Cloning and functional characterization of chicken interleukin-17D

Abstract

The chicken interleukin-17D was cloned from a testis cDNA library prepared from the Korean native chicken. The full-length chicken IL-17D (chIL-17D) cDNA consisted of a 348 nucleotide sequence encoding an open reading frame of 116 amino acids with a predicted molecular mass of 13.3 kDa. Comparison of the deduced amino acid sequence of chIL-17D with homologous proteins from human, mouse and opossum revealed 64%, 53% and 76% identity, respectively, including six conserved cysteine residues present in the mammalian polypeptides. The chIL-17D gene transcript was expressed in a wide range of tissues, and highest levels were in pancreas, thymus and lung. Following Eimeria maxima infection, levels of the chIL-17D mRNA were up-regulated in the intestinal jejunum, bursa, lung, and spleen but decreased in the thymus. Infected chickens also expressed greater levels of chIL-17D mRNA in CD4⁺, CD8⁺ and TCR1⁺ intestinal intraepithelial lymphocytes while decreased expression was seen in TCR2⁺ cells. Treatment of CHCC-OU2 fibroblasts with chIL-17D recombinant protein induced the expression of IL-6 and IL-8. Collectively, these results suggest that chL-17D has structural and functional similarities to mammalian IL-17Ds and that it plays an important role in local gut innate immune responses during experimental coccidiosis.

Introduction

Interleukin-17 (IL-17) was originally described as a cytokine secreted exclusively by activated memory T cells that induced fibroblasts to secrete other cytokines involved in proinflammatory or hematopoietic processes, such as IL-6, IL-8 and granulocyte-colony stimulating factor (G-CSF) (Yao et al., 1995a, Yao et al., 1995b, Broxmeyer, 1996, Fossiez et al., 1996). With the completion of human genome sequences and the availability of public genomic databases, a number of homologous proteins comprising an IL-17 family have been identified, including IL-17A (original IL-17), -17B, -17C, -17D, -17E and -17F (Li et al., 2000, Lee et al., 2001, Starnes et al., 2001, Starnes et al., 2002). The IL-17 families, as well as their cognate receptors, have no sequence similarity to any other known cytokines or receptors and thus appear to represent a distinct ligand-receptor signaling system (Yao et al., 1995a, Kawaguchi et al., 2004). Three members of this family, IL-17A, -17E (IL-25), and -17F, have been the most characterized and have been shown to be proinflammatory in nature (Kawaguchi et al., 2004).

Functional studies of IL-17 cytokines have described a broad range of effects on cells and tissues. IL-17A was expressed by activated CD4⁺ and CD8⁺ T cells, but not resting T cells (Yao et al., 1995a), neutrophils and eosinophils (Teunissen et al., 1998, Chakir et al., 2003, Ferretti et al., 2003). Similarly, IL-17F was produced primarily by activated T cells and monocytes and stimulated the production of IL-6, IL-8 and G-CSF (Hymowitz et al., 2001, Starnes et al., 2001). CD4⁺ helper T cells that produce IL-17A and IL-17F are referred to as T_H-17 cells. T_H-17 cells also produce IL-22, IL-26, interferon-γ, chemokine CCL20 and transcription factor RORγt, but their function in immunity remains to be fully determined (Wilson et al., 2007). IL-17B mRNA was identified in adult pancreas, small intestine and stomach, whereas IL-17C mRNA was not detected by blot hybridization in several adult tissues examined (Li et al., 2000). Neither of these transcripts was found in activated T cells. The IL-17D gene appeared to be most homologous to IL-17B and was preferentially expressed in resting CD4⁺ T cells, skeletal muscle, brain, pancreas, heart, lung and adipose tissue (Starnes et al., 2002). IL-17E was restricted to T_H2 cells and over-expression of this cytokine resulted in the production of IL-4, IL-5, IL-13, and IgE during airway eosinophilia, suggesting that it might be involved in the allergic response (Hurst et al., 2002). Clinically, IL-17 family members have been linked to many disease processes such as rheumatoid arthritis (Kotake et al., 1999, Bush et al., 2001, Chabaud et al., 2001), chronic obstructive pulmonary disease (Linden et al., 2000), psoriasis (Teunissen et al., 1998), and allograft rejection (Van Kooten et al., 1998, Antonysamy et al., 1999).

Nucleotide sequences homologous to human IL-17A, -17B, -17D and -17F have been identified in the chicken genome (Min and Lillehoj, 2002, Kaiser et al., 2005). Among these, only IL-17A has been cloned and characterized (Min and Lillehoj, 2002). In that study, a cDNA encoding chIL-17A was isolated from an expressed sequence tag (EST) library prepared from intestinal intraepithelial lymphocytes (IELs) of chickens infected with Eimeria parasites, the etiologic agent of avian coccidiosis. ChIL-17A mRNA was expressed by activated T cells and its recombinant protein induced chicken embryonic fibroblasts to secrete IL-6. In the current study, we have extended these findings by the identification and characterization of chIL-17D from a testis cDNA library.