Search

Probiotics are essential in the body's nutrients, improving the ratio of meat to meat, immune response, and preventing diseases. In this study, RNA-sequencing (RNA-seq) was used to identify the differentially expressed genes (DEGs), enriched related pathways, and Gene Ontology (GO) terms among blank negative control (NC), supplemented with Bacillus spp. (BS) and commercial probiotic (PC) groups after a 42-day fed supplementation. The results showed that 2005, 1356, and 2189 DEGs were significantly altered in BS vs. NC, PC vs NC, and BS vs PC groups, respectively. On the other hand, 9 DEGs were further validated by qRT-PCR, indicating that the qRT-PCR and RNA-Seq results were more consistent. Therefore, the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs showed that the DEGs were mainly enriched to metabolism signalling pathways (alpha-linolenic acid metabolism, linoleic acid metabolism, tryptophan metabolism, tyrosine metabolism, ether lipid metabolism, and metabolic pathway, etc) and immune response pathways (cytokine-cytokine receptor interaction, MAPK signalling pathway, and intestinal immune network for IgA production, neuroactive ligand-receptor interaction etc). These results will provide a better understanding of the role of probiotics in chicken development and provide basic information on the genetic development of chickens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Exosomes are membrane vesicles containing proteins, lipids, DNA, mRNA, and micro RNA (miRNA). Exosomal miRNA from donor cells can regulate the gene expression of recipient cells. Here, Ri chickens were divided into resistant (Mx/A; BF2/B21) and susceptible (Mx/G; BF2/B13) trait by genotyping of Mx and BF2 genes. Then, Ri chickens were infected with H5N1, a highly pathogenic avian influenza virus (HPAIV). Exosomes were purified from blood serum of resistant chickens for small RNA sequencing. Sequencing data were analysed using FastQCv0.11.7, Cutadapt 1.16, miRBase v21, non-coding RNA database, RNAcentral 10.0, and miRDeep2. Differentially expressed miRNAs were determined using statistical methods, including fold-change, exactTest using edgeR, and hierarchical clustering. Target genes were predicted using miRDB. Gene ontology analysis was performed using gProfiler. Twenty miRNAs showed significantly different expression patterns between resistant control and infected chickens. Nine miRNAs were up-regulated and 11 miRNAs were down-regulated in the infected chickens compared with that in the control chickens. In target gene analysis, various immune-related genes, such as cytokines, chemokines, and signalling molecules, were detected. In particular, mitogen-activated protein kinase (MAPK) pathway molecules were highly controlled by differentially expressed miRNAs. The result of qRT-PCR for miRNAs was identical with sequencing data and miRNA expression level was higher in resistant than susceptible chickens. This study will help to better understand the host immune response, particularly exosomal miRNA expression against HPAIV H5N1 and could help to determine biomarkers for disease resistance.

