Porcine Interleukin-17 and 22 Co-Expressed by Yarrowia lipolytica Enhance Immunity and Increase Protection against Bacterial Challenge in Mice and Piglets

SIMPLE SUMMARY: The abuse of antibiotics leads to the drug resistance of pathogenic bacteria, which greatly threatens the safety of humans and animals. Novel immunoregulatory approaches with safety and effectiveness that promote antimicrobial function and enhance immune response, thus, should be developed as replacements for antibiotics. A recombinant yeast that can express two functional molecules (pig IL-17 and IL-22) was constructed and designated as Po1h-pINA1297-IL-17/22. To evaluate the immunoregulatory activities of the recombinant yeast, Po1h-pINA1297-IL-17/22 was orally fed to 4-week-old female BALB/c mice and then challenged with virulent Salmonella typhimurium. Mucosal, cellular, and humoral immunity were determined by measuring immunoglobulin levels, concentration of cytokines, and the counts of T lymphocytes. The results showed that Po1h-pINA1297-IL-17/22 treated mice had stronger immune responses and resistance to bacterial infection. The subsequent oral inoculation of piglets with Po1h-pINA1297-IL-17/22 confirmed that Po1h-pINA1297-IL-17/22 obviously improved the growth performance and systemic immunity of piglets. The results above indicated that Po1h-pINA1297-IL17/22 could be developed as a promising immunopotentiator to prevent bacterial infections by promotion of immune responses. ABSTRACT: Drug resistance in economic animals to pathogens is a matter of widespread concern due to abuse of antibiotics. In order to develop a safe and economical immunopotentiator to raise the immunity and antibacterial response as a replacement for antibiotics, a recombinant yeast co-expressing pig interleukin-17 (IL-17) and IL-22 was constructed and designated as Po1h-pINA1297-IL-17/22. To evaluate the immunoregulator activities of Po1h-pINA1297-IL-17/22, two experiment groups (oral inoculation with Po1h-pINA1297 or Po1h-pINA1297-IL-17/22) and a negative control group (PBS) were set up using 4-week-old female BALB/c mice (10/group). The level of cytokines, including IL-2, IL-4, IL-10, and IFN-γ, were detected by ELISA, and the circulating CD4+ and CD8+ lymphocytes were quantified by flow cytometry. The IgG and secretory IgA (SIgA) levels in both small intestine and fecal matter were also measured by ELISA. The results indicated that the IgG antibody titer and SIgA concentration increased significantly in the Po1h-pINA1297-IL17/22 group in comparison with the controls (p < 0.05) and so did the cytokine levels in the serum (IL-2, IL-4, IL-10, and IFN-γ). In addition, CD4+ and CD8+ T cells were also obviously elevated in the Po1h-pINA1297-IL17/22 group on 35th day (p < 0.05). After challenge with pathogenic Salmonella typhimurium, the Po1h-pINA1297-IL17/22 group showed a relatively higher survival rate without obvious infectious symptoms. On the contrary, the mortality of control group reached 80% due to bacterial infection. As for the piglet experiment, 30 healthy 7-day piglets were similarly attributed into three groups. The oral inoculation of piglets with Po1h-pINA1297-IL17/22 also markedly improved the growth performance and systemic immunity (up-regulations of IL-4, IL-6, IL-15, IL-17, IL-22, and IL-23). Overall, the results indicated that Po1h-pINA1297-IL17/22 effectively promoted the humoral and cellular immunity against bacterial infection. These proved the promising potential of Po1h-pINA1297-IL-17/22 to be a potent immunopotentiator for the prevention of microbial pathogen infections.