Guarea kunthiana Bark Extract Enhances the Antimicrobial Activities of Human and Bovine Neutrophils

Abstract

Guarea kunthiana is used in folk remedies for the treatment of several diseases including microbial infections. The mechanism behind this phenomenon still needs to be elucidated. Here, we investigated the effect of G. kunthiana bark extract on antimicrobial functions of human and bovine neutrophils as the first line of defense against infections. For this aim, neutrophils were isolated from either human or bovine blood and treated with G. kunthiana bark extract. The antimicrobial activity of the neutrophils against Staphylococcus (S.) aureus and Escherichia (E.) coli was tested in a bacterial survival assay and a fluorescence-based phagocytosis assay. Furthermore, the formation of neutrophil extracellular traps (NETs) was visualized by immunofluorescence microscopy. We show that neutrophils treated with G. kunthiana extract distinctly increased phagocytosis of S. aureus or E. coli. Interestingly, we demonstrate that G. kunthiana bark extract induces the formation of NETs in both cell types. This effect was abolished when treating the cells with diphenyleniodonium chloride (DPI) pointing to a direct implication of the NADPH oxidase-dependent formation of reactive oxygen species in this process. In summary, our data strongly suggest that G. kunthiana bark extract boosts the antimicrobial activities of neutrophils as the first line of defense against invading pathogens.

In Vitro Testing of Crude Natural Plant Extracts from Costa Rica for Their Ability to Boost Innate Immune Cells against Staphylococcus aureus

“…Several authors have shown that NETs act efficiently as antimicrobials against S. aureus [ 8 , 11 , 12 ]. Since there are also some natural products found among the wide class of NET-inducing stimuli [ 6 , 9 ], it was interesting to investigate the NET-stimulating potential of VEOEBA. As shown in Figure 4 , VEOEBA significantly induces the number of NET-releasing neutrophils compared to the vehicle control DMSO after 4 h of incubation, though to a lesser extent than the positive control (PMA-stimulated cells).…”

“…The upper layer with monocytes was transferred into a fresh, sterile 50 mL tube, and washed with PBS before the cells were resuspended in RPMI + 2% fetal calf serum. Neutrophils were harvested and used as previously described [ 6 ].…”

“…The experiment was performed as earlier described [ 6 ]. Briefly, bacteria were grown until logarithmic phase.…”

“…The experiment was performed as described earlier [ 6 ]. Briefly, fixed cells were washed with PBS, permeabilized and blocked with 2% bovine serum albumin (BSA) in 0.2% Triton X-100/PBS.…”

“…Naturally derived products, such as plant extracts, have become the focus of attention not only because they may directly act as an antimicrobial against the pathogen, but they may also be able to boost the host immune system against an infection. Interestingly, we have recently shown that acetone bark extract from Guarea kunthiana (GUKUBA), at a concentration of 1 μg/mL, has the ability to boost the antimicrobial activity of human and bovine neutrophils against bacterial infections [ 6 ].…”