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First Characterization of Chicken Interleukin-9.

ABSTRACT: Interleukin-9 (IL-9) is a pleiotropic cytokine that acts on a variety of cells and tissues, and plays roles in inflammation and infection as well as tumor immunity. While mammalian IL-9s have been widely investigated, avian IL-9 has not yet been identified and characterized. In this study, we cloned chicken IL-9 (chIL-9) and performed a phylogenetic analysis, examined its tissue distribution, characterized the biological functions of recombinant chIL-9 (rchIL-9) and the expression form of natural chIL-9. Phylogenetic analysis showed that chIL-9 has less than 30% amino acid identity with mammalian IL-9s. The chIL-9 mRNA can be abundantly detected only in the testis and thymus, and are significantly up-regulated in peripheral blood mononuclear cells (PBMCs) upon mitogen stimulation. The rchIL-9 was produced by prokaryotic and eukaryotic expression systems and showed biological activity in activating monocytes/macrophages to produce inflammatory cytokines and promoting the proliferation of CD3⁺ T cells. In addition, four monoclonal antibodies (mAbs) and rabbit polyclonal antibody (pAb) against rchIL-9 were generated. Using anti-chIL-9 mAbs and pAb, natural chIL-9 expressed by the activated PBMCs of chickens with a molecular weight of 25kD was identified by Western-blotting. Collectively, our study reveals for the first time the presence of functional IL-9 in birds and lays the ground for further investigating the roles of chIL-9 in diseases and immunity.

SUBMITTER: He S

PROVIDER: S-EPMC9252340 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Molecular and functional characterization of chicken interleukin 1 receptor 2 (chIL-1R2).

Project description:Interleukin-1 receptor type 2 (IL1R2) is a decoy receptor for exogenous IL-1. However, its functional role in chicken immunity is poorly understood. Herein, chicken IL-1R2 (chIL-1R2) was identified and functionally characterized in vivo and in vitro. The chIL-1R2 coding sequence includes 1,236 nucleotides encoding 412 amino acids, is highly conserved, and has a close relationship with its mammalian counterpart. Its extracellular region has three Ig-like domains but no TIR domain for intracellular signaling. Using ELISA, the recombinant chIL-1R2 protein was demonstrated to specifically bind to the chicken IL-1β. ChIL-1R2 mRNA expression was shown to be higher in the spleen, lung, kidney, small intestine, and liver. The expression of chIL-1R2 and chIL-1R1 was significantly upregulated in DF-1 cells treated with poly (I:C), but significantly downregulated in the presence of NF-κB, JNK, and MEK inhibitors, indicating that the NF-κB, JNK, and MEK signaling pathways are required for the transcriptional regulation of chIL-1R1 and chIL-1R2 expression. It is worth noting that while the p30 MAPK pathway was required for chIL-1R1 expression, it was not required for chIL-1R2 expression. Furthermore, chIL-1R2 expression increased as early as day 1, and then significantly decreased until day 3, while chIL-1R1 was dramatically upregulated in four organs of chickens infected with the highly pathogenic avian influenza virus (HPAIV). These findings indicate that chIL-1R1 and chIL-1R2 may play a crucial in innate and adaptive immune responses toward HPAIV infection. In summary the present study showed that chIL-1R2 binds to chIL-1β antibody. ChIL-1R2 expression can be induced by a viral infection, and may be regulated through NF-κB/JNK/MEK-mediated signaling pathways.

| S-EPMC9811199 | biostudies-literature

Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes.

Project description:Compared with mammals, the functionality of chicken cytokines is not well understood because of the unavailability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its 2 receptors, IL-13 receptor (IL-13R)-α1 and IL-13Rα2. In the present study, we developed mouse monoclonal antibodies (mAb) against chIL-13 and further investigated their specificity in detecting endogenously produced chIL-13. Upon characterization of mAb using indirect ELISA and Western blot, the capture ELISA was developed for detecting chIL-13. Neutralizing effects were tested by measuring nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in primary chicken monocytes stimulated with chIL-13, lipopolysaccharide (LPS), chIL-13+LPS, or chIL-13+LPS+mAb. In addition, gene expression of chIL-13Rα1, chIL-13Rα2, and TGF-β1 was tested in chicken monocytes treated with chIL-13 or chIL-13+mAb. Based on indirect ELISA, 5 mAb that detected recombinant chIL-13 were identified, and all of them specifically detected recombinant chIL-13 protein by Western blotting. An optimal signal was obtained with 2 mAb (#9B11 and #10A2) in a pairing assay, and these 2 mAb were used in a capture assay. A neutralization assay further revealed that chIL-13 reduced LPS-stimulated NO production and iNOS expression in monocytes and macrophage cells, and the 2 mAb (#9B11 and #10A2) abrogated these effects. In addition, chIL-13-induced expressions of chIL-13Rα2 and TGF-β1 were neutralized by the 2 mAb. In summary, the present study showed that chIL-13 may be involved in the alternative activation of primary monocytes in chickens and that chIL-13 signaling may be regulated through chIL-13Rα2 binding and TGF-β1 secretion. Importantly, the newly developed anti-chIL-13 mAb will serve as valuable immune reagents for future studies on the biological activity of chIL-13 and its receptors.

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First Whole-Genome Characterization of Avian Nephritis Virus 2 of Broiler Chicken from Para, Brazil.

