"Interleukin-4 (IL-4) regulates alternative activation of macrophages in chickens: a sequential study using novel and specific neutralizing monoclonal antibodies against chicken IL-4"

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

Submitted to: Developmental and Comparative Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2018
Publication Date: 10/29/2018
Citation: Chaudhari, A., Kim, W., Lillehoj, H.S. 2018. "Interleukin-4 (IL-4) regulates alternative activation of macrophages in chickens: a sequential study using novel and specific neutralizing monoclonal antibodies against chicken IL-4". Developmental and Comparative Immunology. 205:72-82.

Interpretive Summary: Limited information on avian immune cells and their function in infectious diseases hinders rapid progress in vaccine development against many important infectious diseases of poultry. In this paper, ARS scientists discuss main characteristics of macrophages, critical cells which play important function in initiating host immune response against pathogens. Host macrophages are the integral part of innate immune responses in mammals as well as in chickens, and they can sense wide variety of stimuli including microbial antigens, apoptotic or cancer cells to facilitate their removal. Activation of macrophages (AAMs), a well-recognized concept, is regulated by Th2 cytokines such as interleukin 4 (IL-4) and IL-13 in mammals. IL-4 is a predominant Th2 cytokine mainly produced by activated T cells, basophils and eosinophils in allergic or parasitic infections but its function in chickens remain unknown. In this paper, chicken IL-4 (chIL-4) was used to investigate its function using new mouse antibodies which were developed at ARS. The newly developed mAbs displayed specific binding with the recombinant chIL-4 with no cross reactivity with human or mouse IL-4 and other cytokines in chickens and indicated a wide applicability in detecting endogenously produced chIL-4. Furthermore, chIL-4 inhibited the NO production by LPS-stimulated HD11 cells. In summary, the results of this paper demonstrated that chIL-4 regulate chicken macrophages via increased arginase activity and expression of M2 markers. Furthermore, the newly developed anti-chIL-4 mAbs may serve as valuable immune reagents to explore basic and applied avian immunology.

Technical Abstract: In mammals, alternative activation of macrophages (AAMs) is well-recognized and is regulated by Th2 cytokines such as interleukin 4 (IL-4) and IL-13. IL-4 regulates growth and differentiation of B Lymphocytes and serves as a key regulator for M2 macrophage phenotype. On their mammalian counterparts, AAMs in chicken macrophages has neither been reported nor the functionality of chicken IL-4 (chIL-4) has been studied till date. Therefore, present study investigated whether chIL-4 induce AAMs in HD11 chicken macrophage cell line using newly developed mouse monoclonal antibodies (mAbs) against chIL-4. The mAbs were first characterized for its specificity and ability to detect chIL-4 by western blot, immunocytochemistry (ICC), flow cytometry and capture ELISA. Upon characterization, a sequential study was carried out to investigate AAMs by measuring the nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression and arginase activity in HD11 cells upon stimulation with chIL-4, lipopolysaccharide (LPS), chIL-4+LPS, chIL-4+LPS+mAbs. Also, the expression of iNOS gene as a M1 marker and expressions of chemokine (C-C motif) ligand 17 (ccl17) and mannose receptor C-type1 (MRC1) genes as M2 marker, were investigated in a time dependent manner. The newly developed mAbs displayed specific binding with the recombinant chIL-4 with no cross reactivity with human or mouse IL-4 and other cytokines in chickens and indicated a wide applicability in detecting endogenously produced chIL-4 by capture ELISA, ICC and flow cytometry. More importantly, our results showed that chIL-4 inhibited the NO production by LPS-stimulated HD11 cells with reduced iNOS expression and increased arginase activity. Additionally, the sequential monitoring of M1 and M2 markers showed that chIL-4 induced robust expression of M2 markers than M1 markers in HD11 cells. All these effects were neutralized by the anti-chIL-4 antibodies. In summary, we clearly demonstrated that chIL-4 regulate AAMs in chicken macrophages via increased arginase activity and expression of M2 markers. Our results also indicate that chIL-4 overrides the LPS functionality in chIL-4+LPS stimulated HD11 cells with an evident M2 polarization. To our knowledge, present study is the first report on M1/M2 paradigm in non-mammalian macrophages. Additionally, the newly developed anti-chIL-4 mAbs may serve as valuable immune reagents to explore basic and applied avian immunology.