CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions
2019; Elsevier BV; Volume: 81; Linguagem: Inglês
10.1016/j.exphem.2019.12.003
ISSN1873-2399
AutoresJennifer L. Rabe, Giovanni Hernandez, James S. Chavez, Taylor Mills, Claus Nerlov, Eric M. Pietras,
Tópico(s)T-cell and B-cell Immunology
Resumo•EPCR and CD34 enrich for long-term repopulating SLAM cells in chronic IL-1 conditions.•EPCR and CD34 identify molecularly distinct SLAM fractions.•EPCR+/CD34– SLAM cells are highly quiescent regardless of IL-1 exposure.•EPCR-SLAM cells possess limited repopulating activity and exhibit Mk priming.•Fgd5 expression marks long-term HSCs minimally impacted by chronic IL-1. 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. 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. Hematopoietic stem cells (HSCs) are tasked with maintaining lifelong blood production, including under nonhomeostatic conditions induced by physiological stresses [1King KY Goodell MA Inflammatory modulation of HSCs: Viewing the HSC as a foundation for the immune response.Nat Rev Immunol. 2011; 11: 685-692Crossref PubMed Scopus (383) Google Scholar]. HSCs must therefore cope with demands brought about by infection, injury, aging, inflammatory disease, and myeloablative therapeutic interventions such as irradiation (IR) and chemotherapy [2Pietras EM Inflammation: A key regulator of hematopoietic stem cell fate in health and disease.Blood. 2017; 130: 1693-1698Crossref PubMed Scopus (212) Google Scholar]. Thus, understanding the impact of these stresses on HSC function and the long-term viability of the HSC pool is crucial for identifying mechanisms that drive pathogenic processes such as leukemogenesis, aging, and bone marrow (BM) failure. Prospective identification and isolation of HSCs under stress conditions by flow cytometry offer opportunities for detailed studies to address the unique biological features of stress hematopoiesis. Phenotypic definitions such as Lineage (Lin)–/cKit+/Sca-1+/Flk2–/CD48–CD150+ surface marker combination (hereafter referred to as SLAM) are commonly used to enrich for HSCs in these studies [3Warr MR Pietras EM Passegue E Mechanisms controlling hematopoietic stem cell functions during normal hematopoiesis and hematological malignancies.Wiley Interdiscip Rev Syst Biol Med. 2011; 3: 681-701Crossref PubMed Scopus (84) Google Scholar, 4Kiel MJ et al.SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2414) Google Scholar, 5Chen J Ellison FM Keyvanfar K et al.Enrichment of hematopoietic stem cells with SLAM and LSK markers for the detection of hematopoietic stem cell function in normal and Trp53 null mice.Exp Hematol. 2008; 36: 1236-1243Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. Surface markers including CD34 and endothelial protein C perceptor (EPCR/CD201) have also been used in combination with the SLAM definition to further enrich for HSC activity [6Balazs AB Fabian AJ Esmon CT Mulligan RC Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow.Blood. 2006; 107: 2317-2321Crossref PubMed Scopus (204) Google Scholar,7Wilson A Laurenti E Oser G et al.Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.Cell. 2008; 135: 1118-1129Abstract Full Text Full Text PDF PubMed Scopus (1374) Google Scholar]. Other methods, such as Hoescht side-population (SP) and rhodamine dye, have been used for further enrichment in combination with SLAM [8Winkler IG Barbier V Wadley R Zannettino AC Williams S Lévesque JP Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: Serially reconstituting hematopoietic stem cells reside in distinct nonperfused niches.Blood. 2010; 116: 375-385Crossref PubMed Scopus (190) Google Scholar, 9Shin JY Hu W Naramura M Park CY High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias.J Exp Med. 2014; 211: 217-231Crossref PubMed Scopus (163) Google Scholar, 10Challen GA Boles N Lin KK Goodell MA Mouse hematopoietic stem cell identification and analysis.Cytometry A. 2009; 75: 14-24Crossref PubMed Scopus (238) Google Scholar]. In addition, a growing body of work has highlighted the functional and molecular heterogeneity at the population and single-cell levels within phenotypic HSC gates [11MacLean AL Smith MA Liepe J et al.Single cell phenotyping reveals heterogeneity among hematopoietic stem cells following infection.Stem Cells. 