Minimal Concentrations of Deoxynivalenol Reduce Cytokine Production in Individual Lymphocyte Populations in Pigs

Abstract

Deoxynivalenol (DON) is a mycotoxin frequently found in cereals, and pigs are one of the most sensitive farm species to DON. The aim of this study was to determine the effects of DON in very low doses on peripheral blood mononuclear cells (PBMC) and on particular lymphocyte subpopulations. The cells were exposed to 1, 10 and 100 ng/mL of DON and lymphocyte viability, proliferation, and cytokine (Interleukin (IL)-1β, IL-2, IL-8, IL-17, Interferon (IFN) γ and tumor necrosis factor (TNF) α production were studied. Cells exposed to DON for 5 days in concentrations of 1 and 10 ng/mL showed higher viability compared to control cells. After 18 h of DON (100 ng/mL) exposure, a significantly lower proliferation after mitogen stimulation was observed. In contrast, an increase of spontaneous proliferation induced by DON (100 ng/mL) was detected. After DON exposure, the expression of cytokine genes decreased, with the exception of IL-1β and IL-8, which increased after 18 h exposure to 100 ng/mL of DON. Among lymphocyte subpopulations, helper T-cells and γδ T-cells exhibiting lower production of IL-17, IFNγ and TNFα were most affected by DON exposure (10 ng/mL). These findings show that subclinical doses of DON lead to changes in immune response.

Keywords: PBMC; animal health; cytokines; deoxynivalenol; immunotoxicity; lymphocytes; pig; subclinical dose.

Figure A1

Gating strategy—Isolation of lymphocyte subpopulations with fluorescence-activated cell sorting (FACS). Doublets and dead cells identified by propidium iodide staining were excluded. Subsequently, T cells subpopulations were defined from all CD3 positive cells. Dot plots show data from one representative sample.

Figure A2

Gatting strategy—Flow cytometry analysis of cytokine production in lymphocyte subpopulations after DON exposure Doublets and dead cells identified by Live/dead probe (Fixable yellow dead cell stain kit for 405 nm excitation) were excluded. Subsequently, T cells subpopulations were defined from all CD3 positive cells and percentage of population positive for cytokine (IFNγ, IL-17 or TNFα) was evaluated. Dot plots show data from one representative sample where IFNγ was stained as cytokine.

Figure 1

Expression of cytokine (IL-2, IL-17, IFNγ and TNFα) mRNA in lymphocyte subpopulations and the impact of DON exposure (10 ng/mL; 18 h). Values are expressed as mean of multiples of the reference gene expression ± standard deviation. Data of samples are from six pigs in biological duplicate. Statistically significant differences between DON exposed samples and control samples are marked with asterisk (* p ≤ 0.05). Tc—Cytotoxic T cells (CD3+γδTcR-CD4-CD8+), gd—γδ T cells (CD3+γδTcR+CD4-CD8+/-), Th—helper T cells (CD3+γδTcR- CD4+CD8-) and DP—double positive T cells (CD3+γδTcR-CD4+CD8+).

Figure 2

Production of cytokines (IL-17, IFNγ and TNFα) at the protein level in lymphocyte subpopulations and the impact of DON exposure (10 ng/mL; 18 h). The level of cytokine production is expressed as median of fluorescence intensity (MFI) for each lymphocyte subpopulation. Data of samples are from 12 pigs. Statistically significant differences between DON exposed samples and control samples are marked with asterisk (* p ≤ 0.05, ** p ≤ 0.01). Tc—Cytotoxic T cells (CD3+γδTcR-CD4-CD8+), gd—γδ T cells (CD3+γδTcR+CD4-CD8+/-), Th—helper T cells (CD3+γδTcR- CD4+CD8-) and DP—double positive T cells (CD3+γδTcR-CD4+CD8+).