Publications by authors named "Eric M Pietras"

46 Publications

Blood stem cell PU.1 upregulation is a consequence of differentiation without fast autoregulation.

J Exp Med 2022 Jan 24;219(1). Epub 2021 Nov 24.

Department of Biosystems Science & Engineering, Eidgenössische Technische Hochschule Zürich, Basel, Switzerland.

Transcription factors (TFs) regulate cell fates, and their expression must be tightly regulated. Autoregulation is assumed to regulate many TFs' own expression to control cell fates. Here, we manipulate and quantify the (auto)regulation of PU.1, a TF controlling hematopoietic stem and progenitor cells (HSPCs), and correlate it to their future fates. We generate transgenic mice allowing both inducible activation of PU.1 and noninvasive quantification of endogenous PU.1 protein expression. The quantified HSPC PU.1 dynamics show that PU.1 up-regulation occurs as a consequence of hematopoietic differentiation independently of direct fast autoregulation. In contrast, inflammatory signaling induces fast PU.1 up-regulation, which does not require PU.1 expression or its binding to its own autoregulatory enhancer. However, the increased PU.1 levels induced by inflammatory signaling cannot be sustained via autoregulation after removal of the signaling stimulus. We conclude that PU.1 overexpression induces HSC differentiation before PU.1 up-regulation, only later generating cell types with intrinsically higher PU.1.
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http://dx.doi.org/10.1084/jem.20202490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8624737PMC
January 2022

Simplified murine multipotent progenitor isolation scheme: Establishing a consensus approach for multipotent progenitor identification.

Exp Hematol 2021 Dec 11;104:55-63. Epub 2021 Oct 11.

Division of Regenerative Medicine, Department of Medicine, Moores Cancer Center, University of California, San Diego, La Jolla, CA.

The mouse hematopoietic system has served as a paradigm for analysis of developmental fate decisions in tissue homeostasis and regeneration. However, multiple immunophenotypic definitions of, and sometimes divergent nomenclatures used to classify, murine multipotent progenitors (MPPs) have emerged in the field over time. This has created significant confusion and inconsistency in the hematology field. To facilitate easier comparison of murine MPP phenotypes between research laboratories, a working group of four International Society for Experimental Hematology (ISEH) members with extensive experience studying the functional activities associated with different MPP phenotypic definitions reviewed the current state of the field with the goal of developing a position statement toward a simplified and unified immunophenotypic definition of MPP populations. In November of 2020, this position statement was presented as a webinar to the ISEH community for discussion and feedback. Hence, the Simplified MPP Identification Scheme presented here is the result of curation of existing literature, consultation with leaders in the field, and crowdsourcing from the wider experimental hematology community. Adoption of a unified definition and nomenclature, while still leaving room for individual investigator customization, will benefit scientists at all levels trying to compare these populations between experimental settings.
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http://dx.doi.org/10.1016/j.exphem.2021.09.007DOI Listing
December 2021

Analysis of Radiation-Induced Changes in Cell Cycle and DNA Damage of Murine Hematopoietic Stem Cells by Multi-Color Flow Cytometry.

Curr Protoc 2021 Aug;1(8):e216

Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, Arkansas.

Exposure of bone marrow to genotoxic stress such as ionizing radiation (IR) results in a rapid decline of peripheral blood cells and stimulates entry of the normally quiescent hematopoietic stem cells (HSCs) into the cell cycle to reconstitute the hematopoietic system. While several protocols have employed flow cytometry analysis of bone marrow cells to study changes in specific cell populations with respect to cell cycle proliferation and/or expression of γ-H2AX, a marker of DNA damage, these parameters were examined in separate panels. Here, we describe a flow cytometry-based method specifically designed to examine cell cycle distribution using Ki-67 and FXCycle violet in combination with γ-H2AX in HSCs and hematopoietic progenitor cells (HPCs) within the same sample. This method is very useful, particularly in studies involving genotoxic stresses such as IR, which substantially reduce the absolute numbers of HSCs and HPCs available for staining. Additionally, we describe several important considerations for the analysis of markers of HSCs in irradiated versus unirradiated samples. Examples include the use of fluorescence minus one (FMO) controls, the gating strategy for markers whose expression is typically impacted by IR such as Sca1, tips for staining of intracellular antigens like Ki67, and ensuring the detection of signal from at least 500 events in each gate to ensure robustness of the results. © 2021 Wiley Periodicals LLC. Basic Protocol: Immunostaining protocol for bone marrow mononuclear cells using a multi-fluorophore panel.
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http://dx.doi.org/10.1002/cpz1.216DOI Listing
August 2021

