scholarly journals Ripk-Mediated Necroptosis Induces Inflammation and Bone Marrow Failure in Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1599-1599
Author(s):  
Justine E. Roderick ◽  
Nicole Hermance ◽  
Matija Zelic ◽  
Matthew Simmons ◽  
Apostolos Polykratis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Type I ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Ifn Γ

Abstract TNF-α and IFN-γ overproduction are features associated with human bone marrow failure syndromes such as Fanconi Anemia (FA) and Aplastic Anemia (AA). Cells from these patients are known to be hypersensitive to TNF-α and IFN-γ-induced cell death. The serine threonine kinases RIPK1 and RIPK3 interact to mediate necroptosis induced by TNF-α, type I or II interferons. We demonstrate that a hematopoietic RIPK1 deficiency results in hematopoietic stem and progenitor cell loss and induction of bone marrow failure. The cell death reflects cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, as Vav-iCre Ripk1fl/fl fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. Hematopoietic failure in these mice is accompanied by increases in serum pro-inflammatory cytokines/chemokines and reduced hematopoietic colony formation in the presence of TNF-α, type I or II interferon. We provide genetic evidence that a RIPK3 deficiency rescues the bone marrow failure and significantly reduces serum cytokine and chemokine levels in Vav-iCre Ripk1fl/fl mice. These data reveal that in the hematopoietic lineage RIPK1 prevents inflammation by suppressing RIPK3 activity and raise the possibility that human bone marrow failure patients may benefit from selective RIPK inhibitors. Disclosures No relevant conflicts of interest to declare.

Isolation and Functional Characterization of Human Erythroid Progenitors: BFU-E and CFU-E

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1028-1028
Author(s):  
Pooja Bhagia ◽  
Narla Mohandas ◽  
Xiuli An
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Functional Characterization ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Erythroid Progenitor ◽  
Expression Levels ◽  
Cd34 Cells

Abstract Abstract 1028 The two committed erythroid progenitor populations that have been functionally defined by colony assays are burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E). While significant progress has been made in defining these two progenitor populations in the murine system, their characterization in the human system is incomplete. To address this issue, we have characterized the dynamic changes in surface expression levels of number of proteins including CD34, c-kit, IL-3R, CD36, CD71, GPA and CD45 during proliferation of purified human CD34+ cells from cord blood during the first phase of the two-phase in vitro erythroid culture system. In the presence of stem cell factor, IL-3 and erythropoietin during this phase, CD34+ cells differentiate first into BFU-E and then into CFU-E during 7 days of culture with peak levels of BFU-E at day 4 and of CFU-E at day 6. During this period of time, the expression levels of CD34 and IL-3R decreased, while that that of CD36 and CD71 increased. CD45 was expressed during the entire 7 day culture period while there was no expression of GPA. Based on these findings, we sorted pure populations of CD34+CD36−IL3-R+ and CD34− CD36+IL-3R− cells and characterized their behavior in colony forming assays. The sorted CD34+CD36−IL3-R+ population gave rise to BFU-E colonies while CD34− CD36+IL-3R− population gave rise CFU-E colonies, both at a purity of over 80%. The identity of the sorted BFU-E and CFU-E cells was further supported by their differential responsiveness to dexamethasone and lenalidomide (Narla A et al Blood 2011), with increased proliferation BFU-E population by dexamethasone and increased proliferation of CFU-E by lenalidomide. These findings were further validated by isolation of pure populations of BFU-E and CFU-E from primary human bone marrow based on the identified markers. The ability to isolate pure populations of human BFU-E and CFU-E progenitors should enable detailed molecular and cellular characterization of these distinct erythroid progenitor populations. Furthermore, enumeration of the number of these progenitor populations in human bone marrow may help in delineating mechanisms of disordered erythropoiesis in various disorders such as bone marrow failure syndromes. Disclosures: No relevant conflicts of interest to declare.

