Supplementary Material for: Bilberry-Derived Anthocyanins Prevent IFN-γ-Induced Pro-Inflammatory Signalling and Cytokine Secretion in Human THP-1 Monocytic Cells

<p><b><i>Background/Aims:</i></b> Anthocyanins are plant-derived dietary components that are highly abundant, for example, in bilberries. We have previously demonstrated that anthocyanins exert anti-inflammatory properties in mouse colitis models and ameliorate disease activity in ulcerative colitis patients. Here, we studied the molecular mechanisms through which anthocyanin-containing bilberry extract (BE) exerts anti-inflammatory effects in human monocytic THP-1 cells. <b><i>Methods:</i></b> THP-1 cells were pre-incubated with BE 20 min prior to TNF-a or IFN-γ (100 ng/ml each) stimulation. Signalling protein activation was studied by Western blotting, mRNA expression by quantitative PCR and cytokine secretion by ELISA. <b><i>Results:</i></b> IFN-γ-induced phosphorylation of STAT1 and STAT3 was significantly reduced by BE co-treatment. Consequently, levels of mRNA expression and/or cytokine secretion of MCP-1, IL-6, TNF-a, ICAM-1, and T-bet were lower with BE co-treatment. In contrast, BE enhanced TNF-a-mediated p65-NF-γB phosphorylation but reduced ERK1/2 phosphorylation. BE co-treatment further increased TNF-a-induced mRNA expression and secretion of NF-γB target genes, such as IL-6, IL-8, and MCP-1, while mRNA levels of ICAM-1 were reduced. <b><i>Conclusions:</i></b> BE co-treatment reduced IFN-γ-induced signal protein activation, pro-inflammatory gene expression, and cytokine secretion, whereas it enhanced TNF-a-induced responses. These findings suggest a distinct role for anthocyanins in modulating inflammatory responses that need to be further studied to fully understand anthocyanin-mediated effects.</p>