Yeojin Hong

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

&NA; 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‐&kgr;B1, TAK1, and MyD88 kinase, which are key regulators of NF‐&kgr;B signaling pathways. Moreover, ChIL‐26 stimulation upregulated mRNA expression of chemokines (CCL4, CCL20, and CXCL14), Th1 (IFN‐&agr;, IFN‐&bgr;, IFN‐&ggr;, IL‐1&bgr;, and IL‐6), Th2 (IL‐4 and IL‐10), and Th17 (IL‐12p40, IL‐17A, and IL‐17F), and the Treg cytokines (TGF‐&bgr;4); 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‐&kgr;B 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‐&kgr;B signaling pathways. HighlightsChicken interleukin‐26 (ChIL‐26), a member of the IL‐10 family.ChIL‐26 activates JAK‐STAT and NF‐&kgr;B1 signaling pathway.ChIL‐26‐induced activation of the chemokines, Th1, Th2, Th17, and Treg cytokines.ChIL‐26 increased Th1 and Th17 protein levels and nitric oxide production.

Characterization and functional analyses of a novel chicken CD8α variant X1 (CD8α1).

We provide the first description of cloning and of structural and functional analysis of a novel variant in the chicken cluster of differentiation 8 alpha (CD8a) family, termed the CD8α X1 (CD8α1) gene. Multiple alignments of CD8α1 with known CD8α and CD8β sequences of other species revealed relatively low conservation of AA residues involved in the specific and unique structural domains among CD8α genes. For example, cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, the O-linked glycosylation sites (XPXX motif) are not found in the chicken CD8α1 sequence, and the A β strand and complementarity-determining region 1 and 2 sequences are poorly conserved between chicken CD8α1 and avian CD8α. Furthermore, the alignment showed that the transmembrane regions show relatively high sequence similarity, whereas the cytoplasmic regions show relatively low similarity, indicating poor conservation. Moreover, the motif (CXCP) that is thought to be responsible for binding the p56 lymphocyte cell kinase subunit (p56) is missing in the CD8α1 sequence. The chicken CD8α1 genomic structure is similar to that of chicken CD8α, but their protein structures differ. Phylogenetic analysis showed that chicken CD8α1 grouped with known avian CD8α sequences but was somewhat distantly related to the CD8α molecules of other species. Moreover, we analyzed the signal transduction and cytokine response to CD8α1 treatment to determine the specific biological functions of chicken CD8α1 in immune cells. The results showed that chicken CD8α1 is a key regulator of the expression of genes that are associated and cooperate with transcription factors in the major histocompatibility complex class I and II promoter regions and activates Janus kinase (JAK) 1/2, signal transducer and activator of transcription (STAT), and suppressor of cytokine signaling (SOCS) 1 signaling-related genes. Immune cells that express functional CD8α1 induce proinflammatory cytokines as well as innate immune responses. Therefore, our data indicate that CD8α1 may have immunoregulatory activity by regulating the expression of proinflammatory or anti-inflammatory cytokines via its effect on immune cells.

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

HighlightsCloning chicken interleukin 23 subunit p19 and IL‐23 complex in chicken.Chicken IL‐23 has proinflammatory properties related to IL‐23R and IL‐12R&bgr;1 receptor expression.Chicken IL‐23p19, IL‐12p40 and IL‐12p35 mRNA expression in various tissues of chickens infected with Salmonella Enteritidis.Chicken IL‐23 activates the JAK/STAT signaling pathway and induced proinflammatory cytokines in immune cells. ABSTRACT This study represents the first description of the cloning of chicken IL‐23p19 (ChIL‐23&agr;) and the function of the IL‐23 complex in birds. Multiple alignment of ChIL‐23&agr; with other known IL‐23&agr; amino acid sequences revealed regions of amino acid conservation. The homologies of ChIL‐23&agr;, IL‐12p35, and similar mammalian subunits ranged between 26% and 42%. ChIL‐23&agr; 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‐&kgr;B, C/EBP&agr;‐&bgr;, c‐Jun, c‐Rel, AP‐1, GATA‐1, and ER promoter sites in ChIL‐23&agr;. Moreover, IL‐23&agr; 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‐12R&bgr;1 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‐12R&bgr;1 receptor expression, and activates the JAK/STAT signaling pathway that results in the interaction of ChIL‐23&agr; with ChIL‐12p40 to form the novel ChIL‐23 complex. Our results provide novel insights into the regulation of immunity, inflammation, and immunopathology.

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-12Rβ1 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.

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 [Mareks 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 (Mareks 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

&NA; 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‐1&bgr;, IL‐6, LITAF, and INF‐&ggr;, but not IL‐17A, and activated MyD88, TAK1, NF‐&kgr;B1, and SOCS1, which are associated with the NF‐&kgr;B 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‐&kgr;B signaling pathway. HighlightsA novel chicken interleukin‐17B (chIL‐17B) was cloned.Chicken IL‐17B expression was upregulated in response to bacterial infection.Chicken IL‐17B functions through the NF‐&kgr;B signaling pathway.ChIL‐17B overexpression activates proinflammatory cytokines in HD11, OU2, and CU91 cells.