TNF-α/Fas-RIP-1–induced cell death signaling separates murine hematopoietic stem cells/progenitors into 2 distinct populations

Blood ◽  
2011 ◽  
Vol 118 (23) ◽  
pp. 6057-6067 ◽  
Author(s):  
Yechen Xiao ◽  
Hongling Li ◽  
Jun Zhang ◽  
Andrew Volk ◽  
Shubin Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Bone Marrow Failure ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
Interacting Protein ◽  
Hematopoietic Stem ◽  
Complete Inactivation ◽  
Bone Marrow Failure Syndromes ◽  
Tnf Α

AbstractWe studied the effects of TNF-α and Fas-induced death signaling in hematopoietic stem and progenitor cells (HSPCs) by examining their contributions to the development of bone marrow failure syndromes in Tak1-knockout mice (Tak1−/−). We found that complete inactivation of TNF-α signaling by deleting both of its receptors, 1 and 2 (Tnfr1−/−r2−/−), can prevent the death of 30% to 40% of Tak1−/− HSPCs and partially repress the bone marrow failure phenotype of Tak1−/− mice. Fas deletion can prevent the death of 5% to 10% of Tak1−/− HSPCs but fails to further improve the survival of Tak1−/−Tnfr1−/−r2−/− HSPCs, suggesting that Fas might induce death within a subset of TNF-α-sensitive HSPCs. This TNF-α/Fas-induced cell death is a type of receptor-interacting protein-1 (RIP-1)–dependent programmed necrosis called necroptosis, which can be prevented by necrostatin-1, a specific RIP-1 inhibitor. In addition, we found that the remaining Tak1−/− HSPCs died of apoptosis mediated by the caspase-8–dependent extrinsic apoptotic pathway. This apoptosis can be converted into necroptosis by the inhibition of caspase-8 and prevented by inhibiting both caspase-8 and RIP-1 activities. We concluded that HSPCs are heterogeneous populations in response to death signaling stimulation. Tak1 mediates a critical survival signal, which protects against both TNF-α/Fas-RIP-1–dependent necroptosis and TNF-α/Fas-independent apoptosis in HSPCs.

scholarly journals A Human Bone Marrow-Derived Stromal Cell Population with Hemogenic Potential

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1201-1201
Author(s):  
Saloomeh Mokhtari ◽  
Evan Joseph Colletti ◽  
Chad Sanada ◽  
Zanetta S. Lamar ◽  
Paul J Simmons ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem

Abstract During ontogeny, definitive hematopoietic stem/progenitor cells (HSC) are thought to arise from vascular endothelial cells, through an endothelial-to-hematopoietic transition, a natural process that occurs in unique, specialized embryonic hemogenic endothelial cells. Developmental studies, and experiments using pluripotent stem cells in an effort to recapitulate this process and thereby gain a better understanding of the emergence of definitive hematopoiesis, have collectively led to the prevailing view that the hemogenic endothelium constitutes a transient population of cells within the embryo that rapidly disappears during development and is absent in the adult. Herein, we provide the first evidence that at early time points of gestation, prior to the establishment of hematopoiesis, a unique subpopulation of Stro-1+ cells present within the inner part of the developing human bone marrow co-expresses APLNR, a marker of angiogenic mesoderm. Moreover, these Stro-1+APLNR+ cells express multiple other markers described for hemogenic endothelium, and subsequently contribute to the vasculature, cartilage, and bone. Importantly, we also show that cells expressing these same markers of primitive mesoderm/hemogenic endothelium persist at low frequency within the adult marrow. These adult-derived cells can be extensively expanded in vitro without loss of potential, but lack hematopoietic colony-forming potential in vitro. However, upon transplantation into a fetal microenvironment, clonally-derived populations of these adult Stro1+ isolated stromal progenitors (SIPs) not only contribute to the vasculature and nascent BM niches, but also efficiently generate, at a clonal level, hematopoietic stem cells (HSC) that are capable of robust, multilineage hematopoietic reconstitution, with generation of both myeloid and lymphoid cells upon serial transplantation. In conclusion, our studies have thus uncovered the latent potential of a highly expandable population of seemingly vestigial adult human somatic cells, whose ontogenic history includes a phenotype identical to that described for hemogenic endothelium. We have also shown that, if provided with the appropriate/necessary inductive factors, these unique adult cells are capable of giving rise to hematopoietic cells that fulfill the gold standard criteria for bona fide HSC. Therefore, these cells could potentially be more amenable to reprogramming technologies, to produce HSC that could be used to treat/cure a broad variety of blood diseases. Disclosures No relevant conflicts of interest to declare.