2017; 35: 2292-2304Crossref PubMed Scopus (12) Google Scholar, 12Haas S Trumpp A Milsom MD Causes and consequences of hematopoietic stem cell heterogeneity.Cell Stem Cell. 2018; 22: 627-638Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13Morita Y Ema H Nakauchi H Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.J Exp Med. 2010; 207: 1173-1182Crossref PubMed Scopus (313) Google Scholar, 14Nestorowa S Hamey FK Pijuan Sala B et al.A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.Blood. 2016; 128: e20-e31Crossref PubMed Scopus (347) Google Scholar, 15Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 16Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (184) Google Scholar], suggesting that markers such as SLAM contain distinct functional compartments within. Moreover, proteins such as CD41 and interferon-γ receptor have been used to distinguish unique functional compartments within the SLAM gate under stress conditions [17Haas S Hansson J Klimmeck D et al.Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.Cell Stem Cell. 2015; 17: 422-434Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar,18Matatall KA Shen CC Challen GA King KY Type II interferon promotes differentiation of myeloid-biased hematopoietic stem cells.Stem Cells. 2014; 32: 3023-3030Crossref PubMed Scopus (75) Google Scholar]. Outcomes common to most studies of inflammatory stress include loss of long-term repopulating activity as measured by transplantation of SLAM HSC, expansion of the phenotypic SLAM HSC compartment, increased cell proliferation, and activation of unique HSC-like subsets, including megakaryocyte (Mk)-biased CD41+ cells, within the SLAM gate [17Haas S Hansson J Klimmeck D et al.Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.Cell Stem Cell. 2015; 17: 422-434Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar,19Pietras EM Lakshminarasimhan R Techner JM et al.Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.J Exp Med. 2014; 211: 245-262Crossref PubMed Scopus (178) Google Scholar, 20Pietras EM Mirantes-Barbeito C Fong S et al.Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.Nat Cell Biol. 2016; 18: 607-618Crossref PubMed Scopus (356) Google Scholar, 21Pietras EM Reynaud D Kang YA et al.Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.Cell Stem Cell. 2015; 17: 35-46Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 22Walter D Lier A Geiselhart A et al.Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells.Nature. 2015; 520: 549-552Crossref PubMed Scopus (393) Google Scholar, 23Essers MA Offner S Blanco-Bose WE et al.IFNalpha activates dormant haematopoietic stem cells in vivo.Nature. 2009; 458: 904-908Crossref PubMed Scopus (981) Google Scholar]. However, a wide variety of stressors including IR, chemotherapy agents such as 5-FU, and inflammatory cytokines can induce phenotypic shifts in key surface markers such as c-Kit and Sca-1 that may lead to contamination of the phenotypic HSC gate with non-HSC cell types [19Pietras EM Lakshminarasimhan R Techner JM et al.Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.J Exp Med. 2014; 211: 245-262Crossref PubMed Scopus (178) Google Scholar,24Domen J Weissman IL Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2.Exp Hematol. 2003; 31: 631-639Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 25Hérault A Binnewies M Leong S et al.Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis.Nature. 2017; 544: 53-58Crossref PubMed Scopus (108) Google Scholar, 26Vazquez SE Inlay MA Serwold T CD201 and CD27 identify hematopoietic stem and progenitor cells across multiple murine strains independently of Kit and Sca-1.Exp Hematol. 2015; 43: 578-585Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar] or exclusion of stem/progenitor cells that may transiently express traditionally excluded lineage markers such as Mac-1 under stress conditions [27Hidalgo A Peired AJ Weiss LA Katayama Y Frenette PS The integrin alphaMbeta2 anchors hematopoietic progenitors in the bone marrow during enforced mobilization.Blood. 