Response Of Hematopoietic Stem Cells Research Articles

Trim47 prevents hematopoietic stem cell exhaustion during stress by regulating MAVS-mediated innate immune pathway

The maintenance of hematopoietic stem cell (HSC) functional integrity is essential for effective hematopoietic regeneration when suffering from injuries. Studies have shown that the innate immune pathways play crucial roles in the stress response of HSCs, whereas how to precisely modulate these pathways is not well characterized. Here, we identify the E3 ubiquitin ligase tripartite motif-containing 47 (Trim47) as a negative regulator of the mitochondrial antiviral-signaling protein (MAVS)-mediated innate immune pathway in HSCs. We find that Trim47 is predominantly enriched in HSCs, and its deficiency impairs the function and survival of HSCs after exposure to 5-flurouracil (5-FU) and irradiation (IR). Mechanistically, Trim47 impedes the excessive activation of the innate immune signaling and inflammatory response via K48-linked ubiquitination and degradation of MAVS. Collectively, our findings demonstrate a role of Trim47 in preventing stress-induced hematopoietic failure and thus provide a promising avenue for treatment of related diseases in the clinic.

Elevated Functional Hematopoietic Stem and Progenitor Cells in the Peripheral Blood of G-CSF-Mobilized Dnmt3a-Mutant Mice

Clonal hematopoiesis (CH) arising from somatic mutations in hematopoietic stem cells (HSCs) is associated with increased risk of developing several non-hematologic and hematologic diseases. In the context of stem cell transplantation, donor HSCs carrying CH-associated somatic mutations can be passed from donor to recipient during stem cell transplantation. Most literature supports that there is no negative impact on recipient survival or outcomes after allogeneic stem cell transplantation (allo-HSCT) from donors with CH. However, it is less clear how allo-HSCT donors with CH respond to common mobilization methods such as Neupogen (G-CSF). As hematopoietic stem and progenitor cells (HSPCs) with CH mutations have been found to respond to external stimuli such as inflammation in a manner distinct from wild-type HSPCs, it is plausible that CH-mutant HSPCs also distinctly respond to G-CSF mobilization. It is important to understand whether the process of mobilization differentially enriches for CH-mutant HSPCs in the mobilized peripheral blood product. To address this gap in knowledge, we used a murine model of Dnmt3a-mutant CH. After five consecutive days of Neupogen (G-CSF) administration, modeling the procedure for human HSC mobilization, we observed that Dnmt3a-mutant mice had significantly increased white blood cell, neutrophil and monocyte counts compared to control mice. We observed a significant increase in cell surface marker-defined HSPCs in the peripheral blood and spleen of G-CSF-treated Dnmt3a-mutant mice compared to G-CSF-treated control mice. Using in vitro colony-forming unit assays, the peripheral blood of G-CSF-treated Dnmt3a-mutant mice was found to contain a greater number of functional hematopoietic progenitors compared to G-CSF-treated control mice. To determine if the peripheral blood of G-CSF-treated Dnmt3a-mutant mice had a greater abundance of functional HSCs, we transplanted equal volumes of mobilized blood from Dnmt3a-mutant and control mice into primary and secondary lethally irradiated wild-type recipient mice. We observed increased long-term engraftment of mobilized blood from Dnmt3a-mutant mice compared to control mice in both primary and secondary recipient animals. At a mechanistic level, HSC mobilization is facilitated by myeloid cells that respond directly to G-CSF via the G-CSF receptor ( Csf1r). In Dnmt3a-mutant mice, we observed increased Csf1r expression in myeloid progenitor and mature myeloid cells compared to control mice, providing a putative mechanism by which increased mobilization is enabled. Together, our data support differential mobilization potential of Dnmt3a-mutant compared to control HSCs in response to G-CSF, which may have implications in the context of allo-HSCT.

Chromatin Remodeler DEK Is a Sensor to Replication Stress and Contributes to HSC Defects in Fanconi Anemia

Hematopoietic stem cells (HSCs) have self-renewal capacity and response for life-long hematopoiesis. Any interference with normal DNA replication leads to replication stress, which drives function decline in HSCs. However, the response of HSCs to replication stress remains largely unknown. Here, draw a transcriptional and epigenetic landscape of HSCs under replication stress. We found that HSCs required opened chromatin to counteract replication stress, along with altered H3K27 acetylation and H3K4 trimethylation. The increased chromatin accessibility was mainly attributed to the reduced chromatin remodelers DEK, while DEK overexpressed HSCs confronted with a strong replication challenge, shown by increased γ-H2AX. Excessive DEK protein impeded proliferation and impaired HSC maintenance (>50% reduction), along with reduced oxidative phosphorylation and protein synthesis. Notably, we found that DEK protein was abnormally accumulated in BM CD34 + cells in Fanconi anemia, which arises from mutations in FA genes essential for replication stress tolerance. Inhibition of DEK substantially recovered HSC proliferation capacity in vitro and engraftment in vivo. At the molecular level, we identified that ATF2 directly promoted the transcription of DEK, especially relying on phosphorylated ATF2 (ser69/71) rather than phosphorylated ATF2 (ser490/498). Rad3-related (ATR) induced the phosphorylation of ATF2 at ser490/498 and mediated the reduction of DEK under replication stress. Nonetheless, knock-down of FA genes (FANCD2) in HSCs resulted in increased phosphorylated ATF2 (ser69/71), which might contribute to DEK accumulation in FA. Collectively, our findings uncover the landscape of HSC response to replication stress, reveal a novel ATR-ATF2-DEK axis helping HSCs counteract replication stress, and suggest inhibition of DEK as a therapeutic utility in FA.

