Dataset on characterization of recombinant interleukin-23α, IL-12p40 and IL-23 complex protein, which activates JAK-STAT signaling pathway in chicken cell lines using immunocytochemical staining.

The data herein is related to the research article entitled "Functional analyses of the interaction of chicken interleukin 23 subunit p19 with IL-12 subunit p40 to form the IL-23 complex" [1] where we demonstrated that the chicken interleukin (IL)-23α, IL-12p40, and IL-23 complex regulates Th1, Th17, and Treg cytokine production through heterodimer receptors as well as a homodimer receptor consisting of IL-12Rß1 and IL-23R, and activates the JAK/STAT signaling pathways. Here, we evaluated the effects of the recombinant chicken IL-23α, IL-12p40, and IL-23 complex protein on cell proliferation and nitric oxide (NO) production in chicken macrophage (HD11) and CU91 T cell lines. In addition, the expression of IL-6, IL-17A, and interferon-γ mRNA were upregulated in vivo and in vitro. Moreover, treatment with the chicken IL-23α, IL-12p40, and IL-23 complex activated phosphorylation of tyrosine and serine residues in JAK2, STAT1, TYK2, and SOCS1 in chicken cell lines.

Functional analyses of the interaction of chicken interleukin 23 subunit p19 with IL-12 subunit p40 to form the IL-23 complex.

This study represents the first description of the cloning of chicken IL-23p19 (ChIL-23α) and the function of the IL-23 complex in birds. Multiple alignment of ChIL-23α with other known IL-23α amino acid sequences revealed regions of amino acid conservation. The homologies of ChIL-23α, IL-12p35, and similar mammalian subunits ranged between 26% and 42%. ChIL-23α consisted of four exons and three introns; similar to those in humans and mice, and limited conservation of synteny between the human and chicken genomes was observed. Using bioinformatics tools, we identified the NF-κB, C/EBPα-ß, c-Jun, c-Rel, AP-1, GATA-1, and ER promoter sites in ChIL-23α. Moreover, IL-23α mRNA was more highly expressed than IL-12p40 and IL-12p35 mRNA in several organs of chickens infected with Salmonella. In addition, ChIL-23 complex are associated with IL-23R, IL-12Rß1 receptors; activate the JAK2/TYK2, STAT1/3, SOCS1 genes, and induced proinflammatory cytokines in immune cells. Collectively, these results indicate that ChIL-23 is a member of the IL-12 family, has proinflammatory properties related to IL-23R and IL-12Rß1 receptor expression, and activates the JAK/STAT signaling pathway that results in the interaction of ChIL-23α with ChIL-12p40 to form the novel ChIL-23 complex. Our results provide novel insights into the regulation of immunity, inflammation, and immunopathology.

Analysis of JAK-STAT signaling pathway genes and their microRNAs in the intestinal mucosa of genetically disparate chicken lines induced with necrotic enteritis.

The JAK-STAT signaling pathway plays a key role in cytokine and growth factor activation and is involved in several cellular functions and diseases. The main objective of this study was to investigate the expression of candidate JAK-STAT pathway genes and their regulators and interactors in the intestinal mucosal layer of two genetically disparate chicken lines [Marek's disease (MD)-resistant line 6.3 and MD-susceptible line 7.2] induced with necrotic enteritis (NE). Through RNA-sequencing, we investigated 116 JAK-STAT signaling pathway-related genes that were significant and differentially expressed between the intestinal mucosa of the two lines compared with respective uninfected controls. About 15 JAK-STAT pathway genes were further verified by qRT-PCR, and the results were in agreement with our sequencing data. All the identified 116 genes were annotated through Gene Ontology and mapped to the KEGG chicken JAK-STAT signaling pathway. To the best of our knowledge, this is the first study to represent the transcriptional analysis of a large number of candidate genes, regulators, and potential interactors in the JAK-STAT pathway of the two chicken lines induced with NE. Several key genes of the interactome, namely, STAT1/3/4, STAT5B, JAK1-3, TYK2, AKT1/3, SOCS1-5, PIAS1/2/4, PTPN6/11, and PIK3, were determined to be differentially expressed in the two lines. Moreover, we detected 68 known miRNAs variably targeting JAK-STAT pathway genes and differentially expressed in the two lines induced with NE. The RNA-sequencing and bioinformatics analyses in this study provided an abundance of data that will be useful for future studies on JAK-STAT pathways associated with the functions of two genetically disparate chicken lines induced with NE.

Differentially expressed JAK-STAT signaling pathway genes and target microRNAs in the spleen of necrotic enteritis-afflicted chicken lines.

