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. 2017 Aug 25;7:9417. doi: 10.1038/s41598-017-09639-7

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© The Author(s) 2017

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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SP induces M2 polarization through early induction of Arginase-1. (a) Mo^BM were collected after 1-h of attachment and differentiated into MΦ^GM-CSF by GM-CSF treatment for 5 d. MΦ^GM-CSF (CD14⁻CD11b⁺CD68⁺CCR7⁺CD206⁺) were defined as M1-like precursors that were differentiated from Mo^BM (CD14⁺CD11b⁺CD68⁻CCR7⁻CD206⁺). For the immunofluorescence staining results (Supplementary Fig. 1a), the number of positive-cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm²) (n = 3). (b) CD14⁺ MACS-isolated monocytes were differentiated with GM-CSF treatment for 5 d, and cells were further characterized as CD11b⁺CD68⁺ macrophages. (Green = CD11b, Red = CD68, Blue = DAPI) (n = 3). (c) Proposed model for MΦ^GM-CSF from Mo^BM with 80% CD14⁺ and 20% CD14⁻ phenotypes. (d) Increased Arginase-1 activity was detected at the indicated time point of SP treatment using the Arginase-1 activity assay, and the results were compared with typical M1/M2-inducing cytokines (n = 3). (e) The increased expression of Arginase-1 was detected after 6 h of SP treatment. The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16a (n = 4). (f–g) Dose-dependent increased activity and expression of Arginase-1 determined after 6 h of SP treatment. This was completely blocked by pretreatment with the NK-1R antagonist RP67580 (1 μM). (f) Arginase-1 activity assay (n = 3). (g) The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16b (n = 4). (h) Increased NO was detected in culture with IFNγ, GM-CSF, or LPS but not with SP, IL-4/13, or IL-10. Quantitative analysis of NO production was performed in conditioned medium from MΦ^GM-CSF that had been treated with SP or cytokines for 1 d. LPS treatment served as a positive control (n = 6). Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 100 μm (b) and 10 μm (high-magnification image of inset in b).