Article start

ArticlePro-inflammatory Aorta-Associated MacrophagesAre Involved in Embryonic Development ofHematopoietic Stem CellsGraphical AbstractHighlightsdYolk-sac-derived macrophages are the most abundanthematopoietic cells in the AGMdCx3cr1 mediates yolk-sac macrophage progenitorrecruitment to the AGM nichedAGM macrophages dynamically interact with emerging intra-aortic hematopoietic cellsdPro-inflammatory AGM macrophages are positive regulatorsof HSC generationAuthorsSamanta Antonella Mariani, Zhuan Li,Siobhan Rice, ...,Chris Sebastiaan Vink,Jeffrey William Pollard, Elaine (S.A.M.), (E.D.)In BriefHSC-independent macrophages derivefrom the early yolk-sac stages ofembryonic hematogenesis. Mariani andcolleagues demonstrate that specific pro-inflammatory embryonic HSC-independent macrophages recruited tothe AGM (AGM-aMs) are crucialcomponents of the AGMmicroenvironment, dynamically interactwith emerging hematopoietic cells, andenhance HSC generation.macrophageprogenitors HSCAGM-associatedCD206+ macrophages Cx3cr1+AGMECIAHCYolk Sac IntravasationPro-inflammatorySignalsHECChemokine attractionMMP mobilizationHSCHPCMariani et al., 2019, Immunity50, 1–14June 18, 2019ª2019 The Author(s). Published by Elsevier Inc.
ImmunityArticlePro-inflammatory Aorta-Associated MacrophagesAre Involved in Embryonic Developmentof Hematopoietic Stem CellsSamanta Antonella Mariani,1,*Zhuan Li,1Siobhan Rice,1Carsten Krieg,2Stamatina Fragkogianni,4Mark Robinson,3Chris Sebastiaan Vink,1Jeffrey William Pollard,4and Elaine Dzierzak1,5,*1Centre for Inflammation Research, The University of Edinburgh, Edinburgh, UK2Medical University of South Carolina, Charleston, SC, USA3University of Zurich, Zurich, Switzerland4MRC Centre for Reproductive Health, The University of Edinburgh, Edinburgh, UK5Lead Contact*, stem cells (HSCs) are generated fromspecialized endothelial cells of the embryonic aorta.Inflammatory factors are implicated in regulatingmouse HSC development, but which cells in theaorta-gonad-mesonephros (AGM) microenvironmentproduce these factors is unknown. In the adult, mac-rophages play both pro- and anti-inflammatory roles.We sought to examine whether macrophages or otherhematopoietic cells found in the embryo prior to HSCgeneration were involved in the AGM HSC-generativemicroenvironment. CyTOF analysis of CD45+AGMcells revealed predominance of two hematopoieticcell types, mannose-receptor positive macrophagesand mannose-receptor negative myeloid cells. Weshow here that macrophage appearance in the AGMwas dependent on the chemokine receptor Cx3cr1.These macrophages expressed a pro-inflammatorysignature, localized to the aorta, and dynamicallyinteracted with nascent and emerging intra-aortic he-matopoietic cells (IAHCs). Importantly, upon macro-phage depletion, no adult-repopulating HSCs weredetected, thus implicating a role for pro-inflammatoryAGM-associated macrophages in regulating thedevelopment of HSCs.INTRODUCTIONHematopoietic stem cell (HSC) transplantation is a curativeregenerative therapy for patients with blood-related disorders.More than 50,000 transplants are carried out annually world-wide. For patients without a histo-compatible donor, the lackof matched HSCs is a serious problem. Given that HSCs donot expand inin vitrocultures, patient-derived induced pluripo-tent stem cells (iPSCs) may be an alternative source for thedenovoproduction of HSCs. Although it is possible to differentiateiPSCs and to reprogram cells into hematopoietic progenitors,the generation of robustin vivorepopulating HSCsin vitrohasnot yet been achieved without genetic manipulation (Doulatovet al., 2013). Thus, an understanding of thein vivomicroenviron-ment in which HSCs are first generated should provide insightsinto improving suchin vitrostrategies.HSCs arise in the AGM (aorta-gonad-mesonephros) region(Medvinsky and Dzierzak, 1996) and other major arteries (deBruijn et al., 2000) through a transdifferentiation process calledendothelial-to-hematopoietic transition (EHT) (Dzierzak andBigas, 2018; Jaffredo et al., 