TNFα-Driven Interplay between Hematopoietic Stem Cells and Antigen-Specific Cytotoxic T Cells

Clonal hematopoiesis (CH) is governed by aberrant hematopoietic stem cell (HSC) clones with leukemia-associated somatic mutations that arise spontaneously during organismal aging or after exposure to genotoxic insults. Cells with a somatic mutation can be eliminated through presentation of neoantigens on MHC class I (MHC-I) to antigen-specific cytotoxic T cells. Indeed, many of the CH-related mutations, such as JAK2V617F, are reported or predicted to elicit antigen-specific cytotoxic T cell response. On the other hand, accumulating evidence suggests that inflammatory environment facilitates clonal expansion of abnormal HSCs. However, much remains to be elucidated on whether HSCs expressing such non-self peptides can be eliminated by antigen-specific cytotoxic T cells and how inflammation affects the T cell-mediated HSC regulation. Our RNA-seq analyses revealed that genes involved in MHC-I-dependent antigen presentations, such as Nlrc5, H2-kb and B2m, are most abundantly expressed in HSCs (Lin -/c-Kit +/Sca-1 +/Flk2 -/CD48 -/CD150 +) among murine bone marrow immature hematopoietic cells. Using transgenic mice that ubiquitously express a model antigen ovalbumin (OVA) and a monoclonal antibody that specifically recognizes MHC-I-OVA complex, we found that while MHC-I protein is abundantly expressed on the surface of hematopoietic cells except granulocytes, HSCs and multipotent progenitor cells (MPPs) possess greater capacity to present intracellular antigens through MHC-I compared to myeloid progenitor and mature cells. Remarkably, in vitro co-culture experiments revealed that whereas OVA-expressing (OVA +) HSCs, MPPs and myeloid progenitors could directly activate OVA-specific OT-I CD8 + T cells through MHC-I-dependent antigen presentation, HSCs are the most susceptible population to the killing effect by cytotoxic T cells. Indeed, co-infused OT-I CD8 + T cells totally abolished the long-term repopulation capacity of OVA + HSCs in a serial transplantation setting, and adoptive transfer of OT-I CD8 + T cells to mixed chimeric mice that harbor both wild-type and OVA + hematopoietic cells completely and specifically eliminated hematopoiesis governed by OVA + HSCs. TNFα is not only a prototypical inflammatory cytokine but also one of the major cytokines that are secreted by CD8 + cytotoxic T cells. Notably, genes directly involved in T cell regulation including Cxcl9, Cd274, and Pdcd1lg2 were induced in HSCs, but not myeloid progenitor cells, upon exposure to TNFα (Yamashita and Passegué, Cell Stem Cell 2019). Indeed, upon co-culturing OT-I CD8 + T cells produced TNFα as well as IFNγ and Granzyme B and OVA + HSCs but not myeloid progenitor cells produced CXCL9 largely in a TNFα receptor-dependent manner. Interestingly, such HSC response to T cell-derived TNFα was critical for naïve CD8 + T cells to rapidly differentiate to effector cytotoxic T cells, as TNFα receptor-deficient ( Tnfr1 -/-Tnfr2 -/-) OVA + HSCs stimulated proliferation and cytokine production of OT-I CD8 + T cells but failed to stimulate Granzyme B production, whereas IFNγ receptor deficiency ( Ifngr -/-) did not have such effect. As a result, Tnfr1 -/-Tnfr2 -/- OVA + HSCs, but not Ifngr -/- OVA + HSCs, could escape from the killing by OT-I CD8 +T cells. In sharp contrast, prior exposure to environmental TNFα, but not to IL-1β or IL-6, before interaction with OT-I CD8 + T cells rendered OVA + HSCs resistant to the killing by OT-I CD8 + T cells largely through upregulation of PD-L1 and PD-L2. Of note, upon 24-month aging or acquisition of Jak2V617F mutation, OVA + HSCs still maintained robust MHC-I-dependent antigen presentation activity and high susceptibility to the killing by OT-I CD8 + T cells, though prior exposure to TNFα allowed for their immune escape. Consequently, adoptive transfer of OT-I CD8 + T cells completely and specifically eliminated Jak2V617F-mutant OVA + HSCs and reversed myeloproliferative neoplasms in the mixed chimeric mice. Taken together, our results reveal the robustness of HSC quality control via MHC-I-dependent antigen presentation to antigen-specific cytotoxic T cells. They also highlight a critical but complex role of TNFα in active interplay between HSCs and antigen-specific cytotoxic T cells as well as immune evasion by HSCs during inflammation. Specifically targeting abnormal HSCs by antigen-specific cytotoxic T cells may pave the way to eradicate mutant HSC clones and prevent CH-associated disease.

Investigation of CD83 As a Novel Human Hematopoietic Stem Cell Inflammatory Activation Marker