2004; 104: 993-1001Crossref PubMed Scopus (41) Google Scholar]. In recent years, numerous reporter mouse lines have also been developed to enrich for HSCs, based on expression of fluorescent reporters driven by promoters of genes including Hoxb5, Fgd5, Vwf, Gprc5c, and Krt18 [28Chapple RH Tseng YJ Hu T et al.Lineage tracing of murine adult hematopoietic stem cells reveals active contribution to steady-state hematopoiesis.Blood Adv. 2018; 2: 1220-1228Crossref PubMed Scopus (44) Google Scholar, 29Cabezas-Wallscheid N Buettner F Sommerkamp P et al.Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.Cell. 2017; 169: 807-823.e19Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 30Gazit R Mandal PK Ebina W et al.Fgd5 identifies hematopoietic stem cells in the murine bone marrow.J Exp Med. 2014; 211: 1315-1331Crossref PubMed Scopus (113) Google Scholar, 31Chen JY Miyanishi M Wang SK et al.Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche.Nature. 2016; 530: 223-227Crossref PubMed Scopus (211) Google Scholar, 32Pinho S Marchand T Yang E Wei Q Nerlov C Frenette PS Lineage-biased hematopoietic stem cells are regulated by distinct niches.Dev Cell. 2018; 44: 634-641.e4Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar]. Together, the capacity of surface markers and reporters to identify functional HSCs under nonhomeostatic conditions such as inflammation has not been extensively evaluated. Hence, careful evaluation and enhancement of commonly used HSC marker/reporter definitions can ensure correct interpretation of experimental results in studies of stress hematopoiesis. In the present study, we used a model of chronic interleukin (IL)-1β exposure to evaluate changes within the SLAM gate during inflammatory stress. We use the markers EPCR and CD34 to coordinately identify four distinct SLAM fractions with unique functional and molecular properties. Strikingly, we find that the relative abundance of these fractions changes significantly following IL-1 treatment, with a sharp reduction in the EPCR+/CD34– fraction, which enriches for the vast majority of HSC activity. Importantly, we find that chronic IL-1 exposure does not substantially change the molecular or proliferative properties of each fraction. Likewise, we find that Fgd5-ZSGreen expression identifies HSCs with equivalent molecular and functional properties regardless of IL-1 exposure, indicating that a functional long-term HSC pool is retained even under chronic inflammatory stress. This work provides critical insights into the dynamic nature of stress hematopoiesis and provides critical insight into improved strategies to identify functional HSCs under nonhomeostatic conditions. Wild-type C57BL/6, CD45.1+ congenic B6.SJL-PtprcaPepcb/BoyJ (Boy/J) mice and Fgd5-ZSGreen-CreERT mice [30Gazit R Mandal PK Ebina W et al.Fgd5 identifies hematopoietic stem cells in the murine bone marrow.J Exp Med. 2014; 211: 1315-1331Crossref PubMed Scopus (113) Google Scholar] were obtained from The Jackson Laboratory and bred in-house. Vwf-GFP mice [33Sanjuan-Pla A Macaulay IC Jensen CT et al.Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.Nature. 2013; 502: 232-236Crossref PubMed Scopus (387) Google Scholar] were a kind gift of Dr. C. Nerlov, Oxford University (Oxford, UK). Both male and female mice were used for experiments. All procedures were performed in accordance with an Institutional Animal Care and Use Committee (IACUC)-approved University of Colorado Anschutz Medical Campus animal protocol (Protocol No. 00091). Recombinant murine IL-1β injections were performed as previously described [20Pietras EM Mirantes-Barbeito C Fong S et al.Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.Nat Cell Biol. 2016; 18: 607-618Crossref PubMed Scopus (356) Google Scholar]. Mice were injected intraperitoneally with 0.5 μg IL-1β (Peprotech) in 100 μL of 0.22-μm sterile-filtered phosphate-buffered saline (PBS)/0.2% bovine serum albumin (BSA), or 100 μL PBS/0.2% BSA alone. Mice were injected daily on alternating sides for 20 days. Transplantation experiments were performed as previously described [20Pietras EM Mirantes-Barbeito C Fong S et al.Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.Nat Cell Biol. 2016; 18: 607-618Crossref PubMed Scopus (356) Google Scholar]. Eight- to twelve-week-old Boy/J mice were lethally irradiated with 11 Gy in a split dose 3 hours apart using a 137Cs fixed-beam source (J. L. Shepherd & Associates, San Fernando, CA) and injected retro-orbitally with 250 donor test cells plus 5 × 105 Sca-1-depleted Boy/J helper BM cells. Mice were maintained on Bactrim in water for 3 weeks following transplantation. Secondary transplantations were performed similarly, but with 500 donor test cells. Donor peripheral blood chimerism was analyzed every 4 weeks by submandibular bleed and collection of blood in 4 mL of ACK buffer (150 mmol/L NH4Cl/10 mmol/L KHCO3) for flow cytometry analyses. 5-Ethynyl-2ʹ-deoxyuridine (EdU) labeling was performed by injecting 2 mg EdU in PBS intraperitoneally at the same time as the final IL-1/PBS injection, and euthanizing mice 24 hours later. At the termination of all experiments, mice were euthanized by CO2 inhalation followed by cervical dislocation according to approved protocols. BM cells were isolated by crushing legs, arms, pelves, and spines from mice in staining medium (SM) consisting of Hanks' buffered saline solution (HBSS) with 2% heat-inactivated fetal bovine serum (FBS). Cells were subsequently incubated on ice in ACK buffer for 3 min to remove erythroid cells, washed with SM, and centrifuged on a Histopaque gradient (Histopaque 1119, Sigma-Aldrich, St. Louis, MO). To enrich c-Kit+ cells for sorting, BM cells were incubated for 20 min with c-Kit microbeads (Miltenyi Biotec, 130-091-224), washed with SM, and enriched on an AutoMACS Pro cell separator (Miltenyi Biotec). For analysis of BM populations, BM cells were flushed with SM from both femurs and tibias from each mouse, treated with ACK, and counted in a ViCell automated counter (Beckman-Coulter) before staining. For hematopoietic stem and progenitor cell analyses, 107 cells were blocked with purified rat IgG (Sigma Aldrich) and stained with PE-Cy5-conjugated lineage antibodies against B220, CD3, CD4, CD5, CD8, Gr1, and Ter119, plus CD34-FITC, EPCR-PE, Mac-1-PE/Cy7, Flk2-Biotin, ESAM-APC, CD48-A700, and c-Kit-APC/Cy7 for 30 min on ice, washed with SM, and stained with Sca-1-BV421, CD41-BV510, strepatavidin-BV605, and CD150-BV785 in a staining buffer composed of 1:3 v/v Brilliant Buffer (BD Biosciences)/SM. Cells were also analyzed for IL-1R (anti-IL-1R-PE) and CD49b (anti-CD49b-PE/Cy7) by substituting EPCR-APC for EPCR-PE. For SLAM chimerism analyses, a variation of this panel was used in which CD41 and Mac-1 were omitted and CD45.1 BV650 and CD45.2-PE/Cy7 were used. For mature BM cell analyses, 5 × 105 cells were stained with Gr-1-Pacific Blue, B220-BV785, CD4-FITC, CD8-PE, Mac-1-PE/Cy7, IgM-APC, CD3-A700, and CD19-A780. Dead cells were excluded by resuspending cells in SM containing 1 µg/mL propidium iodide (PI). Samples were analyzed on a 12-channel, 3-laser FACSCelesta or a 20-channel, 5-laser Fortessa X-20 analyzer running FACSDiva software (Becton-Dickenson). Data were analyzed using FlowJo Version 10 (FlowJo). For cell sorting, anti-CD41 antibodies were not included in the staining cocktail. For sorting experiments, cells were double-sorted to purity on a FACSAria IIu or Aria Fusion cell sorter (Becton-Dickenson). For Ki67/DAPI cell cycle analysis, 107 cells were stained as previously described [34Jalbert E Pietras EM Analysis of murine hematopoietic stem cell proliferation during inflammation.Methods Mol Biol. 2018; 1686: 183-200Crossref PubMed Scopus (6) Google Scholar] using the 12-color panel described above, except using Sca-1-PE/Cy7 and excluding CD41 and Mac-1. After staining, cells were fixed with CytoFix/CytoPerm (BD Biosciences) for 30 min at room temperature (RT), washed with 1 × PermWash buffer (BD Biosciences), permeabilized with Perm Buffer Plus (BD Biosciences), washed with PermWash buffer, re-fixed with CytoFix/CytoPerm for 10 min at RT, and stained with anti-Ki67-PerCP-Cy5.5 for 1 hour at RT before being washed in PermWash. Cells were subsequently incubated with SM containing 2 μg/mL DAPI. For EdU analysis, we found that EPCR was destroyed by the fix/perm regimen. Hence, we double-sorted SLAM fractions from EdU-injected mice and