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Bartonella quintana lipopolysaccharide (LPS): structure and characteristics of a potent TLR4 antagonist for in-vitro and in-vivo applications

View Article: PubMed Central - PubMed

ABSTRACT

The pattern recognition receptor TLR4 is well known as a crucial receptor during infection and inflammation. Several TLR4 antagonists have been reported to inhibit the function of TLR4. Both natural occurring antagonists, lipopolysaccharide (LPS) from Gram-negative bacteria as well as synthetic compounds based on the lipid A structure of LPS have been described as potent inhibitors of TLR4. Here, we have examined the characteristics of a natural TLR4 antagonist, isolated from Bartonella quintana bacterium by elucidating its chemical primary structure. We have found that this TLR4 antagonist is actually a lipooligosaccharide (LOS) instead of a LPS, and that it acts very effective, with a high inhibitory activity against triggering by the LPS-TLR4 system in the presence of a potent TLR4 agonist (E. coli LPS). Furthermore, we demonstrate that B. quintana LPS is not inactivated by polymyxin B, a classical cyclic cationic polypeptide antibiotic that bind the lipid A part of LPS, such as E. coli LPS. Using a murine LPS/D-galactosamine endotoxaemia model we showed that treatment with B. quintana LPS could improve the survival rate significantly. Since endogenous TLR4 ligands have been associated with several inflammatory- and immune-diseases, B. quintana LPS might be a novel therapeutic strategy for TLR4-driven pathologies.

No MeSH data available.


Related in: MedlinePlus

Dose-response of the TLR4 antagonist B. quintana LPS.Human PBMCs were isolated from healthy subjects, using a standard protocol. PBMCs were pre-incubated with a dose-range of B. quintana LPS (10–1000 ng/ml) for 2 h. Thereafter, 10 ng/ml E. coli LPS was added and the PBMCs were cultured for another 24 h. IL-6 was determined by using ELISA (A). (B) Percentage inhibition was calculated using the IL-6 concentration of E. coli LPS exposure as 100%. PBMCs of 6 subjects were used in this experiment. IC50 was 37.04 ng/ml. *P < 0.001, two-sided Mann-Whitney U test.
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f2: Dose-response of the TLR4 antagonist B. quintana LPS.Human PBMCs were isolated from healthy subjects, using a standard protocol. PBMCs were pre-incubated with a dose-range of B. quintana LPS (10–1000 ng/ml) for 2 h. Thereafter, 10 ng/ml E. coli LPS was added and the PBMCs were cultured for another 24 h. IL-6 was determined by using ELISA (A). (B) Percentage inhibition was calculated using the IL-6 concentration of E. coli LPS exposure as 100%. PBMCs of 6 subjects were used in this experiment. IC50 was 37.04 ng/ml. *P < 0.001, two-sided Mann-Whitney U test.

Mentions: The first sets of experiments were designed to investigate whether exposure to B. quintana LPS results in the induction of pro- or anti-inflammatory cytokines by human PBMCs. As shown in Fig. 1, B. quintana LPS itself does not induce the production IL-1β, TNF-α, IL-6 or IL-8. In addition, exposure for 24 h with B. quintana LPS did not result in the production or release of IL-1Ra, or IL-10 by human primary PBMCs (data not shown). However, B. quintana LPS efficiently blocks production of IL-1β, TNF-α, IL-6, IL-8 or after stimulation of human PBMCs with E. coli LPS, indicating the potency of B. quintana LPS as TLR4 antagonist. In addition, we performed dose-response experiments to examine the IC50 of B. quintana LPS for the standard dose of 10 ml E. coli LPS. Figure 2A shows that already 20 ng of B. quintana LPS reduced the IL-6 production. At higher concentrations of B. quintana LPS, 5-fold or higher, a strong suppression of the IL-6 production was stated. Figure 2B demonstrated that B. quintana LPS revealed to have an IC50 of 37.04 ng/ml at a dose of 10 ng/ml ultra pure E. coli LPS. These data confirmed that LPS isolated of B. quintana is a very potent TLR4 antagonist at a low concentration23.


