Nexus Between Immune Responses and Oxidative Stress: The Role of Dietary Hydrolyzed Lignin in ex vivo Bovine Peripheral Blood Mononuclear Cell Response

Abstract

The control of immune responses is particularly critical when an increase of oxidative stress occurs, causing an impairment of immune cell response and a condition of systemic inflammation, named oxinflammation. Nutritional strategies based on the use in the diet of phytochemicals extracted from plants, rich in antioxidants, could help restore the antioxidant/oxidant balance and obtain a modulation of immune response. Lignin represents a valuable resource of phenolic compounds, which are characterized by a corroborated antioxidant effect. To date, there are no studies reporting the effects of lignin in the diet on immune responses and oxidative stress in ruminants. The objective of the present experiment was the evaluation of the dietary inclusion of Pinus taeda hydrolyzed lignin on the ex vivo immune responses and oxidative stress biomarkers by peripheral blood mononuclear cells (PBMCs) isolated from beef steers. In order to test the effect during oxidative stress exposition, cells were treated with hydrogen peroxide (H₂O₂). The proliferation test and the viability assay were carried out on cells, whereas, on supernatants, the cytokine profile and the oxidative stress biomarkers were evaluated. The dietary inclusion with P. taeda hydrolyzed lignin resulted in cytoprotection after H₂O₂ exposition, increasing the number of viable monocytes and decreasing the reactive oxygen/nitrogen species production in supernatants. The cytokine profile indicated the modulatory role of hydrolyzed lignin on immune response, with a concomitant decrease of TNF-α and increase of IL-8 production, which are strictly connected with monocyte activation and antioxidant response pathway. These results demonstrated that hydrolyzed lignin may provide a modulation of oxidative stress and inflammatory response in PBMCs; thus, the P. taeda hydrolyzed lignin could be suggested as an innovative phytochemical in ruminant feed.

Keywords: antioxidant response; cytokines; immune system; oxidative stress; polyphenols; ruminants.

Figure 1

BrdU incorporation in proliferating PBMCs isolated by blood of beef steers supplemented with Pinus taeda hydrolyzed lignin (PTHL) and with a control diet (CON). Cells were unstimulated (USC), stimulated with LPS and ConA (SC, 1 and 5 μg/ml, respectively) and with 4 mM of H₂O₂, and incubated for 24 h at 37°C/5% CO₂.

Figure 2

Imaging of PBMCs from the CON group (A) and from the PTHL group (B), and viability was obtained using the Image-iT® DEAD GreenTM Viability Stain. PBMCs were untreated (UNC), treated with LPS and ConA (SC, 1 and 5 μg/ml, respectively) and with 4 mM of H₂O₂, and incubated for 24 h at 37°C/5% CO₂. Then, PBMCs were stained with Image-iT® DEAD GreenTM viability stain for 30 min, fixed, permeabilized according to the manufacturer's instruction, and imaged on Life Technologies EVOS™ XL Imaging System (Milan, Italy). The nucleus was detected with Hoechst 33342 (blue) in the DAPI/Hoechst channel, and the cell membrane permeability was detected in the FITC/GFP channel (green).

Figure 3

Percentage of viability (A), apoptosis (B) and necrosis (C) of monocyte/lymphocyte stained with supravital exposure to propidium iodide (PI, 50 μg/mL). Cells (1 × 106) were pretreated with LPS and ConA (1 μg/mL and 5 μg/mL, respectively) and with H₂O₂ (4 mM) for 1h, then stained with PI for 30 min at the dark and washed before flow cytometric analysis. The supravital PI exposure on unfixed cells allowed to the simultaneous detection of living (PI negative) apoptotic (PI dim) and necrotic (PI bright) cells.

Figure 4

ROS/RNS production measured as micromolar of DCF (A), total antioxidant capacity measured as micromolar of uric acid equivalents, (B) and Antioxidant/Oxidant Balance (AOB = TAC/ROS-RNS) (C) in supernatant of PBMCs unstimulated (UNC) and stimulated with LPS and ConA (SC, 1 and 5 μg/ml, respectively) and with 4 mM of H₂O₂ for 24 h at 37°C/5% CO₂. *P < 0.05 were considered significant among feeding strategy.

Figure 5

Interleukin (IL)-10 (A), IL-12 (B), IL-8 (C), interferon-(IFN)-γ, (D), tumor necrosis factor (TNF)-α, and (E) secretion by PBMCs unstimulated (UNC) and stimulated with LPS and ConA (SC, 1 and 5 μg/ml, respectively) and with 4 mM of H₂O₂ for 24 h at 37°C/5% CO₂. *P < 0.05 were considered significant among feeding strategy.

Figure 6

Correlation analysis of panel of cytokine, oxidative stress measurements, and cell proliferation of the CON and PTHL group. *P < 0.05.