Gallic acid diminishes pro-inflammatory interferon-γ- and interleukin-17-producing sub-populations in vitro in patients with psoriasis

Abstract

Psoriasis is an inflammation of the skin mediated via the IL-23/Thl17/IL-17 pathway. We have previously demonstrated that the anthocyanin delphinidin diminishes in vitro the IL-17 and IFN-γ production of peripheral monocytes isolated by psoriasis patients (PBMCs). The degradation product of delphinidin is gallic acid (GA). This phenolic acid compound found in fruits, red wine, or green tea exerts pleiotropic antioxidant, anticarcinogenic, antimicrobial, and anti-inflammatory properties. Previous research has demonstrated the inhibitory effect of GA on pro-inflammatory transcription factors, such as STAT3, RORγt, and NF-κB, or cytokines as IL-1β and TNF, which contribute to psoriasis development. We investigated the effect of GA in vitro on PBMCs, which were stimulated ex vivo, from 40 individuals (28 diagnosed with psoriasis vulgaris and 12 healthy controls (HCs)). In our experiments, PBMCs were cultured untreated or were activated in the presence of phorbol 12-myristate 13-acetate/ionomycin with or without GA. We utilized multicolor flow cytometry to assess the production of inteleukin-17 (IL-17) and interferon-γ (IFN-γ) in T and NK cells. GA did not alter the fractions of IL-17- or IFN-γ-producing T and IFN-γ-producing NK cells in HCs. However, in psoriasis patients, the effect of GA on that cell population was significant. Specifically, GA decreased the frequency of IL-17-producing cells within the CD3⁺ (T) and CD3⁺CD4⁺ (Th) compartment; the frequency of IFN-γ-producing cells within the CD3⁺, CD3⁺CD4⁺, and CD3⁺CD4^- (Tc) compartment, and the frequency of IFN-γ-producing cells within the CD3^-CD56⁺ (NK) compartment. Whether GA's effect also appears in vivo needs to be investigated in future.

Keywords: Flow cytometry; NK; NKT; PBMCs; Th1; Th17.

Fig. 1

Subdividing strategy as depicted in representative plots from flow cytometry. Total lymphocytes were sub-gated based on forward/side light scatter characteristics. Next, Th (CD3⁺CD4⁺) and Tc (CD3⁺CD4⁻) sub-populations were characterized utilizing CD3-CD4 staining. Furthermore, using CD3-CD56 staining, NK (CD3⁻CD56⁺) cells were also recognized. Within each cell sub-population IL-17 and IFN-γ expression was identified by IL-17 and IFN-γ staining respectively

Fig. 2

Gallic acid (GA) in vitro pre-treatment diminished fractions of IL-17-producing T (CD3⁺) and Th (CD3⁺CD4⁺) cell subsets that were stimulated by PMA/ionomycin in participants with psoriasis (n = 28) and controls (n = 12). Isolated peripheral blood mononuclear cells were co-cultured with PMA/ionomycin with or without GA. Cells were analyzed following GA pre-treatment for 0.5 h and PMA and ionomycin stimulation. a Representative plots from flow cytometry of GA-treated and -untreated IL-17-producing T and Th cells from patients. b Representative plots from flow cytometry of GA-treated and -untreated IL-17-producing T and Th cells from controls. c Boxplots (showing mean ± SD) depicting that IL-17-producing T and Th cells were significantly inhibited by in vitro GA pre-treatment in patients. ***p ≤ 0.001, ****p ≤ 0.0001 by Wilcoxon signed-rank test or paired t-test

Fig. 3

Gallic acid (GA) in vitro pre-treatment diminished fractions of IFN-γ-producing T (CD3⁺), Th (CD3⁺CD4⁺) and Tc (CD3⁺CD4⁻) cell subsets that were stimulated by PMA and ionomycin in participants with psoriasis (n = 28). Isolated peripheral blood mononuclear cells were co-cultured with PMA/ionomycin with or without GA. Cells were analyzed following GA pre-treatment for 0.5 h and PMA and ionomycin stimulation. a Representative plots from flow cytometry of GA-treated and -untreated IFN-γ-producing T, Th, and Tc cells. b Boxplots (showing mean ± SD) depicting that IFN-γ-producing T, Th, and Tc cells were significantly inhibited by in vitro GA pre-treatment. ****p ≤ 0.0001 by Wilcoxon signed-rank test or paired t-test

Fig. 4

Gallic acid (GA) in vitro pre-treatment diminished the fraction of the IFN-γ-producing ΝΚ (CD3⁻CD56⁺) cell subset that was stimulated by PMA/ionomycin in participants with psoriasis (n = 28). Isolated peripheral blood mononuclear cells were co-cultured with PMA/ionomycin with or without GA. Cells were analyzed following GA pre-treatment for 0.5 h and PMA and ionomycin stimulation. a Representative plots from flow cytometry of GA-treated and -untreated IFN-γ-producing NK cells. b Boxplots (showing mean ± SD) depicting that IFN-γ-producing NK cells were significantly inhibited by in vitro GA pre-treatment, ****p ≤ 0.0001 by Wilcoxon signed-rank test or paired t-test

Fig. 5

Gallic acid (GA) in vitro pre-treatment diminished percentages of Th17-, Th1-, and IFN-γ-producing NK cell subsets that were stimulated by PMA/ionomycin in participants with psoriasis (n = 28). Isolated peripheral blood mononuclear cells were co-cultured with PMA/ionomycin with or without GA. Cells were analyzed following GA pre-treatment for 0.5 h and PMA and ionomycin stimulation. a GA-mediated inhibition on the IFN-γ-producing Th cell percentage was correlated with the inhibition of the fraction of IL-17-producing Th cells. b GA-mediated inhibition on the IFN-γ-producing Th cell percentage was correlated with the inhibition of the fraction of IFN-γ-producing NK cells