Macrophage Cell Line HD11 Research Articles

H9N2 Avian Influenza Virus Downregulates FcRY Expression in Chicken Macrophage Cell Line HD11 by Activating the JNK MAPK Pathway.

The H9N2 avian influenza virus causes reduced production performance and immunosuppression in chickens. The chicken yolk sac immunoglobulins (IgY) receptor (FcRY) transports from the yolk into the embryo, providing offspring with passive immunity to infection against common poultry pathogens. FcRY is expressed in many tissues/organs of the chicken; however, there are no reports investigating FcRY expression in chicken macrophage cells, and how H9N2-infected HD11 cells (a chicken macrophage-like cell line) regulate FcRY expression remains uninvestigated. This study used the H9N2 virus as a model pathogen to explore the regulation of FcRY expression in avian macrophages. FcRY was highly expressed in HD11 cells, as shown by reverse transcription polymerase chain reactions, and indirect immunofluorescence indicated that FcRY was widely expressed in HD11 cells. HD11 cells infected with live H9N2 virus exhibited downregulated FcRY expression. Transfection of eukaryotic expression plasmids encoding each viral protein of H9N2 into HD11 cells revealed that nonstructural protein (NS1) and matrix protein (M1) downregulated FcRY expression. In addition, the use of a c-jun N-terminal kinase (JNK) activator inhibited the expression of FcRY, while a JNK inhibitor antagonized the downregulation of FcRY expression by live H9N2 virus, NS1 and M1 proteins. Finally, a dual luciferase reporter system showed that both the M1 protein and the transcription factor c-jun inhibited FcRY expression at the transcriptional level. Taken together, the transcription factor c-jun was a negative regulator of FcRY, while the live H9N2 virus, NS1, and M1 proteins downregulated the FcRY expression through activating the JNK signaling pathway. This provides an experimental basis for a novel mechanism of immunosuppression in the H9N2 avian influenza virus.

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

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. 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. 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. 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.

Beta-glucans induce cellular immune training and changes in intestinal morphology in poultry.

Beta-glucans are known as biological response modifiers due to their ability to activate the immune system. This research aimed to determine the efficacy and safety of feeding beta-glucans from various sources on the immune status and intestinal morphology of chickens. To this end we used in vitro and in vivo set-ups. In the in vitro set-up the chicken macrophage cell line HD-11 was used to measure the response of the chicken immune cells to beta-glucans extracted from algae and mushrooms on immune-related gene expression and associated activities. Additionally, we conducted two in vivo experiments using either beta-glucans extracted from yeast or mix of yeast and mushrooms beta-glucans as part of the chicks feed in order to test their effects on the chick intestinal morphology. In the in vitro set-up exposure of HD-11 cells to a concentration of 1 mg/ml of algae and mushroom beta-glucans resulted in significantly higher expression of 6 genes (TNFα, IL4, IL6, IL8, IL10, and iNOS2) compared to control. The release of nitrite oxide (NO) to the medium after exposure of HD-11 cells to mushrooms or algae beta-glucans was significantly increased compared to control. Additionally, significantly increased phagocytosis activity was found after exposure of the cells to algae and mushroom beta-glucans. In the in vivo set-up we observed that the length of the villi and the number of goblet cells in the ileum and the jejunum in the beta-glucan fed chicks were significantly augmented compared to control, when the chicks were fed with either yeast or yeast and mushroom beta-glucans mix. In conclusion, dietary supplementation of poultry with beta-glucan exerts significant and positive effects on immune activity and the intestinal morphology in poultry.

Screening of Optimal CpG-Oligodeoxynucleotide for Anti-Inflammatory Responses in the Avian Macrophage Cell Line HD11.