Hematopoietic stem cells (HSC) are responsible for life-long blood production and must balance homeostasis with sudden demand situations such as infections. However, the heterogeneity of molecular programs engaged by acute inflammatory stimuli to activate emergency hematopoiesis at the level of HSC are poorly understood. In a recent study linking human HSC heterogeneity to aging, recovery from severe COVID-19 infection, and clonal hematopoiesis (CH), we discovered two HSC subsets through multiomic analysis, denoted HSC-I and -II, in a xenograft model of inflammation-recovery. While HSC-I had few inflammation-driven molecular changes, HSC-II showed sustained epigenetic and transcriptional alterations in response to dual tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS) challenges despite a 2.5-month recovery period. Importantly, a signature of long-term memory T cells (Akondy et al 2017) was enriched in HSC-II (Zeng, Nagree, et al, in submission). We interrogated the human HSC multiomic signatures uncovered from this human xenograft model to identify potential regulators associated with HSC response to and recovery from acute inflammation. We identified CD83 as a candidate that is differentially expressed between our HSC subsets. Similarly, we found CD83 expression to be enriched in a subset of adult-derived HSCs from patients where CH status was established and enrichment of an HSC-II signature observed (Jakobsen et al, submitted). CD83, first identified as a dendritic cell marker, is expressed on many immune cells, with linkage to controlling murine T regulatory (T-Reg) vs memory cell fate (Doebbeler et al, 2018), but has not previously been shown to be relevant for human HSC biology. Flow cytometry analysis of cord blood (CB) hematopoietic stem and progenitor cells (HSPC) showed CD83 to be low or absent in HSC (Figure A) and their downstream progeny at steady state. However, robust CD83 expression was observed in multiple HSPC subpopulations including long-term (LT-) and short-term (ST-) HSC within 16 hours of ex vivo culture, with highest expression in LT-HSC (Figure A). Importantly, CD83 surface expression on LT-HSC was enhanced following culture with TNFα (Figure A). Bystander effects were excluded by demonstrating similar findings after culture of purified HSC populations. Acute in vivo LPS treatment of mice xenografted with CB-derived HSC was sufficient to upregulate CD83 surface expression after 16 hours on LT-HSC/HSPC. To elucidate the potential function of CD83-expressing HSC/multipotent progenitors (MPP), CD19-CD34+CD38-CD45RA- cells were sorted from xenografts following acute inflammatory challenge for low and high CD83 expression and subjected to bulk RNA sequencing. We found enrichment of a gene expression signature specific to activated human HSCs (Garcia-Prat et al, 2021) within CD83-positive HSC/MPP (Figure B). Strikingly, enrichment of a signature of stimulated vs resting T cells (Galetti et al, 2020) and of effector T cells (Akondy et al 2017) was found in CD83-positive compared to CD83-negative HSC/MPP (Figure B). T cell effector function is in part dictated by metabolic reprogramming. We thus examined reactive oxygen species (ROS) levels in LT-HSC following ex vivo culture with TNFα and found lower ROS levels with CD83 expression. We therefore hypothesize that CD83 may be an ‘effector’ marker defining a distinct HSC subset during homeostatic or inflammatory activation that is subsequently fated to become memory-like HSC-II. We are undertaking studies to determine the mechanistic role of CD83 in HSC function and HSC-II formation via Cas9-sgRNA-mediated knockout or lentivirus-mediated overexpression of CD83. In summary, CD83 represents a long-sought marker of inflammatory activation at the HSC level. CD83 may have significant clinical potential as a biomarker of aberrant activation in gene therapy manufacturing protocols or for HSC transplantation. Future studies will shed light on whether CD83 can functionally regulate the molecular programs underlying human HSC heterogeneity.

Different niches for stem cells carrying the same oncogenic driver affect pathogenesis and therapy response in myeloproliferative neoplasms

Aging facilitates the expansion of hematopoietic stem cells (HSCs) carrying clonal hematopoiesis-related somatic mutations and the development of myeloid malignancies, such as myeloproliferative neoplasms (MPNs). While cooperating mutations can cause transformation, it is unclear whether distinct bone marrow (BM) HSC-niches can influence the growth and therapy response of HSCs carrying the same oncogenic driver. Here we found different BM niches for HSCs in MPN subtypes. JAK–STAT signaling differentially regulates CDC42-dependent HSC polarity, niche interaction and mutant cell expansion. Asymmetric HSC distribution causes differential BM niche remodeling: sinusoidal dilation in polycythemia vera and endosteal niche expansion in essential thrombocythemia. MPN development accelerates in a prematurely aged BM microenvironment, suggesting that the specialized niche can modulate mutant cell expansion. Finally, dissimilar HSC-niche interactions underpin variable clinical response to JAK inhibitor. Therefore, HSC-niche interactions influence the expansion rate and therapy response of cells carrying the same clonal hematopoiesis oncogenic driver.

Genotoxic aldehyde stress prematurely ages hematopoietic stem cells in a p53-driven manner

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.

Both maternal IFNγ exposure and acute prenatal infection with Toxoplasma gondii activate fetal hematopoietic stem cells

Infection directly influences adult hematopoietic stem cell (HSC) function and differentiation, but the fetal hematopoietic response to infection during pregnancy is not well‐studied. Here, we investigated the fetal hematopoietic response to maternal infection with Toxoplasma gondii (T. gondii), an intracellular parasite that elicits Type II IFNγ‐mediated maternal immunity. While it is known that maternal infection without direct pathogen transmission can affect fetal immune development, the effects of maternal IFNγ on developing HSCs and the signals that mediate these interactions have not been investigated. Our investigation reveals that the fetal HSCs respond to T. gondii infection with virulence‐dependent changes in proliferation, self‐renewal potential, and lineage output. Furthermore, maternal IFNγ crosses the fetal–maternal interface, where it is perceived by fetal HSCs. By comparing the effects of maternal IFNγ injection with maternal T. gondii infection, we reveal that the effects of IFNγ treatment mimic some aspects of the fetal HSC response to infection. Moreover, our findings illuminate that the fetal HSC response to prenatal infection is distinct from the adult HSC response to IFNγ‐induced inflammation. Altogether, our data disentangle the role of infection‐induced inflammatory cytokines in driving the expansion of downstream hematopoietic progenitors.

The impact of prenatal inflammation on hematopoietic development.

Inflammation is now recognized as a major regulator of hematopoietic stem cell (HSC) function. Adult hematopoietic stem cells can adaptively modulate hematopoietic output in direct response to acute infection and inflammation. Conversely, prolonged exposure to inflammation can drive impaired HSC function, clonal expansion, and malignant transformation. As compared with adult hematopoiesis, the effects of prenatal inflammation on developing hematopoietic stem cells are understudied. Inflammatory cues directly activate adult HSCs in the bone marrow, but the response of fetal HSCs to maternal inflammation is underexplored. Recent evidence demonstrates that maternal inflammation can be detected by fetal hematopoietic stem and progenitor cells (HSPCs) within the fetal liver and that the same inflammatory cues evoke fundamentally distinct responses during development. The responses of developing stem and progenitor cells and the specialized immune cells they produce have important implications for postnatal hematopoietic output and immune function. We discuss recent insights into the response of fetal hematopoiesis to prenatal inflammation and examine how recent discoveries regarding the contribution of fetal hematopoiesis to the adult hematopoietic system will influence future studies.