Inflammation as a regulator of hematopoietic stem cell function in disease, aging, and clonal selection.

J Exp Med 2021 07 15;218(7). Epub 2021 Jun 15.

Department of Medical Oncology and Hematology, University Hospital Zürich, Zürich, Switzerland.

Inflammation is an evolutionarily selected defense response to infection or tissue damage that involves activation and consumption of immune cells in order to reestablish and maintain organismal integrity. In this process, hematopoietic stem cells (HSCs) are themselves exposed to inflammatory cues and via proliferation and differentiation, replace mature immune cells in a demand-adapted fashion. Here, we review how major sources of systemic inflammation act on and subsequently shape HSC fate and function. We highlight how lifelong inflammatory exposure contributes to HSC inflamm-aging and selection of premalignant HSC clones. Finally, we explore emerging areas of interest and open questions remaining in the field.
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http://dx.doi.org/10.1084/jem.20201541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210622PMC
July 2021

Chronic interleukin-1 exposure triggers selection for Cebpa-knockout multipotent hematopoietic progenitors.

J Exp Med 2021 06;218(6)

Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO.

The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1β (IL-1β), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell-intrinsic competitive advantage; rather chronic IL-1β exposure engendered potent selection for Cebpa loss. Chronic IL-1β augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1β, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention.
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http://dx.doi.org/10.1084/jem.20200560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8094119PMC
June 2021

PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress.

J Exp Med 2021 06;218(6)

Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.
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http://dx.doi.org/10.1084/jem.20201169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056754PMC
June 2021

The anti-inflammatory cytokine interleukin-37 is an inhibitor of trained immunity.

Cell Rep 2021 04;35(1):108955

Department of Medicine, Radboud University Medical Center, Nijmegen, the Netherlands; Department of Medicine, University of Colorado Denver, Aurora, CO 80045, USA. Electronic address:

Trained immunity (TI) is a de facto innate immune memory program induced in monocytes/macrophages by exposure to pathogens or vaccines, which evolved as protection against infections. TI is characterized by immunometabolic changes and histone post-translational modifications, which enhance production of pro-inflammatory cytokines. As aberrant activation of TI is implicated in inflammatory diseases, tight regulation is critical; however, the mechanisms responsible for this modulation remain elusive. Interleukin-37 (IL-37) is an anti-inflammatory cytokine that curbs inflammation and modulates metabolic pathways. In this study, we show that administration of recombinant IL-37 abrogates the protective effects of TI in vivo, as revealed by reduced host pro-inflammatory responses and survival to disseminated candidiasis. Mechanistically, IL-37 reverses the immunometabolic changes and histone post-translational modifications characteristic of TI in monocytes, thus suppressing cytokine production in response to infection. IL-37 thereby emerges as an inhibitor of TI and as a potential therapeutic target in immune-mediated pathologies.
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http://dx.doi.org/10.1016/j.celrep.2021.108955DOI Listing
April 2021

Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion.

Proc Natl Acad Sci U S A 2021 03;118(10)

Department of Medicine, University of Colorado Denver, Aurora, CO 80045;

Interleukin-1β (IL-1β)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1β production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1β expression in cutaneous melanoma samples ( = 469) compared to normal skin ( = 324), with a highly significant correlation between NLRP3 and IL-1β ( < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8 T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy.
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http://dx.doi.org/10.1073/pnas.2000915118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958415PMC
March 2021

MDS-associated SF3B1 mutations enhance proinflammatory gene expression in patient blast cells.