) have been shown to possess immunostimulatory features in both mammals and birds. However, compared to their proinflammatory effects, little is known about the anti-inflammatory responses triggered by CpG-ODN in avian cells. Hence, in this study, the anti-inflammatory response in the chicken macrophage cell line HD11 was characterized under stimulation with five types of CpG-ODNs: CpG-A1585, CpG-AD35, CpG-B1555, CpG-BK3, and CpG-C2395. Single-stimulus of CpG-B1555, CpG-BK3, or CpG-C2395 induced interleukin (IL)-10 expression without causing cell injury. The effects of pretreatment with CpG-ODNs before subsequent lipopolysaccharide stimulation were also evaluated. Interestingly, pretreatment with only CpG-C2395 resulted in high expression levels of IL-10 mRNA in the presence of lipopolysaccharide. Finally, gene expression analysis of inflammation-related cytokines and receptors revealed that pre-treatment with CpG-C2395 significantly reduced the mRNA expression of tumor necrosis factor-α, IL-1β, IL-6, and Toll-like receptor 4. Overall, these results shed light on the anti-inflammatory responses triggered by CpG-C2395 stimulation through a comparative analysis of five types of CpG-ODNs in chicken macrophages. These results also offer insights into the use of CpG-ODNs to suppress the expression of proinflammatory cytokines, which may be valuable in the prevention of avian infectious diseases in the poultry industry.

Campylobacter jejuni infection induces dynamic expression of avian host defense peptides in vitro and in vivo

Campylobacter jejuni is considered as the leading cause of worldwide foodborne bacterial gastroenteritis. Chicken is the main reservoir of C. jejuni. Avian innate immune responses to C. jejuni remain poorly defined. Chicken host defense peptides (HDPs) are the major components of avian innate immune system. This study aimed to characterize the chicken HDPs responses to C. jejuni in vitro and in vivo. In the HD11 macrophage cell line, the HDPs, including AvBD1–2, CATH1–3, AvBD7, AvBD4, and AvBD6, were relatively higher expressed in untreated cells, whereas the expressions were suppressed after C. jejuni infection. In contrast, C. jejuni infection significantly increased the expression of the lower expressed HDPs, such as AvBD3, AvBD5, AvBD8–14, and CATHB1, in untreated cells. In the chicken challenge experiment, the immune tissues of spleens and cecal tonsils were collected from C. jejuni-infected and uninfected chickens at 1, 3 and 15 day post inoculation (DPI). In spleens of C. jejuni-infected chickens, only AvBD14 expression was elevated at 1 DPI. The majority of avian HDPs were significantly up-regulated at 3 DPI and dramatically decreased to the levels of uninfected controls at 15 DPI. In chicken cecal tonsils, only AvBD9 and AvBD14 were significantly up-regulated at 1 DPI with C. jejuni infection. Collectively, C. jejuni infection induced dynamic expression of chicken HDPs in both macrophage HD11 and immune tissues of chickens. Suppression of chicken HDPs expression may be an evasion strategy of C. jejuni for persistent colonization in chicken intestine by circumventing the chicken immune system.

The Outer Membrane Vesicles of Salmonella enterica Serovar Typhimurium Activate Chicken Immune Cells through Lipopolysaccharides and Membrane Proteins.

Salmonella is a common pathogen which can secrete outer membrane vesicles (OMVs). However, the effect of OMVs from Salmonella enterica Serovar Typhimurium (S. Typhimurium) of poultry origin on cells of the chicken innate immune system is not well known. In this study, S. Typhimurium OMVs were first isolated from three different poultry strains of Salmonella, Salmonella CVCC542, SALA, and SALB. In order to investigate the effect of OMVs on the maturation of monocytes into macrophages, both bone marrow-derived (BMD) monocytes and macrophage cell line HD11 cells were used. OMVs promoted the formation of monocyte dendrites in both types of cells, enabled BMD cells to become larger, and stimulated expression of LPS-induced TNF-αfactor (LITAF), IL-6, and inducible nitric oxide synthase (iNOS) genes in HD11 cells. These results demonstrated the capability of OMVs to promote the development of chicken monocytes into macrophages and the maturation of macrophages. In order to study the effect of OMVs on the phagocytosis of macrophages, chicken spleen-derived monocytes and HD11 cells were used. Phagocytosis of FITC-Salmonella and FITC-dextran by these two types of cells was enhanced after stimulation with OMVs. To determine which components in OMVs were responsible for the above observed results, OMVs were treated with proteinase K(PK) or polymyxin B (PMB). Both treatments reduced the phagocytosis of FITC-Salmonella by HD11 cells and chicken spleen mononuclear cells and reduced the secretion of IL-1β, LITAF, and IL-6 cytokines. These results demonstrated that Salmonella OMVs activated chicken macrophages and spleen mononuclear cells and the activation was achieved mainly through lipopolysaccharides and membrane proteins.

