TLR-Mediated Cytokine Gene Expression in Chicken Peripheral Blood Mononuclear Cells as a Measure to Characterize Immunobiotics

Abstract

Immunobiotics are probiotics that promote intestinal health by modulating immune responses. Immunobiotics are recognized by Toll-like receptors (TLRs) and activate cytokine gene expression. This study aimed to characterize cytokine gene expression in the chicken peripheral blood mononuclear cells (PBMC) stimulated with purified TLR ligands and live probiotics. PBMC were isolated from the whole blood. PBMC were stimulated with: lipopolysaccharide (LPS), CpG ODN, Pam3CSK4, Zymosan, galactooligosaccharides (GOS), <i>Lactococcus</i><i>lactis</i> subsp. <i>cremoris</i> (<i>L. lactis</i>), and <i>Saccharomyces cerevisiae</i> at 42.5 °C and 5% CO₂ for 3 h, 6 h, and 9 h. After each time-point, PBMC were harvested for RNA isolation. Relative gene expression was analyzed with RT-qPCR for cytokine genes (<i>IL-1β</i>, <i>IL-2</i>, <i>IL-3</i>, <i>IL-4</i>, <i>IL-6</i>, <i>IL-8</i>, <i>IL-10</i>, <i>IL-12p40</i>, and <i>IFN-ɣ</i>) and reference genes (<i>ACTB</i> and <i>G6PDH</i>). Genes were clustered into pro-inflammatory genes, Th1/Th2 genes, and Th1-regulators. The gene expression differed between treatments in <i>IL1-β</i>, <i>IL-6</i>, <i>IL-8</i>, <i>IL-10</i>, and <i>IL-12p40</i> (<i>p</i> < 0.001). The genes <i>IL-1β</i>, <i>IL-6</i>, and <i>IL-8</i> had the highest fold change of mRNA expression at 3 h in response to TLR ligands. <i>L. lactis</i> up-regulated the pro-inflammatory genes at the 6 h time-point. <i>L. lactis</i> did not activate the anti-inflammatory <i>IL-10</i> gene, but activated <i>IL-12p40</i> at 6 h. Hereby, <i>L. lactis</i> was proven to exert immunostimulatory properties in PBMC.