J Leukoc Biol 2021 07 6;110(1):197-205. Epub 2020 Nov 6.

Department of Immunology and Genomic Medicine, National Jewish Health, Denver, Colorado, USA.

Two factors known to contribute to the development of myelodysplastic syndrome (MDS) and other blood cancers are (i) somatically acquired mutations in components of the spliceosome and (ii) increased inflammation. Spliceosome genes, including SF3B1, are mutated at high frequency in MDS and other blood cancers; these mutations are thought to be neomorphic or gain-of-function mutations that drive disease pathogenesis. Likewise, increased inflammation is thought to contribute to MDS pathogenesis; inflammatory cytokines are strongly elevated in these patients, with higher levels correlating with worsened patient outcome. In the current study, we used RNAseq to analyze pre-mRNA splicing and gene expression changes present in blast cells isolated from MDS patients with or without SF3B1 mutations. We determined that SF3B1 mutations lead to enhanced proinflammatory gene expression in these cells. Thus, these studies suggest that SF3B1 mutations could contribute to MDS pathogenesis by enhancing the proinflammatory milieu in these patients.
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http://dx.doi.org/10.1002/JLB.6AB0520-318RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8612809PMC
July 2021

Extinguishing the Embers: Targeting AML Metabolism.

Trends Mol Med 2021 04 26;27(4):332-344. Epub 2020 Oct 26.

Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address:

Acute myeloid leukemia (AML) is a cancer derived from the myeloid lineage of blood cells, characterized by overproduction of leukemic blasts. Although therapeutic improvements have made a significant impact on the outcomes of patients with AML, survival rates remain low due to a high incidence of relapse. Similar to how wildfires can reignite from hidden embers not extinguished from an initial round of firefighting, leukemic stem cells (LSCs) are the embers remaining after completion of traditional chemotherapeutic treatments. LSCs exhibit a unique metabolic profile and contain metabolically distinct subpopulations. In this review, we detail the metabolic features of LSCs and how thetse characteristics promote resistance to traditional chemotherapy. We also discuss new therapeutic approaches that target metabolic vulnerabilities of LSC to selectively eradicate them.
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http://dx.doi.org/10.1016/j.molmed.2020.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005405PMC
April 2021

ETV6 germline mutations cause HDAC3/NCOR2 mislocalization and upregulation of interferon response genes.

JCI Insight 2020 09 17;5(18). Epub 2020 Sep 17.

Department of Pediatrics, Washington University at St. Louis, St. Louis, Missouri, USA.

ETV6 is an ETS family transcription factor that plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that led to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation was also reflected in patient-derived platelet transcripts and drove aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction.
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http://dx.doi.org/10.1172/jci.insight.140332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526537PMC
September 2020

Tet2-mediated clonal hematopoiesis in nonconditioned mice accelerates age-associated cardiac dysfunction.

JCI Insight 2020 03 26;5(6). Epub 2020 Mar 26.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Clonal hematopoiesis of indeterminate potential is prevalent in elderly individuals and associated with increased risks of all-cause mortality and cardiovascular disease. However, mouse models to study the dynamics of clonal hematopoiesis and its consequences on the cardiovascular system under homeostatic conditions are lacking. We developed a model of clonal hematopoiesis using adoptive transfer of unfractionated ten-eleven translocation 2-mutant (Tet2-mutant) bone marrow cells into nonirradiated mice. Consistent with age-related clonal hematopoiesis observed in humans, these mice displayed a progressive expansion of Tet2-deficient cells in multiple hematopoietic stem and progenitor cell fractions and blood cell lineages. The expansion of the Tet2-mutant fraction was also observed in bone marrow-derived CCR2+ myeloid cell populations within the heart, but there was a negligible impact on the yolk sac-derived CCR2- cardiac-resident macrophage population. Transcriptome profiling revealed an enhanced inflammatory signature in the donor-derived macrophages isolated from the heart. Mice receiving Tet2-deficient bone marrow cells spontaneously developed age-related cardiac dysfunction characterized by greater hypertrophy and fibrosis. Altogether, we show that Tet2-mediated hematopoiesis contributes to cardiac dysfunction in a nonconditioned setting that faithfully models human clonal hematopoiesis in unperturbed bone marrow. Our data support clinical findings that clonal hematopoiesis per se may contribute to diminished health span.
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http://dx.doi.org/10.1172/jci.insight.135204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213793PMC
March 2020

