Interleukin-4 (IL-4) may regulate alternative activation of macrophage-like cells in chickens: A sequential study using novel and specific neutralizing monoclonal antibodies against chicken IL-4

In mammals, alternatively activated macrophages (AAMs) are well-recognized and are produced by stimulation with Th2 cytokines such as interleukin 4 (IL-4) and IL-13. On their mammalian counterparts, AAMs in chickens has neither been reported nor the functionality of chicken IL-4 (chIL-4) has been studied till date. Therefore, present study developed mouse monoclonal antibodies (mAbs) against chIL-4 and used these antibodies to investigate whether chIL-4 induces activation of HD11 chicken macrophage cell line. Upon characterization of mAbs using western blot, immunocytochemistry (ICC), flow cytometry and capture ELISA, activation of HD11 cells was investigated by measuring nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, arginase activity and gene expressions of iNOS, CD80 and CD86 (associated with mammalian M1 phenotype) and chemokine (C-C motif) ligand 17 (ccl17) and mannose receptor C-type1 (MRC1L-A) (as possible chicken M2 markers) in HD11 cells treated with chIL-4, lipopolysaccharide (LPS), chIL-4+LPS, chIL-4+LPS+mAbs. The newly developed mAbs displayed wide applicability in detecting chIL-4 by capture ELISA, ICC and flow cytometry with no cross reactivities with human or mouse IL-4 and other chicken cytokines. Further, our results showed that chIL-4 inhibited NO production by LPS-stimulated HD11 cells and primary monocyte/macrophage cells with reduced iNOS expression and increased arginase activity and, induced robust expression of genes associated with M2 phenotype than M1-related genes. All these effects were neutralized by anti-chIL-4 antibodies. In summary, present study results showed a possible application of anti-chIL-4 mAbs as valuable immune reagents to explore chIL-4 functionality. In addition, our results demonstrated that chIL-4 may override LPS functionality and regulates alternative activation of HD11 cells in chicken via increased arginase activity and expression of M2 associated markers and thus may indicate the possible existence of M1/M2 paradigm in chickens.