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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.


Kinetics of the B. quintana LPS to block the TLR4.Human PBMCs were isolated from healthy subjects, using a standard protocol. (A) PBMCs were pre-incubated with 100 ng/ml or 1000 ng/ml B. quintana LPS for different times before E. coli LPS was added to the culture medium. After 1 hour, 45, 30 and 15 minutes PBMC were exposed to 10 ng/ml E. coli LPS for 24 h. (B) B. quintana LPS was added together with E. coli LPS, thereafter the PBMCs were cultured for additional 24 h, 48 h or 72 h. C, B. quintana LPS was added in a range before (−2 h) and after (+2 h) the cells were exposed to 10 ng/ml E. coli LPS. IL-6 was determined by using ELISA. PBMCs of 4 subjects were used in this experiment. *P < 0.001, two-sided Mann-Whitney U test.
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f5: Kinetics of the B. quintana LPS to block the TLR4.Human PBMCs were isolated from healthy subjects, using a standard protocol. (A) PBMCs were pre-incubated with 100 ng/ml or 1000 ng/ml B. quintana LPS for different times before E. coli LPS was added to the culture medium. After 1 hour, 45, 30 and 15 minutes PBMC were exposed to 10 ng/ml E. coli LPS for 24 h. (B) B. quintana LPS was added together with E. coli LPS, thereafter the PBMCs were cultured for additional 24 h, 48 h or 72 h. C, B. quintana LPS was added in a range before (−2 h) and after (+2 h) the cells were exposed to 10 ng/ml E. coli LPS. IL-6 was determined by using ELISA. PBMCs of 4 subjects were used in this experiment. *P < 0.001, two-sided Mann-Whitney U test.

Mentions: To explore the capacity of B. quintana LPS to bind the TLR4 over time, we examined the minimal pre-incubation time that allows B. quintana LPS to block TLR4 completely. Therefore we pre-incubated PBMCs with 100 ng/ml and 1000 ng/ml B. quintana LPS for 15, 30, 45 or 60 minutes and added 10 ng/ml E. coli LPS. After 24 h the IL-6 concentrations were determined in the supernatants. Figure 5A shows that 15 minutes’ pre-incubation is sufficient for 100 ng/ml of B. quintana LPS to block TLR4 receptor. The rapid binding of B. quintana LPS to TLR4 indicates that this TLR4 inhibitor is efficient.


Bartonella quintana lipopolysaccharide (LPS): structure and characteristics of a potent TLR4 antagonist for in-vitro and in-vivo applications
Kinetics of the B. quintana LPS to block the TLR4.Human PBMCs were isolated from healthy subjects, using a standard protocol. (A) PBMCs were pre-incubated with 100 ng/ml or 1000 ng/ml B. quintana LPS for different times before E. coli LPS was added to the culture medium. After 1 hour, 45, 30 and 15 minutes PBMC were exposed to 10 ng/ml E. coli LPS for 24 h. (B) B. quintana LPS was added together with E. coli LPS, thereafter the PBMCs were cultured for additional 24 h, 48 h or 72 h. C, B. quintana LPS was added in a range before (−2 h) and after (+2 h) the cells were exposed to 10 ng/ml E. coli LPS. IL-6 was determined by using ELISA. PBMCs of 4 subjects were used in this experiment. *P < 0.001, two-sided Mann-Whitney U test.
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Related In: Results  -  Collection

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f5: Kinetics of the B. quintana LPS to block the TLR4.Human PBMCs were isolated from healthy subjects, using a standard protocol. (A) PBMCs were pre-incubated with 100 ng/ml or 1000 ng/ml B. quintana LPS for different times before E. coli LPS was added to the culture medium. After 1 hour, 45, 30 and 15 minutes PBMC were exposed to 10 ng/ml E. coli LPS for 24 h. (B) B. quintana LPS was added together with E. coli LPS, thereafter the PBMCs were cultured for additional 24 h, 48 h or 72 h. C, B. quintana LPS was added in a range before (−2 h) and after (+2 h) the cells were exposed to 10 ng/ml E. coli LPS. IL-6 was determined by using ELISA. PBMCs of 4 subjects were used in this experiment. *P < 0.001, two-sided Mann-Whitney U test.
Mentions: To explore the capacity of B. quintana LPS to bind the TLR4 over time, we examined the minimal pre-incubation time that allows B. quintana LPS to block TLR4 completely. Therefore we pre-incubated PBMCs with 100 ng/ml and 1000 ng/ml B. quintana LPS for 15, 30, 45 or 60 minutes and added 10 ng/ml E. coli LPS. After 24 h the IL-6 concentrations were determined in the supernatants. Figure 5A shows that 15 minutes’ pre-incubation is sufficient for 100 ng/ml of B. quintana LPS to block TLR4 receptor. The rapid binding of B. quintana LPS to TLR4 indicates that this TLR4 inhibitor is efficient.

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.