Immunological characterization of chicken tumor necrosis factor-a (TNF-a) using new sets of monoclonal antibodies specific for poultry TNF

Authors: LU, MINGMIN - US Department Of Agriculture (USDA); LEE, YOUNGSUB - US Department Of Agriculture (USDA); Li, Charles; Lillehoj, Hyun

Submitted to: Developmental and Comparative Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/10/2022
Publication Date: 2/12/2022
Citation: Lu, M., Lee, Y., Li, C.Z., Lillehoj, H.S. 2022. Immunological characterization of chicken tumor necrosis factor-a (TNF-a) using new sets of monoclonal antibodies specific for poultry TNF. Developmental and Comparative Immunology. https://doi.org/10.1016/j.dci.2022.104374.
DOI: https://doi.org/10.1016/j.dci.2022.104374

Interpretive Summary: Lack of information on the nature and the biological function of chicken immune mediators hinders the development of immunological control strategies against poultry diseases. In this paper, ARS scientists investigated chicken tumor necrosis factor that plays an important function in the regulation of numerous biological processes in the context of immune function and disease. Because TNF is secreted by activated chicken lymphocytes and plays a major role in inflammatory responses, ability to measure the level of TNF production in infectious diseases will enhance our understanding of TNF-mediated immunopathology. Because of this, a panel of viable immune reagents and bioassays specific to chicken TNF-a were developed and their specificities were validated using serum samples from Eimeria-infected chickens. Interestingly, augmented serum levels of TNF-a levels were detected at varying time points during Eimeria infection and there was a correlation between intestinal damage and the level of serum TNF-a secretion suggesting significant damage to the intestinal epithelial cells induced by E. tenella infection. These valuable immune reagents will inform on the pleiotropic roles of chicken TNF-a in disease pathogenesis and will facilitate the design of disease control strategies.

Technical Abstract: Tumor necrosis factor-a (TNF-a) is a type II transmembrane protein with either membrane-bound or soluble forms and is a prototypical member of the TNF superfamily. TNF-a is a pleiotropic cytokine associated with the regulation of systemic inflammation and host defense. Chicken TNF-a (chTNF-a) is a long-missed avian ortholog, and its immunological properties remain largely unknown compared to those of its mammalian counterparts. Here, we report the functional characterization and immunomodulatory properties of chTNF-a using a panel of newly developed anti-chTNF-a mouse monoclonal antibodies (mAbs). Using anti-chTNF-a mAbs, we determined the tissue expression of chTNF-a in lymphoid and non-lymphoid organs. A chTNF-a-specific antigen-capture sandwich ELISA was developed using compatible mAb partners by screening and validation of ten different mAbs. Employing 3G11 and 12G6 as capture and detection antibodies, respectively, the levels of native chTNF-a in the circulation of Clostridium perfringens, Eimeria, or dual C. perfringens/Eimeria-infected chickens were determined. Furthermore, intracellular expression of chTNF-a in primary immune cells or cell lines derived from chickens was validated by immunocytochemistry and flow cytometry assays using both 3G11 and 12G6 mAbs. Notably, both 3G11 and 12G6 neutralized chTNF-a-induced nitric oxide production in chicken HD11 cells in vitro. Collectively, our results enhance our understanding of the functional characteristics of chTNF-a, and these anti-chTNF-a mAbs will serve as valuable immune reagents to inform on inflammatory responses and disease pathogenesis in the fundamental and applied studies of avian species.