Comprehensive genome­wide analysis of the chicken heat shock protein family: identification, genomic organization, and expression profiles in indigenous chicken with highly pathogenic avian influenza infection.

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.

MicroRNA expression profiling in the lungs of genetically different Ri chicken lines against the highly pathogenic avian influenza H5N1 virus.

The highly pathogenic avian influenza (HPAI) virus triggers infectious diseases, resulting in pulmonary damage and high mortality in domestic poultry worldwide. This study aimed to analyze miRNA expression profiles after infection with the HPAI H5N1 virus in resistant and susceptible lines of Ri chickens.For this purpose, resistant and susceptible lines of Vietnamese Ri chicken were used based on the A/G allele of Mx and BF2 genes. These genes are responsible for innate antiviral activity and were selected to determine differentially expressed (DE) miRNAs in HPAI-infected chicken lines using small RNA sequencing. A total of 44 miRNAs were DE after 3 days of infection with the H5N1 virus. Computational program analysis indicated the candidate target genes for DE miRNAs to possess significant functions related to cytokines, chemokines, MAPK signaling pathway, ErBb signaling pathway, and Wnt signaling pathway. Several DE miRNA-mRNA matches were suggested to play crucial roles in mediating immune functions against viral evasion. These results revealed the potential regulatory roles of miRNAs in the immune response of the two Ri chicken lines against HPAI H5N1 virus infection in the lungs.

Chicken miR-148a-3p regulates immune responses against AIV by targeting the MAPK signalling pathway and IFN-γ.

MicroRNAs are involved in the immune systems of host animals and play essential roles in several immune-related pathways. In the current study, we investigated the systemic biological function of the chicken miRNA gga-miR-148a-3p on immune responses in chicken lines resistant and susceptible to HPAIV-H5N1. We found that gga-miR-148a expression in the lung tissue of H5N1-resistant chickens was significantly downregulated during HPAIV-H5N1 infection. Overexpression of gga-miR-148a and a reporter construct with wild type or mutant IFN-γ, MAPK11, and TGF-ß2 3' untranslated region (3' UTR)-luciferase in chicken fibroblasts showed that gga-miR-148a acted as a direct translational repressor of IFN-γ, MAPK11, and TGF-ß2 by targeting their 3' UTRs. Furthermore, miR-148a directly and negatively influenced the expression of signalling molecules related to the MAPK signalling pathway, including MAPK11, TGF-ß2, and Jun, and regulated antiviral responses through interferon-stimulated genes and MHC class I and class II genes by targeting IFN-γ. Downstream of the MAPK signalling pathway, several proinflammatory cytokines such as IL-1ß, IFN-γ, IL-6, TNF-α, IFN-ß, and interferon-stimulated genes were downregulated by the overexpression of gga-miR-148a. Our data suggest that gga-miR-148a-3p is an important regulator of the MAPK signalling pathway and antiviral response. These findings improve our understanding of the biological functions of gga-miR-148a-3p, the mechanisms underlying the MAPK signalling pathway, and the antiviral response to HPAIV-H5N1 infection in chickens as well as the role of gga-miR-148a-3p in improving the overall performance of chicken immune responses for breeding disease-resistant chickens.

Analysis of miRNA expression in the trachea of Ri chicken infected with the highly pathogenic avian influenza H5N1 virus.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) is considered a global threat to both human health and the poultry industry. MicroRNAs (miRNA) can modulate the immune system by affecting gene expression patterns in HPAIV-infected chickens. OBJECTIVES: To gain further insights into the role of miRNAs in immune responses against H5N1 infection, as well as the development of strategies for breeding disease-resistant chickens, we characterized miRNA expression patterns in tracheal tissues from H5N1-infected Ri chickens. METHODS: miRNAs expression was analyzed from two H5N1-infected Ri chicken lines using small RNA sequencing. The target genes of differentially expressed (DE) miRNAs were predicted using miRDB. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were then conducted. Furthermore, using quantitative real-time polymerase chain reaction, we validated the expression levels of DE miRNAs (miR-22-3p, miR-146b-3p, miR-27b-3p, miR-128-3p, miR-2188-5p, miR-451, miR-205a, miR-203a, miR-21-3p, and miR-200a-3p) from all comparisons and their immune-related target genes. RESULTS: A total of 53 miRNAs were significantly expressed in the infection samples of the resistant compared to the susceptible line. Network analyses between the DE miRNAs and target genes revealed that DE miRNAs may regulate the expression of target genes involved in the transforming growth factor-beta, mitogen-activated protein kinase, and Toll-like receptor signaling pathways, all of which are related to influenza A virus progression. CONCLUSIONS: Collectively, our results provided novel insights into the miRNA expression patterns of tracheal tissues from H5N1-infected Ri chickens. More importantly, our findings offer insights into the relationship between miRNA and immune-related target genes and the role of miRNA in HPAIV infections in chickens.