EVA1A regulates hematopoietic stem cell regeneration via ER-mitochondria mediated apoptosis

Excessive protein synthesis upon enhanced cell proliferation frequently results in an increase of unfolded or misfolded proteins. During hematopoietic regeneration, to replenish the hematopoietic system, hematopoietic stem cells (HSCs) are activated and undergo a rapid proliferation. But how the activated HSCs respond to the proliferation pressure is still ambiguous; The proper control of the functional reservoir in the activated HSCs remains poorly understood. Here, we show a significant upregulation of EVA1A protein associated with the increase of ER stress during hematopoietic regeneration. Deletion of Eva1a significantly enhances the regeneration capacity of HSCs by inhibiting the ER stress-induced apoptosis. Mechanistically, the expression of EVA1A protein was upregulated by CHOP, and thereby promoted the ER-mitochondria interlinking via MCL1, which resulted in mitochondria-mediated apoptosis. These findings reveal a pathway for ER stress responses of HSCs by the EVA1A mediated apoptosis, which play an important role in HSCs regeneration.

Repair of Limb Ischemia Is Dependent on Hematopoietic Stem Cell Specific-SHP-1 Regulation of TGF-β1.

Hematopoietic stem cell (HSC) therapy has shown promise for tissue regeneration after ischemia. Therefore, there is a need to understand mechanisms underlying endogenous HSCs activation in response to ischemic stress and coordination of angiogenesis and repair. SHP-1 plays important roles in HSC quiescence and differentiation by regulation of TGF-β1 signaling. TGF-β1 promotes angiogenesis by stimulating stem cells to secrete growth factors to initiate the formation of blood vessels and later aid in their maturation. We propose that SHP-1 responds to ischemia stress in HSC and progenitor cells (HSPC) via regulation of TGF-β1. A mouse hind limb ischemia model was used. Local blood perfusion in the limbs was determined using laser doppler perfusion imaging. The number of positive blood vessels per square millimeter, as well as blood vessel diameter (μm) and area (μm2), were calculated. Hematopoietic cells were analyzed using flow cytometry. The bone marrow transplantation assay was performed to measure HSC reconstitution. After femoral artery ligation, TGF-β1 was initially decreased in the bone marrow by day 3 of ischemia, followed by an increase on day 7. This pattern was opposite to that in the peripheral blood, which is concordant with the response of HSC to ischemic stress. In contrast, SHP-1 deficiency in HSC is associated with irreversible activation of HSPCs in the bone marrow and increased circulating HSPCs in peripheral blood following limb ischemia. In addition, there was augmented auto-induction of TGF-β1 and sustained inactivation of SHP-1-Smad2 signaling, which impacted TGF-β1 expression in HSPCs in circulation. Importantly, restoration of normal T GF-β1 oscillations helped in the recovery of limb repair and function. HSPC-SHP-1-mediated regulation of TGF-β1 in both bone marrow and peripheral blood is required for a normal response to ischemic stress.

Abstract LB555: Augmented Txnip-p53-p21 axis preserves Dnmt3a mutant hematopoietic stem cells during inflammatory stress

Abstract Clonal hematopoiesis (CH), the age-related expansion of specific clones in the blood system has been considered as a pre-malignant condition of blood cancers. Approximately 50% of CH manifests from DNMT3A mutations acquired in the hematopoietic stem cells (HSCs), suggesting these mutations may convey a fitness advantage to HSCs during hematopoietic stress. However, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is still lacking. Here, we used interferon-gamma (IFNg) as a model to study the mechanisms by which Dnmt3a mutations enhances HSC fitness under recurrent inflammatory stress. To represent the spectrum of DNMT3A variants found in humans, mouse genetic models were generated by specifically deleting Dnmt3a heterozygously (Vav-Cre;Dnmt3afl/+ = Dnmt3aHET) or homozygously (Vav-Cre;Dnmt3afl/fl = Dnmt3aKO) from the hematopoietic system. A hematopoietic system-specific knockin model analogous to the hotspot point mutation most prevalent in AML (Vav-Cre;Dnmt3aR878H/+ = Dnmt3aR878) was also included in the analysis. Competitive transplantation assays coupled with inflammatory and proliferative challenges suggested that Dnmt3aKO and Dnmt3aR878 HSCs were specifically tolerant of the deleterious effects of IFNg on HSC self-renewal and clonal expansion. When the competition between Dnmt3a-mutant and control HSCs under IFNg exposure was directly quantified in a novel mouse model, we found Dnmt3a-mutant HSCs resisted IFNg-mediated depletion due to an enhanced fitness advantage. Mechanistically, DNA hypomethylation-associated over-expression of Txnip in Dnmt3a-mutant HSCs was identified by coupling single-cell RNA-sequencing and Whole-Genome Bisulfite sequencing. The sustained Txnip levels in Dnmt3aKO HSCs lead to p53 stabilization and upregulation of p21 under IFNg challenge, further correlated with a retained quiescence due to a prolonged G0 exit in response to IFNg exposure. Implementing biochemical studies, we observed Txnip mediated an enrichment of p53 at p21 promoter under IFNg exposure in Dnmt3aKO but not control 32D cells. Knocking down Txnip by shRNA normalized p53 occupancy at p21 promoter and rescued IFNg-associated p21 upregulation in Dnmt3aKO 32D cells. Functionally, knocking down Txnip and p21 sensitized Dnmt3aKO HSCs to IFNg-induced cell cycle activation ex vivo. In vivo, down-regulation of p21 had no effect on control HSCs in response to IFNg exposure, but it completely primed Dnmt3aKO and Dnmt3aR878 HSCs to IFNg-associated exhaustion in a transplantation experiment. In summary, our data suggest an augmented Txnip-p53-p21 axis preserves the functional potential of Dnmt3a-mutant HSCs under inflammatory stress, which highlights a novel mechanism to explain the increased fitness of Dnmt3a-mutant HSCs and supports rationale for developing interventions to mitigate expansion of pre-malignant clones as a method of blood cancer prevention. Citation Format: Christine R. Zhang, Elizabeth Ostrander, Won Kyun Koh, Ostap kukhar, Hamza Celik, Jeffrey Magee, Grant Challen. Augmented Txnip-p53-p21 axis preserves Dnmt3a mutant hematopoietic stem cells during inflammatory stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB555.

