Additional file 1: Figure S1. of Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro

Is showing levels of cytokines secreted by MSCs from three different donors after IL-1α or IL-1β treatment. Secretion of VEGF (A, B) and NGF (C, D) was not modified by any treatments in any of the donors, but secretion of IL-10 showed a non-significant increase in some donors (E, F). Levels of IL-1Ra were high and unchanged after IL-1 treatments (G, H). Changes in the levels of BDNF were different in each donor, showing significance in some donors (I, J) (n = 3 experiments/donor). *p < 0.05, **p < 0.01, ***p < 0.001 vs untreated. (TIF 461 kb

Additional file 3: Figure S3. of Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro

Is showing measurement of cell death and proliferation of BV2 cells in CM treatment experiments. LDH was measured in supernatants (A) and cell lysates (B) as indirect measurements of cell death and proliferation. None of the treatments induced significant cell death or proliferation. (TIF 585 kb

CD4 T cells isolated from IL-1R1^ΔT mice display non-impaired differentiation <i>in vitro</i>.

<p><i>In vitro</i> polarization assay of MACS-purified CD4 T cells activated under Th0, Th17 and Th1 conditions (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161505#sec002" target="_blank">materials and methods</a> section) for 4 days. Data are shown as representative FACS plots gated on VD^-/CD4⁺ cells with average frequencies per group and as mean +SEM of n = 3 of each genotype. Experiments were performed twice with similar results.</p

CD4 T cells isolated from IL-1R1^ΔT mice do not respond to IL-1β administration <i>in vitro</i>.

<p>(A) <i>In vitro</i> proliferation assay of CD4 T cells labeled with violet cell tracer (VCT) and activated with the indicated stimuli (FACS histograms, gated on VD^-/CD4⁺ cells). (B) Proliferation index of cultures supplemented with indicated stimuli as shown in (A). (C) Total numbers of live CD4 T cells harvested from cultures supplemented with indicated stimuli as shown in (A). MACS-purified CD4 T cells from control and IL-1R1^ΔT mice (n = 4 of each genotype) were cultured 4 days under different indicated conditions. Data are (A) representative FACS histograms of control (black) and IL-1R1 deficient (blue) cultures and (B, C) individual values with mean. Experiments were performed twice with the similar results. *p < 0.05, **p < 0.01, ***p < 0.001, N. S.–not significant; two-tailed unpaired t-test.</p

T cell specific deletion of IL-1R1 results in impaired Th17 cell expansion in anti-CD3 treatment model.

<p>(A) Analysis of activation status of CD4 T cells isolated from the spleen after anti-CD3 treatment at indicated time points. Data are shown as representative FACS plots gated on VD^-/CD90.2⁺/CD4⁺ cells with mean frequencies per group ± average deviation; and as mean +SEM of n = 4 of each genotype. (B-C) Analysis of cytokine expression by CD4 T cells isolated from the (B) spleen and (C) IEL compartment of the small intestine after anti-CD3 treatment at indicated time points. Data (B, C) are shown as representative FACS plots gated on VD^-/CD90.2⁺/CD4⁺ cells with mean frequencies per group and as mean +SEM of n = 4 control and n = 5 IL-1R1^ΔT mice analyzed at 48 h, as well as of n = 3 control and n = 4 IL1R1^ΔT mice analyzed at 100 h. Experiments were performed twice with similar results. *p < 0.05; two-tailed unpaired t-test.</p

Conditional deletion of IL-1R1 in T cells.

<p>(A) Schematic representation of targeted murine <i>Il1r1</i> allele with exon 5 flanked by loxP sites. Cre mediated recombination results in excision of exon 5 and leads to the deletion of <i>Il1r1</i> gene. Open squares—numbered exons, triangles—loxP sites. All components are out of scale. (B) FACS analysis of IL-1R1 expression by CD4 T cells (upper row, gated on VD^-/TCR-β⁺/CD4⁺ cells) and γδ-TCR⁺ T cells (lower row, gated on VD^-/γδ-TCR⁺ cells) isolated from the draining lymph nodes of mice immunized with CFA. (C) Mean Fluorescence Intensity (MFI) of IL-1R1 staining by cell populations shown in (B). Data are (B) representative FACS plots and (C) mean +SEM of n = 3 of each genotype, and are representative of three independent experiments. **p < 0.01, ***p < 0.001, N. S.–not significant; two-tailed unpaired t-test.</p

Additional file 3: Figure S3. of Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro

Is showing measurement of cell death and proliferation of BV2 cells in CM treatment experiments. LDH was measured in supernatants (A) and cell lysates (B) as indirect measurements of cell death and proliferation. None of the treatments induced significant cell death or proliferation. (TIF 585 kb