Synergistic effect of co-stimulation of membrane and endosomal TLRs on chicken innate immune responses

Toll-like receptor (TLR) ligands (TLR-Ls) are critical activators of immunity and are successfully being developed as vaccine adjuvants in both mammals and birds. In this study, we investigated the synergistic effect of co-stimulation of membrane and endosomal TLRs on the innate immune responses using chicken bone marrow-derived macrophages (BMMs), and studied the effect of age on the induction of innate responses. BMMs from 1 and 4-week-old birds were stimulated with Pam3Cys-SK4 (PCSK; TLR2), synthetic monophosphoryl lipid A (MPLA), Di[3-deoxy-d-manno-octulosonyl]-lipid A ammonium salt (KLA; TLR4), Gardiquimod, Resiquimod (R848; TLR7), CpG class B and C (TLR21). Nitric oxide (NO) production and mRNA levels of IL-1β, IL-10 and IL-12p40 showed macrophages from 4-week-old birds showed more sensitive responses compared to 1-week-old birds. The most potent TLR-Ls, PCSK, MPLA and CpG B were used to study the effect of co-stimulation on macrophages. Co-stimulation with TLR21 and TLR4 synergistically up-regulated inflammatory-related genes, as well as NO production. However, incubation of splenocytes with PCSK, MPLA and CpG B did not induce cell proliferation. Moreover, treatment with CpG B led to significant cell death.