Deregulated Notch and Wnt signaling activates early-stage myeloid regeneration pathways in leukemia.

J Exp Med 2020 03;217(3)

Columbia Stem Cell Initiative, Department of Genetics and Development, Columbia University, New York, NY.

Targeting commonly altered mechanisms in leukemia can provide additional treatment options. Here, we show that an inducible pathway of myeloid regeneration involving the remodeling of the multipotent progenitor (MPP) compartment downstream of hematopoietic stem cells (HSCs) is commonly hijacked in myeloid malignancies. We establish that differential regulation of Notch and Wnt signaling transiently triggers myeloid regeneration from HSCs in response to stress, and that constitutive low Notch and high Wnt activity in leukemic stem cells (LSCs) maintains this pathway activated in malignancies. We also identify compensatory crosstalk mechanisms between Notch and Wnt signaling that prevent damaging HSC function, MPP production, and blood output in conditions of high Notch and low Wnt activity. Finally, we demonstrate that restoring Notch and Wnt deregulated activity in LSCs attenuates disease progression. Our results uncover a mechanism that controls myeloid regeneration and early lineage decisions in HSCs and could be targeted in LSCs to normalize leukemic myeloid cell production.
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http://dx.doi.org/10.1084/jem.20190787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062512PMC
March 2020

CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions.

Exp Hematol 2020 01 18;81:1-15.e6. Epub 2019 Dec 18.

Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA; Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA. Electronic address:

Hematopoiesis is dynamically regulated to maintain blood system function under nonhomeostatic conditions such as inflammation and injury. However, common surface marker and hematopoietic stem cell (HSC) reporter systems used for prospective enrichment of HSCs have been less rigorously tested in these contexts. Here, we use two surface markers, EPCR/CD201 and CD34, to re-analyze dynamic changes in the HSC-enriched phenotypic SLAM compartment in a mouse model of chronic interleukin (IL)-1 exposure. EPCR and CD34 coordinately identify four functionally and molecularly distinct compartments within the SLAM fraction, including an EPCR/CD34 fraction whose long-term serial repopulating activity is only modestly impacted by chronic IL-1 exposure, relative to unfractionated SLAM cells. Notably, the other three fractions expand in frequency following IL-1 treatment and represent actively proliferating, lineage-primed cell states with limited long-term repopulating potential. Importantly, we find that the Fgd5-ZSGreen HSC reporter mouse enriches for molecularly and functionally intact HSCs regardless of IL-1 exposure. Together, our findings provide further evidence of dynamic heterogeneity within a commonly used HSC-enriched phenotypic compartment under stress conditions. Importantly, they also indicate that stringency of prospective isolation approaches can enhance interpretation of findings related to HSC function when studying models of hematopoietic stress.
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http://dx.doi.org/10.1016/j.exphem.2019.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938677PMC
January 2020

TNF-α-driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging.

Blood 2019 08 16;134(9):727-740. Epub 2019 Jul 16.

Center for Cancer and Blood Disorders, Department of Pediatrics and.

Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.
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http://dx.doi.org/10.1182/blood.2019000200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716075PMC
August 2019

PI3 kinase alpha and delta promote hematopoietic stem cell activation.

JCI Insight 2019 05 23;5. Epub 2019 May 23.

Department of Medicine and.

