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. 2019 Oct 1;9:14050. doi: 10.1038/s41598-019-50320-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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In-vitro analysis of bone marrow-derived macrophages (BMM) polarization upon infection with IOE or E. muris. BMM differentiated from bone marrow cells obtained from naive C57BL/6 mice were kept under non-polarizing conditions and infected with IOE or E. muris (EM). After 24 h, cells were harvested and analyzed by flow cytometry using specific markers of M1/M2 polarization. (A) Flow cytometry analysis of BMM after infection with Ehrlichia. Only CD11b⁺F4/80⁺ live cells (BMM region) were considered in this analysis as shown by the gating strategy represented by flow dot plots. Quantity of MHC class II^hi, iNOS⁺, TNF-α⁺ cells (as percentage) and CD206 (as mean fluorescence intensity - MFI) were assessed within the BMM region. Each marker is accompanied by quantitative analysis considering each studied group (naive, EM or IOE). (B) Representative western blot analysis of protein extracts showing expression of arginase-1, and GAPDH as a load control. Quantitative analyses expressed as relative densitometry was calculated based on GAPDH levels. Full-length blots are presented in Supplementary Fig. S5, from which data were cropped and assembled. (C) Measurement of TGF-β (pg/ml) on the conditioned media of the BMM cultures infected or not with Ehrlichia (time point of 24 h). All quantitative analyses are expressed as mean and standard deviation. Asterisks represent relevant statistical difference between groups. Data is representative of three experimental sets performed individually.