Disease severity grading using light microscopy.

<p>Disease severity grading using light microscopy.</p

Microbiota enhances sensitivity to IMQ-induced skin inflammation in BALB/c and C57BL/6 mice.

<p>(a) Representative macroscopic pictures of healthy (control) and inflamed (IMQ) ear or dorsal skin of BALB/c mice, illustrating the disease severity. Quantification of skin and ear thickness of BALB/c (b) and C57BL/6 (e) mice. Quantification of histopathological score (0–2) after H&E staining of the ear and skin of BALB/c (c) a C57BL/6 (f) mice. (d) Representative H&E-stained ear and skin sections of BALB/c mice (scale bar, 100 μm). The values represent means ± SD as a pool of three independent experiments (n = 10–20 mice per group). *p < 0.05, ***p < 0.001.</p

Absence of microbiota or ATB treatment decreases the percentage of γδ T cells and Th17 cells in spleen or axillary lymph nodes of IMQ-treated mice.

<p>Cells from spleen or axillary lymph nodes were isolated and either analyzed for surface γδTCR or intracellular RORγt or stimulated <i>in vitro</i> for 8 h by PMA and Ionomycin, the last 4h in the presence of Brefeldin A and Monensin, and analyzed for intracellular IL-17A and IFNγ production by flow cytometry. Cells were first gated for live cells and CD3⁺ and subsequently on γδTCR⁺, RORγt⁺ or IL-17⁺ and INFγ⁺ cells as shown on the (a) example of gating strategy. These results from one representative experiment (n = 5–8 mice per group) out of three independent experiments with (b) GF versus CV mice or with (c) ATB-treated versus control mice are quantified in the graphs. Statistical significance was determined by unpaired Student t test; *p < 0.05, **p < 0.01.</p

Absence of microbiota or ATB treatment reduces the production of pro-inflammatory cytokines by T cells from IMQ-treated mice.

<p>Cells isolated from spleen or axillary lymph nodes of (a) GF and CV mice or (b) ATB-treated and control mice were stimulated for 48hours <i>in vitro</i> by plate-bound anti-CD3 antibody and soluble anti-CD28 antibody. Cell culture supernatants were analyzed for IL-17A and INF-γ by ELISA. These data are representative of three independent experiments (n = 5–8 mice per group) with similar results. Statistical significance was determined by unpaired Student t test; *p < 0.05, **p < 0.01.</p

Antibiotic treatment changes the microbiota composition in both BALB/c and C57BL/6 mice.

<p>(a) Comparison of diversity in microbiota between the conventional (CV) and ATB-treated mice using Shannon diversity index. (b) Principal coordinates analysis (PCoA) plot using the unweighted UniFrac distance metric shows the compositional similarity before and after ATB treatment (Day 0, Day 14, Day 21). Each colored orb represents the microbiota composition in feces of one mouse. Each color represents each group of mice at the day 0, 14, 21. (c) The microbial composition in time is displayed as mountain plot before (Day 0) and after ATB treatment (Day 14), and after the induction of skin inflammation (Day 21) in comparison to CV mice. The figure shows (a) means ± SD or (c) means from pool of 5 mice. Statistical significance was determined by unpaired Student t test; *p < 0.05, **p < 0.01.</p

Antibiotic treatment decreases the skin inflammation in both BALB/c and C57BL/6 mice.

<p>Quantification of ear (a) and skin (b) thickness. Quantification of histopathological score (0–2) after H&E staining of the ear (c) a skin (d). The graphs show 18–19 mice per group (a pool of three independent experiments). Statistical significance was determined by unpaired Student t test; *p < 0.05, **p < 0.01.</p