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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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Characterization of chicken interleukin-9 receptor alpha chain.

Project description:Interleukin-9 receptor alpha chain (IL-9Rα) is the ligand-binding subunit of IL-9R that plays roles in IL-9-mediated allergy, inflammation, infection, and tumor immunity. While mammalian IL-9Rαs have been extensively investigated, avian IL-9Rα has not yet been identified and characterized. In this study, we cloned chicken IL-9Rα (chIL-9Rα) and performed a phylogenetic analysis, analyzed its tissue distribution, characterized the expression form of natural chIL-9Rα. Phylogenetic analysis showed that chIL-9Rα has less than 25% amino acid homology with mammalian IL-9Rαs. The chIL-9Rα mRNA was abundantly detected only in heart and mitogen-activated peripheral blood mononuclear cells. Furthermore, 4 monoclonal antibodies (mAbs) against chIL-9Rα were generated using prokaryotic recombinant chIL-9Rα (rchIL-9Rα). Using anti-chIL-9Rα mAbs, natural chIL-9Rα expressed on the splenocytes of chickens was observed by indirect immunofluorescence assay (IFA), and its molecular weight of 51 kDa was identified by Western blotting. Overall, our study reveals for the first time the presence of IL-9Rα in birds, and provides immunological tools for further investigating the roles of chIL-9 in diseases and immunity.

| S-EPMC10432844 | biostudies-literature

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.

| S-EPMC7587669 | biostudies-literature

Research Note: Development and characterization of monoclonal antibodies specific for chicken interleukin-7 receptor α (CD127).

Project description:CD127, also named interleukin-7 receptor (IL-7R), is expressed on various cell types including naive and memory T cells, and plays a critical role in the differentiation and activation of T lymphocytes. The availability of poultry-specific immune reagents to identify and measure chicken CD127 response will enhance fundamental and applied research in poultry immunology. Mouse monoclonal antibodies (MAbs) against chicken CD127 (chCD127) were developed and characterized. More specifically, a 678 bp ectodomain of chCD127 gene was cloned in the pET28a (+) vector and expressed in BL21-AI E. coli competent cells. The recombinant chCD127 protein with a size of 30 KDa which was also recognized by a mouse anti-human CD127 MAb (Clone G-11) was used to immunize mice, and 6 new mouse MAbs which specifically detected chicken CD127 were developed and characterized. Availability of these new sets of chCD127-specific MAbs will facilitate the immunological studies on CD127 in poultry, especially in understanding effector and memory T immune cell responses in normal and diseased states.

| S-EPMC9396400 | biostudies-literature

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 detection and genetic characterization of chicken infectious anemia virus in the Mekong Delta, Vietnam.

Project description:BackgroundChicken infectious anemia (CIA) caused by the CIA virus (CIAV) is considered one of the most important immunosuppressive diseases affecting chickens and recently poses a great economic burden to the poultry industry worldwide.AimThis study aims to identify the presence of CIAV in the Mekong Delta (MD), Vietnam, and to determine genotypes of CIAVs that are currently circulating in this area.MethodsOrgan samples (spleen, liver, and thymus) of 144 chickens suspected with CIA from 47 poultry farms were collected. A total of 47 pooled samples, each containing 2-4 chickens from each farm, were tested for the presence of CIAV.ResultsTwenty out of 47 pooled organ samples (pool of 2-4 chickens per farm) were positive for CIAV using polymerase chain reaction targeting the viral VP1 gene. The VP1 amplicons of eight representative CIAVs were subjected to sequencing and genetic characterization. Phylogenetic analysis based on partial VP1 gene sequence revealed that the CIAVs detected in the MD grouped into different genotypes of II, IIIa, and IIIc together with CIAVs previously detected in the northern Vietnam and other Asian countries. The phylogenetic analysis also confirmed that detected CIAVs genetically differed from vaccine strains. In addition, deduced amino acids of the VP1 identified several critical amino acid substitutions in the VP1 protein that are likely associated with the virulence of CIAV.ConclusionThis is the first report to detect and determine the genetic characterization of the circulating CIAVs in the MD. Therefore, this study provides an important understanding of the evolution of CIAVs and highlights the importance of implementing prompt control measures against CIAVs in the MD and Vietnam.

| S-EPMC10399657 | biostudies-literature

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

Project description:BackgroundThe chicken infectious anemia virus (CIAV) is an important pathogen that causes severe immunosuppression in young chickens.AimsThe study aims to characterize the genotype and full-length sequencing of CIAV strains in Iran.MethodsFirst, the collected thymus samples were investigated by conventional PCR for CIAV detection. Second, one of the CIAV positive samples (UT-Zahraee) was chosen for full genome sequencing.ResultsThroughout 2017, we detected 13 CIAVs isolated from 40 broiler flocks of different provinces of Iran. A comparison of the complete sequences of the genome and homologies of the nucleotides revealed that UT-Zahraee had a high similarity with American and Egyptian CIAV isolates. Moreover, VP1 sequence analysis showed that UT-Zahraee shared high homology with previously reported Iranian CIAV strains, Chinese, and Egyptian isolates.ConclusionThis study is the first report of full genome sequencing of CIAV strain from Iran. It will be beneficial to understand better the epidemiology and molecular characteristics of CIAV circulating in Iran.

| S-EPMC8806177 | 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

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

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

Project description:Both interleukin-17A (IL-17A) and IL-17F are proinflammatory cytokines that have an important role in intestinal homeostasis via receptor signaling. These cytokines have been characterized in chickens, but very little is known about their receptors and their functional activity. We provide here the first description of the sequence analysis, bioactivity, and comparative expression analysis of chicken IL-17RA (chIL-17RA) in chickens infected with Salmonella and Eimeria, two major infectious agents of gastrointestinal diseases of poultry of economic importance. A full-length chIL-17RA cDNA with a 2,568-bp coding region was identified from chicken thymus cDNA. chIL-17RA shares ca. 46% identity with mammalian homologues and 29.2 to 31.5% identity with its piscine counterparts. chIL-17RA transcript expression was relatively high in the thymus and in the chicken macrophage cell line HD11. The chIL-17RA-specific small interfering RNA inhibits interleukin-6 (IL-6), IL-8, and IL-1β mRNA expression in chicken embryo fibroblast cells (but not in DF-1 cells) stimulated with chIL-17A or chIL-17F. Interaction between chIL-17RA and chIL-17A was confirmed by coimmunoprecipitation. Downregulation of chIL-17RA occurred in concanavalin A- or lipopolysaccharide-activated splenic lymphocytes but not in poly(I·C)-activated splenic lymphocytes. In Salmonella- and Eimeria-infected chickens, the expression levels of the chIL-17RA transcript were downregulated in intestinal tissues from chickens infected with two Eimeria species, E. tenella or E. maxima, that preferentially infect the cecum and jejunum, respectively. However, chIL-17RA expression was generally unchanged in Salmonella infection. These results suggest that chIL-17RA has an important role in mucosal immunity to intestinal intracellular parasite infections such as Eimeria infection.

| S-EPMC4187842 | biostudies-literature

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