Unveiling the immunological attributes of chicken tumor necrosis factor-α (TNF-α) using new sets of monoclonal antibodies specific for poultry TNF-α

Abstract

Abstract Tumor necrosis factor-α (TNF-α) is a type II transmembrane protein with either membrane-bound or soluble forms and is a prototypical member of TNF superfamily. TNF-α is a pleiotropic cytokine associated with the regulation of systemic inflammation and host defense. Chicken TNF-α (chTNF-α) is a long missed avian ortholog and the immunological properties of chTNF-α remains largely unknown compared to the mammalian counterpart. Here, we report on the functional characterization and immunomodulatory roles of chTNF-α using a panel of new anti-chTNF-α mouse monoclonal antibodies (mAbs) that we developed. A chTNF-α-specific antigen-capture ELISA was developed using compatible mAb partners via the screening and validation of ten different mAbs (3G11, 4C4, 4F3, 6A52, 6E6, 10E8, 12G6, 12H7, 14H2 and 15G7). Employing 3G11 and 12G6 as capture and detection antibodies, respectively, the levels of native chTNF-α in the circulation of Clostridium perfringens-, Eimeria- or dual C. perfringens/Eimeria-infected chickens were determined. Furthermore, intracellular expressions of chTNF-α in primary cells or cell lines derived from chickens were validated in immunocytochemistry and flow cytometry assays using both 3G11 and 12G6 mAbs. Importantly, both 3G11 and 12G6 neutralized chTNF-α-induced nitric oxide production in chicken HD11 cells in vitro. Collectively, our results provide a better understanding of the functional characteristics of chTNF-α and these anti-chTNF-α mAbs will serve as valuable immunological tools for the fundamental and applied studies in avian species.