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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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(A) Kinetics of association of CFSE-stained S. pyogenes by human neutrophils. Neutrophils and CFSE-stained S. pyogenes were incubated for 5, 10, 20, 30, 60 or 90 min before the reaction was stopped by adding ice-cold PBS and samples were fixed. Flow cytometry was performed using "cell settings" showing only fluorescence associated with neutrophils. Values are expressed as mean ± standard deviation and are obtained from three different experiments carried out with neutrophils isolated from three different donors. (B) The amount of S. pyogenes associated with neutrophils at different timepoints of incubation. The mean fluorescence intensity (MFI) value associated with neutrophils (cell setting) was divided with the MFI for S. pyogenes (bacterial settings) in each experiment. Values are expressed as mean ± standard deviation and are obtained from three different experiments carried out with neutrophils from three different donors.
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Figure 10: (A) Kinetics of association of CFSE-stained S. pyogenes by human neutrophils. Neutrophils and CFSE-stained S. pyogenes were incubated for 5, 10, 20, 30, 60 or 90 min before the reaction was stopped by adding ice-cold PBS and samples were fixed. Flow cytometry was performed using "cell settings" showing only fluorescence associated with neutrophils. Values are expressed as mean ± standard deviation and are obtained from three different experiments carried out with neutrophils isolated from three different donors. (B) The amount of S. pyogenes associated with neutrophils at different timepoints of incubation. The mean fluorescence intensity (MFI) value associated with neutrophils (cell setting) was divided with the MFI for S. pyogenes (bacterial settings) in each experiment. Values are expressed as mean ± standard deviation and are obtained from three different experiments carried out with neutrophils from three different donors.

Mentions: When incubating neutrophils with S. pyogenes a biphasic association was registered with respect to percentage of neutrophils involved (Fig. 10A). This association curve closely resembled that of neutrophils and borreliae (Figs 5, 6, 7). The biphasic shape of the curve was not so prominent as with borreliae, and a plateau level was reached almost immediately in the amount of fluorescent S. pyogenes associated per neutrophil (Fig. 10B).


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)

(A) Kinetics of association of CFSE-stained S. pyogenes by human neutrophils. Neutrophils and CFSE-stained S. pyogenes were incubated for 5, 10, 20, 30, 60 or 90 min before the reaction was stopped by adding ice-cold PBS and samples were fixed. Flow cytometry was performed using "cell settings" showing only fluorescence associated with neutrophils. Values are expressed as mean ± standard deviation and are obtained from three different experiments carried out with neutrophils isolated from three different donors. (B) The amount of S. pyogenes associated with neutrophils at different timepoints of incubation. The mean fluorescence intensity (MFI) value associated with neutrophils (cell setting) was divided with the MFI for S. pyogenes (bacterial settings) in each experiment. Values are expressed as mean ± standard deviation and are obtained from three different experiments carried out with neutrophils from three different donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: (A) Kinetics of association of CFSE-stained S. pyogenes by human neutrophils. Neutrophils and CFSE-stained S. pyogenes were incubated for 5, 10, 20, 30, 60 or 90 min before the reaction was stopped by adding ice-cold PBS and samples were fixed. Flow cytometry was performed using "cell settings" showing only fluorescence associated with neutrophils. Values are expressed as mean ± standard deviation and are obtained from three different experiments carried out with neutrophils isolated from three different donors. (B) The amount of S. pyogenes associated with neutrophils at different timepoints of incubation. The mean fluorescence intensity (MFI) value associated with neutrophils (cell setting) was divided with the MFI for S. pyogenes (bacterial settings) in each experiment. Values are expressed as mean ± standard deviation and are obtained from three different experiments carried out with neutrophils from three different donors.
Mentions: When incubating neutrophils with S. pyogenes a biphasic association was registered with respect to percentage of neutrophils involved (Fig. 10A). This association curve closely resembled that of neutrophils and borreliae (Figs 5, 6, 7). The biphasic shape of the curve was not so prominent as with borreliae, and a plateau level was reached almost immediately in the amount of fluorescent S. pyogenes associated per neutrophil (Fig. 10B).

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