DCs from CD patients exhibit altered levels of phosphorylated STATs after cytokine stimulation.

<p>Heparinized whole blood was stimulated with human recombinant cytokines for 10 minutes, and cells were then immediately fixed, permeabilized, and stained for phosphorylated STATs. CD1c+ mDCs and pDCs were identified as shown in Fig. 1B and 1C, respectively. Light-colored histograms represent unstimulated samples. A representative example of pSTAT staining patterns after cytokine stimulation is shown in a healthy control (A). pDCs from patients exhibit a decreased response to IL-6, whereas IL-10 induces similar levels of pSTAT3 in both groups. In CD1c+ mDCs, pSTAT3 levels after IL-10 stimulation are higher in patients, and no difference is seen in the IL-6 response. IFN-α stimulation results in a lower levels of pSTAT1 and pSTAT3 in both DC subsets in CD, but pSTAT1 responses to IFN-γ stimulation are similar with the healthy controls. Values represent fold increase of median fluorescence intensity over unstimulated control. Infliximab-treated patients are indicated by open circles. Horizontal lines indicate median levels (B).</p

IL-6 modulates pDC maturation.

<p>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. * <i>P</i><0.05; ** <i>P</i><0.01.</p

pDCs express cell-surface IL-6Rα and are not dependent on the presence of serum components for IL-6 signal transduction.

<p>Flowcytometric analysis of IL-6Rα expression on DC subsets was performed in whole blood samples from healthy volunteers. IL-6Rα is expressed on pDCs, although in lower levels than on CD1c+ mDCs (A). PBMCs were isolated from fresh peripheral blood samples from three healthy volunteers in independent experiments. Cells were incubated at 37°C in RPMI 1640 culture medium (supplemented with 2 mM L-glutamine) either with 1/3 vol. of autologous serum or without serum for one hour prior to stimulation with 50 ng/ml of recombinant IL-6 for 10 minutes (in the presence or absence of serum), followed by flowcytometric DC identification and pSTAT3 measurement using similar gating strategy as indicated in Fig. 1. Despite lower IL-6Rα expression on pDCs, IL-6-induced pSTAT3 response in pDCs is not impaired in the absence of autologous serum, whereas mDCs (serving as intrinsic controls) exhibit somewhat reduced responses when stimulated in serum-free conditions. Samples from each donor are indicated with different symbols (B).</p

Expression of maturation markers on DCs.

<p>pDCs and CD1c+ mDCs were identified as shown in Fig. 1, and the expression of maturation markers was assessed by flow cytometry. The expression of CD123 or CD11c is shown to demonstrate the specificity of DC subtype identification. Light-colored histograms represent isotype controls. A representative example of a healthy control is shown (A). pDCs from CD patients exhibit lower expression of HLA-DR, whereas in CD1c+ mDCs no difference between the groups is observed. Low expression of CD40 is seen on both DC subtypes, the levels on CD1c+ mDCs being, however, higher in CD patients. Values represent median fluorescence intensity (MFI) after subtraction of isotype control fluorescence. Horizontal lines indicate medians for each group (B).</p

Patient characteristics in DC enumeration, surface marker expression and STAT phosphorylation studies.

<p>Abbreviations: PD = perianal disease; AZA = azathioprine; IFX = infliximab; 5-ASA = 5-aminosalicylic acid; MTX = methotrexate; 6-MP = 6-mercaptopurine; ESR = erythrocyte sedimentation rate, mm/h; CPR = C-reactive protein, mg/L; CDAI = Crohn's disease activity index, generally interpreted as follows: <150 = inactive disease, ≥150 = active disease; SES-CD = Simple Endoscopic Score for Crohn's disease, generally interpreted as: ≤3 = inactive disease, 4–14 = mild to moderate disease, ≥15 = severe disease.</p

IL-6-treated pDCs promote Th2-type responses.

<p>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).</p

Gating strategy for flow cytometry.

<p>DC subtypes were identified for cell surface molecule analyses as shown. An example of the leukocyte gating first applied for all DC subsets is shown in the upper left corner (A). In pSTAT measurements, CD11c and CD123 were utilized as additional markers for CD1c+ mDCs (B) and pDCs (C), respectively; otherwise, a gating strategy similar to the cell surface marker protocol was used. A representative example of a healthy control is shown.</p

Patients with Crohn’s disease have reduced numbers of pDCs and BDCA3+ mDCs in the peripheral blood.

<p>Both absolute (A) and relative (B) pDC and BDCA3+ mDC counts are decreased in CD. The major blood myeloid DC population expressing CD1c is not significantly different from controls in size. Horizontal lines indicate median levels. Statistically significant negative correlations between the total SES-CD score and relative pDC and BDCA3+ mDC counts are observed in CD patients (C).</p