Chicken miR-26a-5p modulates MDA5 during highly pathogenic avian influenza virus infection.

MicroRNAs play crucial roles in immune-related pathways in host animals. In this study, we aimed to investigate the systemic biological function of gga-miR-26a-5p, a chicken miRNA, in the immune responses to HPAIV H5N1 infection in the Vietnamese Ri chicken line. Our results showed a significant downregulation in gga-miR-26a expression in the lung tissue of Ri chickens during HPAIV H5N1 infection. Overexpression of gga-miR-26a and the reporter construct, either containing the wildtype or mutant melanoma differentiation-associated protein 5 (MDA5) 3' untranslated region (3' UTR)-luciferase, into a chicken fibroblast cell line, revealed that gga-miR-26a can act as a direct translational repressor of MDA5 by targeting the 3' UTRs. Additionally, miR-26a negatively regulated the expression of the signaling molecules related to the MDA5 signaling pathway, including MDA5, mitochondrial antiviral-signaling (MAVS), interferon regulatory factor 7 (IRF7), p38 mitogen-activated protein kinases, and nuclear factor-kappa B (NF-κB). Moreover, downstream of the IRF7 and NF-κB signaling pathway, the proinflammatory cytokines such as IL-1ß, IFN-γ, IFN-α, IFN-ß, and the interferon-stimulated gene (Mx1) were, likewise, downregulated by the overexpression of gga-miR-26a. These findings suggest that gga-miR-26a-5p serves as an important regulator in the MDA5 signaling pathway and antiviral response. Overall, our results contribute to an improved understanding of the biological functions of gga-miR-26a-5p, alongside the mechanisms underlying the MDA5 signaling pathway, and the antiviral response to HPAIV-H5N1 infection in chickens.

HPAI-resistant Ri chickens exhibit elevated antiviral immune-related gene expression.

BACKGROUND: Highly pathogenic avian influenza viruses (HPAIVs) is an extremely contagious and high mortality rates in chickens resulting in substantial economic impact on the poultry sector. Therefore, it is necessary to elucidate the pathogenic mechanism of HPAIV for infection control. OBJECTIVE: Gene set enrichment analysis (GSEA) can effectively avoid the limitations of subjective screening for differential gene expression. Therefore, we performed GSEA to compare HPAI-infected resistant and susceptible Ri chicken lines. METHODS: The Ri chickens Mx(A)/BF2(B21) were chosen as resistant, and the chickens Mx(G)/BF2(B13) were selected as susceptible by genotyping the Mx and BF2 genes. The tracheal tissues of HPAIV H5N1 infected chickens were collected for RNA sequencing followed by GSEA analysis to define gene subsets to elucidate the sequencing results. RESULTS: We identified four differentially expressed pathways, which were immune-related pathways with a total of 78 genes. The expression levels of cytokines (IL-1ß, IL-6, IL-12), chemokines (CCL4 and CCL5), type interferons and their receptors (IFN-ß, IFNAR1, IFNAR2, and IFNGR1), Jak-STAT signaling pathway genes (STAT1, STAT2, and JAK1), MHC class I and II and their co-stimulatory molecules (CD80, CD86, CD40, DMB2, BLB2, and B2M), and interferon stimulated genes (EIF2AK2 and EIF2AK1) in resistant chickens were higher than those in susceptible chickens. CONCLUSIONS: Resistant Ri chickens exhibit a stronger antiviral response to HPAIV H5N1 compared with susceptible chickens. Our findings provide insights into the immune responses of genetically disparate chickens against HPAIV.

Exosome-mediated delivery of gga-miR-20a-5p regulates immune response of chicken macrophages by targeting IFNGR2, MAPK1, MAP3K5, and MAP3K14.

OBJECTIVE: This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. METHODS: Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 µg/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative real-time polymerase chain reaction. RESULTS: Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. CONCLUSION: The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

Small RNA sequencing and profiling of serum-derived exosomes from African swine fever virus-infected pigs.

