Natural pseurotins inhibit proliferation and inflammatory responses through the inactivation of STAT signaling pathways in macrophages
Graphical abstract
Introduction
Toxigenic fungi are ubiquitous in nature and mycotoxins, secondary toxic fungal metabolites, are still unavoidable natural contaminants that occur regularly in worldwide food and feed supplies (Bennett and Klich, 2003; Khlangwiset and Wu, 2010). The most documented toxic effects of ingestion of mycotoxin-contaminated food and feed on humans and farm animals include growth retardation, birth defects, liver and renal failure, carcinogenesis and immunosuppression (Bennett and Klich, 2003; Bhatnagar et al., 2002; Hussein and Brasel, 2001). The most studied toxigenic mycotoxins are aflatoxins because of their carcinogenicity and mutagenicity (Raiola et al., 2015). On the other hand, Aspergillus flavus is known to be able to produce other toxic secondary metabolites which, in addition to aflatoxins, could be of concern for evaluating the safety of the biocontrol strategy (Ehrlich et al., 2015).
Interestingly, among these secondary metabolites there are pseurotins, ergot-like alkaloids, commonly produced by A. flavus (Varga et al., 2011). During the pseurotin biosynthesis, a large number of closely related bioactive compounds, such as azaspirene and synerazol is formed (Maiya et al., 2007; Ishikawa and Ninomiya, 2008). These metabolites are usually not considered to be acutely toxic but are supposed to affect human and farm animal health (Bennett and Klich, 2003). Presently, the contamination of food and feed by alkaloids from this structural family is not being monitored. Nevertheless, intentional food/feed treatment by mold species able to produce pseurotins could result in long-term low-level exposure of humans to these compounds without any information of what their immunotoxicity effects are (Ehrlich et al., 2015). Presently, the contamination of food and feed by alkaloids from spirocyclic family is not being monitored. The importance of pseurotin A contamination of feed on the health of cattle was suggested from the report of Botha et al. (2014) showing pseurotin A as one of the main mycotoxins present in feed of cattle with clinically described neuromycotoxicosis (Botha et al., 2014). Pseurotins have interesting biological activities, including induction of cell differentiation by pseurotin A (Komagata et al., 1996), anti-angiogenic activity (Asami et al., 2008; Igarashi et al., 2004) and inhibition of IgE production of plasma cells (Ishikawa et al., 2009). Recently, Anjum et al., (2017) showed that pseurotin A regulated enzymes of cellular metabolism. But effects of these natural products on innate immune response have not been reported.
Macrophages play a key role in innate immunity, owing to their ability not only to recognize, ingest, and destroy pathogens (e.g. phagocytosis and bacterial killing) but they also contribute to induction of complex immune response (Lojek et al., 2011). Macrophages are activated by molecular motifs conserved within microbial pathogens. The most typical one is Gram-negative bacteria lipopolysaccharide (LPS), known as endotoxins. In response to activation signal, macrophages increase their metabolism and release various inflammatory mediators, such as nitric oxide (NO) formed by inducible nitric oxide synthase (iNOS) and inflammatory cytokines such as interleukin 6 (IL-6), tumor necrosis factor (TNF-α), and various chemokines (Jeong et al., 2016). Complex intracellular signaling pathways are activated upon macrophage stimulation that regulates the inflammatory molecules and immune-related cytotoxic factors that induce inflammatory gene expression in macrophages (Kyriakis and Avruch, 2012). Primarily, LPS activates through the toll-like receptor 4 (TLR4) mitogen-activated protein kinase (MAPK) pathway, such as extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), the p38 subfamily and finally transcription factor such as nuclear factor (NF)-κB. Consequently, robust activation of other signaling pathways such as pathway including Janus kinases (JAK) and signal transduction and transcription (STAT) factors is observed which further potentiates production of inflammatory molecules (Greenhill et al., 2011; Lin et al., 2017). Factors that negatively affect macrophage innate immune response can also suppress proliferation of macrophage precursors and macrophages at the site of inflammation. Disturbance of these key signaling pathways leads to inhibition of a cell cycle that is presented as decrease of expression of specific cyclins that are master regulators of particular parts of the cell cycle. Thus, both cell proliferation and activation of macrophages are regulated by overlapping signaling pathways and are dependent on an appropriate cell metabolic activity. The cell metabolism and mitochondrial respiration particularly play a key role in regulation of macrophage functional response as well as proliferation. Environmental noxes or pharmaceutics can affect any of these factors. However, there is no available information about the effects of natural pseurotins on functions of macrophages.
Therefore, the purpose of this work was to investigate the effects of pseurotins A and D on proliferation and activation of macrophages that brings new knowledge to understanding their potential immunomodulatory effects and possible health hazards.
Section snippets
Materials
Pseurotin A and pseurotin D were purchased from AdipoGen Life Sciences, USA. Lipopolysaccharide from E. coli were purchased from Merck, Germany. AG-490 and Stattic V were purchased from MedChemExpress, Sweden. Stock solutions of pseurotins and inhibitors were prepared in DMSO at 10 mM concentration and frozen at −20 °C. The cell culture medium was used for dilution to working concentrations 100, 250 and 500 μM, which were used for treatment. DMSO at final concentration (less than 0.5%) did not
The effects of pseurotins on macrophage proliferation and cyclins expression
Both pseurotins A and D significantly decreased the amount of RAW 264.7 cells after 48 h (Fig. 1A). Particularly at 50 μM concentrations, pseurotin A decreased the number of cells approximately to 72% and pseurotin D to 64% of non-treated control cells. Moreover, a significantly decreased amount of cells compared to control was observed after exposure to 25 μM pseurotin D for 48 h (Fig. 1A). These data, that were obtained from an assay based on determination of amount of proteins in whole cell
Discussion
This study shows for the first time that natural pseurotin at nontoxic concentrations inhibited RAW264.7 macrophage proliferation and suppressed their responses to LPS-stimulation. These effects were connected with inhibition of activation of several key signaling pathways since pseurotins decreased phosphorylation of several members of the STAT signaling family and EKR1/2 from the MAPK family in LPS activated macrophages. Moreover, pseurotins decreased mitochondrial respiration in macrophages.
Conclusions
In summary, this study has demonstrated for the first time that natural pseurotins inhibited RAW264.7 macrophage proliferation and suppressed RAW264.7 responses to LPS-stimulation. These effects were connected with inhibition of phosphorylation of several members of the STAT family and in minor manner EKR1/2 from the MAPK family in LPS activated macrophages (Fig. 7). Consequently, decreased mitochondrial respiration mediated by pseurotins in macrophages was observed. Additionally, pseurotin D
CRediT authorship contribution statement
Ondrej Vasicek: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Daniela Rubanova: Conceptualization, Methodology, Investigation, Formal analysis. Barbora Chytkova: Investigation, Formal analysis. Lukas Kubala: Conceptualization, Writing - review & editing, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by a project from the Czech Science Foundation 17-18858S. The authors are grateful to Stjepan Uldrijan and Barbora Valcikova for their expert technical assistance.
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