PMC:3279418 / 7229-10327 JSONTXT

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Since the spleen of cpdm mice is markedly enlarged and contains three times as many cells (Fig. 1C), the different percentages of splenic cDC and pDC between WT and mutant mice reflect the increased number of total spleen cells rather than a reduction in cDC and pDC numbers. These data indicate that the Sharpin mutation does not affect the distribution of the examined DC subsets in the spleen.\n10.1371/journal.pone.0031809.g002 Figure 2 Effect of Sharpin mutation on DC subpopulations and maturation.\n(A) Spleens from WT and cpdm mice were isolated and subject to collagenase and DNase digestion. The obtained splenic homogenates were centrifuged over a Percoll gradient (35% and 55% density) for 15 minutes. The bands at the 35%-medium and the 35–55% interface were pooled, washed and stained with a combination of various antibodies to stain different DC subsets, conventional CD11c+CD8α+, CD11c+CD8α− and plasmacytoid DC (CD11c−PDCA-1+).The top panels were gated on FSChiSSClo cells to show separate populations of CD11c+PDCA-1− and CD11c−PDCA-1+ cells. Further gating on the CD11c+PDCA-1− subpopulation gave the bottom panel that showed two distinct pools of CD11c+CD8α+ and CD11c+CD8α− cells. Percentages were calculated based on the parental population and were additionally shown as bar graphs (n = 2) (B). (C) WT and cpdm BMDC (5×105) cells were stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours. The cells were labeled with PE-labeled anti-CD40, anti-CD80, and anti-CD86, and subjected to flow cytometry analysis. The populations shown in histograms were gated on CD11c+ cells. Unstained cells served as negative controls. Results are representative of two independent experiments. BMDC from in vitro cultures functionally resemble non-lymphoid tissue DC and monocyte-derived inflammatory DC [15], [16]. The yields of BMDC from WT and cpdm mice were similar. BMDC were CD11c+ and MHC II+ with low expression of co-stimulatory molecules CD40, CD80, and CD86. The TLR3 ligand poly I:C and the TLR4 ligand LPS each activate overlapping but different signaling pathways and were used to induce DC maturation [17], [18]. Incubation with the TLR agonists for 24 hours resulted in increased expression of CD40, CD80, and CD86 on BMDC; however, there was no difference in the expression levels of these markers between WT and cpdm BMDC (Fig. 2B). Thus, SHARPIN deficiency did not influence the expression of co-stimulatory molecules by BMDC.","divisions":[{"label":"Title","span":{"begin":0,"end":53}},{"label":"Figure 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