Respiratory Treatment and Prevention (Advances in Experimental Medicine and Biology)

2.3 NETs Visualization

Neutrophils were incubated in eight-well
Lab-Tek chamber slides (2.5 104 cells/well;
Nunc, Waltham, MA) with antibiotics and then
NETs formation was stimulated as described
above. Three hours post-stimulation samples
were fixed with 4 % paraformaldehyde and
DNA was stained with Sytox green (Life
Technologies; Waltham, MA). NETs were
visualized using a Leica DMi8 fluorescent micro-
scope equipped with 40x magnification
objective.

2.4 Statistical Analysis

Results are expressed as meansSE of six inde-
pendent experiments performed in triplicates.
Multiple comparisons were made by one-way
ANOVA followed by Dunn’s post-hoc test.
A p-value<0.05 was used to define statistical
significance of differences. Statistical calcu-
lations were performed using a commercial pack-
age of GraphPad Prism v. 5.0 (GraphPad
Software, La Jolla, CA).

3 Results

We isolated neutrophils from fresh blood of
healthy volunteers by density gradient centrifu-
gation. The cells were then treated with 1μl/ml
gentamicin or 10μg/ml cefotaxime, and their
influence on NETs formation was analyzed. As
expected from other studies, both PMA and CI
enhanced NETs formation compared with the
unstimulated neutrophils. The enhancement was
maniefested as an increase in DNA fluorescence
and decondensation of nuclei (decondensation of
chromatin is a step preceding DNA release to the
extracellular space) accompanied by the forma-
tion of DNA-positive web-like threads (Fig.1b,
c, and f).
A quantitative measurement of DNA release
by fluorometry demonstrates that from the two
antibiotics tested, only gentamicin affected

NETs release. The mean fluorescence in

the PMA-stimulated neutrophils was

12875 1257 RFU (relative fluorescence

units) and in the presence of gentamicin it

decreased to 90641585 RFU (p<0.05).

Likewise, DNA release was diminished when

NETs release was CI-stimulated. The mean fluo-

rescence in the CI-treated samples was

6339 1724 RFU and it decreased to

4040 1351 RFU after inhibition with gentami-

cin (p<0.01). Treatment with gentamicin

(Fig.1a, d, and g) diminished NETs formation

when compared with that in the presence of the

PMA and CI stimuli alone (Fig.1a, c, and f)by

30.68.6 % and 33.719.6 %, respectively

(Fig.1a, c, d, f, and g). Cefotaxime did not

influence NETs formation (Fig.1a, e, and h).

These observations were confirmed by fluores-

cent microscopy. We found that the number of

decondensed nuclei and NETs was lower in the

gentamicin-treated than untreated neutrophils.

4 Discussion

This study demonstrates that antibiotics may

affect NETs release by human neutrophils. Neu-

trophil extracellular traps are among major

mechanisms used by neutrophils to eradicate

bacteria. They have been described primarily as

a beneficial tool to entrap and kill pathogens

extracellularly (Brinkmann et al. 2004 ) in the

process called NETosis. Recently, intense

research has addressed the role of NETs in health

and disease. NETs release can contribute to the

pathogenesis of several diseases, including auto-

immune diseases such as systemic lupus

erythematosus (Pieterse et al. 2015 ; Zawrotniak

et al. 2015 ). Thus, a considerable part of the

research is being directed toward identification

of the agents capable of modulating NETs

release. However, the interaction between

antibiotics and release of NETs in humans has

not yet been elucidated.

In the present study we tested two antibiotics,

which belong to different groups: cefotaxime, a

third-generation cephalosporin and gentamicin,

an aminoglycoside. Both are used to treat many

Antibiotics Modulate the Ability of Neutrophils to Release Neutrophil... 49