Hypervirulent FAdV-4 infection induces activation of the NLRP3 inflammasome in chicken macrophages

Fowl adenovirus serotype 4 (FAdV-4) is the primary causative agent of hepatitis–hydropericardium syndrome (HHS) causing great economic losses to the world poultry industry. The exact factors responsible for the pathogenesis of hypervirulent FAdV-4 have not been completely elucidated. Hypervirulent FAdV-4 infection induces inflammatory damages in accompany with a high level of proinflammatory interleukin-1 beta (IL-1β) secretion in a variety of organs. Investigation of the mechanisms underlying hypervirulent FAdV-4-induced IL-1β secretion would contribute to understanding of the pathogenesis of FAdV-4. Here, we investigated whether FAdV-4 infection activates NLRP3 inflammasome in chicken macrophage cell line HD11. The results showed that stimulation of HD11 with hypervirulent FAdV-4 induced NLRP3- and Caspase-1-dependent secretion of IL-1β. Genetic knockdown of NLRP3 or Caspase-1 expression, a critical component of inflammasome, significantly downregulated IL-1β expression, indicating that activation of the NLRP3 inflammasome contributed to the FAdV-4-induced IL-1β secretion. Moreover, ATP signaling and potassium efflux were involved in the process of NLRP3 inflammasome activation. Our data indicated that hypervirulent FAdV-4 infection induces the activation of NLRP3 inflammasome and followed by massive secretion of IL-1β of macrophages, which thereby contribute to the inflamed lesion of tissues.

Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens.

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

DNA aptamer-based rolling circle amplification product as a novel immunological adjuvant

Several agonists to CD40 have shown to induce acquired immune responses. Here, we developed and evaluated the rolling circle amplification (RCA) products that are based on anti-CD40 DNA aptamers as a novel vaccine adjuvant. First, we developed DNA aptamers with specific binding affinity to chicken CD40 extra domain (chCD40ED). Next, we prepared the RCA products that consist of these aptamers to increase the spanning space and overall binding affinity to chCD40ED. Using 8 DNA aptamer candidates, 4 aptamer-based RCA products (aptamer RCAs) were generated, each consisting of two distinct aptamers. We demonstrated that all 4 aptamer RCAs significantly induced the signal transduction in chicken HD11 macrophage cell line (p < 0.05). Finally, we conjugated one of the aptamer RCAs (Aptamer RCA II) to M2e epitope peptide of influenza virus as a model hapten, and the immune complex was injected to chickens. Aptamer RCA II stimulated anti-M2e IgG antibody production to the level significantly higher as compared to the control (M2e epitope alone; p < 0.05). The results of our work suggest that aptamer RCA is a novel platform to boost the efficacy of vaccines, which might find broad applications to other antigens beyond M2e epitope evaluated in this study using chicken infection model.

Antagonistic effect of N-acetyl-L-cysteine against cadmium-induced cytotoxicity and abnormal immune response on chicken peritoneal macrophages

Cadmium is a highly toxic metal threatening human and animal health. N-acetyl-L-cysteine (NAC) was reported to play a positive role in disease treatment and immune regulation. The present study aimed to explore the effect of NAC administration on Cd-induced cytotoxicity and abnormal immune response on chicken peritoneal macrophages. Peritoneal macrophages isolated from Isa Brown male chickens were exposed to CdCl2 (20 or 50 μM) and/or NAC (500 μM) for different time periods. Results showed that Cd caused dose-dependent damage on chicken peritoneal macrophages characterized by morphologic and ultrastructural alterations, increased cell apoptosis, reactive oxygen species accumulation and mitochondrial injury. Cd exposure inhibited phagocytic activity of chicken peritoneal macrophages, and promoted transcriptional status of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in both unactivated macrophages and cells in response to lipopolysaccharide (LPS) stimuli. Pretreatment with 500 μM NAC did not affect growth of normal chicken peritoneal macrophages, while remarkably inhibiting Cd-caused cell death, oxidative stress, and mitochondrial membrane depolarization. NAC pretreatment significantly prevented intracellular Cd2+ accumulation in the Cd-exposed macrophages. Inhibitory effects of NAC on Cd-induced ROS accumulation and mitochondrial injury on chicken macrophages were confirmed in HD-11 macrophage cell line. In addition, NAC pretreatment promoted the phagocytic activity of Cd-exposed chicken peritoneal macrophages, and significantly inhibited expression of pro-inflammatory factors (IL-1β, IL-6 and TNF-α) in both Cd-exposed macrophages and Cd-treated cells in response to LPS stimuli. In conclusion, the present study firstly demonstrated the antagonistic effect of NAC against Cd-caused damage and abnormal immune response on chicken peritoneal macrophages. Protective effect of NAC on chicken macrophages was highly related to its suppression on Cd-induced ROS overproduction, pro-inflammatory reaction and intracellular Cd2+ accumulation.