Effect of dietary antimicrobials on immune status in broiler chickens.

This study evaluated the effects of dietary anticoccidial drugs plus antibiotic growth promoters (AGPs) on parameters of immunity in commercial broiler chickens. Day-old chicks were raised on used litter from a farm with endemic gangrenous dermatitis to simulate natural pathogen exposure and provided with diets containing decoquinate (DECX) or monensin (COBN) as anticoccidials plus bacitracin methylene disalicylate and roxarsone as AGPs. As a negative control, the chickens were fed with a non-supplemented diet. Immune parameters examined were concanavalin A (ConA)-stimulated spleen cell proliferation, intestine intraepithelial lymphocyte (IEL) and spleen cell subpopulations, and cytokine/chemokine mRNA levels in IELs and spleen cells. ConA-induced proliferation was decreased at 14 d post-hatch in DECX-treated chickens, and increased at 25 and 43 d in COBN-treated animals, compared with untreated controls. In DECX-treated birds, increased percentages of MHC2+ and CD4+ IELS were detected at 14 d, but decreased percentages of these cells were seen at 43 d, compared with untreated controls, while increased TCR2+ IELs were evident at the latter time. Dietary COBN was associated with decreased fractions of MHC2+ and CD4+ IELs and reduced percentages of MHC2+, BU1+, and TCR1+ spleen cells compared with controls. The levels of transcripts for interleukin-4 (IL-4), IL-6, IL-17F, IL-13, CXCLi2, interferon-γ (IFN-γ), and transforming growth factorβ4 were elevated in IELs, and those for IL-13, IL-17D, CXCLi2, and IFN-γ were increased in spleen cells, of DECX- and/or COBN-treated chickens compared with untreated controls. By contrast, IL-2 and IL-12 mRNAs in IELs, and IL-4, IL-12, and IL-17F transcripts in spleen cells, were decreased in DECX- and/or COBN-treated chickens compared with controls. These results suggest that DECX or COBN, in combination with bacitracin and roxarsone, modulate the development of the chicken post-hatch immune system.

Comparing the immune responses of two genetically B-complex disparate Fayoumi chicken lines to Eimeria tenella

Abstract The present study was conducted to compare the susceptibility of congenic Fayoumi lines to Eimeria tenella infection and to assess genetic differences in Eimeria egression. Chickens were orally inoculated with 5 × 104 sporulated E. tenella oocysts and challenged with 5 × 106 oocysts on the 10th day after the primary infection. The Fayoumi M5.1 line exhibited higher levels of body weight gain, less oocyst shedding and higher percentages of B and CD4+/CD8+ T cells than the M15.2 chickens. These results demonstrate that M5.1 line is more resistant to E. tenella infection than M15.2 line. Furthermore, the percentage of sporozoite egress from peripheral blood mononuclear cells (PBMCs) was higher in the M5.1 line. The results of this study suggest that enhanced resistance of Fayoumi M5.1 to E. tenella infection may involve heightened cell-mediated and adaptive immunity, resulting in reduced intracellular development of Eimeria parasites.

Leukocyte Immunoglobulin-Like Receptors A2 and A6 are Expressed in Avian Macrophages and Modulate Cytokine Production by Activating Multiple Signaling Pathways

The activating leukocyte immunoglobulin-like receptors (LILRAs) play an important role in innate immunity. However, most of the LILRA members have not been characterized in avian species including chickens. The present study is the first attempt at cloning, structural analysis and functional characterization of two LILRAs (LILRA2 and LILRA6) in chickens. Multiple sequence alignments and construction of a phylogenetic tree of chicken LILRA2 and LILRA6 with mammalian proteins revealed high conservation between chicken LILRA2 and LILRA6 and a close relationship between the chicken and mammalian proteins. The mRNA expression of LILRA2 and LILRA6 was high in chicken HD11 macrophages and the small intestine compared to that in several other tissues and cells tested. To examine the function of LILRA2 and LILRA6 in chicken immunity, LILRA2 and LILRA6 were transfected into HD11 cells. Our findings indicated that LILRA2 and LILRA6 are associated with the phosphorylation of Src kinases and SHP2, which play a regulatory role in immune functions. Moreover, LILRA6 associated with and activated MHC class I, β2-microglobulin and induced the expression of transporters associated with antigen processing but LILRA2 did not. Furthermore, both LILRA2 and LILRA6 activated JAK-STAT, NF-κB, PI3K/AKT and ERK1/2 MAPK signaling pathways and induced Th1-, Th2- and Th17-type cytokines and Toll-like receptors. Collectively, this study indicates that LILRA2 and LILRA6 are essential for macrophage-mediated immune responses and they have the potential to complement the innate and adaptive immune system against pathogens.