Elsevier

Toxicon

Volume 149, July 2018, Pages 20-25
Toxicon

Immunomodulatory effects of selected cyanobacterial peptides in vitro

https://doi.org/10.1016/j.toxicon.2018.04.031Get rights and content

Highlights

  • Immunomodulatory potency significantly differ among cyanobacterial peptides.

  • The cyclic microcystin-RR and -YR have negligible effect on macrophages.

  • New structure-activity relationship is discussed for cyclic microcystins.

  • Newly isolated linear cyanobacterial peptide aeruginosin-865 has no effect on macrophages.

  • Revealed structure specific effects clarify the risks associated with the mixture of cyanobacterial toxins.

Abstract

Cyanobacteria produce many biologically active metabolites synthesized via nonribosomal synthetic pathways such as cyclic microcystins (MCs) and linear aeruginosins (Aers). The present study aimed to investigate the effects of different MC variants and the newly isolated aerugenosin Aer-865 on macrophages, which represent one of the key effector cells within the innate immune responses. Specifically, our study included RAW 264.7 macrophage activation associated with production of cytotoxic and cytostatic nitric oxide (NO) as well as pro-inflammatory mediators like tumor necrosis factor α (TNFα) and interleukin 6 (IL-6). From the compounds investigated, commonly occurring MC variants (-RR, -YR) and Aer-865 had no significant effects within the non-cytotoxic concentrations tested, i.e. 0.001–1 μM for MCs and 0.1–50 μM for Aer-865. In contrast to known immunoactive MC-LR, the negligible immunomodulatory potential of tested MC congeners could be related to their differences in structure. The knowledge of MC structure-specific activities contributes to the understanding of complex toxicity of different MC variants and most importantly their mixtures. This study is one of the first study that evaluate the effect of larger set of cyanobacterial peptides on macrophages and compare their immunomodulatory potential.

Introduction

Cyanobacteria are photosynthetic organism frequently found in the aquatic environment. Due to their potency to produce biologically active secondary metabolites, their massive growth became one of the serious environmental and health problems worldwide. Globally, cyanobacterial dominance in freshwater habitat is increasing and it is expected that a warming climate will exacerbate the frequency and duration of such blooms (Paerl and Huisman, 2009). The communities of the people living the areas with high cyanobacterial densities may be long term exposed to cyanobacterial toxins via poorly treated or untreated water and consumption of contaminated food such as fish and mussels (Chen and Xie, 2007, Henry, 2014, Poste et al., 2011). Cyanobacteria produce many biologically active peptides that are classified into families according to their structural features and include cyclic microcystins (MCs) and linear aeruginosins (Aer) (Welker and von Dohren, 2006). MCs gained public and scientific attention over last three decades due to their common presence in the environment, high concentrations and toxicity (Rastogi et al., 2014). Recent studies documented presence of MCs in blood of people living near contaminated localities (Chen et al., 2009, Li et al., 2011). MC family contains more than hundred structurally similar congeners in which MC-LR, -RR and -YR are the most produced peptides and represent toxins of concern (Carmichael and Boyer, 2016). The hepatotoxic and tumor-promotional potency of MCs has been investigated extensively in the past but other potential chronic impacts of MCs, e.g. on immune system, have been studied to a much lesser extent. The imbalanced immune system is a cause of many inflammatory diseases, hyperactivity and autoimmune reactions and it was also shown to contribute to cancer development (Didonato et al., 2012). Our recent study (Adamovsky et al., 2015) revealed a high potency of MC-LR to stimulate the innate immunity cells macrophages in vitro and suggested the possible mechanism of action. The non-physiological modulation of macrophages functions plays an important role in chronic inflammation. In contrast to acute inflammation, which is usually suppressed after removal of pathogens, long-term activation of macrophages may contribute to diverse local and systemic deleterious effects on cells and tissues. Although MC-LR is the most commonly occurring MC variant, the study of Chen et al. (2009) indicated that other MC variants (-RR and -YR) could have cumulative serum levels higher than the level of MC-LR (Chen et al., 2009). However, no information is currently available on immunomodulatory effects of other variants of MCs.

In addition to MCs, other cyanobacterial peptides become of general interest. For example, Aer-865 has been an attractive research topic because of its potential anti-inflammatory activity together with the inhibitory activity against trypsin-like serine proteases (Ersmark et al., 2008, Kapuścik et al., 2013). Although the anti-inflammatory potency of Aer-865 has been evaluated on human lung microvascular endothelial cells _3rdcrjn (Kapuścik et al., 2013), the interaction of this cyanobacterial peptide with the immune cells has not been studied so far.

The present study aimed to investigate and compare the in vitro effects of two abundant MC variants and Aer-865 (Table 1) on macrophages, which represent one of the key effector cells within the innate immune responses. Specifically, our study includes macrophage activation associated with production of cytotoxic and cytostatic products such as nitric oxide (NO), as well as pro-inflammatory mediators (e.g. tumor necrosis factor α, TNFα and interleukin 6, IL-6). Murine macrophage RAW 264.7 cells have been selected because of broad utilization of this immune model in a range of pharmacological (Tweedie et al., 2009) as well as environmental toxicological studies (Jalava et al., 2007).

Section snippets

Reagents and cell culture

MC-RR and MC-YR was obtained from Enzo Life Sciences, New York with purity ≥95% (HPLC). The stock solution (1 mg/ml) was dissolved in methanol (50%). Aer-865 (MW = 865) was isolated from the cyanobacterial strain Nostoc sp. Lukešová 30/93 and purified in the Institute of Microbiology, Třeboň, Czech Republic (Kapuścik et al., 2013). Sterile phosphate-buffered saline (PBS) was used for preparation of testing concentration. Lyophilized powder of lipopolysaccharide (LPS) from Escherichia coli

Results and discussion

Concentration of tested MCs (MC-RR and MC-YR) were selected in order to describe the effects of MCs in the levels that were detected in sera of individuals living on a lake with high cyanobacterial density (Chen et al., 2009, Li et al., 2011), i.e. adult fishermen (min.-max. 0.045–1.83 nM) and their children (0.4–1.3 nM MC-LR eq.).

All tested substances were initially tested on cell viability with and without co-exposures to a model activator LPS (Fig. 1). In agreement with previous studies (

Acknowledgment

The research was supported by the grant from the Czech National Science Foundation GAČR GP13-27695P. Infrastructure of the RECETOX Centre is supported by the Czech Ministry of Education, Youth and Sports projects No. LO1214 and LM2015051.

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