Exopolysaccharide β-(2,6)-levan-type fructans have a molecular-weight-dependent modulatory effect on Toll-like receptor signalling

Abstract

Scope: Fructans are a group of dietary fibers which are known to have many beneficial effects including immune-modulating effects. A family of fructans are β-(2,6)-linked levan-type fructans that are known to serve as exopolysaccharides in the cell wall of many species of bacteria including commensal bacteria and probiotics. It is still largely unknown whether and how they can serve as immunomodulating molecules.

Results: Microbial β-(2,6)-fructans were found to induce TLR-dependent activation of THP-1 cells, in a dose-dependent fashion. Low molecular weight (M_w), medium M_w and high M_w β-(2,6)-fructans activated both TLR2 and 4 in a dose- and molecular weight-dependent fashion. In addition, it was found that β-(2,6)-fructans were able to inhibit signalling of various TLRs with the strongest effect on TLR5 and 8, which were inhibited by all the β-(2,6)-fructans in a dose- and molecular weight-dependent fashion. The final effect of this activation and inhibition of TLRs on cytokine responses in human dendritic cells (DCs) was minor which may be explained by the counter-activating effects of the different β-(2,6)-linked levan-type fructans on inhibition of TLR signalling in the DCs.

Conclusion: A mechanism by which exopolysaccharide levan β-(2,6)-fructans can be immune-modulating is by impacting TLR signalling. This knowledge could lead to food in which exopolysaccharide levan β-(2,6)-fructans are added for preventing disorders where TLR-signalling is modulated.

Fig. 1. NF-κB/AP-1 activation in THP-1 MD2-CD14 (A) and THP-1 defMyD88 reporter cells (B) by β-(2,6)-fructans. Cells were incubated with 0.5, 1 or 2 mg mL⁻¹ β-fructan. Data are expressed as mean ± standard error of the mean (SEM) (n = 5). For normally distributed data, statistical significance was determined using a One-way ANOVA test with a Dunnett's multiple comparisons post-test. For not-normally distributed data, a Kruskal–Wallis test followed by a Dunn's multiple comparisons test post hoc was done (*P < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 2. Activation of HEK reporter cell lines expressing individual TLRs (A–G) by β-(2,6)-fructans. HEK cell lines were stimulated with 0.5, 1 and 2 mg mL⁻¹ β-fructans. Values are expressed as mean ± standard error of the mean (SEM) (n = 5). Statistical significance was determined using a one-way ANOVA with a Dunnet's multiple comparisons test for normally distributed data and a Kruskal–Wallis test with a Dunn's multiple comparisons test for not normally distributed data (**p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 3. Inhibition of individual TLRs β-(2,6)-fructans. HEK cell lines expressing individual TLRs (A–G) were pre-incubated with 0.5, 1 or 2 mg mL⁻¹ β-(2,6)-fructans before stimulation with their agonists. Values are expressed as mean ± standard error of the mean (SEM) (n = 3–5). Statistical significance was determined using a one-way ANOVA with a Dunnet's multiple comparisons test for normally distributed data and a Kruskal–Wallis test with a Dunn's multiple comparisons test for not normally distributed data (**p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4. Cytokine production by dendritic cells incubated with β-(2,6)-fructans. Dendritic cells were incubated with 0.5 mg mL⁻¹ of β-fructans for 48 h. Subsequently, cytokine levels in the supernatant were determined using Luminex. In addition, the ratio for IL-10/IL-6 was determined. Values are expressed as mean ± standard error of the mean (SEM) (n = 5). Statistical significance was determined using a One-way ANOVA test followed by a Dunnett's multiple comparison test.

Fig. 5. Cytokine production by dendritic cells incubated with Caco-spent medium of β-(2,6)-fructans. Dendritic cells were incubated with of β-(2,6)-fructans CSM. Values are expressed as mean ± standard error of the mean (SEM) (n = 5). Statistical significance was determined using a mixed effects analysis followed by a Dunnett's multiple comparison test. No significant differences were found.