1998). In the mouse embryo, HSCsare generated in a complex and dynamic microenvironmentduring a short period of developmental time (embryonic day [E]10.5–E12.5) (Zovein et al., 2008). Tissues ventral to the AGM(Peeters et al., 2009; Taoudi and Medvinsky, 2007), includingcells of the sympathetic nervous system (Fitch et al., 2012), exerta positive effect on HSC emergence. Moreover, AGM-derivedstromal cell lines produce regulators, such as bone morphoge-netic protein (BMP) and hedgehog (Hh), that support HSC gener-ation and/or maintenance, as demonstrated by transcriptionalprofiling and culture approaches (Charbord et al., 2014; Crisanet al., 2015; Durand et al., 2007; Renstro ̈m et al., 2009).Hematopoietic development occurs in at least three distinctwaves (Dzierzak and Bigas, 2018), with HSCs made only in thelast wave. Macrophages are generated in all three waves, firstin the mouse yolk sac (YS) beginning at E7.5 (Palis et al., 1999;Tober et al., 2007), then in the second YS wave at E8.25 (GomezPerdiguero et al., 2015) from erythro-myeloid progenitors (EMPs)(Frame et al., 2013; Palis et al., 1999), and finally from HSC-derived monocytes. EMPs emigrate to the embryo body, wherethey give rise to hematopoietic cells found in the embryo prior toHSC emergence at E10.5. Because the three distinct waves ofhematopoietic generation temporally overlap, the hematopoieticcells generated in the earlier two waves could be part of themicroenvironment that affects the production of HSCs intraem-bryonically in the third wave (Chen et al., 2011; Espı ́n-Palazo ́net al., 2014; Li et al., 2014; Travnickova et al., 2015).Macrophages from the first two waves function developmen-tally in tissue remodeling (Hume et al., 1983; Leid et al., 2016;Munro et al., 2019). They infiltrate the limbs, mandibular arches,liver, kidneys, and brain between E9.5 and E10.5 and represent2%–5% of total cells in these tissues (Rae et al., 2007). In adultImmunity50, 1–14, June 18, 2019ª2019 The Author(s). Published by Elsevier Inc.1This is an open access article under the CC BY-NC-ND license ( cite this article in press as: Mariani et al., Pro-inflammatory Aorta-Associated Macrophages Are Involved in Embryonic Development of Hemato-poietic Stem Cells, Immunity (2019),
  1. IMMUNI4144_proof.pdf
  2. Pro-inflammatory Aorta-Associated Macrophages Are Involved in Embryonic Development of Hematopoietic Stem Cells
  3. Introduction
  4. Results
  5. Macrophages Are the Most Abundant Hematopoietic Cells in the AGM
  6. Macrophages Accumulate in the AGM Prior to HSC Production
  7. Cx3cr1 Is Involved in Macrophage Progenitor Recruitment to the AGM
  8. AGM-Associated Macrophages Dynamically Interact with Nascent and Emerging IAHCs
  9. Hematopoietic Progenitor Cell Numbers Are Proportional to AGM Macrophage Percentages
  10. AGM HSC Production Is Impaired in the Absence of Macrophages
  11. CD206 Discriminates AGM-Associated Macrophages from Macrophage Progenitors
  12. AGM-Associated CD206+ Macrophages Enhance Endothelial-to-Hematopoietic Transition
  13. AGM-aMs Are Inflammatory and Transcriptionally Distinct from Macrophage Progenitors
  14. Discussion
  15. Supplemental Information
  16. Acknowledgments
  17. Author Contributions
  18. Declaration of Interests
  19. References
  20. STAR★Methods
  21. Key Resources Table
  22. Contact for Reagent and Resource Sharing
  23. Experimental Model and Subject Details
  24. Method Details
  25. Mouse and embryo generation
  26. Mass cytometry (CyTOF)
  27. Mass cytometry statistical analysis
  28. Whole-mount Images
  29. Tissue Sections
  30. Time-lapse ex vivo imaging
  31. AGM explant cultures
  32. CFU-C assay and stem cell transplantation
  33. OP9/OP9-DL1 co-culture system
  34. BLZ945 and Clodronate-liposome treatment
  35. Flow analysis and sorting
  36. Gene Expression Analysis
  37. RNA-seq
  38. Sequencing alignment and Quantification
  39. Statistical analysis for differentially expressed genes
  40. Quantification and Statistical Analysis
  41. Data and Software Availability