Saccharomyces boulardii attenuates inflammatory response induced by Clostridium perfringens via TLR4/TLR15-MyD8 pathway in HD11 avian macrophages

Macrophages are professional phagocytic cells that play a critical role in initiating immune responses by presenting antigen and phagocytic clearance. The macrophages can be targeted for immunomodulation by beneficial microbes, such as probiotics. The aim of this study is to investigate the protective effect of Saccharomyces boulardii against Clostridium perfringens infection in avian macrophage cell line HD11. In this study, HD11 macrophages were prestimulated with S. boulardii for 6 h and then infected with C. perfringens for 3 h. Results showed that S. boulardii enhanced phagocytosis and bactericidal capacity against C. perfringens by HD11 cells. The S. boulardii effectively promoted the mRNA expression of CD80, CD83, and CD197 cell-surface molecules in C. perfringens-infected HD11 cells. Moreover, we found that prestimulation with S. boulardii reduced the mRNA expression of CD40, toll-like receptor [TLR] 4, and TLR15 induced by C. perfringens and thereby downregulated the mRNA expression of myeloid differentiation primary response 88, TNF receptor associated factor 6, nuclear factor kappa-B p65 subunit, and c-Jun N-terminal kinase genes in HD11 cells. The upregulation of cytokines (interleukin [IL]-6, tumor necrosis factor alpha, and IL-10) and inducible nitric oxide synthase mRNA expression in C. perfringens-infected HD11 cells were noticeably inhibited by S. boulardii pretreatment. Conclusively, these results might provide a new insight into the role of S. boulardii in regulating avian immune defense against C. perfringens invasion and immune escape.

EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058

Extraintestinal pathogenic Escherichia coli (ExPEC) shows an enhanced ability to cause infection outside the intestinal tract. Avian pathogenic E. coli (APEC), one type of ExPEC, causes avian colibacillosis, a disease of significant economic importance to poultry producers worldwide that is characterized by systemic infection. Some ExPEC strains as well as other pathogenic enterobacteria produce enterobactin, a catecholate siderophore used to sequester iron during infection. Here, we showed that disruption of enterobactin efflux via outer membrane protein TolC significantly decreased the pathogenicity of APEC strain E058. Furthermore, colonization and persistence assays performed using a chicken infection model showed that the ΔtolC mutant was obviously attenuated (p˂0.001). In contrast, disruption of enterobactin synthesis gene entE and/or the inner membrane transporter gene entS had little effect on pathogenicity. Analysis of growth kinetics revealed a significant reduction in the growth of triple mutant strain E058ΔentEΔentSΔtolC in iron-deficient medium compared with the wild-type strain (p˂0.001), while no growth impairment was noted for the E058ΔtolC mutant in either Luria-Bertani broth or iron-deficient medium. The E058ΔentEΔentSΔtolC mutant also showed significantly decreased virulence compared with single mutant strain E058ΔtolC. Low-copy complementation of strains E058ΔtolC and E058ΔentEΔentSΔtolC with plasmid-borne tolC restored virulence to wild-type levels in the chicken infection model. Macrophage infection assays showed that ingestion of E058ΔtolC by macrophage cell line HD11 cells was reduced compared with ingestion of the E058ΔentEΔentSΔtolC mutant. However, no significant differences were observed between the mutants and the wild-type in a chicken serum resistance assay. Together, these results suggest that EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058 in an iron-deficient environment.

