Limits...
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

Comparison and interaction of B. quintana LPS with polymixin B.Human PBMCs were isolated from healthy subjects, using a standard protocol. (A,B) PMBCs were exposed to a dose range of E. coli LPS (0.0001 to 10 ng/ml) in the presence or absence of B. quintana LPS (100 and 500 ng/ml) or polymixin (B) (3 and 10 μg/ml). TNF-α and IL-6 were determined with ELISA. (C) B. quintana LPS (100 ng/ml or 1000 ng/ml) was pre-incubated with 10 μg/ml polymyxin B for 2 h. Thereafter control medium, B. quintana LPS, polymyxin (B) B. quintana LPS + polymyxin B were added to the PBMCs for 2 h and thereafter the cells were washed 3 times with warm RPMI 1640 medium. After washing, RPMI 1640 medium or E. coli LPS (10 ng/ml) was added and the PBMCs were incubated for 24 h. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037446&req=5

f6: Comparison and interaction of B. quintana LPS with polymixin B.Human PBMCs were isolated from healthy subjects, using a standard protocol. (A,B) PMBCs were exposed to a dose range of E. coli LPS (0.0001 to 10 ng/ml) in the presence or absence of B. quintana LPS (100 and 500 ng/ml) or polymixin (B) (3 and 10 μg/ml). TNF-α and IL-6 were determined with ELISA. (C) B. quintana LPS (100 ng/ml or 1000 ng/ml) was pre-incubated with 10 μg/ml polymyxin B for 2 h. Thereafter control medium, B. quintana LPS, polymyxin (B) B. quintana LPS + polymyxin B were added to the PBMCs for 2 h and thereafter the cells were washed 3 times with warm RPMI 1640 medium. After washing, RPMI 1640 medium or E. coli LPS (10 ng/ml) was added and the PBMCs were incubated for 24 h. 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: It is well known that polymyxin B binds to LPS from several Gram-negative microorganisms and neutralizes the activity. First, we compared B. quintana LPS with polymyxin B to analyze the difference in the neutralizing capacity. Figure 6A,B showed that B. quintana LPS is far more potent to inhibit E. coli LPS mediated TNF-α or IL-6 production by human PBMCs. A concentration of 100 ng/ml B. quintana LPS was equally potent as 10 μg/ml polymyxin B to block 1 or 10 ng/ml of E. coli LPS. Thereafter, we investigated whether polymyxin B was able to bind and inactivate B. quintana LPS. Figure 6C demonstrated that after pre-incubation of B. quintana LPS with 10 μg/ml polymyxin B for 2 hours, the inhibitory capacity of B. quintana LPS was still very high. As control, polymyxin B neutralized the E. coli LPS as expected.


Bartonella quintana lipopolysaccharide (LPS): structure and characteristics of a potent TLR4 antagonist for in-vitro and in-vivo applications
Comparison and interaction of B. quintana LPS with polymixin B.Human PBMCs were isolated from healthy subjects, using a standard protocol. (A,B) PMBCs were exposed to a dose range of E. coli LPS (0.0001 to 10 ng/ml) in the presence or absence of B. quintana LPS (100 and 500 ng/ml) or polymixin (B) (3 and 10 μg/ml). TNF-α and IL-6 were determined with ELISA. (C) B. quintana LPS (100 ng/ml or 1000 ng/ml) was pre-incubated with 10 μg/ml polymyxin B for 2 h. Thereafter control medium, B. quintana LPS, polymyxin (B) B. quintana LPS + polymyxin B were added to the PBMCs for 2 h and thereafter the cells were washed 3 times with warm RPMI 1640 medium. After washing, RPMI 1640 medium or E. coli LPS (10 ng/ml) was added and the PBMCs were incubated for 24 h. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037446&req=5

f6: Comparison and interaction of B. quintana LPS with polymixin B.Human PBMCs were isolated from healthy subjects, using a standard protocol. (A,B) PMBCs were exposed to a dose range of E. coli LPS (0.0001 to 10 ng/ml) in the presence or absence of B. quintana LPS (100 and 500 ng/ml) or polymixin (B) (3 and 10 μg/ml). TNF-α and IL-6 were determined with ELISA. (C) B. quintana LPS (100 ng/ml or 1000 ng/ml) was pre-incubated with 10 μg/ml polymyxin B for 2 h. Thereafter control medium, B. quintana LPS, polymyxin (B) B. quintana LPS + polymyxin B were added to the PBMCs for 2 h and thereafter the cells were washed 3 times with warm RPMI 1640 medium. After washing, RPMI 1640 medium or E. coli LPS (10 ng/ml) was added and the PBMCs were incubated for 24 h. 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: It is well known that polymyxin B binds to LPS from several Gram-negative microorganisms and neutralizes the activity. First, we compared B. quintana LPS with polymyxin B to analyze the difference in the neutralizing capacity. Figure 6A,B showed that B. quintana LPS is far more potent to inhibit E. coli LPS mediated TNF-α or IL-6 production by human PBMCs. A concentration of 100 ng/ml B. quintana LPS was equally potent as 10 μg/ml polymyxin B to block 1 or 10 ng/ml of E. coli LPS. Thereafter, we investigated whether polymyxin B was able to bind and inactivate B. quintana LPS. Figure 6C demonstrated that after pre-incubation of B. quintana LPS with 10 μg/ml polymyxin B for 2 hours, the inhibitory capacity of B. quintana LPS was still very high. As control, polymyxin B neutralized the E. coli LPS as expected.

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