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.

The Alternaria Mycotoxin Alternariol Triggers the Immune Response of IL‐1β‐stimulated, Differentiated Caco‐2 Cells

“…In more detail, the IL‐1β induced transcription of the proinflammatory cytokines IL‐8, IL‐6, and IL‐1β (Figure A) was inhibited after 5 h of incubation as well as the secretion of IL‐8 (Figure ) was reduced after both 5 and 20 h of AOH exposure. Similar effects were reported for RAW 264.7 macrophages, BEAS lung epithelial cells, and THP‐1 cells, where AOH reduced LPS induced immune responses . As underlying mechanism, an inhibition of the LPS induced NF‐κB signaling pathway was reported in THP‐1 macrophages .…”

“…Without additional inflammatory stimulus, AOH displayed a tendency to induce IL‐8 transcription after long‐term incubation (20 h), albeit in a concentration‐independent manner (Figure ). These findings are only in part supported by previous research, as AOH was reported to leave IL‐8 secretion unaffected in human primary macrophages or rather slightly reduce its transcription in THP‐1‐derived macrophages . This discrepancy might arise from differences between cell models of different tissue origin.…”

“…Seven days post‐seeding medium was replaced with medium containing the test substances at required concentrations with a final DMSO concentration of 1%. Test concentrations and incubation times were chosen in accordance to a recent publication on immunomodulatory effects of AOH in THP‐1 macrophages . Briefly, cells were either incubated with AOH alone at concentrations of 0.02, 0.2, 2, and 20 µ m for 5 or 20 h, or cells were first pre‐incubated for 2 h with the test compound and then additionally stimulated with IL‐1β (25 ng µL −1 ) for further 3 or 18 h. Additionally, a concentration of 40 µ m AOH was applied on Caco‐2 cells, as the intestinal epithelial layer is exposed to higher concentrations of food‐associated contaminants in comparison to underlying cells of the lamina propria, e.g., macrophages.…”

“…In detail, the induction of both CD14 and CD11b was reduced by AOH treatment with concomitant induction of CD71 expression, that is usually decreased during the differentiation process . AOH was furthermore reported to not only interfere in inflammatory cytokine signaling in human primary macrophages, RAW 264.7, and differentiated THP‐1 macrophages, but also to reduce the lipopolysaccharide (LPS) induced immune response in THP‐1 as well as RAW 264.7 cells . The observed immunosuppressive potential of AOH was very recently in part attributed to its potent inhibition of the LPS‐induced NF‐κB (nuclear factor kappa B) activation …”

“…miRNAs were not only reported to tune and decrease translational efficiency, but also to destabilize or cleave target protein‐coding mRNA . Indeed, inflammation‐related miRNAs 155 and 146a were significantly induced and repressed, respectively, by AOH treatment (20 µ m ) in LPS stimulated, differentiated THP‐1 macrophages . The interpretation of the specific function of a miRNA, in general, is utterly complex as hundreds of targets can be regulated by a single miRNA.…”