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Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils.

Tuominen-Gustafsson H, Penttinen M, Hytönen J, Viljanen MK - BMC Microbiol. (2006)

Bottom Line: In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility.Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain.The results also indicate a clear difference in the association with neutrophils between virulent and non-virulent borrelial strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, FI-20520 Turku, Finland. hetugu@utu.fi

ABSTRACT

Background: Species of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi) cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis) which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry.

Results: In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii), or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain.

Conclusion: These results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The results also indicate a clear difference in the association with neutrophils between virulent and non-virulent borrelial strains.

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Localisation of CFSE-stained Bb-HP incubated with human neutrophils. Confocal microscopy was used to demonstrate intracellular localisation of CFSE-stained Bb-HP. Neutrophils and CFSE-stained Bb-Hp were incubated for 5 min (A) or 30 min (B) before the reaction was stopped by adding ice-cold PBS and samples were fixed. The white arrow on panel (A) indicates a borrelia bacterium attached to neutrophil surface. In panel (C), neutrophils were pre-treated for 10 min with 5 μg/ml Cytochalasin B before addition of CFSE-stained Bb-HP for 30 min. Images shown are "stack images" taken at a defined cross section of the neutrophil, as close to the centre of the cell as possible. The white bar represents 10 μm. Images are representative of two to three samples carried out with neutrophils from two to three different donors.
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Figure 4: Localisation of CFSE-stained Bb-HP incubated with human neutrophils. Confocal microscopy was used to demonstrate intracellular localisation of CFSE-stained Bb-HP. Neutrophils and CFSE-stained Bb-Hp were incubated for 5 min (A) or 30 min (B) before the reaction was stopped by adding ice-cold PBS and samples were fixed. The white arrow on panel (A) indicates a borrelia bacterium attached to neutrophil surface. In panel (C), neutrophils were pre-treated for 10 min with 5 μg/ml Cytochalasin B before addition of CFSE-stained Bb-HP for 30 min. Images shown are "stack images" taken at a defined cross section of the neutrophil, as close to the centre of the cell as possible. The white bar represents 10 μm. Images are representative of two to three samples carried out with neutrophils from two to three different donors.

Mentions: To be able to localise CFSE-stained Bb-HP associated with neutrophils, some experiments were performed using confocal microscopy of fixed samples. After 5 min of incubation, some of the neutrophil-associated CFSE-stained Bb-HP were bound to the plasma membrane of the cells (Fig. 4A). After 30 min of incubation, when looking at the localisation of fluorescent material at different cross-sections of the neutrophils, most fluorescence appeared to be inside the cells (Fig. 4B). The fluorescent material inside the cell was appearing in a "spotted" fashion, and intact Bb-HP could not be detected, suggesting localization of fluorescence in phagosomes. After pre-treatment of neutrophils with Cytochalasin B, inhibiting phagocytosis, a dramatic inhibition of intracellular localisation could be detected (Fig. 4C). Similar data were obtained with neutrophils incubated with CFSE-stained Ba or Bg (data not shown).


Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils.

Tuominen-Gustafsson H, Penttinen M, Hytönen J, Viljanen MK - BMC Microbiol. (2006)

Localisation of CFSE-stained Bb-HP incubated with human neutrophils. Confocal microscopy was used to demonstrate intracellular localisation of CFSE-stained Bb-HP. Neutrophils and CFSE-stained Bb-Hp were incubated for 5 min (A) or 30 min (B) before the reaction was stopped by adding ice-cold PBS and samples were fixed. The white arrow on panel (A) indicates a borrelia bacterium attached to neutrophil surface. In panel (C), neutrophils were pre-treated for 10 min with 5 μg/ml Cytochalasin B before addition of CFSE-stained Bb-HP for 30 min. Images shown are "stack images" taken at a defined cross section of the neutrophil, as close to the centre of the cell as possible. The white bar represents 10 μm. Images are representative of two to three samples carried out with neutrophils from two to three different donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1621068&req=5

Figure 4: Localisation of CFSE-stained Bb-HP incubated with human neutrophils. Confocal microscopy was used to demonstrate intracellular localisation of CFSE-stained Bb-HP. Neutrophils and CFSE-stained Bb-Hp were incubated for 5 min (A) or 30 min (B) before the reaction was stopped by adding ice-cold PBS and samples were fixed. The white arrow on panel (A) indicates a borrelia bacterium attached to neutrophil surface. In panel (C), neutrophils were pre-treated for 10 min with 5 μg/ml Cytochalasin B before addition of CFSE-stained Bb-HP for 30 min. Images shown are "stack images" taken at a defined cross section of the neutrophil, as close to the centre of the cell as possible. The white bar represents 10 μm. Images are representative of two to three samples carried out with neutrophils from two to three different donors.
Mentions: To be able to localise CFSE-stained Bb-HP associated with neutrophils, some experiments were performed using confocal microscopy of fixed samples. After 5 min of incubation, some of the neutrophil-associated CFSE-stained Bb-HP were bound to the plasma membrane of the cells (Fig. 4A). After 30 min of incubation, when looking at the localisation of fluorescent material at different cross-sections of the neutrophils, most fluorescence appeared to be inside the cells (Fig. 4B). The fluorescent material inside the cell was appearing in a "spotted" fashion, and intact Bb-HP could not be detected, suggesting localization of fluorescence in phagosomes. After pre-treatment of neutrophils with Cytochalasin B, inhibiting phagocytosis, a dramatic inhibition of intracellular localisation could be detected (Fig. 4C). Similar data were obtained with neutrophils incubated with CFSE-stained Ba or Bg (data not shown).

Bottom Line: In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility.Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain.The results also indicate a clear difference in the association with neutrophils between virulent and non-virulent borrelial strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, FI-20520 Turku, Finland. hetugu@utu.fi

ABSTRACT

Background: Species of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi) cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis) which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry.

Results: In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii), or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain.

Conclusion: These results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The results also indicate a clear difference in the association with neutrophils between virulent and non-virulent borrelial strains.

Show MeSH
Related in: MedlinePlus