Many cytokines and chemokines that are important for hematopoiesis activate the PI3K signaling pathway. Because this pathway is frequently mutated and activated in cancer, PI3K inhibitors have been developed for the treatment of several malignancies, and are now being tested in the clinic in combination with chemotherapy. However, the role of PI3K in adult hematopoietic stem cells (HSCs), particularly during hematopoietic stress, is still unclear. We previously showed that the individual PI3K catalytic isoforms P110α or P110β have dispensable roles in HSC function, suggesting redundancy between PI3K isoforms in HSCs. We now demonstrate that simultaneous deletion of P110α and P110δ in double knockout (DKO) HSCs uncovers their redundant requirement in HSC cycling after 5-fluorouracil (5-FU) chemotherapy administration. In contrast, DKO HSCs are still able to exit quiescence in response to other stress stimuli, such as LPS. We found that DKO HSCs and progenitors have impaired sensing of inflammatory signals ex vivo, and that levels of IL1-β and MIG are higher in the bone marrow after LPS than after 5-FU administration. Furthermore, exogenous in vivo administration of IL1-β can induce cell cycle entry of DKO HSCs. Our findings have important clinical implications for the use of PI3K inhibitors in combination with chemotherapy.
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http://dx.doi.org/10.1172/jci.insight.125832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629136PMC
May 2019

Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis.

Haematologica 2020 03 17;105(3):585-597. Epub 2019 May 17.

Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.
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http://dx.doi.org/10.3324/haematol.2018.197210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049366PMC
March 2020

Hematopoietic Stem Cells Rock Around The Clock: Circadian Fate Control via TNF/ROS Signals.

Cell Stem Cell 2018 10;23(4):459-460

Division of Hematology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address:

Hematopoietic stem cell function is closely tied to circadian rhythms. In this issue of Cell Stem Cell, Golan et al. (2018) identify crosstalk between circadian hormone signals, the inflammatory cytokine TNF, and bone marrow macrophages as a key regulator of HSC proliferation, differentiation, and self-renewal in the bone marrow.
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http://dx.doi.org/10.1016/j.stem.2018.09.002DOI Listing
October 2018

Zinc Finger Protein 521 Regulates Early Hematopoiesis through Cell-Extrinsic Mechanisms in the Bone Marrow Microenvironment.

Mol Cell Biol 2018 09 15;38(17). Epub 2018 Aug 15.

Department of Biomedical Sciences, National Jewish Health, Denver, Colorado, USA

Zinc finger protein 521 (ZFP521), a DNA-binding protein containing 30 Krüppel-like zinc fingers, has been implicated in the differentiation of multiple cell types, including hematopoietic stem and progenitor cells (HSPC) and B lymphocytes. Here, we report a novel role for ZFP521 in regulating the earliest stages of hematopoiesis and lymphoid cell development via a cell-extrinsic mechanism. Mice with inactivated genes () possess reduced frequencies and numbers of hematopoietic stem and progenitor cells, common lymphoid progenitors, and B and T cell precursors. Notably, ZFP521 deficiency changes bone marrow microenvironment cytokine levels and gene expression within resident HSPC, consistent with a skewing of hematopoiesis away from lymphopoiesis. These results advance our understanding of ZFP521's role in normal hematopoiesis, justifying further research to assess its potential as a target for cancer therapies.
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http://dx.doi.org/10.1128/MCB.00603-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094055PMC
September 2018

Ezh2 spares KitL from the cutter.

Authors:
Eric M Pietras

Blood 2018 05;131(20):2180-2181

University of Colorado Anschutz Medical Campus.

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http://dx.doi.org/10.1182/blood-2018-04-841890DOI Listing
May 2018

Analysis of Murine Hematopoietic Stem Cell Proliferation During Inflammation.

Methods Mol Biol 2018 ;1686:183-200

Division of Hematology, Department of Medicine, University of Colorado Anschutz Medical Campus, 12700 East 19th Ave, Research Complex 2, Mail Stop F754, Aurora, CO, 80045, USA.

