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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.


Time course of the TLR4 inhibitor after removal of the B. quintana LPS.Human PBMCs were isolated from healthy subjects, using a standard protocol. PBMCs were pre-incubated with 100 ng/ml or 1000 ng/ml B. quintana LPS for 2 h. Thereafter the PBMCs were extensively washed (3 times) to removed the non-bound B. quintana LPS. After the washing step, 10 ng/ml purified E. coli LPS was added. Cytokines were determined after 24 h, 48 h or 72 h of culture by standard ELISA for IL-1β (A), TNF-α (B), IL-6 (C) and IL-8 (D). PBMCs of 6 healthy donors were examined. *P < 0.001, two-sided Mann-Whitney U test.
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f4: Time course of the TLR4 inhibitor after removal of the B. quintana LPS.Human PBMCs were isolated from healthy subjects, using a standard protocol. PBMCs were pre-incubated with 100 ng/ml or 1000 ng/ml B. quintana LPS for 2 h. Thereafter the PBMCs were extensively washed (3 times) to removed the non-bound B. quintana LPS. After the washing step, 10 ng/ml purified E. coli LPS was added. Cytokines were determined after 24 h, 48 h or 72 h of culture by standard ELISA for IL-1β (A), TNF-α (B), IL-6 (C) and IL-8 (D). PBMCs of 6 healthy donors were examined. *P < 0.001, two-sided Mann-Whitney U test.

Mentions: In order to investigate the kinetics of the bindings capacity of B. quintana LPS to TLR4 and whether removing of the TLR4 inhibitor has an effect on blockade TLR4 function, we pre-incubated human PBMCs with B. quintana LPS for 1 hour. Two different approaches were investigated. The first approach was that B. quintana LPS was continuously present during the exposure to E. coli LPS and in the second approach we removed the B. quintana LPS by thorough washing (3 times). Thereafter the PBMCs were exposed to E. coli LPS and cells were incubated for additional 24 h, 48 h or 72 h hours. At each time point the cells were microscopically checked and the supernatant was collected to measure IL-1β, IL-6, IL-8, or TNF-α. Figure 3 shows that B. quintana LPS blocks the cytokine production by E. coli LPS at least for a period of 72 h. Cytokine production by human PBMCs is reduced for more than 90% over this exposure period, when the B. quintana LPS is present in the culture medium. The second approach in which B. quintana LPS was removed after 1 hour by repeated washing, identical effects on the neutralizing capacity of B. quintana LPS were seen (Fig. 4): Almost complete inhibition of the E. coli LPS-induced cytokine production after 72 h culture in the presence of 10 ng/ml of this classical TLR4 agonist. Thus, our data show that the blocking of TLR4 by B. quintana LPS is strong and stable for at least 72 hours.


Bartonella quintana lipopolysaccharide (LPS): structure and characteristics of a potent TLR4 antagonist for in-vitro and in-vivo applications
Time course of the TLR4 inhibitor after removal of the B. quintana LPS.Human PBMCs were isolated from healthy subjects, using a standard protocol. PBMCs were pre-incubated with 100 ng/ml or 1000 ng/ml B. quintana LPS for 2 h. Thereafter the PBMCs were extensively washed (3 times) to removed the non-bound B. quintana LPS. After the washing step, 10 ng/ml purified E. coli LPS was added. Cytokines were determined after 24 h, 48 h or 72 h of culture by standard ELISA for IL-1β (A), TNF-α (B), IL-6 (C) and IL-8 (D). PBMCs of 6 healthy donors were examined. *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

f4: Time course of the TLR4 inhibitor after removal of the B. quintana LPS.Human PBMCs were isolated from healthy subjects, using a standard protocol. PBMCs were pre-incubated with 100 ng/ml or 1000 ng/ml B. quintana LPS for 2 h. Thereafter the PBMCs were extensively washed (3 times) to removed the non-bound B. quintana LPS. After the washing step, 10 ng/ml purified E. coli LPS was added. Cytokines were determined after 24 h, 48 h or 72 h of culture by standard ELISA for IL-1β (A), TNF-α (B), IL-6 (C) and IL-8 (D). PBMCs of 6 healthy donors were examined. *P < 0.001, two-sided Mann-Whitney U test.
Mentions: In order to investigate the kinetics of the bindings capacity of B. quintana LPS to TLR4 and whether removing of the TLR4 inhibitor has an effect on blockade TLR4 function, we pre-incubated human PBMCs with B. quintana LPS for 1 hour. Two different approaches were investigated. The first approach was that B. quintana LPS was continuously present during the exposure to E. coli LPS and in the second approach we removed the B. quintana LPS by thorough washing (3 times). Thereafter the PBMCs were exposed to E. coli LPS and cells were incubated for additional 24 h, 48 h or 72 h hours. At each time point the cells were microscopically checked and the supernatant was collected to measure IL-1β, IL-6, IL-8, or TNF-α. Figure 3 shows that B. quintana LPS blocks the cytokine production by E. coli LPS at least for a period of 72 h. Cytokine production by human PBMCs is reduced for more than 90% over this exposure period, when the B. quintana LPS is present in the culture medium. The second approach in which B. quintana LPS was removed after 1 hour by repeated washing, identical effects on the neutralizing capacity of B. quintana LPS were seen (Fig. 4): Almost complete inhibition of the E. coli LPS-induced cytokine production after 72 h culture in the presence of 10 ng/ml of this classical TLR4 agonist. Thus, our data show that the blocking of TLR4 by B. quintana LPS is strong and stable for at least 72 hours.

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.