A complex proinflammatory cascade mediates the activation of HSCs upon LPS exposure in vivo

AbstractInfections are a key source of stress to the hematopoietic system. While infections consume short-lived innate immune cells, their recovery depends on quiescent hematopoietic stem cells (HSCs) with long-term self-renewal capacity. Both chronic inflammatory stress and bacterial infections compromise competitive HSC capacity and cause bone marrow (BM) failure. However, our understanding of how HSCs act during acute and contained infections remains incomplete. Here, we used advanced chimeric and genetic mouse models in combination with pharmacological interventions to dissect the complex nature of the acute systemic response of HSCs to lipopolysaccharide (LPS), a well-established model for inducing inflammatory stress. Acute LPS challenge transiently induced proliferation of quiescent HSCs in vivo. This response was not only mediated via direct LPS-TLR4 conjugation on HSCs but also involved indirect TLR4 signaling in CD115+ monocytic cells, inducing a complex proinflammatory cytokine cascade in BM. Downstream of LPS-TLR4 signaling, the combined action of proinflammatory cytokines such as interferon (IFN)α, IFNγ, tumor necrosis factor-α, interleukin (IL)-1α, IL-1β, and many others is required to mediate full HSC activation in vivo. Together, our study reveals detailed mechanistic insights into the interplay of proinflammatory cytokine-induced molecular pathways and cell types that jointly orchestrate the complex process of emergency hematopoiesis and HSC activation upon LPS exposure in vivo.

Deleterious effect of bone marrow-resident macrophages on hematopoietic stem cells in response to total body irradiation

AbstractBone marrow (BM) resident macrophages interact with a population of long-term hematopoietic stem cells (LT-HSCs) but their role on LT-HSC properties after stress is not well defined. Here, we show that a 2 Gy-total body irradiation (TBI)-mediated death of LT-HSCs is associated with increased percentages of LT-HSCs with reactive oxygen species (ROS) and of BM resident macrophages producing nitric oxide (NO), resulting in an increased percentage of LT-HSCs with endogenous cytotoxic peroxynitrites. Pharmacological or genetic depletion of BM resident macrophages impairs the radio-induced increases in the percentage of both ROS+ LT-HSCs and peroxynitrite+ LT-HSCs and results in a complete recovery of a functional pool of LT-HSCs. Finally, we show that after a 2 Gy-TBI, a specific decrease of NO production by BM resident macrophages improves the LT-HSC recovery, whereas an exogenous NO delivery decreases the LT-HSC compartment. Altogether, these results show that BM resident macrophages are involved in the response of LT-HSCs to a 2 Gy-TBI and suggest that regulation of NO production can be used to modulate some deleterious effects of a TBI on LT-HSCs.

3176 – MAC-1 MARKS A QUIESCENT AND FUNCTIONALLY SUPERIOR HSC SUBSET DURING REGENERATION

Hematopoietic stem cells are capable of sustaining a lifelong production of mature blood cells as well as rapid hematopoietic regeneration upon acute stress. Mouse hematopoietic stem cells (HSCs) at steady state have been extensively defined both molecularly and functionally. When exposed to regenerative stress, however, HSCs undergo several immunophenotypical changes that limit the possibilities of isolating them at high purity. As a consequence, actively regenerating HSCs have remained less defined, and it is of great interest to identify markers that specifically label activated HSCs to gain further knowledge about their molecular and functional properties. Macrophage-1 antigen (Mac-1), a cell surface marker typically expressed by myeloid cells, has been shown to be upregulated on HSCs in response to the cytotoxic drug 5-fluorouracil (5FU). Here, we assessed the expression of Mac-1 on HSCs during regeneration following transplantation and observed a transient but distinct increase in Mac-1 expression during the early reconstitution phase 4 weeks after transplantation. Remarkably, from serial transplantation experiments, we found that the functional reconstitution potential was highly enriched in this Mac-1+ portion of the HSC pool. A similar correlation between Mac-1 expression and functional HSC activity was seen following 5-FU treatment. Moreover, in contrast to previous reports, we found that Mac-1 expression inversely correlates with cell cycling, and global transcriptome analysis showed that regenerating Mac-1+ HSCs share molecular features with stem cells with low mitotic history and low historic progeny output. Taken together, our results suggest that Mac-1 expression marks predominantly quiescent and functionally superior HSCs during early regeneration. Hematopoietic stem cells are capable of sustaining a lifelong production of mature blood cells as well as rapid hematopoietic regeneration upon acute stress. Mouse hematopoietic stem cells (HSCs) at steady state have been extensively defined both molecularly and functionally. When exposed to regenerative stress, however, HSCs undergo several immunophenotypical changes that limit the possibilities of isolating them at high purity. As a consequence, actively regenerating HSCs have remained less defined, and it is of great interest to identify markers that specifically label activated HSCs to gain further knowledge about their molecular and functional properties. Macrophage-1 antigen (Mac-1), a cell surface marker typically expressed by myeloid cells, has been shown to be upregulated on HSCs in response to the cytotoxic drug 5-fluorouracil (5FU). Here, we assessed the expression of Mac-1 on HSCs during regeneration following transplantation and observed a transient but distinct increase in Mac-1 expression during the early reconstitution phase 4 weeks after transplantation. Remarkably, from serial transplantation experiments, we found that the functional reconstitution potential was highly enriched in this Mac-1+ portion of the HSC pool. A similar correlation between Mac-1 expression and functional HSC activity was seen following 5-FU treatment. Moreover, in contrast to previous reports, we found that Mac-1 expression inversely correlates with cell cycling, and global transcriptome analysis showed that regenerating Mac-1+ HSCs share molecular features with stem cells with low mitotic history and low historic progeny output. Taken together, our results suggest that Mac-1 expression marks predominantly quiescent and functionally superior HSCs during early regeneration.

