Anh Duc Truong

High-throughput sequencing reveals differing immune responses in the intestinal mucosa of two inbred lines afflicted with necrotic enteritis.

We investigated the necrotic enteritis (NE)-induced transcripts of immune-related genes in the intestinal mucosa of two highly inbred White Leghorn chicken lines, line 6.3 and line 7.2, which share the same MHC haplotype and show different levels of NE susceptibility using high-throughput RNA sequencing (RNA-Seq) technology. NE was induced by the previously described co-infection model using Eimeria maxima and Clostridium perfringens. The RNA-Seq generated over 38 million sequence reads for Mareks disease (MD)-resistant line 6.3 and over 40 million reads for the MD-susceptible line 7.2. Alignment of these sequences with the Gallus gallus genome database revealed the expression of over 29,900 gene transcripts induced by NE in these two lines, among which 7,841 genes were significantly upregulated and 2,919 genes were downregulated in line 6.3 chickens and 6,043 genes were significantly upregulated and 2,764 genes were downregulated in NE-induced line 7.2 compared with their uninfected controls. Analysis of 560 differentially expressed genes (DEGs) using the gene ontology database revealed annotations for 246 biological processes, 215 molecular functions, and 81 cellular components. Among the 53 cytokines and 96 cytokine receptors, 15 cytokines and 29 cytokine receptors were highly expressed in line 6.3, whereas the expression of 15 cytokines and 15 cytokine receptors was higher in line 7.2 than in line 6.3 (fold change ≥ 2, p<0.01). In a hierarchical cluster analysis of novel mRNAs, the novel mRNA transcriptome showed higher expression in line 6.3 than in line 7.2, which is consistent with the expression profile of immune-related target genes. In qRT-PCR and RNA-Seq analysis, all the genes examined showed similar responses to NE (correlation coefficient R=0.85-0.89, p<0.01) in both lines 6.3 and 7.2. This study is the first report describing NE-induced DEGs and novel transcriptomes using RNA-seq data from two inbred chicken lines showing different levels of NE susceptibility. These findings provide important insights into our current knowledge of host-pathogen interaction and the nature of host genes that can serve as NE resistance markers for molecular breeding.

RNA-seq Profiles of Immune Related Genes in the Spleen of Necrotic Enteritis-afflicted Chicken Lines

The study aimed to compare the necrotic enteritis (NE)-induced transcriptome differences between the spleens of Marek’s disease resistant chicken line 6.3 and susceptible line 7.2 co-infected with Eimeria maxima/Clostridium perfringens using RNA-Seq. Total RNA from the spleens of two chicken lines were used to make libraries, generating 42,736,296 and 42,617,720 usable reads, which were assembled into groups of 29,897 and 29,833 mRNA genes, respectively. The transcriptome changes were investigated using the differentially expressed genes (DEGs) package, which indicated 3,255, 2,468 and 2,234 DEGs of line 6.3, line 7.2, and comparison between two lines, respectively (fold change ≥2, p<0.01). The transcription levels of 14 genes identified were further examined using qRT-PCR. The results of qRT-PCR were consistent with the RNA-seq data. All of the DEGs were analysed using gene ontology terms, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the DEGs in each term were found to be more highly expressed in line 6.3 than in line 7.2. RNA-seq analysis indicated 139 immune related genes, 44 CD molecular genes and 150 cytokines genes which were differentially expressed among chicken lines 6.3 and 7.2 (fold change ≥2, p<0.01). Novel mRNA analysis indicated 15,518 novel genes, for which the expression was shown to be higher in line 6.3 than in line 7.2 including some immune-related targets. These findings will help to understand host-pathogen interaction in the spleen and elucidate the mechanism of host genetic control of NE, and provide basis for future studies that can lead to the development of marker-based selection of highly disease-resistant chickens.

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.

The novel chicken interleukin 26 protein is overexpressed in T cells and induces proinflammatory cytokines

In the present study, we describe the cloning and functional characterization of chicken interleukin 26 (ChIL-26). ChIL-26, a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by T cells. The ChIL-26 cDNA encodes an 82-amino-acid protein whose amino acid sequence has 22.63, 46.31 and 43.15% homology with human IL-26, pig IL-26 and canary IL-26, respectively. ChIL-26 signals through a heterodimeric receptor complex composed of the IL-20R1 and IL-10R2 chains, which are expressed primarily in the CU91 T cell line as well as CD4+ and CD8+ T cells. Recombinant ChIL-26 protein induced Th1 cytokines (IL-16 and IFN-γ), Th2 cytokines (IL-4, IL-6 and IL-10), Th17 cytokines (IL-17A, IL-17D, and IL-17F), and chemokine transcripts (mainly CCL3, CCL4, CCL5, CCL20 and CXCL13) in the CU91 T cell line and in CD4+ and CD8+ T cells, however IL-18 was not expressed in the CU91 T cell line. Taken together, the data demonstrates that T cells express the functional ChIL-26 receptor complex and that ChIL-26 modulates T cell proliferation and proinflammatory gene expression. To the best of our knowledge, this is the first report of cloned ChIL-26. We evaluated its functional roles, particularly in the pathogenic costimulation of T cells, which may be significantly associated with the induction of cytokines.

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.

Expression analysis of cytosolic DNA-sensing pathway genes in the intestinal mucosal layer of necrotic enteritis-induced chicken

Necrotic enteritis (NE) is a serious problem to the poultry farms, which report NE outbreaks more than once per year, as a result of the inappropriate use of antibiotics in the feed. The NE affected bird die rapidly as a result of various pathophysiological complications in the intestine and immune system. Also, several studies have reported that the genes exclusively related to intestine and immune functions are significantly altered in response to NE. In this study, NE was induced in two genetically disparate chicken lines that are resistant (line 6.3) and sensitive (line 7.2) to avian leukosis and Mareks disease. The intestinal mucosal layer was collected from NE-induced and control chickens, and subjected to RNA-sequencing analysis. The involvement of differentially expressed genes in the intestinal mucosal layer of line 6.3 and 7.2 with the immune system-related pathways was investigated. Among the identified immune system-related pathways, a candidate pathway known as chicken cytosolic DNA-sensing pathway (CDS pathway) was selected for further investigation. RNA-sequencing and pathway analysis identified a total of 21 genes that were involved in CDS pathway and differentially expressed in the intestinal mucosal layer of lines 6.3 and 7.2. The expression of CDS pathway genes was further confirmed by real-time qPCR. In the results, a majority of the CDS pathway genes were significantly altered in the NE-induced intestinal mucosal layer from lines 6.3 and 7.2. In conclusion, our study indicate that NE seriously affects several genes involved in innate immune defense and foreign DNA sensing mechanisms in the chicken intestinal mucosal layer. Identifying the immune genes affected by NE could be an important evidence for the protective immune response to NE-causative pathogens.

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.