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1 Supplementary Information Distinct bone marrow-derived and tissue resident macrophage lineages proliferate at key stages during inflammation. 1 Luke C. Davies, 1 Marcela Rosas, 2 Stephen J. Jenkins, 1 Chia-Te Liao, 1 Martin J. Scurr, 3 Frank Brombacher, 4 Donald J. Fraser, 2 Judith E. Allen, 1 Simon A. Jones and 1 Philip R. Taylor 1 Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff, CF14 4XN, UK. 2 Centre for Immunity, Infection and Evolution, and the Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JT, UK. 3 International Center for Genetic Engineering and Biotechnology, Cape Town component, South Africa & Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa 4 Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Heath Park, Cardiff, CF14 4XN, UK. 1

2 F4/8-PE-TxR Cells (x1 6 ) Ly6B Log Comp % >2N DNA Cell counts (x1 6 ) % SG 2 M % Ki67 high, 4N DNA % phh3 +, 4N DNA Supplementary Figures A 3 Ly-6B + Ly-6B B ** *** P< P<.1 P<.1 *** C F48 Log Comp Res MØ Ly-6B Inf MØ Ly-6B-A647 Ly-6B + Inf MØ Mo D Supplementary Figure S1. MØ proliferation zymosan and thioglycollate peritonitis. A) High dose zymosan peritonitis (2x1 7 particles), recruits more Inf MØ than low dose peritonitis, but Ly-6B + and Ly-6B Inf MØ are present in similar proportions to the low dose model (compare with Figure 1c). B) These graphs show proliferation in Ly-6B + and Ly-6B Inf MØ 72 hours after the induction of peritonitis with a higher dose (2x1 7 ) of zymosan particles. When subdivided based on Ly-6B expression there was a clear enrichment of cells in cell cycle amongst the Ly-6B expressing cells. The levels of proliferation within the two populations of Inf MØ was measured by the proportion of cells with >2N DNA content (left), >2N DNA content with Ki67 expression ( SG2/M, middle), phospho-histone H3 + phase of mitosis ( phh3 + ; right). Data is expressed as mean±sem of 5 mice per group (7-9 week old female 129S6 2

3 mice) and is representative of 2 similar experiments. Data were analyzed by a paired t-test. C) Representative flow-cytometric analysis of peritoneal MØ 7 days after intraperitoneal administration of thioglycollate broth. At this time point cells with the phenotype of Res MØ and Ly-6B + Inf MØ are present. D) Quantification of MØ numbers (left) and proliferation (right) in the peritoneal cavity of thioglycollate challenged (i.p.) mice. Data shown is 7 days after induction of peritonitis and representative of 5 mice from one of two independent experiments (9-11 week old 129S6 mice). 3

4 M-CSF (ng) Naïve 48 hour postzymosan Supplementary Figure S2. Measurement of M-CSF in naïve and intraperitoneally challenged mice. M-CSF in the lavage fluid of naïve mice and mice 48 hours after the induction of peritonitis with 2x1 6 zymosan particles. Data is adjusted for lavage volume and expressed as M-CSF in the peritoneal cavity. Each symbol represents an individual 6 week old C57BL/6 female mouse and data are pooled from 2 identical experiments. The horizontal bars denote the group means. 4

5 Mitosis % % mitotic Ki67+ % % Ki67 + Naive 48hr 72hr ZIP Naive (r 2 =.96; n.s.) 48 hours (r 2 =<.1; ) 72 hours (r 2 =.53; n.s.) *** Supplementary Figure S3. Considerations for the use of Ki67 as a measure of cell division. Naïve mice and mice 48 and 72 hours after the induction of peritonitis with 2x1 6 zymosan particles were analyzed for evidence of proliferation using ki67 expression and indicators of active mitosis (4N DNA content associated with phh3 or Ki67 high phenotype 18 ). In this model, a marked increase in mitotic events is transient at the 48 hour time point 18. High Ki67 expression in Res MØ of mice did not always correlate with the levels of mitosis, indicating that definitive measures of S, G 2 /M phases of cell cycle were most reliable in identifying MØ proliferation. All mice used were 6-7week female C57BL/6 and each symbol represents an individual mouse. 5

6 F4/8 F4/8 Donor cell number Donor cells (% of transfered transferred) cells) A B Ly-6B Ly-6B Time after transfer (hours) Time (hours) after transfer Supplementary Figure S4. Adoptive transfer of Ly-6B + Inf MØ into congenic mice during peritonitis.a) Ly-6B + Inf MØ (1.5x1 5 ) from CD45.2 (129S6) mice were purified by flow-cytometry (FACS Aria III) and transferred into 129S6 CD45.1 congenic mice. The cells were purified from donors 3 days after induction of peritonitis with 2x1 6 zymosan particles and transferred into recipients at the same stage of an identical peritoneal challenge. The flow-cytometry plots indicated (A) are gated on CD11b + F4/8 + monocytes and MØ (after excluding eosinophils as detailed in the main text (and 18 ) and any residual neutrophils via their Ly-6G expression). The density plots show the recipients monocytes and MØ, the superimposed contour plots show the adoptively transferred cells 24 hours (left panel) and 9 hours (right panel) after adoptive transfer (~days 4 and 7 after induction of zymosan peritonitis). The quandrants delineate expression of Ly-6B and the higher F4/8 expression of Ly-6B + Inf MØ. B) The persistence of adoptive transferred cells expressed as a % of the transferred cell number. Data shown represents the mean±sem of 4 mice per time point. 6

Distinct bone marrow-derived and tissue-resident macrophage lineages proliferate at key stages during inflammation

Distinct bone marrow-derived and tissue-resident macrophage lineages proliferate at key stages during inflammation Received 18 Sep 212 Accepted 12 Apr 213 Published 21 May 213 DOI: 1.138/ncomms2877 Distinct bone marrow-derived and tissue-resident macrophage lineages proliferate at key stages during inflammation Luke

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