Changes in body weight of chow and HFD-fed C57Bl/6 and BALB/c mice during 32 weeks of the experiment.

<p>(A) Body weights of chow-fed C57Bl/6 mice at 16, 24 and 32 weeks of age were significantly lower than that of BALB/c mice. Body weight gain and weight gain as a percentage of initial body weight were significantly lower in chow-fed C57Bl/6 mice. (B) Body weights of HFD-fed C57Bl/6 mice at 24 and 32 weeks of age were significantly higher than that of BALB/c mice. Body weight gain and weight gain as a percentage of initial body weight were significantly higher in HFD-fed C57Bl/6 mice. The results are shown as the means ± SEM of 9–10 animals per group. *p<0.05. The results are representative of two experiments.</p

EAE after passive transfer of ST2^−/− MOG_35–55 stimulated cells.

a<p>Passive transfer of ST2^−/− MOG_35–55 specific mononuclear cells was performed in ST2^+/+ and ST2^−/− mice two experiments/group, with five mice/experiment, giving a minimum total, n = 10 mice per group.</p>b<p>Mean cumulative score is calculated as the mean of cumulative daily scores for each mouse. Mean maximal score is calculated as the mean of most severe EAE score for each mouse. The mean cumulative and mean maximal scores include all mice within a group, even those not afflicted by EAE.</p

ST2 deletion leads to inflammatory phenotype of APC after EAE induction.

<p>Mononuclear cells were isolated from lymph nodes of ST2^−/− and WT BALB/c mice 4, 7 and 9 days after immunization and analyzed by flow cytometry. (A) Percentages of subpopulation of dendritic cells, CD11c⁺CD11b⁺CD8⁻, in the lymph nodes 4, 7 and 9 days after immunization are presented. (B) Expression of markers of activation, CD86 and MHC II, on dendritic cells was performed at day 4. Histograms were gated on CD11c⁺CD11b⁺CD8⁻ cells. (C) Percentages of CD11c⁺CD11b⁺CD8⁻ cells positive for IL-1, IL-12 and IL-6 in lymph nodes 4 days after immunization is presented. Data are presented from 3 independent experiments as the mean+SD of representative experiment (n = 23 in both groups). Statistical significance was tested by Student’s t-test.</p

ST2 deletion leads to the induction of inflammatory T helper lymphocytes in draining lymph nodes.

<p>Mononuclear cells from draining lymph nodes of immunized ST2^−/− BALB/c and BALB/c WT mice were isolated on day 9. (A) Flow cytometry was used to evaluate IL-17, IFN-γ, TNF-α, GM-CSF and IL-10 containing CD4⁺ T cells. Data are presented as individual percentages of all mice, 10 mice per group. (B) Expression of activation marker CD69 on cells isolated from lymph nodes and peripheral blood 4 and 7 days after immunization was evaluated by flow cytometry. Plots are gated on lymphocyte population. Mean values of CD4⁺CD69⁺ cells are obtained from two experiments with 16 mice per group (C) Flow cytometric analysis of expression of chemokine receptors CCR6, CXCR3 and CXCR5 on CD4⁺ lymphocytes was performed on cells isolated from lymph nodes (4 days after immunization) and peripheral blood (7 days after immunization). Percentages of double positive cells CD4⁺CCR6⁺, CD4⁺CXCR3⁺ and CD4⁺CXCR5⁺ are presented (upper panel) and chemokine receptor expression in CD4⁺ population of lymph node cells (lower panel). Data are presented as the mean+SD, 16 mice per group (*P<0.05). Statistical significance was tested by Student’s t-test.</p

Primers used for qRT-PCR analysis.

<p>Primers used for qRT-PCR analysis.</p

Liver gene expression in chow and HFD-fed C57Bl/6 and BALB/c mice.

<p>(A) Expression of genes related to lipid metabolism in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of LXRα and PPARγ was significantly lower in C57Bl/6 mice mice on HFD. (B) Expression of profibrogenic genes in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of procollagen and TGF-β was significantly higher in C57Bl/6 mice on both diets. The results are shown as the means ± SEM of 9–10 animals per group. *p<0.05. The results are representative of two experiments.</p

Spinal cord and brain infiltrates from ST2^−/− BALB/c mice contain inflammatory T helper lymphocytes.

