Additional file 1 of The relationship between sarcopenia and metabolic dysfunction-associated fatty liver disease among the young and middle-aged populations

Supplementary Material

The new and special phenotype of DC cells in AT of ND and HFD mice.

<p>(A) The DCs that infiltrated into AT are immature DCs. Multicolor FCM analyses were performed on single cell suspensions that were stained with specific mAb against FITC-CD11c and PE-CD40, CD80, CD86 MHCI and MHCII, cells were gated on the CD11c population. Numbers in histograms represent the geometric mean fluorescence of the gated cells. Open histograms indicate isotype control. (B) The ATDCs of HFD mice have the relatively stable phenotype with or without LPS stimulation. The ATDCs were incubated with or without LPS (100 ng/ml) stimulation at 37°C for 12 hours and stained with specific mAb against FITC-CD11c and PE-CD40, CD80, CD86, MHCI and MHCII. Numbers in histograms represent the geometric mean fluorescence of the gated CD11c cells. (C) The ATDCs of HFD mice have the relatively phagocytosis ability with or without LPS stimulation. The ATDCs were incubated with or without LPS stimulation and HcRed-expressed <i>E.coli</i> at 37°C for 4 hours. Data are representative of three independent experiments.</p

Interplay between ATDCs and CD4⁺T cells.

<p>(A,B) 2×10⁵ ATDCs or SPDCs were isolated from HFD mice AT and SP by microbeads method and co-cultured them with 1×10⁶ CD4⁺T cells that isolated from 6-8 weeks old male C57BL/6 mice for 7 days. The single cell suspensions of the co-culture system which was in the presence or absence of anti-IL-6, anti-IL-23 or isotype IgG were stained with specific mAb against FITC-CD4 and PE-IL-17. And the multicolor FCM analyses were performed on the single cell suspensions. (C) The concentration of IL-17 in the supernatant were examined by ELISA method. Data are presented as mean ± SD of 3 samples pooled from three independent experiments. Student's t-test was used for the statistical analysis. **<i>p</i><0.01, *<i>p</i><0.05.</p

There are more DCs in HFD mice AT than ND mice AT.

<p>(A) CD11c⁺ DCs expression in HFD mice AT. CD11c⁺ DCs in HFD mice AT were analyzed using flow cytometry. And proportions of CD11c⁺ DC in the AT of HFD mice (n = 6) and ND mice (n = 6). (B) Morphology of DC-like cells in HFD mice AT under transmission electron microscope. (C) The CD11c⁺ DCs in HFD mice AT were negative for CD3 and B220. Open histograms indicate isotype control, numbers in histograms represent the geometric mean fluorescence. (D, E) The CD11c⁺ DCs in HFD mice AT are different from macrophages. (D) The phagocytosis ability of ATDCs and macrophages assessed by incubating with HcRed-expressed <i>E.coli</i> at 37°C for 4 hours. Numbers in histograms represent the geometric mean fluorescence. (E) The CD11c⁺ DCs and macrophages from HFD mice spleen or adipose tissues were stained with specific mAb against F4/80, cells were gated on the CD11c⁺ population, and numbers in histograms represent the geometric mean fluorescence of the gated cells. All data are shown as the mean ± SD of 6 samples pooled from three independent experiments. Student's t-test was used for the statistical analysis. ***<i>p</i><0.001.</p

The ATDCs expressed higher levels of IL-6, TGF-β and IL-23 than SPDCs.

<p>The ATDCs and SPDCs of HFD mice were isolated by microbeads method, and then incubated them with PMA and ionomycin at 37°C for 4 hours. mRNA levels of IL-6, TGF-β, IL-23p19, IL-12p40 and IL-12p35 in the ATDCs and SPDCs were examined by qRT-PCR method. The concentration of IL-6, IL-23 in the supernatant were examined by ELISA method. Data are presented as mean ± SD of 6 samples pooled from three independent experiments. Student's t-test was used for the statistical analysis. **<i>p</i><0.01, *<i>p</i><0.05.</p

HFD mice AT expresses higher levers of ATDCs chemo-attractant CCL20 than ND mice.

