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. 2018 Jul 5;8:10172. doi: 10.1038/s41598-018-28234-y

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© The Author(s) 2018

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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AM-like cells phagocytosed bloodborne bacteria. Inactivated FITC-labelled pseudomonas bacteria were injected in the jugular vein of anesthetized pigs. Ten minutes after, blood was collected on heparinized tubes and proceeded to Ficoll isolation of peripheral blood mononuclear cells (PBMC), animals were euthanized and cells from broncho-alveolar lavages and parenchyma were collected and stained for FACS analysis of DC and Mϕ. (a) Dot plot gating strategy of lung DC and Mϕ as previously described⁴ and histograms of phagocytosed bacteria (FITC) signal in the different gated populations. The PBMC gate 4 contained no event. One representative out of 4 independent experiments. (b) Plotting of the 4 independent experiments. Statistics: Mann-Whitney test *p < 0.05. (c) Confocal imaging of parenchyma from FITC-bacteria injected animals. Upper left, DAPI (white), FITC-bacteria (green), CD163 (red) and MHC-II (blue) staining. Upper right, magnification of the squared zone of the upper left picture. Arrows indicate PIM/AM-like cells (extra-alveolar CD163pos/MHC-IIpos) having internalized FITC-bacteria. Lower left, DAPI (white), FITC-bacteria (green), ve-Cadherin (red) and CD163 (blue) staining. Lower right, magnification of the squared zone of the lower left picture. Arrows indicate PIM/AM-like cells (extra-alveolar CD163pos) localized in ve-Cadherin rich area (lung capillaries) and having internalized FITC-bacteria. Alveoli are visualized by dashed lines. Images are representative of 3 independent experiments.