BCL-Xl Driven Accumulation of Dysfunctional Mitochondria in Aged Stromal Cells Impairs the Haematopoietic Stem Cell Response to Stress

The burden of infections is known to increase with age. Not only is ageing associated with greater susceptibility to infections but also an increase in subsequent morbidity and mortality. The bone marrow (BM) niche is essential for the body's response to infection. Haematopoietic stem and progenitor cells (HSPCs) heavily rely on their supporting BM microenvironment to effectively expand and differentiate in response to stress and infection (1). The role of senescent cells has been explored in a number of age-related diseases including acute myeloid leukaemia (2). Here we explore the role of senescence during natural ageing in the BM microenvironment, the mechanism which drives this and how this impacts on the metabolic health of HSPCs.BM was isolated from aged (18-24 months) and young (8-12 weeks) C57Bl/6 mice and flow cytometry was used to compare mitochondrial membrane potential (ΔΨm) and mitochondrial ROS in HSPCs. Results show that HSPCs from aged animals increase in numbers and accumulate mitochondria with low membrane potential with lower mitochondrial ROS. Next, young and aged mice were treated with lipopolycaccharide (LPS). Metabolic analysis revealed that HSPCs from young mice increase metabolism of mitochondrial TCA cycle substrates in response to LPS whereas aged HSPCs continued to rely on glycolysis. When HSPCs from aged C57Bl/6 mice (CD45.2+) were FACS purified and adoptively transferred into young PepCboy (CD45.1+) mice, thus removing them from the aged BM microenvironment, they were able to recover their mitochondrial health and showed an improved metabolic response to treatment with LPS. Furthermore, qRT-PCR analysis of p16 and p21 expression in HSPCs and mesenchymal stromal cells (MSC) showed that MSCs, but not HSPCs, acquire a senescent phenotype in aged mice, and depletion of senescent cells in the p16-3MR mouse model (3) allowed recovery of HSPC mitochondrial function and response to LPS. Mechanistically, we found a significant upregulation of the anti-apoptotic protein BCL-XL in MSCs of aged mice. This has previously been described to drive the senescent phenotype and prevent apoptosis in senescent cells. By over-expressing GFP-tagged BCL-XL in MSCs and then co-culturing them with LSKs we were able to show that BCL-XL is transferred from MSCs to HSPCs in vitro. Finally, we demonstrated that targeting BCL-XL in vivo, using the senolytic drug ABT-263, in aged mice can restore the HSPC metabolic response to stress resulting in upregulation of TCA cycle metabolism.In conclusion, we show that the aged BM microenvironment is responsible for the HSPC metabolic shortfall resulting in impaired response to stress. Targeting the senescent cells in the environment restored the HSPC metabolic function and their response to infection in aged mice. This suggests that manipulation of the ageing BM microenvironment can help to improve the body's response to infection.1. Mistry JJ, Marlein CR, Moore JA, Hellmich C, Wojtowicz EE, Smith JGW, et al. ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection. Proc Natl Acad Sci U S A. 2019;116(49):24610-9.2. Abdul-Aziz AM, Sun Y, Hellmich C, Marlein CR, Mistry J, Forde E, et al. Acute myeloid leukemia induces protumoral p16INK4a-driven senescence in the bone marrow microenvironment. Blood. 2019;133(5):446-56.3. Demaria M, Ohtani N, Youssef SA, Rodier F, Toussaint W, Mitchell JR, et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev Cell. 2014;31(6):722-33. DisclosuresNo relevant conflicts of interest to declare.

The Tetraspanin CD53 Protects Hematopoietic Stem Cells Following Cellular Stress through Induction of DREAM Complex-Mediated Quiescence

