Table 1.
Cell densities and positive controls used in reporter cell stimulations.
Figure 1.
HPAEC profiles and HPSEC elution patterns of different β2→1-fructan formulations.
Figure A depicts the fructose (F) and glucose (G) monomers, dimers, and fructan oligomers present in the ITF formulations. GFn chains are terminated by a glucose molecule, and Fn chains consist of only fructose moieties. In both cases, n represents the number of fructose moieties in the chain. Figure B depicts the elution patterns as a measure for molecular weight distribution profiles of the four different β2→1-fructan formulations in kDa.
Figure 2.
Induction of cytokines by ITF I, in a dose range of 0 to 100 µg/ml.
Statistical significance levels were determined with a parametric Student’s t-test for unpaired observations (two-tailed). Mean and SEM of cytokine production is plotted as percentage of controls, which were set to 100% (n = 4). Panels A to F show the results for IL-1Ra, IL-1β, IL-6, IL-10, IL-12, and TNF-α respectively.
Figure 3.
Induction of cytokines by β2→1-fructan formulations with different chain length (DP range), at 0, 1, and 100 µg/ml.
Statistical significance levels were determined with a non-parametric Mann-Whitney U-test for unpaired observations (two-tailed). Mean and SD of cytokine expression are plotted as percentage of controls (represented by 0 µg/ml), which were set to 100% (n = 6). Panel A to D represent cytokine expression induced by ITF I to IV respectively.
Figure 4.
Ratio of IL-10/IL-12 upon incubation of PBMCs with different chain length β2→1-fructans.
PBMCs (n = 6) were stimulated with 1 µg/ml (panel A) and 100 µg/ml (panel B) β2→1-fructans for 24h. Statistical significance levels were determined with a non-parametric Mann-Whitney U-test for unpaired observations (two-tailed). Mean and SD of the IL-10/IL-12 ratios is plotted for the different β2→1-fructans as percentage of controls, which were set to 100% (n = 6) and horizontal bars indicate the significant differences between β2→1-fructan treatments.
Figure 5.
NF-κB/AP-1 activation in THP-1 MD2-CD14 and THP-1 defMyD reporter cells.
Statistical significance levels were determined with a non-parametric Mann-Whitney U-test for unpaired observations (two-tailed). Mean and SD of NF-κB/AP-1 activation by β2→1-fructans of different chain lengths (ITF I, II, III and IV) are plotted as percentage of negative controls (unstimulated cells), which were set to 100% for both cell lines. LPS stimulation was used as a common positive control for TLR4/MyD88 signalling to NF-κB/AP-1 in the functional THP-1 MD2-CD14 cell line. TriDAP was used as a positive control in THP-1 DefMyD cells, which induces MyD88-independent signalling to NF-κB/AP-1. Panel A to D represent cytokine expression induced by ITF I to IV respectively. Only concentrations which induced activation in the THP-1 MD1-CD14 cells are shown for comparison of these concentrations in the THP-1 DefMyD cells.
Figure 6.
NF-κB/AP-1 activation of HEK cell lines overexpressing separate TLRs or NODs.
Statistical significance levels were determined with a non-parametric Mann-Whitney U-test for unpaired observations (two-tailed). Mean and SD of NF-κB/AP-1 activation in HEK cell lines stimulated with β2→1-fructans for 24 h are plotted as percentage of unstimulated controls, which were set to 100%. Dosages are plotted in µg/ml. Endotoxin free H2O was used as an additional negative control and per cell line the relevant positive controls were applied as mentioned in Table 1.