African swine fever (ASF) virus (ASFV) is responsible for one of the most severe swine diseases worldwide, with a morbidity rate of up to 100%; no vaccines or antiviral medicines are available against the virus. Exosomal miRNAs from individual cells can regulate the immune response to infectious diseases. In this study, pigs were infected with an ASFV Pig/HN/07 strain that was classified as acute form, and exosomal miRNA expression in the serum of infected pigs was analyzed using small RNA sequencing (small RNA-seq). Twenty-seven differentially expressed (DE) miRNAs were identified in the ASFV-infected pigs compared to that in the uninfected controls. Of these, 10 were upregulated and 17 were downregulated in the infected pigs. All DE miRNAs were analyzed using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the DE miRNAs were found to be highly involved in T-cell receptor signaling, cGMP-PKG signaling, Toll-like receptor, MAPK signaling, and mTOR signaling pathways. Furthermore, the Cytoscape network analysis identified the network of interactions between DE miRNAs and target genes. Finally, the transcription levels of four miRNA genes (ssc-miR-24-3p, ssc-miR-130b-3p, ssc-let-7a, and ssc-let-7c) were examined using quantitative real-time PCR (qRT-PCR) and were found to be consistent with the small RNA-seq data. These DE miRNAs were associated with cellular genes involved in the pathways related to immune response, virus-host interactions, and several viral genes. Overall, our findings provide an important reference and improve our understanding of ASF pathogenesis and the immune or protective responses during an acute infection in the host.

African swine fever is a viral disease caused by African swine fever virus (ASFV) which induces a big threat to the pig industry in the world. To date, there are no vaccines or antiviral medicines against the ASFV. Therefore, it is important to improve the understanding of the pathogenesis of ASFV and host­pathogen interaction using miRNA that may regulate genes related to the immune system. This study aimed to investigate the differentially expressed (DE) miRNA in serum-derived exosomes from African swine fever virus infected pigs. We successfully infected pigs with an ASFV Pig/HN/07 strain and identified the DE miRNAs in serum-derived exosomes using small RNA sequencing. Our results showed that total of 27 miRNAs were differentially expressed in serum-derived exosomes from ASFV-infected pigs. We analyzed the small RNA sequencing results using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and found that most DE miRNA may regulate the expression of genes related with the immune response pathway (T-cell receptor signaling pathway, cGMP-PKG signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, etc.).

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

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-1ß. 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-1ß antibody. ChIL-1R2 expression can be induced by a viral infection, and may be regulated through NF-κB/JNK/MEK-mediated signaling pathways.

Genome­wide identification, organization, and expression profiles of the chicken fibroblast growth factor genes in public databases and Vietnamese indigenous Ri chickens against highly pathogenic avian influenza H5N1 virus infection.

OBJECTIVE: Fibroblast growth factors (FGFs) play critical roles in embryo development, and immune responses to infectious diseases. In this study, to investigate the roles of FGFs, we performed genome-wide identification, expression, and functional analyses of FGF family members in chickens. METHODS: Chicken FGFs genes were identified and analyzed by using bioinformatics approach. Expression profiles and Hierarchical cluster analysis of the FGFs genes in different chicken tissues were obtained from the genome-wide RNA-seq. RESULTS: A total of 20 FGF genes were identified in the chicken genome, which were classified into seven distinct groups (A-F) in the phylogenetic tree. Gene structure analysis revealed that members of the same clade had the same or similar exon-intron structure. Chromosome mapping suggested that FGF genes were widely dispersed across the chicken genome and were located on chromosomes 1, 4-6, 9-10, 13, 15, 28, and Z. In addition, the interactions among FGF proteins and between FGFs and mitogen­activated protein kinase (MAPK) proteins are limited, indicating that the remaining functions of FGF proteins should be further investigated in chickens. Kyoto encyclopedia of genes and genomes pathway analysis showed that FGF gene interacts with MAPK genes and are involved in stimulating signaling pathway and regulating immune responses. Furthermore, this study identified 15 differentially expressed genes (DEG) in 21 different growth stages during early chicken embryo development. RNA-sequencing data identified the DEG of FGFs on 1- and 3-days post infection in two indigenous Ri chicken lines infected with the highly pathogenic avian influenza virus H5N1 (HPAIV). Finally, all the genes examined through quantitative real-time polymerase chain reaction and RNA-Seq analyses showed similar responses to HPAIV infection in indigenous Ri chicken lines (R2 = 0.92- 0.95, p<0.01). CONCLUSION: This study provides significant insights into the potential functions of FGFs in chickens, including the regulation of MAPK signaling pathways and the immune response of chickens to HPAIV infections.