Maintenance of quiescence is necessary for optimal hematopoietic stem cell (HSC) function. The absence of fine-tuned cycling regulation of HSCs can result in impaired hematopoiesis, bone marrow (BM) failure, or malignant transformation. While various factors, including inflammatory cytokines and chemotherapy, have been identified to induce HSC cycling, the mechanisms regulating HSC return to quiescence are unclear.Herein, we profiled HSCs in response to a variety of inflammatory stressors (cytokines, toll-like receptor agonists, mobilizing agents, and chemotherapeutics) and found markedly upregulated expression of CD53 in both mouse and human HSCs, in some cases by as much as 70-fold higher expression over vehicle-treated controls. CD53 is a tetraspanin, a type of transmembrane protein involved in plasma membrane organization and regulation of processes such as cellular migration, adhesion, and signaling. CD53 has been shown to be asymmetrically segregated in HSCs, with CD53-enriched HSCs believed to be more stem-like; however, there is no current proposed mechanism to explain the association between CD53 and stem cell quality.To understand the role of CD53 in HSC quality, we generated a CD53 knockout mouse, and profiled HSC phenotype and function. Under homeostatic conditions, Cd53 -/- HSC number and frequency are normal as compared to wild type (WT) mice. However, we found Cd53 -/-HSCs to have significantly impaired function, particularly in response to inflammatory stimuli. Cd53 -/- BM failed to engraft as well as WT BM (45% chimerism vs 63%, p<0.05) in competitive transplants, and this deficit was exacerbated when G-CSF-mobilized HSCs from the spleen were transplanted (47% vs 77%, p<0.0001). Analysis of cycling status during and after G-CSF stimulation found that Cd53 -/- HSCs, particularly those that mobilize to the spleen, undergo cell division twice as frequently as WT HSCs (22% vs 10%, p<0.05). Treatment of WT and Cd53 -/- mice with serial doses of the chemotherapeutic 5-fluorouracil resulted in significantly accelerated death in Cd53 -/- mice (median survival 14 days vs 28 days, p<0.0001), supporting that loss of CD53 is associated with dysregulated HSC quiescence.Transcriptomic sequencing revealed significant upregulation of genes associated with cell cycling and division in G-CSF-treated Cd53 -/- HSCs compared to WT controls. Notably, these differentially expressed genes are targets of the dimerization partner, RB-like, E2F and multi-vulval class B (DREAM) complex, a newly described transcriptional regulator that represses cell cycling-associated genes, suggesting that CD53 promotes DREAM-mediated quiescence in stressed HSCs. We performed CUT&Tag profiling of Rbl2/p130, a DREAM complex subunit, in HSCs and found that loss of CD53 was associated with decreased complex binding (consistent with enhanced transcription of cell cycle genes). Proximity labeling studies demonstrated that CD53 interacts with cell cycle machinery proteins, including Rbl2/p130, and Western blots of sorted HSCs from Cd53 -/- and WT mice treated with G-CSF showed decreased expression of DREAM complex components (Rbl1/p107 and Rbl2/p130) in the absence of CD53. Finally, enforced activation of DREAM and HSC quiescence using the CDK4/6 inhibitor palbociclib rescued the Cd53 -/-HSC repopulating defect. Together, these data derive a novel mechanism whereby CD53 regulates HSC quiescence through regulating DREAM complex binding during stress-induced cycling. DisclosuresNo relevant conflicts of interest to declare.

Txnip enhances Fitness of Dnmt3a-Mutant Hematopoietic Stem Cells Via p21

Clonal hematopoiesis (CH) refers to the age-related expansion of specific clones in the blood system and manifests from somatic mutations acquired in hematopoietic stem cells (HSCs). Approximately 50% of CH variants occur in the gene DNMT3A. While DNMT3A-mutant CH becomes almost ubiquitous in aging humans, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is still lacking. Here, we used interferon-gamma (IFNg) as a model to study the mechanisms by which Dnmt3a mutations increase HSC fitness under recurrent hematopoietic stress.To represent the spectrum of DNMT3A variants found in humans, mouse genetic models were generated; Dnmt3a heterozygous (Vav-Cre; Dnmt3afl/+ = Dnmt3aHET) and homozygous (Vav-Cre; Dnmt3afl/fl = Dnmt3aKO) hematopoietic loss-of-function, and a knock-in model analogous to the hotspot point mutation most prevalent in AML (Vav-Cre; Dnmt3aR878H/+ = Dnmt3aR878).When Dnmt3a-mutant cells were competitively transplanted with wild-type (WT) competitor bone marrow (BM) cells and challenged with different inflammatory and proliferative stressors, Dnmt3aKO and Dnmt3aR878 HSCs were specifically resistant to the deleterious effects of IFNg on HSC self-renewal and clonal expansion. This insensitivity was also confirmed in a humanized mouse model where human CD34 + cord blood cells edited with DNMT3A-targeting gRNAs were xenografted into recipient mice and episodically exposed to human recombinant IFNg. DNMT3A mutant cells maintained their clone size, whereas AAVS1-targeted cells (control) were depleted over serial transplantation. These data suggest that Dnmt3a-mutant HSCs, mimicked DNMT3A-mutated human HSCs and are specifically resistant to IFNg-mediated depletion. One explanation for the observed resistance is that Dnmt3a-mutant HSCs have a fitness advantage under IFNg challenge. Therefore, we generated a novel mouse model to directly quantify the competition between Dnmt3a-mutant and WT HSCs. 10% donor BM cells (CD45.2; WT or Dnmt3a-mutant), 10% WT competitor BM cells (CD45.1/2; Ubc-GFP+) and 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA-IFNg) that express IFNg by doxycycline were transplanted into CD45.1 recipient mice. To normalize the effect of doxycycline, chimera made with 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA) were also transplanted into recipients. Our result from this transplantation experiment showed Dnmt3a-mutant HSCs resisted IFNg-mediated depletion due to an enhanced fitness advantage. Genetic ablation of IFNgr1 from Dnmt3a-mutant mice revealed that IFNg signaling is cell-intrinsically required by clonal expansion of Dnmt3a-mutant HSCs. In parallel, when HSCs were transplanted into IFNg-deficient recipient mice, clonal expansion of Dnmt3aKO HSCs but not WT HSCs was significantly compromised, suggesting IFNg signaling is also cell-extrinsically crucial for the clonal expansion of Dnmt3a-mutant HSCs in vivo. Mechanistically, DNA hypomethylation-associated over-expression of Thioredoxin-interacting protein (Txnip) in Dnmt3a-mutant HSCs was identified by coupling single-cell RNA-sequencing and Whole-Genome Bisulfite sequencing. The sustained Txnip levels in Dnmt3aKO HSCs led to p53 stabilization and upregulation of p21 under IFNg challenge, further correlated with a retained quiescence and resistance to apoptosis in response to IFNg exposure. Implementing biochemical studies, we observed Txnip mediated an enrichment of p53 at p21 promoter under IFNg exposure in Dnmt3aKO but not WT 32D murine myeloid cell line. Knocking down Txnip by shRNA normalized p53 occupancy at p21 promoter and rescued IFNg-associated p21 upregulation in Dnmt3aKO 32D cells. Functionally, knocking down Txnip and p21 re-sensitized Dnmt3aKO HSCs to IFNg-induced cell cycle activation and apoptosis. In vivo, down-regulation of p21 had no effect on WT HSCs in response to IFNg exposure, but it completely primed Dnmt3aKO and Dnmt3aR878 HSCs to IFNg-induced exhaustion in a transplantation experiment.Taken together, our data highlighted a Txnip-p53-p21 pathway that preserves the functional potential of Dnmt3a-mutant HSCs under conditions of inflammatory stress, which suggests a novel mechanism to explain the increased fitness of Dnmt3a-mutant HSCs and supports rationale for developing interventions to mitigate expansion of pre-malignant clones as a method of blood cancer prevention. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Haploid Kmt2c Deletions Enhance Hematopoietic Stem Cell Mobilization in Response to Granulocyte-Colony Stimulating Factor

