Development and characterization of monoclonal antibodies for chicken interleukin-13 and their neutralizing effect in chicken primary monocytes

Authors: CHAUDHARI, ATUL - US Department Of Agriculture (USDA); KIM, WOOHYUN - US Department Of Agriculture (USDA); Lillehoj, Hyun

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/8/2020
Publication Date: 2/1/2020
Citation: Chaudharia, A., Kim, W., Lillehoj, H.S. 2020. Development and characterization of monoclonal antibodies for chicken interleukin-13 and their neutralizing effect in chicken primary monocytes. Veterinary Immunology and Immunopathology. https://doi.org/10.1016/j.psj.2019.10.023.
DOI: https://doi.org/10.1016/j.psj.2019.10.023

Interpretive Summary: Limited information about host proteins that mediate innate immunity in poultry hinders progress in poultry disease research including vaccine development. In this report ARS scientists report a new progress in understanding one of chicken cytokines that mediates innate immunity in poultry. IL-13 is a cytokine which is produced from monocytes or macrophages and IL-13 mediates anti-inflammatory effect by suppressing the production of pro-inflammatory mediators via suppression of nuclear factor 'B (NF-'B) pathway. Although the expression of chIL-13 receptors have been reported in chickens, the effect of chIL-13 on their expression has not been reported. Therefore, in the present study, ARS scientists developed antibodies which detect chicken IL-13 protein and further investigated their specificity in detecting endogenously produced IL-13. These new immune reagents will be used to detect inflammatory response in chicken serum to follow infection response of poultry in the farms and to investigate the function of innate immune system in poultry.

Technical Abstract: Compared to mammals, functionality of chicken cytokines is not well understood due to the lack of availability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its two receptors, IL-13 receptor (IL-13R)-a1 and IL-13Ra2. The functionality of chicken IL-13 (chIL-13) has not been studied thoroughly due to lack of the anti-chIL-13 antibodies. In the present study, we developed mouse monoclonal antibodies (mAbs) against chIL-13 and further investigated their specificity in detecting endogenously produced chIL-13. Upon characterization of mAbs using indirect ELISA and Western blot, the capture ELISA was developed for detecting circulating serum chIL-13 levels. The neutralizing effect was tested by measuring nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in primary monocytes stimulated with chIL-13, lipopolysaccharide (LPS), chIL-13+LPS, or chIL-13+LPS+mAbs. Also, gene expressions of chIL-13Ra1, chIL-13Ra2 and TGF-ß1 were tested in chicken monocytes treated with chIL-13, or chIL-13+mAbs. Based on the indirect ELISA, five mAbs that detected recombinant chIL-13, were identified and all of them specifically detected recombinant chIL-13 protein by Western blot. The two mAbs (#9B11 and #10A2) showed an optimal signal in a pairing assay and the capture assay based on these two mAbs successfully detected chIL-13 in the serum of Eimeria tenella-infected chickens. Further, neutralization assay revealed that chIL-13 reduced LPS-stimulated NO production and iNOS expression in monocytes/macrophage cells and the two mAbs (#9B11 and #10A2) abrogated these effects. Also, the chIL-13-induced expressions of chIL-13Ra2 and TGF-ß1 were neutralized by two mAbs. To summarize, present study showed that chIL-13 may be involved in alternative activation of primary monocytes in chickens and chIL-13 signaling may be regulated through chIL-13Ra2 binding and TGF-ß1 secretions. Importantly, the newly developed anti-chIL-13 mAbs will serve as valuable immune reagents for future studies on biological activity of chIL-13 and its receptors.