Berry Extracts and Their Bioactive Compounds Mitigate LPS and DNFB-Mediated Dendritic Cell Activation and Induction of Antigen Specific T-Cell Effector Responses

Abstract

Berries have gained widespread recognition for their abundant natural antioxidant, anti-inflammatory, and immunomodulatory properties. However, there has been limited research conducted thus far to investigate the role of the active constituents of berries in alleviating contact hypersensitivity (CHS), the most prevalent occupational dermatological disease. Our study involved an ex vivo investigation aimed at evaluating the impact of black raspberry extract (BRB-E) and various natural compounds found in berries, such as protocatechuic acid (PCA), proanthocyanidins (PANT), ellagic acid (EA), and kaempferol (KMP), on mitigating the pathogenicity of CHS. We examined the efficacy of these natural compounds on the activation of dendritic cells (DCs) triggered by 2,4-dinitrofluorobenzene (DNFB) and lipopolysaccharide (LPS). Specifically, we measured the expression of activation markers CD40, CD80, CD83, and CD86 and the production of proinflammatory cytokines, including Interleukin (IL)-12, IL-6, TNF-α, and IL-10, to gain further insights. Potential mechanisms through which these phytochemicals could alleviate CHS were also investigated by investigating the role of phospho-ERK. Subsequently, DCs were co-cultured with T-cells specific to the OVA_323-339 peptide to examine the specific T-cell effector responses resulting from these interactions. Our findings demonstrated that BRB-E, PCA, PANT, and EA, but not KMP, inhibited phosphorylation of ERK in LPS-activated DCs. At higher doses, EA significantly reduced expression of all the activation markers studied in DNFB- and LPS-stimulated DCs. All compounds tested reduced the level of IL-6 in DNFB-stimulated DCs in Flt3L as well as in GM-CSF-derived DCs. However, levels of IL-12 were reduced by all the tested compounds in LPS-stimulated Flt3L-derived BMDCs. PCA, PANT, EA, and KMP inhibited the activated DC-mediated Interferon (IFN)-γ and IL-17 production by T-cells. Interestingly, PANT, EA, and KMP significantly reduced T-cell proliferation and the associated IL-2 production. Our study provides evidence for differential effects of berry extracts and natural compounds on DNFB and LPS-activated DCs revealing potential novel approaches for mitigating CHS.

Keywords: antioxidants; berries; contact hypersensitivity; immune response.

Figure 1

Expression of p-ERK and ERK in LPS-stimulated, BRB-E, protocatechuic acid, proanthocyanidins, ellagic acid, and kaempferol pretreated BMDCs. (A–E) Representative western blot images of p-ERK, ERK, and GAPDH protein levels in either LPS non-stimulated or LPS-stimulated DCs pretreated with BRB-E, PCA, PANT, EA, and KMP. Cell lysates were collected from DCs after 6 h LPS (100 ng/mL) stimulation. Average protein intensity of western blot bands was measured for different groups using ImageJ. (A–E) Decreased protein intensity of p-ERK in DCs after treatment with (A) BRB-E, (B) PCA, (C) PANT, and (D) EA. Data are represented as mean ± SEM relative to the GAPDH control (N = 3 per group). * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001 for comparisons between the LPS to no LPS and the natural compound treatment groups using one-way ANOVA.

Figure 2

Effects of BRB-E, protocatechuic acid, proanthocyanidins, ellagic acid, and kaempferol on DNFB- and LPS-stimulated DC activation markers in Flt3L-derived BMDCs. (A) Gating strategy utilized to identify CD11b⁺ and CD11c⁺ cells. (B) Percent viability of DCs after treatment with natural compounds measured by propidium iodide staining. (C–F) Histograms and Mean fluorescent intensity (MFI) showing the population of cells positive for activation markers (C) CD40, (D) CD80, (E) CD83, and (F) CD86 expression by CD11c+ dendritic cells stimulated with DNFB and LPS. Solid lines represent the lower doses and shaded lines represent higher doses for each compound. Data are represented as mean ± SEM. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001 for comparisons between the DNFB/LPS and the natural compound treatment groups using one-way ANOVA.

