Journal: PLoS ONE

Article Title: Dendritic Cells from Crohn’s Disease Patients Show Aberrant STAT1 and STAT3 Signaling

doi: 10.1371/journal.pone.0070738

Figure Lengend Snippet: Human pDCs from healthy volunteers were either treated with 50 ng/ml of IL-6 or cultured only in the presence of 10 ng/ml of IL-3 (to maintain pDC viability), with or without stimulation with CpG, for two days. IL-6 inhibits the phenotypic maturation induced by the culture with IL-3. In contrast, IL-6 further upregulates CD40 expression on pDCs when stimulated with CpG-rich hypomethylated oligodeoxynucleotides (mimicking fragments of microbial DNA). Freshly isolated pDCs at d0 consistently had high levels of HLA-DR expression and typically low levels of CD86 and CD40, whereas the staining intensities for CD80 and ICOS-L were at the isotype control level (data not shown). % expression values (in the bottom right part of the figure) were calculated for each experiment ((expression in the presence of IL-6/expression in the absence of IL-6) ×100) and presented as mean ± SD. Flow cytometry data from five independent experiments are presented. * P <0.05; ** P <0.01.

Article Snippet: Median purity of the pDC fraction was 93.8%, as determined by flow cytometry (data not shown). pDCs were washed and plated at a density of 1.25×10 5 cells/ml on U-bottom 96-well plates (Nunc, Roskilde, Denmark) in culture medium consisting of RPMI 1640, supplemented with 10% FBS, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, antibiotics, and 10 ng/ml of recombinant human IL-3 (R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, Expressing, Isolation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Dendritic Cells from Crohn’s Disease Patients Show Aberrant STAT1 and STAT3 Signaling

doi: 10.1371/journal.pone.0070738

Figure Lengend Snippet: pDCs were matured for two days with IL-3 and the indicated treatments (IL-6 and CpG). Allogeneic naive CD4+ T cells were primed for six days with the pDCs and then restimulated for 16 hours with anti-CD3 and anti-CD28 antibodies. The levels of mRNA transcripts were analyzed by RT-qPCR. Induction of IL-4 is significantly enhanced by the IL-6 treatment of pDCs in the presence of the TLR9 ligand CpG. In addition, the expression of IL-10 mRNA transcripts is higher (A). The concentrations of Th signature cytokines in culture supernatants were determined. IFN-γ to IL-4, i.e., Th1/Th2 ratio, is reduced by the IL-6 treatment of pDCs both in the presence and absence of CpG during the pDC maturation (B).

Article Snippet: Median purity of the pDC fraction was 93.8%, as determined by flow cytometry (data not shown). pDCs were washed and plated at a density of 1.25×10 5 cells/ml on U-bottom 96-well plates (Nunc, Roskilde, Denmark) in culture medium consisting of RPMI 1640, supplemented with 10% FBS, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, antibiotics, and 10 ng/ml of recombinant human IL-3 (R&D Systems, Minneapolis, MN).

Techniques: Quantitative RT-PCR, Expressing