Marilen Benner

GESTATIONAL ANTIBIOTICS AFFECT IMMUNITY 193 7 group, Figure 3A). Percentages of CD4 + CCR6 + Ror γ T + Th17 cells were lower in the spleens of mice treated with antibiotics (1.4% ± 0.1% compared to 1.1% ± 0.1% in the control group, Figure 3B) but no such differences were observed in other tissues. A significantly lower percentage of splenic CD4 + CD25 + FOXP3 + regulatory T cells (Treg) was observed in the antibiotic-treated group (5.3% ± 0.4% compared to 6.7% ± 0.3% in the control group, Figure 3C), but not in other compartments (Supplementary Figure S3C). Overall, T cell activation as observed through CD25 and CD69 expression was not affected in any of the tissues (Supplementary Figure S3D). Functional assessment of maternal lymphocytes through splenocytes stimulated ex vivo with LPS revealed only limited alterations of detectable cytokine levels in the supernatant. IL-6 production was significantly affected by the antibiotic treatment (Figure 3D) and a trend towards a higher IL-22 production was observed (p 0.061, Figure 3E). No significant differences were observed for any of the other cytokines analyzed (IFN γ, TNF α , IL-1 β , IL-10, IL-2). Control Antibiotics A C B D E Th17 Splenocytes (24h LPS) Splenocytes (24h LPS) Th2 Placenta Spleen Peritoneal cavity MLN ILN IL-22 IL-6 Placenta %CD4+CCR6+RorγT+ of T cells pg/ml pg/ml %CD4+T1/ST2+ of T cells * 0 25 50 75 100 ns (0.41) 0.0 2.5 5.0 7.5 10.0 ns (0.6) 0 20 40 ns (1) 0 20 40 * 0.0 2.5 5.0 7.5 10.0 ns (0.061) 0 25 50 75 100 * 0 500 1000 1500 ns (0.5) 0.0 2.5 5.0 7.5 10.0 Regulatory T cells Spleen ** %CD4+CD25+FOXP3+ of T cells 0.0 2.5 5.0 7.5 10.0 Figure 3. Univariate analysis comparing T cell subsets of antibiotic-treated and control mice. (A) Frequencies of T helper 2 cells within T cells isolated from placental tissue, staining CD4+T1ST2+. (B) Splenic CD4+CD25+FOXP3+ regulatory T cells as frequency of total T cells. (C) CD4+CCR6+ROR γ T+ T helper 17 cells isolated from spleen, peritoneal cavity lavage fluid, inguinal lymph nodes, mesenteric lymph nodes, and placenta. Data were compared by Mann-Whitney/Wilcoxon tests (non-parametric), * p < 0.05 **p < 0.01. ILN: Inguinal lymph nodes, MLN: mesenteric lymph nodes, ns: non-significant.