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Pre/Pro-B Cells Generate Macrophage Populations During Pre/pro-B cells generate macrophage populations PNAS PLUS during homeostasis and inflammation Tatsiana Audzevicha, Rachael Bashford-Rogersb, Neil A. Mabbottc,d, Dan Framptone, Tom C. Freemanc,d, Alexandre Potocnikf, Paul Kellamb, and Derek W. Gilroya,1 aCentre for Clinical Pharmacology and Therapeutics, Division of Medicine, University College London, London WC1E 6JJ, United Kingdom; bDepartment of Medicine, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0XY, United Kingdom; cThe Roslin Institute, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, United Kingdom; dRoyal (Dick) School of Veterinary Sciences, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, United Kingdom; eDivision of Infection and Immunity, University College London, London WC1E 6AE, United Kingdom; and fInsitute of Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3FL, United Kingdom Edited by Harinder Singh, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH and accepted by Editorial Board Member Ruslan Medzhitov March 31, 2017 (received for review October 6, 2016) Most tissue-resident macrophages (Mφs) are believed to be derived publications (16, 18, 19), mature B cells do not differentiate into prenatally and are assumed to maintain themselves throughout Mφs. Instead, we found a population of cells within the early pro- life by self-proliferation. However, in adult mice we identified a B-cell/fraction B-cell compartment with non-rearranged B-cell progenitor within bone marrow, early pro-B cell/fraction B, that receptor (BCR) genes that exit bone marrow under steady state, differentiates into tissue Mφs. These Mφ precursors have non- circulate in peripheral blood, and populate serous cavities where rearranged B-cell receptor genes and coexpress myeloid (GR1, CD11b, they differentiate into Mφs, thereby contributing to the overall and CD16/32) and lymphoid (B220 and CD19) lineage markers. During pool of tissue Mφs alongside embryonically derived cells. Hence, steady state, these precursors exit bone marrow, losing Gr1, and in addition to local proliferation, we now further refine our un- enter the systemic circulation, seeding the gastrointestinal system derstanding of how tissue Mφs numbers are maintained during as well as pleural and peritoneal cavities but not the brain. While adulthood. Moreover, these data report cells with origins that in these tissues, they acquire a transcriptome identical to embryoni- are distinct from embryonic Mφ precursors but nonetheless dif- cally derived tissue-resident Mφs. Similarly, these Mφ precursors also ferentiate into cells with equivalent phenotypes. CELL BIOLOGY enter sites of inflammation, gaining CD115, F4/80, and CD16/32, and become indistinguishable from blood monocyte-derived Mφs. Thus, Results we have identified a population of cells within the bone marrow Mφs with a Potential B-Cell Origin Detected Using Mb1-iCre/Rosa26R-YFP Reporter Mice. Analysis of cells from the peritoneal cavity of naive early pro-B cell compartment that possess functional plasticity to dif- ++ ++ ferentiate into either tissue-resident or inflammatory Mφs, depend- wild-type mice revealed a population of F4/80 CD11b Mφs ing on microenvironmental signals. We propose that these precursors that also expressed CD19 (Fig. 1A). These cells constitute ∼0.8% represent an additional source of Mφ populations in adult mice dur- of total Mφs, suggesting a potential developmental relationship ing steady state and inflammation. between mononuclear phagocytes and B cells. Crossing Mb1-iCre (20) with Rosa26-YFP (21) mice allowed us to investigate this homeostasis | inflammation | macrophages potential further, because Mb1-dependent Cre-recombinase ex- pression is induced in B cells during the very early pro-B stage (20). Thus, tissue Mφs that express YFP must have been derived from B onocytes and macrophages (Mφs) maintain tissue ho- cells. In naive Mb1-iCre/Rosa26-YFP mice a significant proportion Mmeostasis and orchestrate immune-mediated responses to of Mφs expressed YFP, including ∼25% of peritoneal Mφs, 4–10% infection/injury. During embryonic hematopoiesis, monocytes arise of pleural cavity Mφs, and 1–2.5% of intestinal Mφs, with very few from myeloid precursors in fetal liver, whereas during adulthood they are derived from bone marrow progenitors. Circulating Significance monocytes migrate to sites of infection/injury where they differ- entiate into Mφs (1, 2) and under certain circumstances can also replenish Mφs in the colon (3). In contrast, the majority of resident In this report we provide evidence of a source of macrophage φ Mφ populations are embryonically derived and are proposed to (M ) populations that are derived from unique biphenotypic maintain their numbers by local proliferation after birth (4–9). early pro-B cells with non-rearranged B-cell receptors. These However, in the course of our studies we identified a population of early precursors give rise to