The mycotoxin alternariol suppresses lipopolysaccharide-induced inflammation in THP-1 derived macrophages targeting the NF-κB signalling pathway

Abstract

Alternariol (AOH) is a secondary metabolite formed by black mold of the genus Alternaria alternata. Due to limited hazard and occurrence data, AOH is still considered as an "emerging mycotoxin" and, as such, not monitored and regulated yet. Recent studies indicate immunosuppressive effects in vitro by altering the expression of CD molecules and proinflammatory cytokines, which are indispensable in mounting an innate immune response. However, the mode of action by which AOH exerts its immunosuppressive effects has not been unraveled yet. The present study aimed to characterise the impact of AOH on the nuclear factor kappa B (NF-κB) pathway, the expression of NF-κB target cytokines and involved regulatory microRNAs (miRNAs). In THP-1 derived macrophages, AOH (1-20 µM) was found to suppress lipopolysaccharide (LPS)-induced NF-κB pathway activation, decrease secretion of the proinflammatory cytokines IL-8, IL-6, TNF-α and to induce secretion of the anti-inflammatory IL-10. Thereby, a distinct pattern of cytokine mRNA levels was monitored, varying between short- and long-term exposure. Concomitantly, AOH (2-20 µM) affected the transcription levels of miR-146a and miR-155 in LPS-stimulated THP-1 derived macrophages dose-dependently by down- and upregulation, respectively. In contrast, transcription of miR-16 and miR-125b, two other immune-related miRNAs, was not modulated. In the absence of a LPS stimulus, AOH (20 µM) did not affect basal NF-κB activity, but increased IL-10 transcription. Collectively, our results indicate, that AOH itself does not induce a proinflammatory immune response in human macrophages; however, in an inflamed environment it possesses the ability to repress inflammation by targeting the NF-κB signalling pathway and regulatory miRNAs.

Keywords: Alternariol; Immune response; Mycotoxin; NF-κB; miRNA.

Fig. 1

Cytotoxic effects of AOH in LPS and non-stimulated differentiated THP-1 derived macrophages measured with the Alamar Blue^® assay. Differentiated LPS-stimulated THP-1 cells were preincubated with AOH for 2 h and afterwards stimulated with LPS (10 ng/ml) for 3 h (a) and 18 h (b). Differentiated non-stimulated THP-1 cells were exposed to AOH for 5 h (c) and 20 h (d). THP1-Lucia™ NF-κB cells were preincubated with AOH for 2 h and stimulated with LPS for further 18 h (e). Fluorescence intensity was calculated as the percent of treated cells over control cells [treated with LPS or the solvent control) × 100 (T/C, %). Results are normalized to LPS or the solvent control (0.1% DMSO], respectively, expressed as mean ± SD of T/C (%). Statistical significances between varying concentrations of AOH were evaluated by one-way ANOVA and Holm–Bonferroni test (a–d p < 0.05), significances compared to LPS/solvent control were calculated with a two-sample t test (*p; **p; ***p < 0.05, 0.01, 0.001); n = 3–7 independent experiments

Fig. 2

Activity of NF-κB in LPS-stimulated THP1-Lucia NF-κB cells. THP1-Lucia NF-κB cells were preincubated with AOH for 2 h followed by an 18 h LPS challenge (10 ng/ml). Luminescence intensity of the expressed luciferase protein was measured by the NF-κB reporter gene assay and calculated as the percent of treated cells over control cells (treated with LPS) × 100 (T/C, %). Results are normalized to LPS and are expressed as mean ± SD of T/C (%). Statistical significances between varying concentrations of AOH were evaluated by one-way ANOVA and Holm–Bonferroni test (a–d p < 0.001) and significances compared to LPS were calculated with a two-sample t test (*p; **p; ***p < 0.05, 0.01, 0.001); n = 3–6 independent experiments

Fig. 3

Relative gene transcription levels of IL-8, IL-6, TNF-α and IL-10 in LPS- and non-stimulated THP-1 derived macrophages after AOH exposure. Differentiated LPS-stimulated THP-1 cells were preincubated with AOH for 2 h and afterwards stimulated with LPS (10 ng/ml) for 3 h (a) and 18 h (b). Differentiated non-stimulated THP-1 cells were exposed to AOH for 5 h (c) and 20 h (d). Relative transcript levels were measured with qRT-PCR. Results are expressed as mean ± SD of the relative gene transcription ($2^{-\mathrm{\Delta }\mathrm{\Delta }{\text{C}}_T}$) and normalized either to LPS or the solvent control (0.1% DMSO). Statistical significances between varying concentrations of AOH were evaluated by Kruskal–Wallis ANOVA and significances compared to LPS/solvent control were calculated by Mann–Whitney U test; n = 3–7 independent experiments

Fig. 4

Cytokine secretion levels of IL-8, IL-6, TNF-α and IL-10 in LPS-stimulated THP-1 derived macrophages after AOH exposure measured with ProcartaPlex™ Multiplex Immunoassay. THP-1 macrophages were preincubated with AOH for 2 h followed by an 18 h LPS challenge (10 ng/ml). Cytokine protein levels were calculated as percent of treated cells over control cells (treated with LPS) × 100 (T/C, %) and are expressed as mean ± SD of T/C (%) normalized to LPS. Statistical significances between varying concentrations of AOH were evaluated by Kruskal–Wallis ANOVA and significances compared LPS were calculated by Mann–Whitney U test; n = 3 independent experiments

Fig. 5

Transcription levels of miR-16, -125b, -146a and -155 in LPS-stimulated THP-1 derived macrophages after AOH exposure. THP-1 macrophages were preincubated with AOH for 2 h followed by an 18 h LPS challenge (10 ng/ml). Relative transcript levels were measured with qPCR. Results are expressed as mean ± SD of the relative gene transcription ($2^{-\mathrm{\Delta }\mathrm{\Delta }{\text{C}}_T}$) normalized to LPS-stimulated macrophages. Statistical significances between varying concentrations of AOH were evaluated by one-way ANOVA and Holm–Bonferroni test (a–c p < 0.001) and significances compared to LPS were calculated with a two-sample t test (*p; **p; ***p < 0.05, 0.01, 0.001); n = 3–5 independent experiments

Fig. 6

The involvement of NF-κB in AOH-mediated immunosuppression. A suggested cellular pathway illustrating the consequences of AOH-induced NF-κB suppression on cytokine and miRNA expression resulting in a suppressed immune response