mixed the cells with 1 × 105 B220+ carrier cells harvested from the spleen, and EdU was visualized using the Click-iT Plus EdU Flow Cytometry Assay Kit (Thermo Fisher Scientific) containing the AlexaFluor 488 picolyl azide fluorochrome according to the kit instructions. Fix, perm/wash, and click reagents were diluted according to the manufacturer's instructions, and sorted cells were fixed for 15 min at RT, washed in PBS/1% BSA, permeabilized for 15 min at RT, washed, and incubated with click reagents for 30 min at RT. Cells were then washed in perm/wash before analysis. For all flow cytometry applications, cells were analyzed on a four-laser, 20-channel LSRII analyzer running FACSDiva software (Becton-Dickenson) and analyzed using FlowJo. For all procedures listed above, antibody clone, manufacturer, catalogue number, and dilution information are contained in Supplementary Table E1 (online only, available at www.exphem.org.Supplementary Table E1Antibodies used in this study. Antibody information including target, fluor, manufacturer, catalog number, clone and dilution used in this study.TargetFluorManufacturerCatalogCloneDilutionB220APC-Cy7BioLegend105826RA3-6821:200B220PE-Cy5BioLegend103201RA3-6821:800c-KitAPC-Cy7BioLegend1058262B81:800CD150BV786BioLegend115937TC15-12F12.21:100CD19APC-Cy7BioLegend1155236D51:400CD3PE-Cy5BioLegend15-0031-8117A21:100CD34FITCeBioscience11-0341-85RAM341:25CD4PE-Cy5eBioscience15-0051-81GK1.51:1600CD41BV510BioLegend133923MW/reg301:400CD45APC-Cy7BD Biosciences55765930-F111:400CD45.2FITCBioLegend1098061041:400CD45.1PE-Cy7BD Biosciences560578A201:400CD48AlexaFluor 700BioLegend103416HM48-11:100CD49bPE-Cy7BioLegend103517HMα21:200CD5PE-Cy5BioLegend10061053-7.31:800CD8PE-Cy5BioLegend10071053-6.71:800ESAMAPCBioLegend1362051G81:200FcγRPerCP-eFluor710eBioscience46-0161-82931:1600Flk2BiotineBioscience13-1351-82RMV71:400Gr-1Pacific BlueBioLegend108430RB6-8C51:800Gr-1PE-Cy5BioLegend108410RB6-8C51:800IgMAPCeBioscience17-5790-82II/411:200IL-1RPEBiolegend113505JAMA-1471:50Ki67PerCP-eFluor710eBioscience46-5698-80SolA151:400Ly6CBV605BD Biosciences6077610AL211:600Mac-1PE-Cy7BioLegend101215M1/701:800Sca-1Pacific BlueBioLegend108120D71:400Sca-1PE-Cy7BioLegend108113D71:400StreptavidinBV605BioLegend405229NA1:100Ter119PE-Cy5BioLegend116201ter1191:400 Open table in a new tab For gene expression analyses, pools of 100 cells were sorted into wells of a DNase- and RNase-free 96-well plate (Applied Biosystems) containing 5 μL CellsDirect 2 × reaction buffer (Invitrogen), centrifuged for 5 min at 500g, snap-frozen on dry ice, and stored at –80°C until use. RNA was reverse-transcribed using Superscript III Taq polymerase (Invitrogen) and pre-amplified for 18 rounds with a custom 96-target DeltaGene (Fluidigm) primer panel on a PCR cycler (Eppendorf). Excess primers were removed from the pre-amplified product by incubation with Exonuclease-1 (New England Biolabs), and cDNA samples were diluted in DNA buffer. Primers and cDNAs mixed with SsoFast Sybr Green Master Mix (BioRad) were subsequently loaded onto a Fluidigm 96.96 Dynamic Array IFC and run on a BioMark HD system (Fluidigm). Data were subsequently analyzed using Fluidigm Gene Expression Software and normalized to Gusb. Relative changes were subsequently calculated using the ΔΔCt approach. Unsupervised clustering of Gusb-normalized ΔΔCt values, with Gusb removed along with poorly performing Ebf1 and Hoxa2 primer sets, was performed using average linkage. Clustering and principal component analysis (PCA) and heatmap generation were performed using ClustVis software (biit.cs.ut.ee/clustvis). PCA and PCA loading plots were generated using Prism 8 (Graphpad, San Diego, CA) from data generated by ClustVis. Statistical analyses were performed using Prism 8 software (GraphPad). p Values were determined using either a Mann–Whitney U test for bivariate comparisons or two-way analysis of variance (ANOVA) for multivariate comparisons. p Values ≤ 0.05 were considered to indicate statistical significance. To address the impact of chronic IL-1-driven inflammatory signaling on functional heterogeneity in the HSC-enriched SLAM (LSK/Flk2–/CD48–/CD150+/ESAM+) HSC faction, we injected mice intraperitoneally each day for 20 days with or without 0.5 μg of recombinant murine IL-1β. Consistent with our prior published results, chronic IL-1 treatment induced expansion of myeloid cells coincident with contraction of B-cell populations in the BM (Supplementary Figure E1A–C, online only, available at www.exphem.org), as well as expansion of SLAM HSC and multipotent progenitor (MPP)-2 and MPP3 populations [21Pietras EM Reynaud D Kang YA et al.Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.Cell Stem Cell. 2015; 17: 35-46Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar,35Cabezas-Wallscheid N Klimmeck D Hansson J et al.Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.Cell Stem Cell. 2014; 15: 507-522Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar] (Supplementary Figure E1D,E, online only, available at www.exphem.org). To study IL-1-induced changes in the SLAM compartment, we re-analyzed cells in this compartment using EPCR and CD34, which have previously been used to distinguish functional HSCs in the SLAM fraction [7Wilson A Laurenti E Oser G et al.Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.Cell. 2008; 135: 1118-1129Abstract Full Text Full Text PDF PubMed Scopus (1374) Google Scholar,36Kent DG Copley MR Benz C et al.Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.Blood. 2009; 113: 6342-6350Crossref PubMed Scopus (238) Google Scholar]. Coordinate use of EPCR and CD34 allowed us to prospectively identify four distinct phenotypic compartments within the SLAM gate (Figure 1A). Strikingly, the relative abundance of these compartments was altered significantly following chronic IL-1 exposure, with a significant decrease in the frequency of EPCR+/CD34– cells, alongside twofold expansion in the frequency of the EPCR–/CD34– compartment (Figure 1B). Despite decreased frequency, the absolute number of EPCR+/CD34– cells in the BM was unchanged (Figure 1C), whereas increased absolute numbers of the EPCR–/CD34– fraction substantially contributed to the overall numerical expansion of SLAM cells observed after chronic IL-1 exposure (Figure 1C; Supplementary Figure E1D, online only, available at www.exphem.org).Figure 1The EPCR+/CD34– SLAM fraction enriches for functional HSCs. (A) Experimental design and representative fluorescence-activated cell sorting (FACS) plots of SLAM cells from mice treated with or without IL-1 for 20 days fractionated by EPCR and CD34 expression. (B,C) Frequency (B) and absolute number (C) of EPCR/CD34 SLAM cell fractions (n = 9 or 10 per group). (D–F) Long-term engraftment of purified SLAM cells fractionated by EPCR and CD34 expression, from mice treated with or without IL-1 for 20 days: (D) experimental design; (E) donor chimerism in peripheral blood (PB); (F) phenotypic SLAM compartment of recipient mice at the indicated time points (N = 18–20/group, compiled from two independent experiments). (G) Donor PB and (H) SLAM chimerism after secondary transplant of donor-derived SLAM cells from primary recipient mice in (D) (n = 18–20/group, compiled from two independent experiments). (I–K) Long-term engraftment of SLAM and EPCR+/CD34– SLAM cells from mice treated with or without IL-1 for 20 days: (I) experimental design; (J) donor chimerism in peripheral blood (PB); (K) phenotypic SLAM compartment of recipient mice at the indicated time points (N = 9 or 10 per group, representative of two independent experiments). °°p < 0.01 versus –IL-1 EPCR+/CD34– SLAM cells by one-way analysis of variance. *p < 0.05, **p < 0.01, ***p < 0.001 versus –IL-1 condition in each fraction by Mann–Whitney U test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To interrogate the functional properties of SLAM fractions defined by CD34 and EPCR expression in homeostatic and chronic inflammatory conditions, we first isolated each fraction from mice treated with or without IL-1 for 20 days, transplanted them into lethally irradiated CD45.1+ recipient mice, and analyzed long-term repopulating activity by bleeding recipient animals every 4 weeks (Figure 1D; Supplementary Figure E2A, online only, available at www.exphem.org). Notably, the EPCR+/CD34– fraction contained the majority of long-term repopulating
Referência(s)