Normally, quiescent hematopoietic stem cells (HSC) rapidly enter the cell cycle following exposure to inflammatory stimuli. The analysis of HSC cell cycle activity in murine bone marrow during inflammation is often complicated by the relative rarity of HSCs and shifts in Sca-1, a key cell surface marker used to identify HSCs. Here, we report a method to analyze HSC proliferation and cell cycle distribution under inflammatory conditions. Our approach uses EdU incorporation and Ki67 staining coupled with DNA content quantification by DAPI. We also incorporate the surface marker ESAM to help minimize the potential for contaminating events that may confound analysis in the HSC compartment.
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http://dx.doi.org/10.1007/978-1-4939-7371-2_14DOI Listing
June 2018

Inflammation: a key regulator of hematopoietic stem cell fate in health and disease.

Authors:
Eric M Pietras

Blood 2017 10 5;130(15):1693-1698. Epub 2017 Sep 5.

Division of Hematology, Department of Medicine, and Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO.

Hematopoietic stem cells (HSCs) are responsible for lifelong production of blood cells. At the same time, they must respond rapidly to acute needs such as infection or injury. Significant interest has emerged in how inflammation regulates HSC fate and how it affects the long-term functionality of HSCs and the blood system as a whole. Here we detail recent advances and unanswered questions at the intersection between inflammation and HSC biology in the contexts of development, aging, and hematological malignancy.
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http://dx.doi.org/10.1182/blood-2017-06-780882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639485PMC
October 2017

The importance of tying up loose (DNA) ends.

Authors:
Eric M Pietras

Blood 2017 05;129(18):2457-2458

UNIVERSITY OF COLORADO ANSCHUTZ MEDICAL CAMPUS.

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http://dx.doi.org/10.1182/blood-2017-03-773184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418642PMC
May 2017

Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis.

Nature 2017 04 29;544(7648):53-58. Epub 2017 Mar 29.

The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Medicine, Division of Hematology/Oncology, University of California San Francisco, San Francisco, California 94143, USA.

Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1β, G-CSF, TGFβ and CXCL4) and activation of an inducible Irf8 and β-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.
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http://dx.doi.org/10.1038/nature21693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5383507PMC
April 2017

ISSLS PRIZE IN BASIC SCIENCE 2017: Intervertebral disc/bone marrow cross-talk with Modic changes.

Eur Spine J 2017 05 31;26(5):1362-1373. Epub 2017 Jan 31.

Department of Orthopaedic Surgery, University of California San Francisco, 513 Parnassus Ave, S-1164, San Francisco, CA, 94143, USA.

Study Design: Cross-sectional cohort analysis of patients with Modic Changes (MC).

Objective: Our goal was to characterize the molecular and cellular features of MC bone marrow and adjacent discs. We hypothesized that MC associate with biologic cross-talk between discs and bone marrow, the presence of which may have both diagnostic and therapeutic implications.

Background Data: MC are vertebral bone marrow lesions that can be a diagnostic indicator for discogenic low back pain. Yet, the pathobiology of MC is largely unknown.

Methods: Patients with Modic type 1 or 2 changes (MC1, MC2) undergoing at least 2-level lumbar interbody fusion with one surgical level having MC and one without MC (control level). Two discs (MC, control) and two bone marrow aspirates (MC, control) were collected per patient. Marrow cellularity was analyzed using flow cytometry. Myelopoietic differentiation potential of bone marrow cells was quantified to gauge marrow function, as was the relative gene expression profiles of the marrow and disc cells. Disc/bone marrow cross-talk was assessed by comparing MC disc/bone marrow features relative to unaffected levels.

Results: Thirteen MC1 and eleven MC2 patients were included. We observed pro-osteoclastic changes in MC2 discs, an inflammatory dysmyelopoiesis with fibrogenic changes in MC1 and MC2 marrow, and up-regulation of neurotrophic receptors in MC1 and MC2 bone marrow and discs.

Conclusion: Our data reveal a fibrogenic and pro-inflammatory cross-talk between MC bone marrow and adjacent discs. This provides insight into the pain generator at MC levels and informs novel therapeutic targets for treatment of MC-associated LBP.
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http://dx.doi.org/10.1007/s00586-017-4955-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409869PMC
May 2017

Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal.