KMT2C is one of several tumor suppressor genes deleted in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) as part of larger chromosome 7q deletions. These deletions are particularly common in therapy-related MDS/AML, raising the question of whether loss of one or more 7q genes conveys a selective advantage to hematopoietic stem cells (HSCs) in the setting of chemotherapy-induced stress. We recently showed that haploid Kmt2c deletions do indeed enhance HSC self-renewal capacity. However, the deletions do not drive HSCs into cycle; instead, a proliferative stress such as chemotherapy is required to create a context in which Kmt2c deletion convey a selective advantage. We have also identified a mechanism, to explain this phenotype. Kmt2c encodes MLL3, a SET domain protein that binds enhancers and facilitates transcription. We have shown that Kmt2c/MLL3 deletions impairs enhancer recruitment during HSC differentiation, therefore blunting HSC commitment.Our findings suggest that acquired or pre-existing 7q (KMT2C) deletions may select for HSCs that could give rise to MDS/AML in the setting of autologous-transplantation. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that is often used to expedite neutrophil recovery after chemotherapy and to mobilize HSCs for collection and transplantation. We considered the possibility that 7q deletions, and KMT2C deletions in particular, may promote disproportionate mobilization of the mutant HSCs in response to G-CSF. To test this, we treated Kmt2c f/f; Vav1-Cre and Kmt2c f/+; Vav1-Cre mice with G-CSF, and we assessed HSC mobilization to the spleen and bone marrow. A far greater proportion of HSCs with heterozygous and homozygous Kmt2c deletions mobilized in response to G-CSF, relative to wild type HSCs. Kmt2c deletion also enhanced colony forming unit frequency in the peripheral blood after G-CSF treatment. Total body HSC numbers did not change in the body after G-CSF treatment on any genetic background, indicating that Kmt2c deletions enhanced HSC mobilization in response to G-CSF rather than self-renewal.To more faithfully recapitulate clinical conditions, we used Fgd5-CreER to delete a single Kmt2c allele in only a minority of HSCs. We then tested whether the mutant HSCs mobilized more efficiently than wild type HSCs. Surprisingly, Kmt2c deletions did not enhance HSC mobilization in this context. This raised the question of whether Kmt2c deletion in a non-HSC population could promote HSC mobilization in the Kmt2c f/+; Vav1-Cre mice. Indeed, analysis of mice chimeric for wild type and Kmt2c f/+; Vav1-Cre bone marrow suggested that Kmt2c non-cell autonomously regulates HSC mobilization. Finally, Kmt2c deletions did not enhance mobilization following exposure to plerixafor, a CXCR4 antagonist that acts directly on HSCs to promote mobilization rather than indirectly via monocyte populations, as occurs with G-CSF. Additional studies are needed to elucidate the mechanism by which Kmt2c non-cell autonomously regulates HSC mobilization. Our findings provide reassurance that, in a clinical setting, rare KMT2C-mutant HSCs will not disproportionately mobilize prior to apheresis. Furthermore, the data suggest that transient inhibition of MLL3, or its targets, may enhance HSC mobilization and negate selective advantages that 7q-deleted HSCs may acquire after chemotherapy treatment. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Stem Cells in Wound Healing Response.

Significance:Emerging evidence has shown a link between the status of hematopoietic stem cells (HSCs) and wound healing responses. Thus, better understanding HSCs will contribute to further advances in wound healing research.Recent Advances:Myeloid cells such as neutrophils and monocyte-derived macrophages are critical players in the process of wound healing. HSCs actively respond to wound injury and other tissue insults, including infection and produce the effector myeloid cells, and a failing of the HSC response can result in impaired wound healing. Technological advances such as transcriptome at single-cell resolution, epigenetics, three-dimensional imaging, transgenic animals, and animal models, have provided novel concepts of myeloid generation (myelopoiesis) from HSCs, and have revealed cell-intrinsic and -extrinsic mechanisms that can impact HSC functions in the context of health conditions.Critical Issues:The newer concepts include—the programmed cellular fate at a differentiation stage that is used to be considered as the multilineage, the signaling pathways that can activate HSCs directly and indirectly, the mechanisms that can deteriorate HSCs, the roles and remodeling of the surrounding environment for HSCs and their progenitors (the niche).Future Directions:The researches on HSCs, which produce blood cells, should contribute to the development of blood biomarkers predicting a risk of chronic wounds, which may transform clinical practice of wound care with precision medicine for patients at high risk of poor healing.