MicroRNA gga-miR-10a-mediated transcriptional regulation of the immune genes in necrotic enteritis afflicted chickens

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 MyD88 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.

Salmonella-containing vacuole development in avian cells and characteristic of cigR in Salmonella enterica serovar Pullorum replication within macrophages

Salmonella enterica serovar Pullorum (S. Pullorum) is one of the host-restricted serotypes causing systemic infection in poultry. Survival in macrophages is one of the mechanisms underlying the persistent infection of S. Pullorum in hosts. Formation of Salmonella-containing vacuole (SCV) is essential for bacteria to be concealed in macrophages. In this study, confocal microscopy was applied to detect the SCV development by S. Pullorum in the chicken hepatocellular carcinoma cell line LMH and the macrophage cell line HD-11, respectively. The results showed that macrophages were more appropriate for the SCV maturation during S. Pullorum infection compared to epithelial cells. We evaluated the role of the CigR effector protein in the formation of SCVs. CigR is a membrane-binding protein, which is one of the type III secretion system 2 (T3SS2) effectors encoded within Salmonella pathogenicity island 3 (SPI3). The deletion of cigR in S. Typhimurium and S. Pullorum enhanced bacterial virulence to both mice and chickens. To analyze the influence of CigR on the SCV development during S. Pullorum infection, this study compared the formation of SCVs by using S. Pullorum C79-13 and the cigR deleted strain C79-13ΔcigR. Compared to the wild type strain, the loss of cigR gene in strain C79-13ΔcigR caused a four-fold increase in the recruitment ability of the SCV marker protein Rab7 in infected macrophages after 30 min, 5 h, and 16 h post-infection. In addition, infection and proliferation of strain C79-13ΔcigR in the avian macrophage cell line HD-11 was higher than that of the wild type strain. Our findings suggest that CigR is an anti-virulence effector inhibiting replication of S. Pullorum and SCV development in macrophages.

Enhanced Replication of Virulent Newcastle Disease Virus in Chicken Macrophages Is due to Polarized Activation of Cells by Inhibition of TLR7.

Newcastle disease (ND), caused by infections with virulent strains of Newcastle disease virus (NDV), is one of the most important infectious disease affecting wild, peridomestic, and domestic birds worldwide. Vaccines constructed from live, low-virulence (lentogenic) viruses are the most accepted prevention and control strategies for combating ND in poultry across the globe. Avian macrophages are one of the first cell lines of defense against microbial infection, responding to signals in the microenvironment. Although macrophages are considered to be one of the main target cells for NDV infection in vivo, very little is known about the ability of NDV to infect chicken macrophages, and virulence mechanisms of NDV as well as the polarized activation patterns of macrophages and correlation with viral infection and replication. In the present study, a cell culture model (chicken bone marrow macrophage cell line HD11) and three different virulence and genotypes of NDV (including class II virulent NA-1, class II lentogenic LaSota, and class I lentogenic F55) were used to solve the above underlying questions. Our data indicated that all three NDV strains had similar replication rates during the early stages of infection. Virulent NDV titers were shown to increase compared to the other lentogenic strains, and this growth was associated with a strong upregulation of both pro-inflammatory M1-like markers/cytokines and anti-inflammatory M2-like markers/cytokines in chicken macrophages. Virulent NDV was found to block toll-like receptor (TLR) 7 expression, inducing higher expression of type I interferons in chicken macrophages at the late stage of viral infection. Only virulent NDV replication can be inhibited by pretreatment with TLR7 ligand. Overall, this study demonstrated that virulent NDV activates a M1-/M2-like mixed polarized activation of chicken macrophages by inhibition of TLR7, resulting in enhanced replication compared to lentogenic viruses.

