Effects of different TLR ligands on the expression of proinflammatory cytokines and avian β-defensins in the uterine and vaginal tissues of laying hens

The immune response in the lower part of the hen oviduct is of crucial importance to protect the oviductal tissues from infection by microorganisms colonizing the cloaca. The aim of this study was to examine whether different TLRs can recognize their ligands to induce expression of proinflammatory cytokines and avian β-defensins (AvBDs) in the uterus and vagina of laying hens. The mucosal tissues of the uterus and vagina were collected, cultured in TCM-199 medium and stimulated with or without different ligands of TLRs, namely Pam3CSK4 (TLR2), poly I:C (TLR3), flagellin (TLR5), R848 (TLR7), and CpG-ODN (TLR21) and incubated for 3 h. The expression of IL1B in the uterus and vagina was upregulated by all TLR ligands tested. The expression of IL6 in the uterus and vagina was upregulated by poly I:C and CpG-ODN, and it was also upregulated by Pam3CSK4 in the uterus and by R848 in the vagina. The expression of AvBD10 was upregulated by poly I:C in the uterus and by flagellin in the vagina. On the other hand, the AvBD10 expression was downregulated by CpG-ODN in the uterus and by R848 in the vagina, whereas its expression was not affected by Pam3CSK4 in both tissues. The expression of AvBD12 in the uterus and vagina was not affected by any TLR ligands except for CpG-ODN, which downregulated its expression in the vagina. These results suggest that TLR2, 3, 5, 7, and 21 in the uterine and vaginal tissues are functionally active in inducing proinflammatory cytokines in response to their specific ligands, although the effect on the expression of AvBDs is limited. Proinflammatory cytokines induced by interaction of TLRs with their ligands may play roles in the defense against infectious microorganisms.