Analysis of Differentially Expressed Genes in Necrotic Enteritis-infected Fayoumi Chickens using RNA Sequencing

Truong, Anh Duc,Hong, Yeojin,Ban, Jihye,Park, Boyeong,Hoang, Thanh C.,Hong, Yeong H.,Lillehoj, Hyun S. Japan Poultry Science Association 2017 Journal of Poultry Science Vol.54 No.2

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

Anh Duc Truong,홍영호,Hyun S. Lillehoj 아세아·태평양축산학회 2015 Animal Bioscience Vol.28 No.10

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 immunerelated 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 diseaseresistant chickens.

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

Truong, Anh Duc,Park, Boyeong,Ban, Jihye,Hong, Yeong Ho BioMed 2016 VETERINARY RESEARCH Vol.47 No.-

<P>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<SUP>+</SUP> and CD8<SUP>+</SUP> 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<SUP>+</SUP> and CD8<SUP>+</SUP> 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.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s13567-016-0342-0) contains supplementary material, which is available to authorized users.</P>

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

Truong, Anh Duc,Hong, Yeojin,Rengaraj, Deivendran,Lee, Janggeun,Lee, Kyungbaek,Hong, Yeong Ho Elsevier 2018 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.87 No.-

<P><B>Abstract</B></P> <P>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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Cloning and sequence analysis of chicken interleukin 11 (chIL-11). </LI> <LI> chIL-11 has an anti-inflammatory related to IL-11RA and IL-6ST receptor expression. </LI> <LI> chIL-11 activated and regulated JAK-STAT, NF-κB, PI3K/AKT, and MAPK signaling pathways. </LI> <LI> chIL-11 inhibited nitric oxide production, affected proliferation of both tested cell lines. </LI> <LI> chIL-11 inhibited Th1, Th17 cytokine and enhanced Th2 cytokine production. </LI> </UL> </P>

TGF-β Signaling and miRNAs Targeting for BMP7 in the Spleen of Two Necrotic Enteritis-Afflicted Chicken Lines

Anh Duc Truong,Yeojin Hong,Janggeun Lee,Kyungbaek Lee,Hyun S. Lillehoj,Yeong Ho Hong 한국가금학회 2017 韓國家禽學會誌 Vol.44 No.3

Transforming growth factor beta (TGF-β) signaling pathways are involved in the regulation of proliferation, differentiation, immunity, survival, and apoptosis of many cells. The aim of this study was to investigate the differential expression of TGF-β-related genes, and their interactions and regulators in the spleen of two genetically disparate chicken lines (Marek’s disease resistant line 6.3 and Marek’s disease-susceptible line 7.2) induced with necrotic enteritis (NE) by Eimeria maxima and Clostridium perfringens infection. By using high-throughput RNA-sequencing, we investigated 76 TGF-β-related genes that were significantly and differentially expressed in the spleens of the chickens. Approximately 20 TGF-β pathway genes were further verified by qRT-PCR, and the results were consistent with our RNA sequencing data. All 76 identified genes were analyzed through Gene Ontology and mapped onto the KEGG chicken TGF-β pathway. Our results demonstrated that several key genes, including TGF-β1-3, bone morphogenetic proteins (BMP)1-7, inhibitor of differentiation (ID) proteins ID1-3, SMAD1-9, and Jun, showed a markedly differential expression between the two chicken lines, relative to their respective controls. We then further predicted 24 known miRNAs that targeted BMP7 mRNA from 139 known miRNAs in the two chicken lines. Among these, six miRNAs were measured by qRT-PCR. In conclusion, this study is the first to analyze most of the genes, interactions, and regulators of the TGF-β pathway in the innate immune responses of NE afflicted chickens.

Interleukin-26 regulates Th1, Th2 and Th17 cytokines through the JAK/STAT and NF-κB signaling pathways

Anh Duc Truong,Yeojin Hong,Janggeun Lee,Kyungbaek Lee,Hyun S. Lillehoj,Yeong Ho Hong 한국가금학회 2017 한국가금학회 정기총회 및 학술발표회 Vol.2017 No.11

Analysis of JAK-STAT signaling pathway genes and their microRNAs in the intestinal mucosa of genetically disparate chicken lines induced with necrotic enteritis

Truong, Anh Duc,Rengaraj, Deivendran,Hong, Yeojin,Hoang, Cong Thanh,Hong, Yeong Ho,Lillehoj, Hyun S. Elsevier 2017 Veterinary immunology and immunopathology Vol. No.

<P><B>Abstract</B></P> <P>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 [Marek’s 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.</P>

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

Truong, Anh Duc,Hong, Yeojin,Lee, Janggeun,Lee, Kyungbaek,Kil, Dong Yong,Lillehoj, Hyun S.,Hong, Yeong Ho MDPI AG 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.6

<P>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.</P>

TGF-β Signaling and miRNAs Targeting for BMP7 in the Spleen of Two Necrotic Enteritis-Afflicted Chicken Lines

Truong, Anh Duc,Hong, Yeojin,Lee, Janggeun,Lee, Kyungbaek,Lillehoj, Hyun S.,Hong, Yeong Ho The Korean Society of Poultry Science 2017 韓國家禽學會誌 Vol.44 No.3

Transforming growth factor beta ($TGF-{\beta}$) signaling pathways are involved in the regulation of proliferation, differentiation, immunity, survival, and apoptosis of many cells. The aim of this study was to investigate the differential expression of $TGF-{\beta}$-related genes, and their interactions and regulators in the spleen of two genetically disparate chicken lines (Marek's disease resistant line 6.3 and Marek's disease-susceptible line 7.2) induced with necrotic enteritis (NE) by Eimeria maxima and Clostridium perfringens infection. By using high-throughput RNA-sequencing, we investigated 76 $TGF-{\beta}$-related genes that were significantly and differentially expressed in the spleens of the chickens. Approximately 20 $TGF-{\beta}$ pathway genes were further verified by qRT-PCR, and the results were consistent with our RNA sequencing data. All 76 identified genes were analyzed through Gene Ontology and mapped onto the KEGG chicken $TGF-{\beta}$ pathway. Our results demonstrated that several key genes, including $TGF-{\beta}$1-3, bone morphogenetic proteins (BMP)1-7, inhibitor of differentiation (ID) proteins ID1-3, SMAD1-9, and Jun, showed a markedly differential expression between the two chicken lines, relative to their respective controls. We then further predicted 24 known miRNAs that targeted BMP7 mRNA from 139 known miRNAs in the two chicken lines. Among these, six miRNAs were measured by qRT-PCR. In conclusion, this study is the first to analyze most of the genes, interactions, and regulators of the $TGF-{\beta}$ pathway in the innate immune responses of NE afflicted chickens.

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

Anh Duc Truong,홍여진,이장근,이경백,Ha Thi Thanh Tran,Hoang Vu Dang,Viet Khong Nguyen,Hyun S. Lillehoj,홍영호 아세아·태평양축산학회 2019 Animal Bioscience Vol.32 No.5

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 β2-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, β2-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.