Interleukins (ILs) regulate cell surface antigens known as activation markers, which have distinct functional roles. However, the regulation of major histocompatibility complex (MHC) class I, MHC class II, and related genes by cytokines in chickens is not well understood. In the present study, we evaluated the influence of certain recently discovered chicken interleukins-i.e., IL-11, IL-12B, IL-17A, IL-17B, IL-26, and IL-34-on the expression and regulation of genes related to MHC class I, MHC class II, and the associated proteins in an HD11 chicken macrophage cell line. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunocytochemical, and flow cytometric analyses to assess dose- and time-dependent expression in the HD11 cell line and found that the ILs induced MHC class I, MHC class II, and associated protein. As NF-κB is actively involved in cell activation and is constitutively activated in many immune cells, we also determined whether NF-κB regulates MHC class I, MHC class II, and related gene expression in the HD11 cell line. The NF-κB inhibitor sulfasalazine (Sz) dose-dependently inhibited MHC class I and MHC class II in the HD11 cell line. Sz also downregulated the expression of MHC class I, MHC class II, and the associated proteins in the IL-induced HD11 cell line. The expression of MHC class I, MHC class II, and associated genes was accompanied by the Sz-sensitive degradation of the p65 (RelA) and p50 subunits of NF-κB and IκBα. Our results indicate that the different effects of each IL on the expression of genes related to MHC class I, MHC class II, and the associated proteins are involved with the regulation of the dose and duration of antigenic peptide presentation and, thus, also influence Th1, Th2, and Th17 production., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are small non-coding RNAs that contribute to host immune response as post-transcriptional regulation. The current study investigated the biological role of the chicken (Gallus gallus) microRNA-200a-3p (gga-miR-200a-3p), using 2 necrotic enteritis (NE) afflicted genetically disparate chicken lines, 6.3 and 7.2, as well as the mechanisms underlying the fundamental signaling pathways in chicken. The expression of gga-miR-200a-3p in the intestinal mucosal layer of NE-induced chickens, was found to be upregulated during NE infection in the disease-susceptible chicken line 7.2. To validate the target genes, we performed an overexpression analysis of gga-miR-200a-3p using chemically synthesized oligonucleotides identical to gga-miR-200a-3p, reporter gene analysis including luciferase reporter assay, and a dual fluorescence reporter assay in cultured HD11 chicken macrophage cell lines. Gga-miR-200a-3p was observed to be a direct transcriptional repressor of ZAK, MAP2K4, and TGFβ2 that are involved in mitogen-activated protein kinase (MAPK) pathway by targeting the 3'-UTR of their transcripts. Besides, gga-miR-200a-3p may indirectly affect the expression of protein kinases including p38 and ERK1/2 at both transcriptional and translational levels, suggesting that this miRNA may function as an important regulator of the MAPK signaling pathway. Proinflammatory cytokines consisting of IL-1β, IFN-γ, IL-12p40, IL-17A, and LITAF belonging to Th1 and Th17-type cytokines, were upregulated upon gga-miR-200a-3p overexpression. These findings have enhanced our knowledge of the immune function of gga-miR-200a-3p mediating the chicken immune response via regulation of the MAPK signaling pathway and indicate that this miRNA may serve as an important biomarker of diseases in domestic animals.

The inhibitory leukocyte immuno-globulin-like receptors (LILRBs) play an important role in innate immunity. Currently, no data exist regarding the role of LILRB4 and LILRB5 in the activation of immune signaling pathways in mammalian and avian species. Here, we report for the first time, the cloning and structural and functional analyses of chicken LILRB4-5 genes identified from 2 genetically disparate chicken lines. Comparison of LILRB4-5 amino acid sequences from lines 6.3 and 7.2 with those of mammalian proteins revealed 17 to 62% and 19 to 29% similarity, respectively. Phylogenetic analysis indicated that the chicken LILRB4-5 genes were closely associated with those of other species. LILRB4-5 could be subdivided into 2 groups having distinct immunoreceptor tyrosine-based inhibitory motifs, which bind to Src homology 2-containing tyrosine phosphatase 2 (SHP-2). Importantly, LILRB4-5 also upregulated the major histocompatibility complex (MHC) class I and β2-microglobulin gene expression as well as the expression of transporter associated with antigen processing 1-2, which play an important role in MHC class I activation. Our results indicate that LILRB4-5 are transcriptional regulators of the MHC class I pathway components and regulate innate immune responses. Furthermore, LILRB4-5 could activate the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway genes in macrophages and induce the expression of chemokines and T helper (Th)1, Th2, and Th17 cytokines. Our data suggest that LILRB4-5 are innate immune receptors associated with SHP-2, MHC class I, and β2-microglobulin. Additionally, they activate the JAK/STAT signaling pathway and control the expression of cytokines in macrophages., (© 2019 Poultry Science Association Inc.)