<p>Mononuclear cells were isolated from spinal cord (A) and brain tissue (B) of mice killed at the peak of the disease and analyzed by flow cytometry after intracellular staining for inflammatory cytokines. The total numbers of CD4⁺ cells containing IL-17, IFN-γ, TNF-α and GM-CSF are calculated as the product of the average number of CNS mononuclear cells harvested per mouse from pooled brains and spinal cords. Presented data are from representative experiment, 12 mice in each group (means+SD). Statistical significance was tested by Student’s t-test.</p

ST2^−/− BALB/c mice have similar infiltration in CNS as C57BL/6 mice.

<p>Fourteen days after immunization mononuclear cells were isolated from spinal cords and brains and counted (A). Pooled mononuclear cells isolated from brains and spinal cords (three per group, four samples in group, 12 mice per group) were used for flow cytometric analysis of percentages (B) and total cell numbers (C) of CD4⁺, CD8⁺, CD11c⁺, and F4/80⁺ cells. Absolute numbers were calculated as the product of the average number of CNS mononuclear cells harvested per mouse pooled from brains and spinal cords. The data are from representative experiment (mean+SD *P<0.05 and #P<0.005). Statistical significance was tested by Student’s t-test.</p

Adipocyte size and phenotypic analysis of immune cells in VAT in chow and HFD-fed C57Bl/6 and BALB/c mice.

<p>(A) Representative images of hematoxylin-eosin staining (original magnification x40, scale bar = 50μm) of paraffin-embedded visceral adipose tissue sections. Mean adipocyte diameter was significantly higher in 8-weeks old chow-fed C57Bl/6 mice, while significantly lower in 32 weeks old chow and HFD-fed mice, compared with diet-matched BALB/c mice. (B) Number of CD11c⁺F4/80^- DCs in VAT is significantly higher in chow- and HFD-fed C57Bl/6 mice compared with diet-matched BALB/c mice at 32 weeks of age. (C) Number of F4/80⁺ macrophages is significantly higher in HFD-fed C57Bl/6 mice compared to HFD-fed BALB/c mice at 32 weeks of age. (D) Number of VAT associated CD3⁺ lymphocytes is significantly higher in HFD-fed C57Bl/6 mice compared to HFD-fed BALB/c mice at 32 weeks of age. (E) Number of VAT associated CD3⁺CXCR3⁺ lymphocytes is significantly higher in HFD-fed C57Bl/6 mice compared to HFD-fed BALB/c mice at 32 weeks of age. Gating strategy and representative FACS plots of CD3⁺CXCR3⁺ cells in VAT of C57Bl/6 and BALB/c mice on HFD. The results are shown as the means ± SEM of 9–10 animals per group. *p<0.05. The results are representative of two experiments.</p

Primed ST2^−/− T cells induce EAE in WT and ST2^−/− recipient.

<p>The donor cells were isolated from popliteal lymph nodes 9 days after foot pad immunization of ST2^−/− BALB/c and BALB/c WT mice with MOG/CFA emulsion. Donor cells were restimulated with MOG_35–55 peptide (50 µM) for 72 hours and 1×10⁷ were transfer to ST2^−/− and WT BALB/c mice. Mice were observed every day after injection for disease onset and clinically scored. Data are presented as the mean of two independent experiments with 5 animals in each group in each experiment (n = 10 in both groups * P<0.05) (A). Lymph node's mononuclear cells harvested from donor mice and restimulated <i>in vitro</i> with MOG_35–55 peptide (50 µM) for 72 hours are analyzed for expression of inflammatory cytokines (B) and markers of Th1 and Th2 differentiation (C). Data are presented as the mean+SD from representative experiment with 10 animals in each group. Histograms and mean values presented in the graphs are gated on CD4⁺ cells (C). Levels of proinflammatory cytokines were measured in cell culture supernatants by ELISA and CBA. Data are presented as the mean+SD of two independent experiments, 10 animals per group (D). Statistical significance was tested by Student’s t-test.</p