TNFα-Induced Apoptosis of Hematopoietic Progenitors Is the Major Cause of Bone Marrow Failure Observed In TAK-1 Knockout Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1557-1557
Author(s):  
Yechen Xiao ◽  
Hongling Li ◽  
Peter Breslin ◽  
Shubin Zhang ◽  
Wei Wei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Knockout Mice ◽  
Bone Marrow Failure ◽  
In Vitro Studies ◽  
Mutant Mice ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Induced Apoptosis

Abstract Abstract 1557 In bone marrow (BM) hematopoietic stem cells/progenitors (HSC/Ps), the apoptotic machinery is tightly controlled by a complex interplay between intrinsic signals and stimuli from the surrounding microenvironment, inducing a dynamically balanced network between pro-apoptotic and anti-apoptotic influences. Disruption of this balance can result in hematopoietic disorders such as various BM failure syndromes. Studies have suggested that tumor necrosis factor-α (TNF-α) and Fas-ligand induce programmed cell death and/or differentiation of HSC/Ps, thus exercising negative regulation over hematopoiesis. However, whether the apoptotic cell death induced by these factors plays a role in BM failure syndromes remains ambiguous. We have reported that transforming growth factor beta-activated kinase-1 (TAK-1) plays an essential role in the survival of HSC/Ps. Mice with TAK-1 deletion in BM hematopoietic cells develop BM failure due to the apoptotic death of mutant HSC/Ps. We have taken advantage of the dramatic phenotypes of this mutant mouse line to closely examine TNF-α and Fas signaling in order to understand which of these is related to the induction of apoptosis of HSC/Ps, and to what degree. To do so, we generated TAK-1/TNFR1a, TAK-1/TNFR1b and TAK-1/Fas compound-mutant mice in order to evaluate which signaling system, when inactivated, permitted the rescue the BM failure defects observed in TAK-1 knockout mice, and the degree to which it did so, using both in vivo and in vitro studies. We found that, as was the case with TAK-1 knockout mice, TAK-1/TNFR1b and TAK-1/Fas compound-mutant mice died within 8 to 10 days after induction of TAK-1 deletion with exactly the same type of BM failure observed in TAK-1 knockout mice. In vitro studies indicated that neither TNFR1b nor Fas deletion was able to protect cells from apoptosis, nor could they rescue the colony-forming ability of TAK-1 mutant HSC/Ps. However, TAK-1/TNFR1a-mutant mice appeared to be healthy one month after induction of TAK1 deletion. By careful analysis, we found that TNFR1a deletion partially rescued the BM failure phenotype of TAK-1 knockout mice. The total numbers of nucleated BM and splenic cells in TAK-1/TNFR1a- mutant mice are approximately 54.7% and 83.8% (respectively) of those of their wild-type littermate controls. These percentages represent significant increases comparing to their littermates with TAK-1 deletion only (7.5% and 17% of WT control). In vitro studies demonstrated that TNFR1a deletion restored colony-forming ability in 20–30% of TAK1-knockout HSC/Ps. Currently, we are in the process of analyzing the hematopoietic phenotypes of TAK-1/TNFR1a/1b triple-mutant mice in order to determine whether the complete inactivation of TNF-α signaling further reverses the hematopoietic defects seen in TAK1-knockout mice. Our study demonstrated that TNF-α, via its receptor 1a-induced apoptosis, contributes substantially to the loss of HSC/Ps in TAK-1 knockout mice. Our results also suggest that pro-apoptotic signaling other than TNF-α is also involved in the BM failure syndrome observed in TAK-1 mutant mice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IFNγ-Induced Necroptosis Contributes to Hematopoietic Stem and Progenitor Cell Death and Bone Marrow Failure

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1485-1485
Author(s):  
Joanne O'Donnell ◽  
Justine E. Roderick ◽  
Manolis Pasparakis ◽  
Michelle Kelliher
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Progenitor Cell ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Elevated Serum ◽  
Type Ii ◽  
Cell Type ◽  
Receptor Ligands ◽  
Hematopoietic Stem

