BYCRBA and vitexin deplete cholesterol in human neutrophils

Cyclodextrins, such as MβCD have been widely used to deplete cholesterol from cellular membranes (Brogden et al., 2015, 2017; Zidovetzkiet al., 2007). Here we used MβCD as positive control. Primary blood-derived neutrophils were treated with MβCD, BYCRBA or vitexin for 2 h. The effect of MβCD, BYCRBA, or vitexin on the cholesterol content of the cells was determined by HPLC analysis (Fig.8). Treatment of human neutrophils with MβCD and BYCRBA resulted in a significant twofold decrease in cholesterol concentration after 2 h of incubation. Treatment with vitexin also resulted in a significant decrease in cholesterol content, however less than that seen after BYCRBA and MβCD treatment.

Ctr

MCD

BYCRBA

Vitexin 0 . 0

0 . 5 1 . 0 1 . 5

µg cholesterol/million cells

*

*

*

Figure 8: BYCRBA and vitexin (10µg/ml) lead to a significant depletion in cholesterol after 2 hours incubation compared to the untreated control. MβCD was used as a positive control for cholesterol depletion (n=6). * = P≤0.05, one tailed, paired Student’s T-Test.

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aureus. Since neutrophils are the most abundant antimicrobial active cells in blood, tthese results support the finding that treatment of neutrophils with BYCRBA or vitexin increases the capability of these cells to reduce the growth of S. aureus.

Interestingly, there was no effect on the phagocytic uptake of bacteria or oxidative burst. However, NETs have been recently described by several authors to contribute to the antimicrobial effect of neutrophils against S. aureus. NETs have been described as a novel phagocytosis-independent antimicrobial process of neutrophils (Brinkmann et al., 2004). Those traps consist of nuclear DNA fibres and are released by cells into the extracellular milieu to entrap and kill bacteria. Here we show that BYCRBA extracts and vitexin both boost the formation of NETs. In good correlation to these data, both plant extracts also reduce the cholesterol level of the cells.

Interestingly, BYCRBA showed stronger cholesterol depleting capabilities compared to vitexin, however further research must be conducted to determine which additional compounds in the crude BYCBRA preparation are responsible (Figure 8). Depletion of cholesterol by methyl-beta-cyclodextrin (MβCD) (Neumann et al., 2014) or statins has been recently described to mediate ROS-independent NET-formation (Chow et al., 2011). In line with this finding, no alteration of oxidative burst was found after treatment of neutrophils with vitexin or BYCRBA.

Both BYCRBA and vitexin have shown to exhibit antioxidant activity (An et al., 2012).

Here, we also tested antioxidative activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as substrate: Whereas 100 µg/ml of vitexin causes 50% inhibition of DPPH, only 10 µg/ml of BYCRBA are enough to cause 50% inhibition of DPPH (data not shown).

Thus, vitexin has lower antioxidant activity, which may be due to the fact that BYCRBA has additional active compounds besides vitexin (de Souza et al., 2018).

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Altogether, these data indicated that treatment of immune cells with BYCRBA plant extracts e.g. vitexin might have beneficial effects against S. aureus infections.

However, the detailed cellular processes need to be characterized and also in vivo effects need to be evaluated in future work.

5. Acknowledgments

This work was supported by a grant from Friedrich-Ebert-Stiftung, Germany. The authors are grateful to the Monteverde Cloud Forest Preserve and the Tropical Science Center for granting permission to collect plant materials under a cooperative rights agreement and to the Commission for the Development of Biodiversity of Costa Rica’s Ministry of the Environment, Energy, and Telecommunications for Research Permit R-001-2006-OT-CONAGEBIO.

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Chapter VI

Discussion

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75 1. Discussion

We are daily exposed to disease triggers caused by pathogens like bacteria or viruses. Our general challenge is now to eradicate the infectious disease that can be pandemic. In case of an infection, the immunity system in our body is immediately activated to fight and eliminate the pathogens. The immune system consists of two main classes: the innate and the adaptive immune system. Neutrophils represent a key element of the innate immune system that is involved in the host defense against many common microorganisms. One of the most eligible functions of neutrophils is phagocytosis to eliminate invading pathogens. In addition, formation of antimicrobial DNA-based extracellular traps (NETs), degranulation and cytokine release can be alternative strategies of neutrophils to support their function in the immune response (Pietrocola et al., 2017). However, bacterial pathogens have developed strategies to avoid that defense mechanism.

Many studies have been performed on different types of bacteria to understand the strategies that enable bacteria to evade entrapment by NETs. One of these studies focused on S. aureus. It has been found that S. aureus degrades NETs. Once S.

aureus is entrapped into the NETs, it converts NETs to deoxyadenosine, which triggers the caspase-3 mediated death of immune cells (Thammavongsa et al.,2013).

Another example came from pulmonary infection: Once the pulmonary cells become infected, S. pneumoniae produces virulence factors (EndA) that degrade DNA and allow S. pneumoniae to escape from NETs (Beiter et al., 2006; Zhu et al., 2013).

Furthermore, studies on Group A Streptococcus (GAS) revealed that these bacteria have DNase activity encoded by the Sda1gene. This activity is sufficient to degrade NETs and to escape from the entrapment by NETs (Buchanan et al., 2006).

The combination of immune evasion with emergence of antibiotic resistance still nowadays leads to major problems in the health systems. An alternative approach to using antibiotics is needed to overcome the threat of this resistance. Herbal products, which are used traditionally in the treatment of a lot of diseases including bacterial infections, are of interest to search for new therapeutic targets. The literature supports the usages of natural compounds as antioxidant, anti-inflammatory, anticancer as well as antibiotic agent. Nevertheless, therapeutic capabilities of plant and animal source were used long ago without isolation of pure compounds. Thus, the plant species supply definite physiological effects from a huge number of compounds such as alkaloids, terpenoids, flavonoids, glycosides and phenolics.

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These chemical compounds are known as a source of secondary metabolites with a lot of interesting biological activities (Andriani et al., 2015). Looking back to the history, plants are the major source of medicine (Prince et al., 2011). Many flavonoids have bioactive properties such as antimicrobial or insecticidal and pharmacological properties. Therefore especially flavonoids are very important in medicine as therapeutics (Panche et al., 2016). To date many traditional medicines are used to support our immune system against bacterial infection.

The goal of this study was to characterize the ability of selected natural products to stimulate innate immune cells as neutrophils. However, first, we tested the antimicrobial activity of DMSO- and retinoic acid-differentiated HL-60 cells as an alternative model of human neutrophils against the pathogen S. aureus in comparison to primary human blood-derived neutrophils. But the data revealed that HL-60 do not serve as alternative model (chapter 2).

Then, one of our initial studies (chapter 3) showed that acetone bark extract from Guarea kunthiana (GUKUBA) has the ability to boost the host defense of human and bovine neutrophils against bacterial infections (Jerjomiceva et al., 2016) Therefore, the next aim was to screen for novel natural products that have antimicrobial activity for their capability to boost the immune cells against bacterial infections (chapter 4).

In addition, our interest was to study the detailed effects of selected natural products on innate immune-stimulating functions, such as phagocytosis, NETs release, and degranulation (chapter 5).

1. HL-60 cells as alternative model of primary blood-derived

Im Dokument Exploring natural products to boost the host innate immune system against bacterial infections (Seite 80-90)