Background: B cell lymphoma (BCL) families play an important role in apoptosis as a growth factor, cell death programming, cytokine expression and immune-related genes expression., Objectives: In this study, to investigate the roles of BCLs, we performed genome-wide identification, expression and functional analyses of the BCL family in chicken., Methods: Chicken BCLs genes were identified and analyzed by using bioinformatics approach. Expression profiles and Hierarchical cluster analysis of the BCLs genes in different chicken tissues were obtained from the genome-wide RNA-seq in the GEO, and Cluster and Java Treeview, respectively., Results: A total of 16 BCLs genes were identified from the chicken genome, which could be further classified into five distinct groups in the phylogenetic tree. On the other hand, the interaction among BCLs proteins and between BCLs proteins with NF-κB subunits are limited, indicating that the remaining the functions of BCLs protein could be investigated in chicken. Moreover, KEGG pathway analysis indicated that BCL gene family was involved in regulation of apoptotic and immune response. Finally, BCL gene family was differentially expressed in chicken tissues, pathogen infection and growth stages of early chicken early embryo., Conclusion: This study provides significant insights into the potential functions of BCLs in chicken, including the regulation of apoptosis, cell death and expression of immune-related genes.

Among the eight forms of vitamin E, the liver preferentially releases α-tocopherol into the circulation and it is distributed to the non-liver tissues. In the hepatocytes, alpha tocopherol transfer protein (TTPA) specifically recognizes α-tocopherol with 2R-configuration and facilitates its intracellular transfer. The identification and characterization of TTPA expression have not been demonstrated in avian species. Therefore, the objectives of this study were to identify avian TTPAs, to compare the sequence conservation, phylogenetic relationship, protein interactions, and disease associations of chicken TTPA with those of human and vertebrate TTPA, and to characterize the tissue expression of the TTPA gene in chickens fed diets supplemented with different amounts of α-tocopherol. Our results suggest that the chicken TTPA was highly conserved with the human and vertebrate TTPA, and consisted of a cellular retinaldehyde binding protein and TRIO guanine exchange factor (CRAL_TRIO) domain. Feeding diets supplemented with increasing amounts of α-tocopherol (25 IU/Kg, 50 IU/Kg, or 100 IU/Kg) to broiler chickens had no effects on growth performance compared with feeding basal diets containing no supplemental α-tocopherol. The expression of TTPA gene was detected high in the liver of chickens in response to dietary α-tocopherol concentrations, whereas its expression was very low or undetectable in the non-liver tissues. In conclusion, the chicken TTPA protein sequence is highly conserved with other avian and vertebrate TTPA protein sequences. The higher expression of TTPA gene in the chicken liver in response to dietary α-tocopherol concentrations may suggest its crucial role in transporting α-tocopherol in the chicken liver., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The activating leukocyte immunoglobulin-like receptors (LILRAs) play an important role in innate immunity. However, most of the LILRA members have not been characterized in avian species including chickens. The present study is the first attempt at cloning, structural analysis and functional characterization of two LILRAs (LILRA2 and LILRA6) in chickens. Multiple sequence alignments and construction of a phylogenetic tree of chicken LILRA2 and LILRA6 with mammalian proteins revealed high conservation between chicken LILRA2 and LILRA6 and a close relationship between the chicken and mammalian proteins. The mRNA expression of LILRA2 and LILRA6 was high in chicken HD11 macrophages and the small intestine compared to that in several other tissues and cells tested. To examine the function of LILRA2 and LILRA6 in chicken immunity, LILRA2 and LILRA6 were transfected into HD11 cells. Our findings indicated that LILRA2 and LILRA6 are associated with the phosphorylation of Src kinases and SHP2, which play a regulatory role in immune functions. Moreover, LILRA6 associated with and activated MHC class I, β2-microglobulin and induced the expression of transporters associated with antigen processing but LILRA2 did not. Furthermore, both LILRA2 and LILRA6 activated JAK-STAT, NF-κB, PI3K/AKT and ERK1/2 MAPK signaling pathways and induced Th1-, Th2- and Th17-type cytokines and Toll-like receptors. Collectively, this study indicates that LILRA2 and LILRA6 are essential for macrophage-mediated immune responses and they have the potential to complement the innate and adaptive immune system against pathogens.