either tissue resident- or monocyte- φ biphenotypic Mφs in the serous cavities of naive mice that coex- derived M s during homeostasis and inflammatory responses, press the B-lineage marker CD19 and the Mφ markers CD11b and thereby demonstrating functional plasticity depending on the environmental cues in adult mice. We suggest that these find- F4/80. Interestingly, revised models of hematopoiesis (10, 11) and φ evidence of developmental plasticity within hematopoietic precur- ings significantly advance and expand our understanding of M biology and hematopoiesis, the plasticity of hematopoietic pre- sors suggest a closer developmental relationship between myeloid φ and lymphoid lineages than previously appreciated (10–14). Con- cursors, and the heterogeneity of M subsets. sequently, because there is convincing evidence that lymphocyte-to- φ Author contributions: D.W.G. designed research; T.A. and A.P. performed research; R.B.-R., M differentiation can occur in vitro under artificial experimental N.A.M., D.F., T.C.F., and P.K. contributed new reagents/analytic tools; N.A.M., D.F., T.C.F., and settings (15–19), we determined whether the biphenotypic cells D.W.G. analyzed data; and D.W.G. wrote the paper. we observed in the peritoneal and pleural cavities and in the gas- The authors declare no conflict of interest. trointestinal system were derived from B lymphocytes or from This article is a PNAS Direct Submission. H.S. is a guest editor invited by the Editorial precursors within the B-cell lineage. This latter derivation would Board. challenge the existing view that local self-proliferation is the only Freely available online through the PNAS open access option. means by which tissue-resident Mφs maintain their numbers Data deposition: Microarray data are archived under ArrayExpress accession number throughout adulthood. E-MTAB-1878. Using a range of B-cell lineage-specific transgenic reporter mice 1To whom correspondence should be addressed. Email: [email protected]. and single-cell PCR, ImageStream/polychromatic flow cytometry, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and adoptive transfer studies, we show that, contrary to previous 1073/pnas.1616417114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1616417114 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 + A YFP MφsinMb1-iCre/Rosa26R-YFP Mice Do Not Arise from Mature B Cells. The hematopoietic development of B cells includes seven – ′– Mac stages (A CandC F), which are defined by stage-specific cell- 53.0 CD19+ F4-80+ surface marker expression and BCR gene rearrangement status F4/80 0.84 CD11b (22, 23). Rearrangement of Ig genes, which is initiated during 0" 0" bone marrow development at the pro-B-cell stage/fraction B stage and is completed in pre-B/fraction C′ B cells (23), leads to surface 0" 0" expression of BCRs of unique DNA sequence and enables B-cell F4/80 CD19 development and survival (24). This distinctive feature of B-cell + development was exploited to determine whether YFP Mφs B Gated Gated on on ssue-residenttissue-resident Mφs Mφs carry V-DJ rearrangements in their IgH locus as evidence of their origin from mature/pre B cells. Peritoneum Pleural Cavity Intestine Before embarking on single-cell PCR to determine whether + YFP Mφs carry V-DJ rearrangements, we ensured that the cell populations we intended to sort by FACS were single cells and not + YFP+ doublets or clusters of MφswithYFP B cells. Previous studies F4/80 F4/80 F4/80 24.5 YFP+ have reported biphenotypic murine peritoneal Mφs coexpressing 0" YFP+ 0" 1.97 0" 7.28 F4/80 and CD11b along with surface IgM, CD5, CD19, and/or B220(25–27) but have not considered the possibility that cell 0" 0" 0" YFP YFP YFP doublets/clusters might generate potential false-positive data. ImageStream analysis of the murine peritoneum identified three ++ ++ − − Spleen Liver Brain distinct cell types within the CD11b F4/80 Mφ gate: CD19 YFP − + + + (embryonic Mφs) and CD19 YFP and CD19 YFP cells (Fig. 2A). We suspect that these biphenotypic Mφ coex- CD11b CD11b YFP+ CD11b YFP+ pressing CD11b, F4/80, YFP, and CD19 are intermediates in YFP+ 0.2 0.3 the transition of B cells to Mφs. However, ImageStream also 0" 0" 0" 0.63 revealed significant numbers of doublets (Mφ/B-cell clusters) + + within this intermediate CD19 YFP Mφ population, with 0" 0" 0" YFP YFP YFP single biphenotypic cells constituting only ∼16.74% of the total + + φ ∼ φ CD19 YFP M s (Fig. 2B); thus 80% of the cells within the Fig. 1. M s with a potential B-cell origin detected in Mb1-iCre/Rosa26R-YFP biphenotypic population are aggregates of Mφs and B cells. See reporter mice. (A) Flow cytometric analysis of wild-type C57BL6 mice show- ++ ++ ing CD19 expression on peritoneal CD11b F4/80 Mφs. (B) Equivalent SI Appendix, Fig. S3.1 for further data on identifying clusters/ doublets. analysis of Mb1-iCre/Rosa26-YFP mice showing the YFP expression pattern in − − φ Therefore, we sorted CD19 YFP (embryonic Mφs) and M s from various tissues.
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