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. 2020 Nov 5;10:19120. doi: 10.1038/s41598-020-76109-y

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© The Author(s) 2020

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 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/.

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Abolishment of protective effect of butyrate upon Treg depletion. Hyperglycemic NOD female mice (n = 8) were treated with sodium butyrate (150 mM) in drinking water for 6 weeks and randomly divided into two groups. The treatment group (n = 4) received two intraperitoneal injections of anti-CD25 antibody (0.5 mg in PBS), while the control group (n = 4) received PBS alone, one week apart. (a) The frequency of Tregs (CD4 + FoxP3 + T cells) gated on live CD3 + CD4 + T cells in colon, MLN, PP and spleen were assessed by flow cytometry (n = 4 per group). (b) Representative flow cytometry plots show the frequency of CD4 + FoxP3 + T cells in colon of PBS treated and anti-CD25 treated NOD mice. (c) The graph shows the blood glucose levels of hyperglycemic NOD mice at day 0 and 6 weeks after butyrate treatment (n = 8). The graph also shows the blood glucose levels of mice treated with PBS (n = 4) and anti-CD25 (n = 4) at 7th (after the 1st dose of injections) and 8th week (after the 2nd dose of injections). (d) The pancreas were assessed for insulitis at 8th week post treatment in NOD mice. Representative photomicrographs of H&E stained pancreas sections of PBS administered (left) and anti-CD25 administered (right) groups at 40 × magnification; scale bars indicate 100 µm. Statistical significance was determined by Mann–Whitney U test, with P < 0.05 considered significant. Data are shown as mean ± SEM.