Nat Cell Biol 2016 06 25;18(6):607-18. Epub 2016 Apr 25.

The Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Medicine, Division of Hematology/Oncology, University of California San Francisco, San Francisco, California 94143, USA.

Haematopoietic stem cells (HSCs) maintain lifelong blood production and increase blood cell numbers in response to chronic and acute injury. However, the mechanism(s) by which inflammatory insults are communicated to HSCs and their consequences for HSC activity remain largely unknown. Here, we demonstrate that interleukin-1 (IL-1), which functions as a key pro-inflammatory 'emergency' signal, directly accelerates cell division and myeloid differentiation of HSCs through precocious activation of a PU.1-dependent gene program. Although this effect is essential for rapid myeloid recovery following acute injury to the bone marrow, chronic IL-1 exposure restricts HSC lineage output, severely erodes HSC self-renewal capacity, and primes IL-1-exposed HSCs to fail massive replicative challenges such as transplantation. Importantly, these damaging effects are transient and fully reversible on IL-1 withdrawal. Our results identify a critical regulatory circuit that tailors HSC responses to acute needs, and is likely to underlie deregulated blood homeostasis in chronic inflammation conditions.
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http://dx.doi.org/10.1038/ncb3346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884136PMC
June 2016

Functionally Distinct Subsets of Lineage-Biased Multipotent Progenitors Control Blood Production in Normal and Regenerative Conditions.

Cell Stem Cell 2015 Jul 18;17(1):35-46. Epub 2015 Jun 18.

The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Division of Hematology/Oncology, Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA. Electronic address:

Despite great advances in understanding the mechanisms underlying blood production, lineage specification at the level of multipotent progenitors (MPPs) remains poorly understood. Here, we show that MPP2 and MPP3 are distinct myeloid-biased MPP subsets that work together with lymphoid-primed MPP4 cells to control blood production. We find that all MPPs are produced in parallel by hematopoietic stem cells (HSCs), but with different kinetics and at variable levels depending on hematopoietic demands. We also show that the normally rare myeloid-biased MPPs are transiently overproduced by HSCs in regenerating conditions, hence supporting myeloid amplification to rebuild the hematopoietic system. This shift is accompanied by a reduction in self-renewal activity in regenerating HSCs and reprogramming of MPP4 fate toward the myeloid lineage. Our results support a dynamic model of blood development in which HSCs convey lineage specification through independent production of distinct lineage-biased MPP subsets that, in turn, support lineage expansion and differentiation.
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http://dx.doi.org/10.1016/j.stem.2015.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542150PMC
July 2015

Pro-inflammatory cytokines: emerging players regulating HSC function in normal and diseased hematopoiesis.

Exp Cell Res 2014 Dec 19;329(2):248-54. Epub 2014 Aug 19.

The Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Medicine, Division of Hematology/Oncology, University of California San Francisco, San Francisco, CA 94143, USA. Electronic address:

Hematopoiesis is the hierarchical process in which all lineages of blood cells are produced by self-renewing hematopoietic stem cells (HSCs) in the bone marrow (BM). While the regulatory factors that maintain proper HSC function and lineage output under normal conditions are well understood, significantly less is known about how HSC fate is regulated in response to inflammation or disease. As many blood disorders are associated with overproduction of pro-inflammatory cytokines, significant interest has emerged in understanding the impact of these factors on HSC function. In this review we highlight key advances demonstrating the impact of pro-inflammatory cytokines on the biology of HSCs and the BM niche, and address ongoing questions regarding their role in normal and pathogenic hematopoiesis.
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http://dx.doi.org/10.1016/j.yexcr.2014.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4250307PMC
December 2014

Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells.

Nature 2014 Aug 30;512(7513):198-202. Epub 2014 Jul 30.

The Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Medicine, Hem/Onc Division, University of California San Francisco, San Francisco, California 94143, USA.

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.
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http://dx.doi.org/10.1038/nature13619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456040PMC
August 2014
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