Project description:Our results show the first full-genome characterization of avian nephritis virus 2 recovered from stools of broiler chickens at a commercial farm located in Benevides, Pará, Brazil. Nucleotide analyses of whole-genome sequences showed the isolate to be a strain of Avastrovirus 2 in the family Astroviridae.

| S-EPMC6013626 | biostudies-literature

First Isolation and Molecular Characterization of Chicken Astrovirus and Avian Nephritis Virus in Chickens in Bangladesh.

Project description:Chicken astrovirus (CAstV) and avian nephritis virus (ANV) are enteric viruses of poultry and have infected a wide range of poultry species worldwide, causing runting-stunting syndrome (RSS), which requires virus screening and results in serious economic damage. No confirmed cases have been reported from Bangladesh. In the present study, CAstV and ANV were monitored in Bangladesh. We monitored samples for CAstV and ANV and compared their genomic sequences to other reference strains. We found 8/31 flocks (25.8%) were positive for CAstV, 6/31 flocks (19.3%) had mixed infection of CAstV and ANV, and 1 flock (3.2%) was positive for ANV. Only ANV and a combination of CAstV and ANV were found in broilers and broiler breeders, but CAstV was found in all types of chickens. We isolated two of each from CAstV and ANV through specific pathogen-free chicken embryonated eggs via the yolk sac route. Phylogenetic analysis based on the ORF1b conserved region of CAstV and ANV suggested that the locally circulating strain was closely related to the strains isolated from India and Brazil. This report is the first molecular characterization of CAstV and ANV in Bangladesh. This study highlights that CAstV and ANV are circulating in Bangladeshi poultry.

| S-EPMC8672296 | biostudies-literature

Molecular characterization and the first full sequencing genome of chicken infectious anemia virus (CIAV) in Iran.

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| S-EPMC8806177 | biostudies-literature

Downregulation of chicken interleukin-17 receptor A during Eimeria infection.

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| S-EPMC4187842 | biostudies-literature

Four Cysteine Residues Contribute to Homodimerization of Chicken Interleukin-2.

Project description:Interleukin-2 (IL-2) is a pleiotropic cytokine regulating the immune and nervous systems. Mammalian and bird IL-2s have different protein sequences, but perform similar functions. In the current study, two bands were detected by immunoblotting using an antibody against freshly purified chicken IL-2 (chIL-2). The molecular weight of the larger band was approximately twice as much of the chIL-2 monomer, although a chIL-2 complex or homodimer has never been reported. To explain this intriguing result, several dissociation reagents were used to examine the intermolecular forces between components of the proposed chIL-2 complex. It was found that intermolecular disulphide bond promotes homodimerization of chIL-2. Subsequently, mutation of Cys residues of chIL-2 revealed that mutation of all four Cys residues disrupted homodimerization, but a single, dual, or triple Cys mutation failed to disrupt homodimerization, suggesting that all four Cys residues on chIL-2 contribute to this dimerization. Functional analysis showed that both monomeric and dimeric chIL-2 consisting of either wild type or mutant chIL-2 were able to stimulate the expansion of CD4+ T cell in vivo or in vitro, and effectively bind to chIL-2 receptor. Overall, this study revealed that the recombinant chIL-2 purified from either Escherichia coli (E. coli) or Spodoptera frugiperda (Sf9) cells could homodimerize in vitro, with all four Cys residues on each chIL-2 protein contributing to this homodimerization, and dimerization and Cys mutation not impacting chIL-2 induced stimulation of chicken CD4+ T cells.

| S-EPMC6888268 | biostudies-literature

Isolation and Characterization of Chicken NPAS3.

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| S-EPMC3214776 | biostudies-literature

Molecular characterization of chicken anemia virus circulating in chicken flocks in egypt.

Project description:Introduction. Although many previous studies reported detection of chicken anemia virus (CAV) in Egypt since 1990, genomic characterization of this circulating CAV has not been published. In the present study, four nucleotide sequences of detected CAV were genetically characterized. Methods. These nucleotide sequences were obtained from commercial chicken flocks in two different locations of Egypt during 2010. The target region for sequencing was 675?bp nucleotide of partial coding region of VP1 protein. The nucleotide and deduced amino acid sequences of the detected CAV were aligned and compared to worldwide CAV isolates including commonly used vaccine strains. Phylogenetic analysis of these sequences was also carried out. Results. Our results showed that all the Egyptian CAV sequences were grouped in one group with viruses from diverse geographic regions. This group is characterized by amino acids profile (75)I, (97)L, (139)Q, and (144)Q in VP1. The phylogenetic and amino acid analyses of deduced amino acid indicated that the detected CAV sequences differ from CAV vaccine strains. Conclusion. This is the first report that describes molecular characterization of circulating CAV in Egypt. The study showed that the detected CAV, in Egypt are field viruses and unrelated to vaccine strains.

| S-EPMC4180199 | biostudies-literature

Characterization of the chicken beta-globin insulator.

Project description:Insulators, first identified in Drosophila, are DNA sequence elements that shield a promoter from nearby regulatory elements. We have previously reported that a DNA sequence at the 5' end of the chicken beta-globin locus can function as an insulator. It is capable of shielding a reporter gene from the activating effects of a nearby mouse beta-globin locus control region element in the human erythroleukemic cell line K562. In this report, we show that most of the insulating activity lies in a 250-bp CpG island (core element), which contains the constitutive DNase I-hypersensitive site (5'HS4). DNA binding assays with the core sequence reveal a complex protein binding pattern. The insulating activity of the core element is multiplied when tandem copies are used. Although CpG islands are often associated with promoters of housekeeping genes, we find little evidence that the core element is a promoter. Furthermore, the insulator differs from a promoter in its ability to block the locus control region effect directionally.

| S-EPMC19555 | biostudies-literature

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