Activation of Toll-like receptor 2 and Toll-like receptor 2/6 by lactobacilli

Abstract number: O355

Vankerckhoven V., Van Voorden S., Hens N., Goossens H., Molenberghs G., Wiertz E.

Objectives: Toll-like receptors function as key regulators of both innate and adaptive immunity. Lactobacilli modulate the immune system in different ways. The aim of this study was to examine Toll-like receptor (TLR2, TLR2/6 and TLR4) signalling induced by clinical and probiotic Lactobacillus strains.

Methods: A total of 45 Lactobacillus strains (19 L. paracasei and 26 L. rhamnosus) of different origin (22 probiotic, 2 faecal, and 21 clinical) were tested for TLR2, TLR2 in combination with TLR6, and TLR4. TLR signalling was measured as relative IL-8 promotor activation in transfected human embryonic kidney (HEK) 293 cells. IL-8 concentrations were measured using an enzyme-linked immunosorbent assay. Heat-killed Listeria monocytogenes (HKLM) was used as positive control in all assays, whereas PAM3, PAM2, and LPS were used as positive controls for, respectively, TLR2, TLR2/6, and TLR4. All assays were performed at least in duplicate. Linear mixed model analyses and stepwise model selection were used to identify the statistically significant effects. Random effects were used to account for heterogeneity across and homogeneity within isolates. P < 0.05 was considered statistically significant.

Results: HEK-TLR2 and HEK-TLR2/6, but not HEK-TLR4, cells released IL-8 upon stimulation with UV-inactivated lactobacilli, which was enhanced by co-transfection with CD-14. Interestingly, the production of IL-8 was shown to be variable for the different Lactobacillus isolates. Although similar results were seen for all isolates for TLR2 and TLR2/6, IL-8 production was significantly higher for TLR2 (8.4 log pg/ml) compared to TLR2/6 (6.05 log pg/ml) (P < 0.0001). No significant differences in IL-8 production were seen between clinical and probiotic isolates. However, L. rhamnosus isolates induced a significantly higher IL-8 production compared to L. paracasei isolates in both cell lines, 7.88 and 6.84 log pg/ml, respectively (P = 0.0004). Intra-isolate correlation was found significant (P < 0.0022).

Conclusions: Our study shows that lactobacilli activate both TLR2 and TLR2 in combination with TLR6. Our results also indicate that heterodimerisation of TLR2 with TLR6 does not lead to an improved recognition of lactobacilli. Furthermore, taking intra-isolate correlation into consideration proved to be important. Finally, our results suggest that differences in immunomodulation by lactobacilli may be related to differential signalling through TLRs, including TLR2 and TLR2/6.