The JAK signal transducer and STAT signaling pathway is an important regulator of cell proliferation, differentiation, survival, motility, apoptosis, immune response, and development. In this study, we used RNA-Sequencing, qRT-PCR, and bioinformatics tools to investigate the differential expression of JAK-STAT pathway genes, their interactions, and regulators in the spleen of two genetically disparate chicken lines (Marek's disease-resistant line 6.3 and MD-susceptible line 7.2) induced necrotic enteritis (NE) disease by co-infection with Eimeria maxima and Clostridium perfringens. Using RNA-Seq analysis, we identified a total of 116 JAK-STAT pathway genes that were differentially expressed in the spleen of these chickens. All of the identified genes were analyzed through clustering, mapping to the KEGG chicken JAK-STAT pathway, and the Pathway Studio program. Of the 116 JAK-STAT pathway genes, 20 were further verified by qRT-PCR. According to the RNA-Seq results, several key genes, including STAT1-6, JAK1-3, TYK2, AKT1, AKT3, SOCS1-5, PIAS1, PIAS2, PIAS4, SHP1, SHP2, and PIK3, showed marked differential expression in the two lines, relative to their respective controls. Moreover, the RNA-Seq results of many key genes were highly correlated with the qRT-PCR results. Finally, we predicted 63 mature miRNAs that variably target JAK-STAT pathway genes and are differentially expressed in the spleen of chickens of both lines. To the best of our knowledge, this study is the first to analyze most of the genes, interactions, and regulators of the JAK-STAT pathway in the innate immune response to NE disease in chickens.

Molecular cloning of chicken interleukin-17B, which induces proinflammatory cytokines through activation of the NF-κB signaling pathway.

Interleukin (IL)-17B is a little known member of the IL-17 cytokine family, which plays an important role in immunity by regulating the expression of proinflammatory cytokines. In this study, we determined the coding sequence and biological functions of a novel chicken IL-17B (chIL-17B). The full-length chIL-17B coding sequence includes 567 nucleotides encoding 188 amino acids, which was identified in small intestinal epithelial cells. The chIL-17B protein shares 96.48% amino acid sequence identity with turkey, 92.57% with duck, and 44.92-64.06% with mammalian IL-17B proteins. ChIL-17B shares three exons and two introns with mammals, turkey, and duck. Moreover, IL-17B mRNA was more highly expressed than IL-17A mRNA in several organs of chickens infected with Salmonella and was upregulated in chicken cell lines following LPS stimulation. In addition, in chicken cell lines, chIL-17B induced the mRNA expression of several proinflammatory cytokines, including IL-1ß, IL-6, LITAF, and INF-γ, but not IL-17A, and activated MyD88, TAK1, NF-κB1, and SOCS1, which are associated with the NF-κB signaling pathway. Taken together, chicken interleukin-17B plays a critical role in host defense against the bacterial pathogens, and regulates proinflammatory cytokines by activating the NF-κB signaling pathway.

Chicken IL-26 regulates immune responses through the JAK/STAT and NF-κB signaling pathways.

Chicken interleukin 26 (ChIL-26), a member of the IL-10 family, is expressed in T cells and can induce expression of proinflammatory cytokines. We examined the response of signal transduction pathways to ChIL-26 stimulation in the chicken T (CU91), macrophage (HD11), and fibroblast (OU2) cell lines. ChIL-26 activated JAK2 and TYK2 phosphorylation, as well as activation of STAT1, STAT3, and SHP2 via tyrosine/serine residues. We also showed that ChIL-26 activates the phosphorylation of NF-κB1, TAK1, and MyD88 kinase, which are key regulators of NF-κB signaling pathways. Moreover, ChIL-26 stimulation upregulated mRNA expression of chemokines (CCL4, CCL20, and CXCL14), Th1 (IFN-α, IFN-ß, IFN-γ, IL-1ß, and IL-6), Th2 (IL-4 and IL-10), and Th17 (IL-12p40, IL-17A, and IL-17F), and the Treg cytokines (TGF-ß4); additionally, it increased Th1 and Th17 protein levels and nitric oxide production but did not affect cell proliferation. Together, these results suggest that ChIL-26-induced activation of chemokines, Th1, Th2, and, Th17, and the Treg cytokines is mediated through JAK/STAT and NF-κB signaling pathways in chicken T, macrophage, and fibroblast cell lines. These results indicate a key role for ChIL-26-induced polarization of the immune response and could reveal new therapeutic approaches for use in combination with molecules that activate T and macrophage cells via activation JAK/STAT and NF-κB signaling pathways.