Identification of duck liver-expressed antimicrobial peptide 2 and characterization of its bactericidal activity.

OBJECTIVE: This study was conducted to identify duck liver-expressed antimicrobial peptide 2 (LEAP-2) and demonstrate its antimicrobial activity against various pathogens. METHODS: Tissue samples were collected from 6 to 8-week-old Pekin ducks (Anas platyrhynchos domesticus), total RNA was extracted, and cDNA was synthesized. To confirm the duck LEAP-2 transcript expression levels, quantitative real-time polymerase chain reaction was conducted. Two kinds of peptides (a linear peptide and a disulfide-type peptide) were synthesized to compare the antimicrobial activity. Then, antimicrobial activity assay and fluorescence microscopic analysis were conducted to demonstrate duck LEAP-2 bactericidal activity. RESULTS: The duck LEAP-2 peptide sequence showed high identity with those of other avian species (>85%), as well as more than 55% of identity with mammalian sequences. LEAP-2 mRNA was highly expressed in the liver with duodenum next, and then followed by lung, spleen, bursa and jejunum and was the lowest in the muscle. Both of LEAP-2 peptides efficiently killed bacteria, although the disulfide-type LEAP-2 showed more powerful bactericidal activity. Also, gram-positive bacteria was more susceptible to duck LEAP-2 than gram-negative bacteria. Using microscopy, we confirmed that LEAP-2 peptides could kill bacteria by disrupting the bacterial cell envelope. CONCLUSION: Duck LEAP-2 showed its antimicrobial activity against both gram-positive and gram-negative bacteria. Disulfide bonds were important for the powerful killing effect by disrupting the bacterial cell envelope. Therefore, duck LEAP-2 can be used for effective antibiotics alternatives.

Chicken novel leukocyte immunoglobulin-like receptor subfamilies B1 and B3 are transcriptional regulators of major histocompatibility complex class I genes and signaling pathways.

OBJECTIVE: The inhibitory leukocyte immunoglobulin-like receptors (LILRBs) play an important role in innate immunity. The present study represents the first description of the cloning and structural and functional analysis of LILRB1 and LILRB3 isolated from two genetically disparate chicken lines. METHODS: Chicken LILRB1-3 genes were identified by bioinformatics approach. Expression studies were performed by transfection, quantitative polymerase chain reaction. Signal transduction was analyzed by western blots, immunoprecipitation and flow cytometric. Cytokine levels were determined by enzyme-linked immunosorbent assay. RESULTS: Amino acid homology and phylogenetic analyses showed that the homologies of LILRB1 and LILRB3 in the chicken line 6.3 to those proteins in the chicken line 7.2 ranged between 97%-99%, while homologies between chicken and mammal proteins ranged between 13%-19%, and 13%-69%, respectively. Our findings indicate that LILRB1 and LILRB3 subdivided into two groups based on the immunoreceptor tyrosine-based inhibitory motifs (ITIM) present in the transmembrane domain. Chicken line 6.3 has two ITIM motifs of the sequence LxYxxL and SxYxxV while line 7.2 has two ITIM motifs of the sequences LxYxxL and LxYxxV. These motifs bind to SHP-2 (protein tyrosine phosphatase, non-receptor type 11) that plays a regulatory role in immune functions. Moreover, our data indicate that LILRB1 and LILRB3 associated with and activated major histocompatibility complex (MHC) class I and ß2-microglobulin and induced the expression of transporters associated with antigen processing, which are essential for MHC class I antigen presentation. This suggests that LILRB1 and LILRB3 are transcriptional regulators, modulating the expression of components in the MHC class I pathway and thereby regulating immune responses. Furthermore, LILRB1 and LILRB3 activated Janus kinase2/tyrosine kinase 2 (JAK2/TYK2); signal transducer and activator of transcription1/3 (STAT1/3), and suppressor of cytokine signaling 1 genes expressed in Macrophage (HD11) cells, which induced Th1, Th2, and Th17 cytokines. CONCLUSION: These data indicate that LILRB1 and LILRB3 are innate immune receptors associated with SHP-2, MHC class I, ß2-microglobulin, and they activate the Janus kinase/signal transducer and activator of transcription signaling pathway. Thus, our study provides novel insights into the regulation of immunity and immunopathology.

Interleukin-34 Regulates Th1 and Th17 Cytokine Production by Activating Multiple Signaling Pathways through CSF-1R in Chicken Cell Lines.

Interleukin-34 (IL-34) is a newly recognized cytokine with functions similar to macrophage colony-stimulating factor 1. It is expressed in macrophages and fibroblasts, where it induces cytokine production; however, the mechanism of chicken IL-34 (chIL-34) signaling has not been identified to date. The aim of this study was to analyze the signal transduction pathways and specific biological functions associated with chIL-34 in chicken macrophage (HD11) and fibroblast (OU2) cell lines. We found that IL-34 is a functional ligand for the colony-stimulating factor receptor (CSF-1R) in chicken cell lines. Treatment with chIL-34 increased the expression of Th1 and Th17 cytokines through phosphorylation of tyrosine and serine residues in Janus kinase (JAK) 2, tyrosine kinase 2 (TYK2), signal transducer and activator of transcription (STAT) 1, STAT3, and Src homology 2-containing tyrosine phosphatase 2 (SHP-2), which also led to phosphorylation of NF-κB1, p-mitogen-activated protein kinase kinase kinase 7 (TAK1), MyD88, suppressor of cytokine signaling 1 (SOCS1), and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Taken together, these results suggest that chIL-34 functions by binding to CSF-1R and activating the JAK/STAT, nuclear factor κ B (NF-κB), and mitogen-activated protein kinase signaling pathways; these signaling events regulate cytokine expression and suggest roles for chIL-34 in innate and adaptive immunity.

Identification and functional characterization, including cytokine production modulation, of the novel chicken Interleukin-11.

Interleukin (IL)-11 plays an important role in the immune system. However, IL-11 has not yet been characterized in avian species, including chickens. This study is the first to clone and functionally characterize chicken IL-11 (chIL-11). Multiple alignments and phylogenetic tree comparisons of chIL-11 with IL-11 proteins from other species revealed high levels of conservation and a close relationship between chicken and Japanese quail IL-11. Our results demonstrate that chIL-11 was a functional ligand of IL-11RA and IL-6ST in chicken HD11 and OU2 cell lines, as well as activated and regulated JAK-STAT, NF-κB, PI3K/AKT, and MAPK signaling pathways in chicken cell lines. In addition, chIL-11 inhibited nitric oxide production, affected proliferation of both tested cell lines, inhibited Type 1 and 17 T helper (Th) cytokine and IL-26, IL-12, and IL-17A-induced interferon-γ production, and enhanced Th2 cytokine (IL-4 and IL-10) production. Taken together, functional analysis of chIL-11 revealed it bound to IL-11RA and IL-6ST and activated the JAK-STAT, NF-κB, and MAPK signaling pathways, which resulted in modulation of Th1/Th17 and Th2 cytokine production in chicken HD11 and OU2 cell lines. Overall, this indicates chIL-11 has a role in both the innate and adaptive immune system.

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.

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.