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. 2019 Jul 2;9:9559. doi: 10.1038/s41598-019-45966-7

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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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Topical instillation of Z-FL reduces CD68+ cell abundance and expression of MHC II (H2-Ab1) in LG, but does not affect LG lymphocytic infiltration. 14–15 week old male NOD mice were treated twice a day for 6 weeks with 200 µM Z-FL. (A) LG were obtained and assessed for area occupied by lymphocytic infiltration. Topical treatment with Z-FL was no different from vehicle. (n = 13, 14 for vehicle, drug groups respectively); (B) LG lysates were prepared and assessed for H2-Ab1 gene expression. Treatment with Z-FL significantly reduced gene expression relative to vehicle (n = 7/group, P = 0.04). Expression of H2-Ab1 was normalised to endogenous Gapdh; (C) LG were obtained, sectioned, assessed by immunofluorescence for CD68, and quantified. The percentage of CD68+ cells in areas of lymphocytic infiltration were significantly lower upon treatment with Z-FL compared to vehicle alone (n = 3/groups, P = 0.04); (D) Representative LG images of CD68+ cell immunostaining in areas of lymphocytic infiltration quantified in (C). CD68 (Green) labels pan-macrophages, dendritic cells and progenitor monocytes and DAPI (Blue) labels nuclei. Scale bar = 10 µm. Data represent mean ± SD. A two-tailed, unpaired Student’s t-test was used to compare treatments.