Supernatant of SFA-exposed moDC induces reduced migration of activated moDC and CD4+ T cells.

<p>moDCs were generated in the presence of GM-CSF and IL-4 and activated for 12 h with LPS. CD4 T cells were isolated by microbead-sorting and activated for 16 h with CD3/CD28 mAbs. DC supernatant from SFA- or vehicle-exposed moDCs was harvested 12 h after LPS activation and added to the lower chamber of the transwell. The “SFA carry over control” consisted of supernatant of control-treated moDCs+1 µM SFA. Migration of cells was quantitated by flow cytometry. (A) Activated moDCs were inserted in the upper chamber of the transwell and migration was analysed after 4 h. (B) Activated CD4⁺-T cells were set in the upper chamber of the transwell and migration was analysed after 4 h. The spontaneous migration of cells was subtracted from the results (mean DCs: 1673; T cells: 8676). The results are representative for n = 9 (A) and n = 5 (B) independent experiments. Mean (± SEM) **p<0.01, *p<0.05 versus vehicle.</p

Dose-dependent suppression of CCL5, CCL17 and CCL19 in moDCs by SFA.

<p>Human moDC were exposed on day 5 with 10, 50 100, 250 and 500 nM of SFA and 4 hours later stimulated with 100 ng/mL LPS. CCL5 (A), CCL7 (B) and CCL19 (C) production were analyzed after 12 h stimulation by ELISA. Mean (± SEM) of n = 3 (A) and n = 4 (B, C) independent experiments.</p

Rapid suppression of moDC chemokine production by SFA.

<p>Human moDCs were generated in the presence of GM-CSF and IL-4. 100 nM SFA or drug vehicle were added 4 h before stimulation with 100 ng/mL LPS. 12 hours later the supernatant was collected. CCL5 (A), CCL17 (B), CCL19 (C), CXCL9 (D), CXCL10 (E) and CCL1 (F) were analyzed by ELISA. The results are representative of n = 6 (A,D), n = 7 (E, F), n = 8 (C), and n = 11 (B) independent experiments (Mean ± SEM). **p<0.01, *p<0.05 versus vehicle.</p

SFA inhibits cytokine-cytokine receptor interactions.

<p>Human moDCs were treated with 1 µM SFA or vehicle for 1 hour and total RNA was prepared after 12 h stimulation with 1 µg/mL LPS. The cDNA were labelled with Cy3- and Cy5-fluorescent dyes for microarray hybridization. Chart summarizes the results of the pathway impact analysis. Numbers indicate the impact factor. The impact factor is calculated based on the normalized fold of gene expression change, the number and amount of perturbation of genes downstream from it and the proportion of differentially regulated genes in the respective pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018406#pone.0018406-Khatri1" target="_blank">[25]</a>.</p

Phenotypic maturation of LPS-matured moDC.

<p>*Median fluorescence intensity determined by calibrated flow cytometer before and after LPS stimulation (100 ng/ml LPS; 12 h; n = 4).</p

SFA-regulated genes in the cytokine-cytokine receptor pathway.

<p>*lods (log odds ratio for differential expression given an expectation of 1% regulated genes on the array.</p

moDC chemokine suppression by SFA is independent of cylophilin A binding.

<p>To analyze whether DC chemokine suppression by SFA is dependent on cyclophilin A binding competitive experiments with a 100-fold molar excess of CsA (A) and with a 10-fold molar excess of a cyclophilin-binding nonimmunosuppressive derivative of CsA, 4_Cs (B) were performed. 4-Cs has been reported to efficiently inhibit SFA cyclophilin A binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018406#pone.0018406-Zenke1" target="_blank">[5]</a>. Human moDC were pre-exposed to 10 µM CSA and 1 hour later to 100 nM SFA (A). With respect to 4-Cs, moDC were pre-exposed to 2500 nM 4-Cs and 1 hour later to 250 nM SFA (B). After 4 hours, moDCs were stimulated with LPS (100 ng/ml) and DC chemokine production were analyzed after 12 h by ELISA. In the presence of a 100-fold molar excess of CsA or a 10-fold molar excess of 4-Cs, SFA's DC chemokine inhibitory activity was not abrogated. In contrast, 10 µM CSA did not inhibit CCL5 (F) or CCL17 (G) moDC production and only moderately inhibited CCL19 (H) expression. 4-Cs did not inhibit CCL5, CCL17 or CCL19 production. Mean (± SEM) of n = 3–6 independent experiments. **p<0.01, *p<0.05 versus vehicle.</p

In vivo administration of SFA inhibits migration of FITC-labelled CD11c+ DCs.

<p>Mice were injected i.p. with SFA (10 mg/kg/day, 2days), CsA (10 mg/kg/day, 2 days) or vehicle. On day two, abdomen of mice were FITC-painted. The inguinal lymph nodes were removed 24 h later and CD11c+FITC+ migrated cells quantitated by flow cytometry. (A) Dotplot analysis of CD11c+ FITC+ DCs. Numbers indicate percentages of CD11c+FITC+ DCs. (B) Mean (± SEM) numbers of CD11c+ FITC+ DCs in SFA-injected versus vehicle-treated controls. Results are representative for n = 3–5 independent experiments. *p<0.05 versus vehicle.</p

moDC chemokine suppression by SFA is unique in direct comparison to CsA and dexamethasone.

<p>Human moDC were exposed to 100 nM SFA, CsA, dexamethasone or vehicle. After 4 h moDCs were stimulated with LPS (100 ng/ml) and CCL5, CCL17, CCL19, CXCL9, CXCL10 were analyzed by ELISA after 12 h. In contrast to CsA and dexamethasone, SFA markedly inhibited CCL5 (A), CCL17 (B) and CCL19 expression (C).</p

SFA suppresses moDC migration to CCL19 in a CCR7 independent manner and inhibits CD38 expression.

<p>Human moDCs were exposed to SFA, CsA or vehicle and matured for 12 h (A) or 24 h (B–D) with LPS (100 ng/ml). (A) SFA-exposed moDC (1 µM), CsA-exposed moDC (1 µM) or control moDC were added to the upper chamber of the transwell and migrated to CCL19 in the lower chamber as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018406#s4" target="_blank">Materials and Methods</a>. Control experiments included the spontaneous migration in the absence of CCL19. The results indicate number of migrated DC (mean ± SEM). (B–DF) Surface CCR7 and CD38 expression of human moDCs exposed to 1 µM SFA, 1 µM CsA (B, C) or drug vehicle was analyzed by flow cytometry with mAbs. (D) SFA but not CsA inhibits CD38 expression on matured CD1a+ moDC. The results are representative for of n = 6 (A) and n = 3 (B–C) independent experiments (mean ± SEM). The results in D are representative for n = 5–8 (100–500 nM SFA, CsA) and n = 2 (1000 nM SFA, CsA) independent experiments *p<0.05 ; **p<0.01 versus drug-vehicle.</p