Examination of Novel Immunomodulatory Effects of L-Sulforaphane

Abstract

The dietary isothiocyanate L-sulforaphane (LSF), derived from cruciferous vegetables, is reported to have several beneficial biological properties, including anti-inflammatory and immunomodulatory effects. However, there is limited data on how LSF modulates these effects in human immune cells. The present study was designed to investigate the immunomodulatory effects of LSF (10 µM and 50 µM) on peripheral blood mononuclear cell (PBMC) populations and cytokine secretion in healthy adult volunteers (n = 14), in the presence or absence of bacterial (lipopolysaccharide) and viral (imiquimod) toll-like receptor (TLRs) stimulations. Here, we found that LSF reduced pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and chemokines monocyte chemoattractant protein (MCP)-1 irrespective of TLR stimulations. This result was associated with LSF significantly reducing the proportion of natural killer (NK) cells and monocytes while increasing the proportions of dendritic cells (DCs), T cells and B cells. We found a novel effect of LSF in relation to reducing cluster of differentiation (CD) 14⁺ monocytes while simultaneously increasing monocyte-derived DCs (moDCs: lineage-Human Leukocyte Antigen-DR isotype (HLA-DR)⁺CD11b^low-high CD11c^high). LSF was also shown to induce a 3.9-fold increase in the antioxidant response element (ARE) activity in a human monocyte cell line (THP-1). Our results provide important insights into the immunomodulatory effects of LSF, showing in human PBMCs an ability to drive differentiation of monocytes towards an immature monocyte-derived dendritic cell phenotype with potentially important biological functions. These findings provide insights into the potential role of LSF as a novel immunomodulatory drug candidate and supports the need for further preclinical and phase I clinical studies.

Keywords: L-sulforaphane; anti-inflammatory effects; cruciferous vegetables; dendritic cells; immune cells; immunomodulatory effects; monocytes.

Figure 1

LSF effect on cytokine and chemokine secretion. Cytokine and chemokine measurements in supernatants from healthy adult PBMCs (n = 14) following pre-treatment with 10 µM or 50 µM LSF or its metabolites (LSF-cys, LSF-GSH, LSF-NAc) for 24 h before stimulation with LPS (10 ng/mL) (A) or IMQ (5 mg/mL) (B) for a further 24 h. The medium control was used as the control group for comparisons with the TLR-stimulated groups; the DMSO control was used as the vehicle control for comparison with the LSF treatments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the respective stimulation controls, assessed by a non-parametric Wilcoxon signed-rank test. The median ± IQR range is displayed. Legend: • Control, • LPS, • IMQ, • LSF, • LSF-cys, • LSF-GSH, • LSF-NAc (10 µM are open circles and 50 µM are closed circles).Abbreviation: IL, interleukin; IMQ, imiquimod; IQR, inter-quartile range; LPS, lipopolysaccharide; LSF, L-sulforaphane; LSF-cys, LSF-cysteine; LSF-GSH, LSF-glutathione; LSF-NAc, LSF-N-Acetyl-L-cysteine; MCP-1, monocyte chemoattractant protein -1; PBMC, peripheral blood mononuclear cells; RANTES, Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted; TNF-α, tumour necrosis factor-alpha.

Figure 2

The effect of LSF on adaptive immune cell populations. Flow cytometric gating strategy (A) and the proportion of T cells (B) and B cells (C) (n = 9) displayed as a proportion of total lymphocyte populations. The median ± IQR range is presented. The medium control was used as the control group for comparisons with the TLR-stimulated groups; the DMSO control was used as the vehicle control for comparison with the LSF treatments. * p < 0.05, ** p < 0.01 compared to the respective untreated control, with statistical analysis performed using a non-parametric Wilcoxon signed-rank test. Legend: • Control/LPS/IMQ, 10 µM LSF, • 50 µM LSF, • DMSO. Abbreviation: CD, cluster of differentiation; DMSO, dimethyl sulfoxide; IMQ, imiquimod; IQR, inter-quartile range; LPS, lipopolysaccharide; LSF, L-sulforaphane; TLR, toll-like receptor.