Comparative analysis of exosomal miRNAs derived from lipopolysaccharide and polyinosinic-polycytidylic acid-stimulated chicken macrophage cell line.

Exosomes play important roles in cellular communication by delivering exosomal proteins and nucleic acid molecules to cells. In particular, exosomal miRNAs can modulate various biological processes in recipient cells by repressing target gene expression. In this study, to identify the composition of exosomal miRNAs and their regulatory mechanisms against bacterial and viral infections, profiles of exosomal miRNAs from lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly(I:C))-stimulated chicken macrophage cell line (HD11) were analyzed by small RNA sequencing. Exosomes were purified after stimulation with LPS (1 µg/mL) and poly(I:C) (50 µg/mL) for 24 h. Then, exosomal RNA were analyzed for small RNA sequencing using the HiSeq 2500 System. Thirty six differentially expressed miRNAs (DE miRNAs) were obtained by comparing LPS-stimulated exosomes (LPS-EXO) and unstimulated exosomes (CTRL-EXO), 42 DE miRNAs in poly(I:C)-stimulated exosomes (POLY-EXO) and CTRL-EXO, and 45 DE miRNAs in LPS-EXO and POLY-EXO. Target genes of DE miRNAs were predicted using miRDB and TargetScan. KEGG pathway analysis showed that most of the target genes were related to mitogen-activated protein kinase and Wnt signaling pathways. Moreover, results of qRT-PCR for miRNAs (gga-miR-142-3p, gga-miR-19a-3p, gga-miR-21-3p, gga-miR-301a-3p, gga-miR-338-3p, and gga-miR-3523) were consistent with the sequencing results. This study will provide knowledge about immuno-regulatory mechanisms of exosomal miRNAs derived from macrophages against pathological insults such as bacterial and viral infections.

Profiling and analysis of exosomal miRNAs derived from highly pathogenic avian influenza virus H5N1-infected White Leghorn chickens.

Exosomes are small cell membrane-derived vesicles; they play important roles as mediators of cell-to-cell communication via delivery of their contents, such as proteins and microRNAs (miRNAs). In particular, exosomal miRNAs regulate the gene expression of recipient cells by inhibiting the expression of target mRNAs. In this study, we investigated the miRNA expression profiles of highly pathogenic avian influenza virus (HPAIV) H5N1-infected White Leghorn chickens and analyzed the functions of their target genes. After 3 d of infection with A/chicken/Vietnam/NA-01/2019 (H5N1), exosomes were isolated from the blood serum of White Leghorn chickens for small RNA sequencing. We accordingly identified 10 differentially expressed miRNAs (DE miRNAs; 5 upregulated and 5 downregulated) by comparing the exosomes derived from infected and noninfected chickens. The target genes of DE miRNAs were predicted using miRDB and TargetScan for Gene Ontology and KEGG pathway enrichment analyses. A majority of the target genes was found to be associated with the MAPK signaling pathway; several immune-related genes were identified as being regulated by these DE miRNAs. Moreover, we predicted DE miRNA binding sites in HPAIV RNA segments using the RNAhybrid algorithm. The findings of this study provide a theoretical basis for gaining insights into the regulatory mechanisms of exosomal miRNAs in response to HPAIV H5N1 infection and the identification of novel vaccine candidates.

Influenza A pathway analysis of highly pathogenic avian influenza virus (H5N1) infection in genetically disparate Ri chicken lines.

Influenza A/H5N1 virus is a highly pathogenic (HPAIV) and contagious zoonotic virus that can be transmitted to humans. In the present study, infection with this virus and differential gene expression analyses were carried out with genetically resistant and susceptible Ri chicken lines that are native to Vietnam. A total of 20 four-week-old Ri chickens from each line were inoculated with the highly pathogenic H5N1 avian influenza virus (5 chickens/group). On day 3 post-infection, the total tracheal RNA was sequenced. Differentially expressed genes in the influenza A pathway, including signaling pathway-related genes, were validated by reverse transcription quantitative real-time PCR (RT-qPCR). The resistant and susceptible lines showed significant differences in gene expression and multiple biological functions. Compared to expression in the susceptible line, in the resistant line, the expression of MX1, STAT1, IRF7, and TLR3 was upregulated, while that of BF1 was downregulated. Genes from the interferon (IFN) and cytokine families, which regulate the immune system, were highly expressed in the HPAIV infected resistant line. Finally, significant differences were observed in the expression of genes encoding components of the Jak-STAT and TLR signaling pathways between the two chicken lines. Collectively, our findings suggest that HPAIV-resistant and -susceptible Ri chicken lines differed in immunity upon infection. Understanding the regulation of immune pathways against HPAIV will help to better understand the mechanisms of immune regulation in chickens.

