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. 2021 Mar 25;19:83. doi: 10.1186/s12951-021-00830-7

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Reaction of immune cell subsets after stimulation with supernatant from degraded CCL25-loaded/unloaded PLGA particles. CCL25-loaded (CL) and unloaded (NL) PLGA particles were allowed to degrade for either 21 or 63 days and particle supernatant was collected and used for in vitro stimulation assays with human PBMCs and macrophages. Changes in the cytokine/chemokine release pattern of PBMCs and macrophages were analyzed with a LEGENDplex cytokine array. Additionally, macrophages were analyzed by immunofluorescence staining and flow cytometry for the expression of surface markers indicating activation or polarization. a Cytokine array of PBMCs stimulated over 5 days with 21- and 63-day CL and NL PLGA particle supernatants. Cytokines with detectable differences in fold change are IL-8, MCP-1 and IL-1β. Data is shown as the mean fold change + SD normalized to the negative control (red dotted line) (n = 3). b Cytokine array of M0/M1 macrophages stimulated over 1 day with 21- and 63-day CL and NL PLGA particle supernatants. Cytokines with detectable differences in fold change are IL-8 and MCP-1. Data is shown as mean fold change + SD normalized to the negative control (red dotted line) (n = 4). c The migration of immune cell subsets towards a dilution series of particle degradation supernatant is displayed as the mean + SD of the number of migrated cells (n = 4). d/e Flow cytometric analysis of M0/M1 macrophage polarization surface markers after 1 day of stimulation with 21- and 63-day CL and NL PLGA particle supernatants. Increased CD80 and HLA-DR indicates M1 polarization (d), while increased CD163 and CD206 indicates M2 polarization (e). Results are displayed as mean of the fold change + SD normalized to the control (red dotted line), (n = 4). Statistical analysis was performed using either a student’s T-test or the Mann–Whitney-U test depending on whether data followed normal distribution. Individual p-values are given above the bars if lower than p = 0.20. CL CCL25-loaded, NL non-loaded, NC negative control, ΜΦ Macrophage