Figure 3

The effect of LSF on innate immune cell populations. Gating strategy (A) and flow cytometric results (n = 9) are displayed as a proportion of total lymphocyte populations (B). The median ± IQR is displayed. The medium control was used as the control group for comparisons with the TLR-stimulated groups; the DMSO control was used as the vehicle control for comparison with the LSF treatments. * p < 0.05, ** p < 0.01 compared to the respective untreated control using a non-parametric Wilcoxon signed-rank test. Legend: • Control/LPS/IMQ, 10 µM LSF, • 50 µM LSF, • DMSO. Abbreviation: CD, cluster of differentiation; DC, dendritic cell, DMSO, dimethyl sulfoxide; HLA-DR, Human Leukocyte Antigen–DR isotype; IMQ, imiquimod; IQR, inter-quartile range; Lin, lineage; LPS, lipopolysaccharide; LSF, L-sulforaphane; moDC, monocyte-derived dendritic cell; NK, natural killer cell; TLR, toll-like receptor.

Figure 4

LSF effect on monocyte and dendritic cell populations. The effect of 10 µM or 50 µM LSF on DC populations in healthy adult PBMCs (n = 8) over 6, 24, or 48 h. PBMC viability (n = 8) (A) and flow cytometric results of monocytes (B) and DC subsets (C) displayed as a proportion of total lymphocyte populations, with the median ± IQR displayed. DMSO was used as a vehicle control for LSF treatments. * p < 0.05, ** p < 0.01 compared to the untreated DMSO control using a non-parametric Wilcoxon signed-rank test. Legend: • Control, 10 µM LSF, • 50 µM LSF, • DMSO. Abbreviation: CD, cluster of differentiation; DC, dendritic cell, DMSO, dimethyl sulfoxide; HLA-DR, Human Leukocyte Antigen – DR isotype; IQR, inter-quartile range; Lin, lineage; LPS, lipopolysaccharide; LSF, L-sulforaphane; moDC, monocyte-derived dendritic cell; PBMC, peripheral blood mononuclear cells; pDC, plasmacytoid dendritic cells.

Figure 5

LSF effect on cytokine and chemokine secretion over time. Cytokine and chemokine measurements in supernatants from healthy adult PBMCs (n = 8) following treatment with 10 µM or 50 µM LSF for 6 h, 24h, or 48 h. The median ± IQR are displayed. DMSO was used as a vehicle control for LSF treatments. * p < 0.05, ** p < 0.01 compared to the untreated DMSO control using a non-parametric Wilcoxon signed-rank test. Legend: • Control, 10 µM LSF, • 50 µM LSF, • DMSO. Abbreviation: DMSO, dimethyl sulfoxide; IL, interleukin; IQR, inter-quartile range; LSF, L-sulforaphane; MCP-1, monocyte chemoattractant protein-1; RANTES, PBMC, peripheral blood mononuclear cells; Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted; TNF-α, tumour necrosis factor-alpha.

Figure 6

Association between reduced CD14+ cells and increased DC populations. Correlation between CD14⁺ cells and DC subsets: (A) total DCs, (B) moDCs, and (C) pDCs after 24 h of LSF (10 µM and 50 µM) treatment. Each datapoint is an individual sample across each of the three groups (n = 8/group). A Pearson’s correlation was performed. Abbreviation: CD, cluster of differentiation; DC, dendritic cell, LSF, L-sulforaphane; moDC, monocyte-derived dendritic cell; pDC, plasmacytoid dendritic cells.

Figure 7

The effect of 10 µM or 50 µM LSF on Nrf2-ARE activity in THP-1 monocytes. Cell viability (A) and ARE-luciferase reporter activity after 6 h (B) and 24 h (C) of LSF treatment are shown. Data was collected as three independent experiments. The mean ± 95% CI are displayed. DMSO was used a vehicle control for LSF treatments. * p < 0.05, **** p < 0.0001 compared to the untreated DMSO control cells using the Student’s t-test. Legend: • Control, 10 µM LSF, • 50 µM LSF, • DMSO. Abbreviation: ARE, antioxidant response element; CI, confidence interval; DMSO; dimethyl sulfoxide; LSF, L-sulforaphane.