<p>(A) mRNA levels of CCR6 were determined by RT-PCR method. mRNA levels of CCL20(B), MIP-1(C) and IL-8(D) were determined by qRT- PCR method on HFD mice AT and ND mice AT relative to β-actin. Data are presented as mean ± SD of 5 samples pooled from three independent experiments. Student's t-test was used for the statistical analysis. *<i>p</i><0.05, N.S no significance.</p

Higher level of Th17 cells were existed in obesity adipose tissues.

<p>(A) The CD4⁺T cells of HFD and ND mice AT were isolated by microbeads method and then incubated them with PMA and ionomycin at 37°C for 4 hours. mRNA levels of IL-17 in the HFD and ND mice AT were examined by qRT-PCR method. (B) mRNA levels of RORγt in the HFD and ND mice AT were examined by qRT-PCR method. Data are presented as mean ± SD of 3 samples pooled from three independent experiments. Student's t-test was used for the statistical analysis. **<i>p</i><0.01, *<i>p</i><0.05.</p

Increased GITRL expression on BMDCs by WGP impairs Treg-mediated suppressive effect and enhances Teff proliferation.

<p>Murine CD4⁺CD25⁻ Teff and CD4⁺CD25⁺ Treg cells were purified from C57BL/6 mice splenocytes as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046936#s4" target="_blank">Materials and Methods</a></i>. BMDCs were stimulated with WGP (100 µg/ml) or not for 24 h. Cells were harvested, treated with mitomycin C and then co-cultured at a 1∶5 ratio with CD4⁺CD25⁻ Teff and CD4⁺CD25⁺ Treg cells (A) or Teff cells (B) in the presence of anti-CD3 mAb with or without anti-GITRL blocking antibody or control Igs for 72 h, Treg and Teff cells were added at 1∶1 ratio. Wells were pulsed with 1 µCi/well [³H]-thymidine for the last 16 h. Data are expressed as means ± SD. ***P<0.001, **P<0.01, *P<0.05. N.S. represents no significance between columns.</p

WGP alters the suppressive capacity of regulatory T cells in spleens.

<p>Groups of mice (n = 6) bearing established Lewis lung carcinoma were treated as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046936#s4" target="_blank">Materials and Methods</a></i>. (A, B, C) Single cell suspensions from spleens (A), draining lymph nodes (B) and tumor tissues (C) were stained with fluorochrome labeled mAbs to evaluate the proportions of CD4⁺CD25⁺Foxp3⁺ Tregs. Cells were gated on CD4⁺ T cells. RNAs from tumor specimens were extracted and Foxp3 mRNA expression was analyzed by qRT-PCR. (D) Splenic CD4⁺CD25⁺ Tregs isolated from tumor-bearing mice treated with or without WGP were co-cultured with CD4⁺CD25⁻ Teffs from wild type C57BL/6 mice in the presence of anti-CD3 mAb and anti-CD28 mAb for 72 h. Wells were pulsed with 1 µCi/well [³H]-thymidine and analyzed as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046936#s4" target="_blank">Materials and Methods</a></i>. **P<0.01, *P<0.05, N.S. represents no significance between columns.</p

WGP treatment up-regulates GITRL expression on DCs in tumor-bearing C57BL/6 mice.

<p>(A) Groups of mice (n = 6) bearing established Lewis lung carcinoma were treated as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046936#s4" target="_blank">Materials and Methods</a></i>. Tumors were measured with a caliper at indicated time. (B) The proportions of CD11c⁺ DCs in spleens, draining lymph nodes (dLN) and tumor tissues from WGP-treated or untreated mice were analyzed using flow cytometry. (C) Expression of GITRL on CD11c⁺ cells in spleens, draining lymph nodes and tumor sites treated with or without WGP were assessed by flow cytometry. Histograms are gated on CD11c⁺ cells (isotype: solid gray). RNAs extracted from spleens, draining lymph nodes and tumors were subjected to qRT-PCR for GITRL expression. Numbers indicate percentages of events in gate (dot plot) or geometric mean fluorescence intensity (GeoMFI; histograms). Results are expressed as mean ± SD. ***P<0.001, **P<0.01, *P<0.05, ^###P<0.001, N.S. represents no significance between columns.</p