Exosomes from H5N1 avian influenza virus-infected chickens regulate antiviral immune responses of chicken immune cells.

Exosomes (membrane-derived vesicles) enable intracellular communication by delivering lipids, proteins, DNA, and RNA from one cell to another. Highly pathogenic avian influenza virus (HPAIV) H5N1 causes considerable economic loss in the poultry industry and poses a public health concern. The host innate immune system defends against H5N1 infection by activating antiviral immune responses. This study aimed to demonstrated that immunomodulatory effects of exosomes from HPAIV H5N1-infected White Leghorn chickens on chicken macrophages, fibroblasts, T cell, and B cell lines. The expression of type I interferons (IFN-α and -ß) were highly upregulated in immune-related cell lines after treatment with exosomes derived from H5N1-infected chickens. Levels of pro-inflammatory cytokines, such as IFN-γ, IL-1ß, and CXCL8, were also elevated by the exosomes. The mitogen-activated protein kinase (MAPK) signaling pathway was stimulated in immune-related cells by such exosomes via phosphorylation of extracellular regulated kinases 1/2 and p38 signaling molecules. Furthermore, the H5N1 viral proteins, nucleoprotein (NP) and non-structural protein (NS1), were packaged in exosomes and successfully transferred to non-infected immune-related cells. Therefore, exosomes from H5N1-infected chickens induced pro-inflammatory cytokine expression and stimulated the MAPK signaling pathway by delivering key viral proteins. These findings would aid better understanding of the mechanism underlying the modulation of antiviral immune responses of host immune-related cells by viral-protein-carrying exosomes and support their further application as a novel exosome-based H5N1 AIV vaccine platform.

The highly pathogenic H5N1 avian influenza virus induces the mitogen-activated protein kinase signaling pathway in the trachea of two Ri chicken lines.

OBJECTIVE: The highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the mitogen-activated protein kinase (MAPK) signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines. METHODS: Resistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, the tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing. RESULTS: A gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. A total of 1,794 DEGs were observed between control and H5N1-infected resistant Ri chickens, 432 DEGs between control and infected susceptible Ri chickens, and 1,202 DEGs between infected susceptible and infected resistant Ri chickens. The expression levels of MAPK signaling pathway-related genes (including MyD88, NF-κB, AP-1, c-fos, Jun, JunD, MAX, c-Myc), cytokines (IL-1ß, IL-6, IL-8), type I interferons (IFN-α, IFN-ß), and IFN-stimulated genes (Mx1, CCL19, OASL, and PRK) were higher in H5N1-infected than in non-infected resistant Ri chickens. MyD88, Jun, JunD, MAX, cytokines, chemokines, IFNs, and IFN-stimulated expressed genes were higher in resistant-infected than in susceptible-infected Ri chickens. CONCLUSION: Resistant Ri chickens showed higher antiviral activity compared to susceptible Ri chickens, and H5N1-infected resistant Ri chickens had immune responses and antiviral activity (cytokines, chemokines, interferons, and IFN-stimulated genes), which may have been induced through the MAPK signaling pathway in response to H5N1 infection.

Cytokine-cytokine receptor interactions in the highly pathogenic avian influenza H5N1 virus-infected lungs of genetically disparate Ri chicken lines.

OBJECTIVE: The highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry as well as the economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for HPAIV resistance. Therefore, in this study, we investigated gene expression related to cytokine-cytokine receptor interactions by comparing resistant and susceptible Ri chicken lines for avian influenza virus infection. METHODS: Ri chickens of resistant (Mx/A; BF2/B21) and susceptible (Mx/G; BF2/B13) lines were selected by genotyping the Mx dynamin like GTPase (Mx) and major histocompatibility complex class I antigen BF2 genes. These chickens were then infected with influenza A virus subtype H5N1, and their lung tissues were collected for RNA sequencing. RESULTS: In total, 972 differentially expressed genes (DEGs) were observed between resistant and susceptible Ri chickens, according to the gene ontology and Kyoto encyclopedia of genes and genomes pathways. In particular, DEGs associated with cytokine-cytokine receptor interactions were most abundant. The expression levels of cytokines (interleukin-1ß [IL-1ß], IL-6, IL-8, and IL-18), chemokines (C-C Motif chemokine ligand 4 [CCL4] and CCL17), interferons (IFN-γ), and IFN-stimulated genes (Mx1, CCL19, 2'-5'-oligoadenylate synthaselike, and protein kinase R) were higher in H5N1-resistant chickens than in H5N1-susceptible chickens. CONCLUSION: Resistant chickens show stronger immune responses and antiviral activity (cytokines, chemokines, and IFN-stimulated genes) than those of susceptible chickens against HPAIV infection.