Abstract RIPK1 has important kinase-dependent and kinase-independent scaffolding functions that prevent or activate necroptosis or apoptosis. Complete RIPK1 deficiency results in cell death and widespread inflammation yet tissue specific RIPK1 deletion can result in apoptosis, necroptosis and/or systemic inflammation, depending on the cell type. We have previously demonstrated that a hematopoietic RIPK1 deficiency results in constitutive activation of RIPK3 and MLKL and induction of necroptosis (Roderick et al, PNAS, 2014). These mice exhibit elevated serum TNFα and IFNγ levels, hematopoietic stem and progenitor cell (HSPC) loss, and ultimately succumb to bone marrow failure (BMF). When mice with a hematopoietic RIPK1 deficiency were placed on a RIPK3 deficient background, plasma pro-inflammatory cytokine and chemokine levels were reduced, HSPC numbers increased and BMF was significantly delayed. These mouse genetic data demonstrate that necroptotic death contributes to BMF in the mouse. To identify the receptor/ligands that trigger necroptosis, we generated mice with a hematopoietic RIPK1-deficiency on the Tnfr1-/-and Tnfr1-/-Tnfr2-/-genetic backgrounds. An absence of TNF signaling failed to prevent necroptosis and consequently, vav-iCre Ripk1f/f Tnfr1-/- Tnfr2-/-mice succumbed to BMF. Because type II interferons can also induce necroptosis, we generated vav-iCre Ripk1f/f Ifngr1-/- mice. These mice appear phenotypically normal demonstrating that an absence of IFNγ signaling prevents HSPC necroptosis and BMF.Collectively, these data may implicate IFNγ-mediated, RIPK3-dependent necroptosis in human BMF syndromes and raise the intriguing possibility that the progressive HSPC elimination observed in these patients reflects in part, IFNγ-induced necroptotic death. Disclosures No relevant conflicts of interest to declare.

A Stro-1+ human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic stem/progenitor cells in a serum-free culture system

2006 ◽  
Vol 34 (10) ◽  
pp. 1353-1359 ◽  
Author(s):  
Raquel Gonçalves ◽  
Cláudia Lobato da Silva ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
Graça Almeida-Porada
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Culture System ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Serum Free

scholarly journals The Effectiveness of Rapamycin Combined with Eltrombopag in Murine Models of Immune-Mediated Bone Marrow Failure

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Rong Fu ◽  
Shaoxue Ding ◽  
Xiaowei Liang ◽  
Tian Zhang ◽  
Zonghong Shao
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Standard Dose ◽  
Bone Marrow Failure ◽  
Statistical Significance ◽  
Murine Models ◽  
Immune Mediated ◽  
Tnf Α ◽  
Significant Difference ◽  
Ifn Γ