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 (Marek's 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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-1β, 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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-β, IFN-γ, IL-1β, and IL-6), Th2 (IL-4 and IL-10), and Th17 (IL-12p40, IL-17A, and IL-17F), and the Treg cytokines (TGF-β4); 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

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 Marek's 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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Heat shock proteins (HSPs) function as molecular chaperones with critical roles in chicken embryogenesis, immune response to infectious diseases, and response to various environmental stresses. However, little is known on HSP genes in chicken. In this study, to understand the roles of chicken HSPs, we performed genome-wide identification, expression, and functional analyses of the HSP family genes in chicken. Results: A total of 76 HSP genes were identified in the chicken genome, which were further classified into eight distinct groups (I-VIII) based on phylogenetic tree analysis. The gene-structure analysis revealed that the members of each clade had the same or similar exon-intron structures. Chromosome mapping suggested that HSP genes were widely dispersed across the chicken genome, except in chromosomes 16, 18, 22, 25, 26, and 28–32, which lacked chicken HSP genes. On the other hand, the interactions among chicken HSPs were limited, indicating that the remaining functions of HSPs could be investigated in chicken. Moreover, KEGG pathway analysis showed that the HSP gene family was involved in the regulation of heat stress, apoptotic, intracellular signaling, and immune response pathways. Finally, RNA sequencing data revealed that, of the 76 chicken HSP genes, 46 were differentially expressed at 21 different growth stages in chicken embryos, and 72 were differentially expressed on post-infection day 3 in two indigenous Ri chicken lines infected with highly pathogenic avian influenza. Conclusions: This study provides significant insights into the potential functions of HSPs in chicken, including the regulation of apoptosis, heat stress, chaperone activity, intracellular signaling, and immune response to infectious diseases. [ABSTRACT FROM AUTHOR]

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-β, 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-12Rβ1 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-12Rβ1 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. [ABSTRACT FROM AUTHOR]

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 Marek's 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. [ABSTRACT FROM AUTHOR]

miRNAs are involved in both adaptive and innate immune systems of host animals; and play important roles in many immune-related pathways. The systemic biological roles of gga-miR-10a-5p chicken microRNA on immune response were investigated in two necrotic enteritis (NE) induced chicken lines, Marek's disease (MD) resistant (line 6.3) and susceptible (line 7.2). We determined the expression patterns of gga-miR-10a in the intestinal mucosal layer of chickens upon NE induction, and identified the target genes (MyD88 , and SKP1) related to the host immune response to pathogens. We found that gga-miR-10a expression in the intestinal mucosal layer of MD-resistant chicken line 6.3 gga-miR-10a was significantly down-regulated (p < 0.01) during NE. Overexpression analysis of gga-miR-10a and reporter gene analysis using a wild- or mutant-type MyD88 3′ untranslated region (3′ UTR)-luciferase construct in chicken macrophage cell line HD11 and chicken fibroblast cell line OU2 showed that gga-miR-10a acted as a direct translational repressor of MyD8 8 by targeting the 3′ UTR of this gene. Furthermore, miR-10a indirectly negatively influenced the expression of signaling molecules related to the MyD88-dependent pathway, including TRAF6, TAK1, and NF-κB1 at both transcriptional and translational levels. Downstream of the MyD88-dependent pathway, several proinflammatory cytokines such as IL-1β, IFN-γ, IL-12p40, TNFSF15, and LITAF were down-regulated by overexpression of gga-miR-10a. These results suggest that gga-miR-10a is an important regulator of the Toll-like receptor signaling pathway. The findings of this study improve our understanding of the biological functions of miR-10a and the mechanisms underlying the TLR signaling pathway upon NE afflicted chickens, as well improving the overall understanding of the immune system function in domestic animals. • Gga-miR-10a significantly down-regulated in the resistant line 6.3 compared to the susceptible line 7.2 induced by NE. • Gga-miR-10a directly suppressed the expression level of its target gene MyD88. • Gga-miR-10a inhibited the expression of the signaling molecules as the downstream of MyD88-dependent pathway. • Gga-miR-10a indirectly repressed Th1, and pro-inflammatory cytokine production. [ABSTRACT FROM AUTHOR]