Bartonella quintana lipopolysaccharide (LPS): structure and characteristics of a potent TLR4 antagonist for in-vitro and in-vivo applications
Dose-response of the TLR4 antagonist B. quintana LPS.Human PBMCs were isolated from healthy subjects, using a standard protocol. PBMCs were pre-incubated with a dose-range of B. quintana LPS (10–1000 ng/ml) for 2 h. Thereafter, 10 ng/ml E. coli LPS was added and the PBMCs were cultured for another 24 h. IL-6 was determined by using ELISA (A). (B) Percentage inhibition was calculated using the IL-6 concentration of E. coli LPS exposure as 100%. PBMCs of 6 subjects were used in this experiment. IC50 was 37.04 ng/ml. *P < 0.001, two-sided Mann-Whitney U test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037446&req=5

f2: Dose-response of the TLR4 antagonist B. quintana LPS.Human PBMCs were isolated from healthy subjects, using a standard protocol. PBMCs were pre-incubated with a dose-range of B. quintana LPS (10–1000 ng/ml) for 2 h. Thereafter, 10 ng/ml E. coli LPS was added and the PBMCs were cultured for another 24 h. IL-6 was determined by using ELISA (A). (B) Percentage inhibition was calculated using the IL-6 concentration of E. coli LPS exposure as 100%. PBMCs of 6 subjects were used in this experiment. IC50 was 37.04 ng/ml. *P < 0.001, two-sided Mann-Whitney U test.
Mentions: The first sets of experiments were designed to investigate whether exposure to B. quintana LPS results in the induction of pro- or anti-inflammatory cytokines by human PBMCs. As shown in Fig. 1, B. quintana LPS itself does not induce the production IL-1β, TNF-α, IL-6 or IL-8. In addition, exposure for 24 h with B. quintana LPS did not result in the production or release of IL-1Ra, or IL-10 by human primary PBMCs (data not shown). However, B. quintana LPS efficiently blocks production of IL-1β, TNF-α, IL-6, IL-8 or after stimulation of human PBMCs with E. coli LPS, indicating the potency of B. quintana LPS as TLR4 antagonist. In addition, we performed dose-response experiments to examine the IC50 of B. quintana LPS for the standard dose of 10 ml E. coli LPS. Figure 2A shows that already 20 ng of B. quintana LPS reduced the IL-6 production. At higher concentrations of B. quintana LPS, 5-fold or higher, a strong suppression of the IL-6 production was stated. Figure 2B demonstrated that B. quintana LPS revealed to have an IC50 of 37.04 ng/ml at a dose of 10 ng/ml ultra pure E. coli LPS. These data confirmed that LPS isolated of B. quintana is a very potent TLR4 antagonist at a low concentration23.

View Article: PubMed Central - PubMed

ABSTRACT

The pattern recognition receptor TLR4 is well known as a crucial receptor during infection and inflammation. Several TLR4 antagonists have been reported to inhibit the function of TLR4. Both natural occurring antagonists, lipopolysaccharide (LPS) from Gram-negative bacteria as well as synthetic compounds based on the lipid A structure of LPS have been described as potent inhibitors of TLR4. Here, we have examined the characteristics of a natural TLR4 antagonist, isolated from Bartonella quintana bacterium by elucidating its chemical primary structure. We have found that this TLR4 antagonist is actually a lipooligosaccharide (LOS) instead of a LPS, and that it acts very effective, with a high inhibitory activity against triggering by the LPS-TLR4 system in the presence of a potent TLR4 agonist (E. coli LPS). Furthermore, we demonstrate that B. quintana LPS is not inactivated by polymyxin B, a classical cyclic cationic polypeptide antibiotic that bind the lipid A part of LPS, such as E. coli LPS. Using a murine LPS/D-galactosamine endotoxaemia model we showed that treatment with B. quintana LPS could improve the survival rate significantly. Since endogenous TLR4 ligands have been associated with several inflammatory- and immune-diseases, B. quintana LPS might be a novel therapeutic strategy for TLR4-driven pathologies.

No MeSH data available.


Related in: MedlinePlus