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Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Rossi O, Pesce I, Giannelli C, Aprea S, Caboni M, Citiulo F, Valentini S, Ferlenghi I, MacLennan CA, D'Oro U, Saul A, Gerke C - J. Biol. Chem. (2014)

Bottom Line: GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line.We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation.The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.

View Article: PubMed Central - PubMed

Affiliation: From the Novartis Vaccines Institute for Global Health and.

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TLR blocking experiments. 200,000 human PBMC cells were stimulated with GMMA from different strains. After a 4-h incubation, IL-6 release was measured by ELISA and plotted as average of duplicates with standard deviation. A, IL-6 release after stimulation with 0.0001–1000 ng/ml (10-fold steps) of GMMA from different strains. The rectangle highlights the concentration of GMMA used in blocking experiments. B–E, cells were incubated with 25 μg/ml anti-TLR4 (dark red graphs), 15 μg/ml anti-TLR2 (green graphs), or both (violet graphs) for 30 min before exposure to 1 or 10 ng of GMMA from Ss−p − OAg ΔmsbB (B); Sf2a−p − OAg ΔmsbB (C); Ss−p − OAg ΔhtrB (D); and Sf2a−p − OAg ΔhtrB (E). The graphs from experiments without blocking are shown in the same color as in A. A representative result of three independent experiments is shown.
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Figure 5: TLR blocking experiments. 200,000 human PBMC cells were stimulated with GMMA from different strains. After a 4-h incubation, IL-6 release was measured by ELISA and plotted as average of duplicates with standard deviation. A, IL-6 release after stimulation with 0.0001–1000 ng/ml (10-fold steps) of GMMA from different strains. The rectangle highlights the concentration of GMMA used in blocking experiments. B–E, cells were incubated with 25 μg/ml anti-TLR4 (dark red graphs), 15 μg/ml anti-TLR2 (green graphs), or both (violet graphs) for 30 min before exposure to 1 or 10 ng of GMMA from Ss−p − OAg ΔmsbB (B); Sf2a−p − OAg ΔmsbB (C); Ss−p − OAg ΔhtrB (D); and Sf2a−p − OAg ΔhtrB (E). The graphs from experiments without blocking are shown in the same color as in A. A representative result of three independent experiments is shown.

Mentions: With the objective of identifying the TLRs that contribute to the residual activation by GMMA with lipid A modification, PBMC were incubated with TLR2 and/or TLR4 blocking antibodies before stimulation with 1 and 10 ng/ml of GMMA, concentrations chosen to give a significant but not saturating increase of IL-6 (Fig. 5). The three GMMA with penta-acylated lipid A, Ss−p − OAg ΔhtrB (Fig. 5D), Ss−p − OAg ΔmsbB (Fig. 5B), and Sf2a−p − OAg ΔmsbB (Fig. 5C), gave similar results as follows: IL-6 production was substantially reduced following incubation with TLR2 blocking antibody (70–90%) but either no reduction or minimal reduction was observed with TLR4 blocking antibody (10–30%) suggesting residual activity was principally due to TLR2 activation. With the Ss−p − OAg ΔmsbB and Sf−p − OAg ΔmsbB GMMA, a small reduction was obtained with the TLR4 blocking antibody alone (Ss−p − OAg ΔmsbB, p = 0.0234; Sf2a−p − OAg ΔmsbB, p = 0.0078 in four experiments) suggesting that there was still residual TLR4 activation. Further reduction was achieved with the combination of TLR2 and TLR4 blocking antibodies compared with TLR2 blocking antibody alone. With the Ss−p − OAgΔhtrB GMMA, no effect from TLR4 blocking was observed.


Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Rossi O, Pesce I, Giannelli C, Aprea S, Caboni M, Citiulo F, Valentini S, Ferlenghi I, MacLennan CA, D'Oro U, Saul A, Gerke C - J. Biol. Chem. (2014)

TLR blocking experiments. 200,000 human PBMC cells were stimulated with GMMA from different strains. After a 4-h incubation, IL-6 release was measured by ELISA and plotted as average of duplicates with standard deviation. A, IL-6 release after stimulation with 0.0001–1000 ng/ml (10-fold steps) of GMMA from different strains. The rectangle highlights the concentration of GMMA used in blocking experiments. B–E, cells were incubated with 25 μg/ml anti-TLR4 (dark red graphs), 15 μg/ml anti-TLR2 (green graphs), or both (violet graphs) for 30 min before exposure to 1 or 10 ng of GMMA from Ss−p − OAg ΔmsbB (B); Sf2a−p − OAg ΔmsbB (C); Ss−p − OAg ΔhtrB (D); and Sf2a−p − OAg ΔhtrB (E). The graphs from experiments without blocking are shown in the same color as in A. A representative result of three independent experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4155660&req=5

Figure 5: TLR blocking experiments. 200,000 human PBMC cells were stimulated with GMMA from different strains. After a 4-h incubation, IL-6 release was measured by ELISA and plotted as average of duplicates with standard deviation. A, IL-6 release after stimulation with 0.0001–1000 ng/ml (10-fold steps) of GMMA from different strains. The rectangle highlights the concentration of GMMA used in blocking experiments. B–E, cells were incubated with 25 μg/ml anti-TLR4 (dark red graphs), 15 μg/ml anti-TLR2 (green graphs), or both (violet graphs) for 30 min before exposure to 1 or 10 ng of GMMA from Ss−p − OAg ΔmsbB (B); Sf2a−p − OAg ΔmsbB (C); Ss−p − OAg ΔhtrB (D); and Sf2a−p − OAg ΔhtrB (E). The graphs from experiments without blocking are shown in the same color as in A. A representative result of three independent experiments is shown.
Mentions: With the objective of identifying the TLRs that contribute to the residual activation by GMMA with lipid A modification, PBMC were incubated with TLR2 and/or TLR4 blocking antibodies before stimulation with 1 and 10 ng/ml of GMMA, concentrations chosen to give a significant but not saturating increase of IL-6 (Fig. 5). The three GMMA with penta-acylated lipid A, Ss−p − OAg ΔhtrB (Fig. 5D), Ss−p − OAg ΔmsbB (Fig. 5B), and Sf2a−p − OAg ΔmsbB (Fig. 5C), gave similar results as follows: IL-6 production was substantially reduced following incubation with TLR2 blocking antibody (70–90%) but either no reduction or minimal reduction was observed with TLR4 blocking antibody (10–30%) suggesting residual activity was principally due to TLR2 activation. With the Ss−p − OAg ΔmsbB and Sf−p − OAg ΔmsbB GMMA, a small reduction was obtained with the TLR4 blocking antibody alone (Ss−p − OAg ΔmsbB, p = 0.0234; Sf2a−p − OAg ΔmsbB, p = 0.0078 in four experiments) suggesting that there was still residual TLR4 activation. Further reduction was achieved with the combination of TLR2 and TLR4 blocking antibodies compared with TLR2 blocking antibody alone. With the Ss−p − OAgΔhtrB GMMA, no effect from TLR4 blocking was observed.

Bottom Line: GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line.We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation.The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.

View Article: PubMed Central - PubMed

Affiliation: From the Novartis Vaccines Institute for Global Health and.

Show MeSH
Related in: MedlinePlus