Immunomodulatory effects of poly(I:C)-stimulated exosomes derived from chicken macrophages.

Exosomes are small membrane vesicles that contain proteins and nucleic acids derived from secretory cells and mediate intracellular communication. Immune cell-derived exosomes regulate immune responses and gene expression of recipient cells. Macrophages recognize viral dsRNA via Toll-like receptor 3, thereby inducing the activation of transcription factors such as interferon regulatory factor 3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In this study, we aimed to identify the immunomodulatory functions of exosomes derived from chicken macrophages (HD11) stimulated with polyinosinic-polycytidylic acid (poly[I:C]); exosomes were then delivered into HD11 cells and CU91 chicken T cells. Exosomes purified from poly(I:C)-activated macrophages stimulated the expression of type I interferons, proinflammatory cytokines, anti-inflammatory cytokines, and chemokines in HD11 and CU91 cells. Moreover, poly(I:C)-stimulated exosomes induced the NF-κB signaling pathway by phosphorylating TAK1 and NF-κB1. Therefore, we suggest that after the activation of Toll-like receptor 3 ligands following infection with dsRNA virus, chicken macrophages regulate the immune response of naive macrophages and T cells through the NF-κB signaling pathway. Furthermore, poly(I:C)-activated exosomes can be potentially utilized as immunostimulators.

Exosomal miRNA profiling from H5N1 avian influenza virus-infected chickens.

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.

Exosomes of lipopolysaccharide-stimulated chicken macrophages modulate immune response through the MyD88/NF-κB signaling pathway.

Exosomes are small membrane-extracellular vesicles produced from multivesicular bodies and play a role in cell-to-cell signaling. Exosomes from immune cells can regulate immune responses of recipient cells by releasing their contents. In the immune system, macrophages recognize lipopolysaccharides (LPSs) of gram-negative bacteria by toll-like receptor 4 (TLR4) and intracellular pathways, such as NF-κB pathway, are activated, inducing proinflammatory cytokine expression. However, no studies have investigated the functions of exosomes in chicken macrophages. The purpose of this study was to demonstrate the immunoregulatory functions of LPS-activated exosomes in chicken immune systems. Therefore, chicken macrophages cells (HD11) were activated with LPS, and exosomes were purified. The LPS-activated exosomes enhanced the gene expression of cytokines and chemokines, including IL-1ß, IFN-γ, IFN-α, IL-4, CCL4, CCL17, and CCL19, in naive chicken macrophages. Furthermore, LPS-activated exosomes induced the MyD88/NF-κB signaling pathway. Therefore, as an immune response against gram-negative bacterial infection, LPS-activated chicken macrophages can release exosomes that are delivered to inactivated macrophages by regulating the expression of immune-related genes and the MyD88/NF-κB signaling pathway. In the future, LPS-stimulated exosomes may be utilized as an immune stimulator.

Chicken avian ß-defensin 8 modulates immune response via the mitogen-activated protein kinase signaling pathways in a chicken macrophage cell line.

Defensins are antimicrobial peptides composed of 3 conserved disulfide bridges, a ß-sheet, and both hydrophobic and cationic amino acids. In this study, we aimed to demonstrate the immunomodulation role of avian ß-defensin 8 (AvBD8) in a chicken macrophage cell line. Chicken AvBD8 stimulated the expression of proinflammatory cytokines (IL-1ß, interferon gamma, and IL-12p40) and chemokines (CCL4, CXCL13, and CCL20) in macrophages. Furthermore, by Western blotting and immunocytochemistry, we confirmed that AvBD8 activated the mitogen-activated protein kinase signaling pathway via extracellular regulated kinases 1/2 and p38 signaling molecules. Overall, AvBD8 plays a crucial role in host defense as not only an antimicrobial peptide but also an immunomodulator by activating the mitogen-activated protein kinase signaling pathway and inducing the expression of proinflammatory cytokines and chemokines.