Figure 3

Effects of BRB-E, protocatechuic acid, proanthocyanidins, ellagic acid, and kaempferol on the production of IL-12 by stimulated DCs. Supernatants were collected from DCs after 24 h of LPS (100 ng/mL) and 18 h of DNFB (5 µg/mL) stimulation. (A–D) IL-12 (pg/mL) production by DCs after treatment with BRB-E, PCA, PANT, EA, and KMP as determined by ELISA: (A) GM-CSF-derived LPS-stimulated DCs (B) Flt3L-derived LPS-activated DCs. (C) GM-CSF-derived DNFB-stimulated DCs. (D) Flt3L-derived DNFB-activated DCs (N = 4 per group). Data are represented as mean ± SEM. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001 for comparisons between the LPS/DNFB to no LPS/no DNFB and the LPS/DNFB to natural compound treatment groups using one-way ANOVA.

Figure 4

Effects of BRB-E, protocatechuic acid, proanthocyanidins, ellagic acid, and kaempferol on IL-6 production by stimulated DCs. Supernatants were collected from DCs after 24 h of LPS (100 ng/mL) and 18 h of DNFB (5 µg/mL) stimulation. (A–D) IL-6 (pg/mL) production by DCs after treatment with BRB-E, PCA, PANT, EA, and KMP as determined by ELISA. (A) GM-CSF-derived LPS-stimulated DCs (F-JB) Flt3L-derived LPS-activated DCs. (C) GM-CSF-derived DNFB-stimulated DCs. (D) Flt3L-derived DNFB-activated DCs (N = 4 per group). Data are represented as mean ± SEM. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001 for comparisons between the LPS/DNFB to no LPS/no DNFB and LPS/DNFB to natural compound treatment groups using one-way ANOVA.

Figure 5

Proanthocyanidins, ellagic acid, and kaempferol abrogate antigen-specific T-cell proliferation and IL-2 levels induced by LPS-stimulated dendritic cells. T-cells isolated from OT-II TCR transgenic mice were stained with CFSE and co-cultured with DCs for 72 h and analyzed for proliferation by flow cytometry. (A) Gating strategy utilized to identify the different generation of proliferating T-cells. (B) Peaks from various negative controls, positive controls and treatment groups used in the assay. (C,D) Proliferation and division index shows that PANT, EA, and KMP significantly lowered the Ag-specific T-cell proliferation ex vivo. (E) Number of proliferating cells were significantly lowered on treatment with PANT, EA, and KMP. (F) IL-2 level (pg/mL) production was significantly reduced after treatment with PANT, EA, and KMP in supernatants collected from DC:T-cell co-cultures as determined by ELISA but remained unchanged after BRB-E and PCA treatment (N = 2 per group). Data are represented as mean ± SEM ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001 for comparisons between the LPS to no LPS and LPS to natural compound treatment groups using one-way ANOVA.

Figure 6

Effect of BRB-E, protocatechuic acid, proanthocyanidins, ellagic acid, and kaempferol on IFN-γ and perforin production during antigen specific T--cell activation induced by LPS-stimulated DCs. (A) Gating strategy utilized to identify CD4+ and CD8+ T-cells. (B) Decreased IFN- -γ (pg/mL) production by T-cells co-cultured with DCs in different treatment groups determined by ELISA. (C) Frequency of IFN-γ expression by CD4+CD8+ cells in no LPS, LPS and treatment groups, determined by flow cytometry. (D) IFN-γ expression by CD4+ and CD8+ cells in the studied groups. (E) Frequency of Perforin expression by CD4+CD8+ cells in no LPS, LPS, and treatment groups, determined by flow cytometry. (F) Perforin expression by CD4+ and CD8+ cells in different groups (N = 2 per group). Data are represented as mean ± SEM ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001 for comparisons between the LPS to no LPS and LPS to natural compound treatment groups using one-way ANOVA.

Figure 7

IL-17 is reduced by protocathechuic acid, proanthocyanidins, ellagic acid, and kaempferol during antigen specific T-cell activation induced by LPS-stimulated DCs. IL-17 production (pg/mL) by OT-II T-cells stimulated with OVA_323–339 peptide in presence or absence LPS and natural compounds (N = 2 per group) *** p-value < 0.001; **** p-value < 0.0001 for comparisons between the LPS to no LPS and LPS to natural compound treatment groups using one-way ANOVA.