/− HSCs subjected to tangible stress in vivo. Finally, we investigated the sensitivity of Foxo3a−/− mice to weekly 5-fluorouracil treatment in vivo. Although 60% of Foxo3a+/+mice survived for at least 4 weeks post-injection, all Foxo3a−/− mice were dead in 4 weeks. It suggests that Foxo3a protects hematopoietic cells from destruction by cell cycle-dependent myelotoxic agent. Taken together, our results demonstrate that Foxo3a plays a pivotal role in maintaining HSC quiescence and stress resistance.

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TGF-β signaling–deficient hematopoietic stem cells have normal self-renewal and regenerative ability in vivo despite increased proliferative capacity in vitro

Blood ◽

10.1182/blood-2003-04-1300 ◽

2003 ◽

Vol 102 (9) ◽

pp. 3129-3135 ◽

Cited By ~ 110

Author(s):

Jonas Larsson ◽

Ulrika Blank ◽

Hildur Helgadottir ◽

Jon Mar Björnsson ◽

Mats Ehinger ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Negative Regulator ◽

Type I ◽

Hematopoietic Stem ◽

Knock Out

Abstract Studies in vitro implicate transforming growth factor β (TGF-β) as a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. In vivo studies have been hampered by early lethality of knock-out mice for TGF-β isoforms and the receptors. To directly assess the role of TGF-β signaling for hematopoiesis and hematopoietic stem cell (HSC) function in vivo, we generated a conditional knock-out model in which a disruption of the TGF-β type I receptor (TβRI) gene was induced in adult mice. HSCs from induced mice showed increased proliferation recruitment when cultured as single cells under low stimulatory conditions in vitro, consistent with an inhibitory role of TGF-β in HSC proliferation. However, induced TβRI null mice show normal in vivo hematopoiesis with normal numbers and differentiation ability of hematopoietic progenitor cells. Furthermore HSCs from TβRI null mice exhibit a normal cell cycle distribution and do not differ in their ability long term to repopulate primary and secondary recipient mice following bone marrow transplantation. These findings challenge the classical view that TGF-β is an essential negative regulator of hematopoietic stem cells under physiologic conditions in vivo.

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Scribble Is a Negative Regulator of Interferon-I Dependent Notch1 Activation in Adult Hematopoietic Stem Cells and Progenitors

Blood ◽

10.1182/blood-2021-151046 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 299-299

Author(s):

Abhishek K Singh ◽

Mark J Althoff ◽

Saimul Islam ◽

Ashley M Wellendorf ◽

Jose A. Cancelas

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Molecular Mechanisms ◽

South Florida ◽

Negative Regulator ◽

Poly I:C ◽

Hematopoietic Stem ◽

Cellular Quiescence

Abstract Hematopoietic stem cells (HSC) are highly quiescent cells with the ability to rapidly enter cell cycle and differentiate through changes in their polarity and the disposition of intracellular molecular fate determinants in response to microenvironment (ME) cues. Interferons type 1 (IFN-I) are ME cytokines produced during the physiological response mounted to combat a viral infection. In bone marrow hematopoiesis, IFN-I induces activation and proliferation of HSC. Clinically, patients treated with IFN-I, as well as individuals suffering from IFN-I associated chronic disease, often exhibit sustained hematological cytopenias and HSC failure. The precise molecular mechanisms that govern HSC behavior in response to IFN-I are still unclear. Our data highlights that Scribble deficient HSC are less sensitive to IFN-I mediated activation. By using hematopoietic specific deletion of Scribble in murine hematopoiesis (Vav-Cre;Scribble KO); we demonstrated that Scribble deficient LSK CD150 +/CD48 - HSC are less responsive to polyinositide-polycytidine (pI:C) induced IFN-I mediated activation and retain cellular quiescence (G0:45±5.4% vs 63±2.7% in WT and Scribble KO, respectively, p<0.05). IFN-I induced upregulation of Sca-1 expression was also significantly hampered in Scribble deficient HSC. Functionally, serial transplantation experiments demonstrated that in response to poly I:C, Scribble deficient HSC display increased competitive repopulating potential (26±1.3% vs 38±1.2% BM chimerism for WT and KO BM in secondary recipients and 38±2.5% vs 48±2.7% BM chimerism in tertiary recipients, p<0.01). The maintenance of cellular quiescence and function for Scribble deficient HSC are independent of canonical IFN-I driven STAT-1 signaling, as we report no differences in STAT-1 activation, nuclear translocation or the expression of STAT-1 canonical target genes in response to pI:C. Unsupervised transcriptomics analysis of Scribble-deficient HSC supported dysregulation of Notch signaling. Furthermore, Scribble deficiency in non-activated LSK HSC and progenitors (HSPC) was associated with constitutive activation and cleavage of Notch1 (Notch1 ICD;~3 fold) at levels comparable to IFN-I mediated activation of WT HSPC. However, Scribble deficient HSPC did not exhibit further Notch1 cleavage activation upon in vivo IFN-I induction. Pharmacological in vivo γ-secretase inhibition (YO-01027) prevented the protective effect of Scribble deficiency on IFN-I dependent loss of HSC quiescence. These data indicate that Notch1 activation, and subsequent cleavage, is indispensable for Scribble deficient HSC quiescence in response to IFN-I. Active Cdc42 is a critical regulator of HSC quiescence and fate, and previous studies have demonstrated that Scribble controls HSC asymmetric division potential and fate through the PDZ mediated scaffolding of cytosolic Yap1 with activated Cdc42 (Cdc42-GTP). Next to determine whether poly I:C mediated Notch1 cleavage linked with Cdc42 activity, we analyzed the protein interactions between cleaved Notch1 and Cdc42-GTP in relation with Scribble. Our findings revealed that Scribble associates with non-cleaved, membrane bound Notch but upon in vivo IFN-I induction Notch1 is cleaved, activated and translocates with Scribble-free, activated Cdc42 to the nucleus of HSC. Deletion of HSC Scribble associated with a reduced (~45%, p<0.001) proximity interaction between cleaved Notch1 and Cdc42-GTP. Collectively our findings identify that Scribble controls IFN-I mediated HSPC activation through induction of Notch1 cleavage and Cdc42 activity, and highlight such interaction as a new potential target to dampen inflammation driven HSC exhaustion. Disclosures Cancelas: Cerus Co: Research Funding; TerumoBCT: Research Funding; Hemanext: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytosorbents Inc: Research Funding; Fresenius-Kabi LLC: Research Funding; Westat Inc: Research Funding; Vascular Solutions Inc.: Research Funding; Hemerus LLC: Research Funding; University of South Florida/MEQU Inc: Research Funding.

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In vivo time-lapse imaging shows diverse niche engagement by quiescent and naturally activated hematopoietic stem cells

Blood ◽

10.1182/blood-2013-10-534859 ◽

2014 ◽

Vol 124 (1) ◽

pp. 79-83 ◽

Cited By ~ 42

Author(s):

Narges M. Rashidi ◽

Mark K. Scott ◽

Nico Scherf ◽

Axel Krinner ◽

Jens S. Kalchschmidt ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Acute Infection ◽

Time Lapse ◽

Hematopoietic Stem ◽

Key Points ◽

Time Lapse Imaging ◽

Bone Marrow Niches

Key Points Normally, engrafting HSCs reside and oscillate within confined bone marrow niches. HSCs harvested from mice bearing acute infection are migratory and interact with larger niches.

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Faculty Opinions recommendation of Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.734776713.793573818 ◽

2020 ◽

Author(s):

Ivan Martin

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Lineage Commitment ◽

Hematopoietic Stem ◽

Clonal Level

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Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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