Recent research has found that Rapamycin (Rapa) was an effective therapy in mouse models of immune-mediated bone marrow failure. However, it has not achieved satisfactory effect in clinical application. At present, many studies have confirmed that Eltrombopag (ELT) combined with IST can improve the curative effect of AA patients. Then whether Rapa combined with Elt in the treatment of AA will be better than single drug application. In this study, we tested efficacy of Rapa combined with Elt as a new treatment in mouse models of immune-mediated bone marrow failure. Compared with AA group, the whole blood cell count of Rapa+Elt group increased significantly (Figure 1A) (P<0.05). Survival of mice of Rapa+Elt group was significantly higher than that in the Rapa group (p <0.01)(Figure 1B).There was no obvious difference in the numbers of NK cells and their subsets were noted in Rapa group,CsA group and Rapa+Elt group.The expression of NKG2D on peripheral functional NK cells was up-regulated in CsA group, Rapa group and Rapa+Elt group compared with AA group (P<0.05). But there was no significant difference between effect of Rapa and CsA on the function of NK cells (Figure 1C).When Rapa combined with Elt, the expression of CD80 and CD86 are down-regulated more compared to Rapa group, but there is no statistical significance. Although these results suggested that Rapa+Elt had no statistical significance effect on numbers of mDC and expression of its functional molecule CD80 and CD86, the combined therapy still indicated that there is a potential synergy with immunosuppressant on AA mice to improve its outcome (Figure 1D).The results showed that CD4+/CD8+ ratio in CsA group, Rapa group, Rapa + Elt group had an obvious elevation than AA group (all P<0.05). But there were no significant difference among the three groups on the CD4+/CD8+ ratio (Figure 1E,1F). As for INF-gamma, Rapa can reduce the secretion of IFN-γ from CD8+T cells with efficacy similar to that of the standard dose of CsA, and had a better outcome when combined with Elt in bone marrow failure mice (Figure 1E,1G).CsA group, Elt group, Rapa group, Rapa + Elt group showed notable increased ratio of Tregs compared with AA group, among which there were only Rapa group, Rapa + Elt group showed statistical significance(P<0.05). for IL-10/Tregs ratio, Rapa group and Rapa +Elt group were superior to than CsA group(P<0.05) (Figure 1H,1I).Rapa+Elt group and Rapa showed more lower level of IFN-γ compared with CsA group, and there was significant difference in Rapa+Elt group(P<0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO and TNF-α, the Rapa+Elt group showed more significant effect than Rapa or Elt alone(Figure1J). Thus, Rapa+Elt significantly down-regulated cytokines related to Th1 immune responses, such as IFN-γ, and upregulated cytokines related to Th2 immune responses, such as IL-10. To some extent, Rapa combined with Elt has a synergistic effect with CsA and Rapa alone in AA treatment. Conclusions In this study, Although Rapa combined with Elt had no significant improvement effect on the number and function of NK cells and their subsets, mDCs, and CD4+/CD8+ ratio in AA mice compared with Rapa alone, the Rapa+Elt can increase the secretion of IL-10 of Tregs and the number of Tregs, but has no significant effect on the number of Treg cells compared to with Rapa alone. Compared with AA group, the level of plasma IFN-γ, IL-2 and TNF-α decreased significantly (P<0.05), but IL-10, IL-4, IL-5 and IL-1β increased significantly in Rapa group(P<0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO and TNF-α, the Rapa+Elt group showed more significant effect than Rapa alone. intervention treatment with Rapa in combination Elt in the AA mouse model more obviously ameliorated pancytopenia, improved bone marrow cellularity, and extended animal survival in a manner comparable to the standard dose of CsA and Rapa alone. Combination therapy support potential clinical utility in aplastic anemia treatment, which may further improve the efficacy of AA patients. Keywords: Rapamycin, Eltrombopag, murine models, bone marrow failure Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals Eltrombopag maintains human hematopoietic stem and progenitor cells under inflammatory conditions mediated by IFN-γ

Blood ◽  
2019 ◽  
Vol 133 (19) ◽  
pp. 2043-2055 ◽  
Author(s):  
Luigi J. Alvarado ◽  
Heather D. Huntsman ◽  
Hai Cheng ◽  
Danielle M. Townsley ◽  
Thomas Winkler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Binding Site ◽  
Bone Marrow Failure ◽  
Interferon Γ ◽  
General Mechanism ◽  
Clinical Activity ◽  
Critical Pathway ◽  
Hematopoietic Stem ◽  
Therapeutic Implications ◽  
Ifn Γ

Abstract The proinflammatory cytokine interferon-γ (IFN-γ) has been implicated in human hematopoietic stem and progenitor cell (HSPC) depletion in immune-mediated bone marrow failure syndromes. We show that IFN-γ specifically prevents full engagement of thrombopoietin (TPO), a primary positive regulator of HSPC survival, to its receptor (c-MPL) via steric occlusion of the low-affinity binding site, contributing to perturbation of TPO-induced signaling pathways and decreased survival of human HSPCs. Eltrombopag, a synthetic small molecule mimetic of TPO that interacts with c-MPL at a position distinct from the extracellular binding site of TPO, bypasses this inhibition, providing an explanation for its clinical activity in bone marrow failure, despite already elevated endogenous TPO levels. Thus, IFN-γ–mediated perturbation of TPO:c-MPL complex formation and the resulting inhibition of a critical pathway of growth factor cell signaling may represent a general mechanism by which IFN-γ impairs the function of human HSPCs. This understanding could have broad therapeutic implications for various disorders of chronic inflammation.

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