Chicken IL-17A is expressed in αβ and γδ T cell subsets and binds to a receptor present on macrophages, and T cells

IL-17A as important cytokine in host defense has been analysed intensively and various homologous have been identified. To further gain insight into the functional properties of chicken (gg) IL-17A its expression profile was analysed by intracellular cytokine staining. In splenocytes and peripheral blood mononuclear cells gg IL-17A was detected in subsets of CD4+ T cells and γδ T cells. In contrast the gg IL-17A producing populations in intestinal intraepithelial lymphocytes were characterized as either CD3+CD25+ cells or γδ T cells. Furthermore, using FLAG tagged gg IL-17A, binding to its receptor was demonstrated on the macrophage cell line HD11. In peripheral blood IL-17A binding activity was found on αβ and γδ T cell subsets, monocytes and a distinct population of CD25high cells. Treatment of HD11 cells with gg IL-17A induced IL-6 mRNA expression and nitric oxide production. These results demonstrate the presence of a αβ T helper17 cell subset and IL-17 producing γδ T cells in the chicken.

Evaluation of the Immunomodulatory Activity of the Chicken NK-Lysin-Derived Peptide cNK-2

Chicken NK-lysin (cNK-lysin), the chicken homologue of human granulysin, is a cationic amphiphilic antimicrobial peptide (AMP) that is produced by cytotoxic T cells and natural killer cells. We previously demonstrated that cNK-lysin and cNK-2, a synthetic peptide incorporating the core α-helical region of cNK-lysin, have antimicrobial activity against apicomplexan parasites such as Eimeria spp., via membrane disruption. In addition to the antimicrobial activity of AMPs, the immunomodulatory activity of AMPs mediated by their interactions with host cells is increasingly recognized. Thus, in this study, we investigated whether cNK-lysin derived peptides modulate the immune response in the chicken macrophage cell line HD11 and in chicken primary monocytes by evaluating the induction of chemokines, anti-inflammatory properties, and activation of signalling pathways. cNK-2 induced the expression of CCL4, CCL5 and interleukin(IL)-1β in HD11 cells and CCL4 and CCL5 in primary monocytes. We also determined that cNK-2 suppresses the lipopolysaccharide-induced inflammatory response by abrogating IL-1β expression. The immunomodulatory activity of cNK-2 involves the mitogen-activated protein kinases-mediated signalling pathway, including p38, extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinases, as well as the internalization of cNK-2 into the cells. These results indicate that cNK-2 is a potential novel immunomodulating agent rather than an antimicrobial agent.

A Bioprocessed Polysaccharide from Lentinus edodes Mycelia Cultures with Turmeric Protects Chicks from a Lethal Challenge of Salmonella Gallinarum

Our previous studies demonstrated that a bioprocessed polysaccharide (BPP) isolated from Lentinus edodes mushroom mycelia cultures supplemented with black rice bran can protect mice against Salmonella lipopolysaccharide-induced endotoxemia and reduce the mortality from Salmonella Typhimurium infection through upregulated T-helper 1 immunity. Here, we report that a BPP from L. edodes mushroom mycelia liquid cultures supplemented with turmeric (referred to as BPP-turmeric) alters chicken macrophage responses against avian-adapted Salmonella Gallinarum and protects chicks against a lethal challenge from Salmonella Gallinarum. In vitro analyses revealed that the water extract of BPP-turmeric (i) changed the protein expression or secretion profile of Salmonella Gallinarum, although it was not bactericidal, (ii) reduced the phagocytic activity of the chicken-derived macrophage cell line HD-11 when infected with Salmonella Gallinarum, and (iii) significantly activated the transcription expression of interleukin (IL)-1β, IL-10, tumor necrosis factor α, and inducible nitric oxide synthase in response to various Salmonella infections, whereas it repressed that of IL-4, IL-6, interferon-β, and interferon-γ. We also found that BPP-turmeric (0.1 g/kg of feed) as a feed additive provided significant protection to 1-day-old chicks infected with a lethal dose of Salmonella Gallinarum. Collectively, these results imply that BPP-turmeric contains biologically active component(s) that protect chicks against Salmonella Gallinarum infection, possibly by regulating macrophage immune responses. Further studies are needed to evaluate the potential efficacy of BPP-turmeric as a livestock feed additive for the preharvest control of fowl typhoid or foodborne salmonellosis.

Novel MicroRNA Involved in Host Response to Avian Pathogenic Escherichia coli Identified by Deep Sequencing and Integration Analysis.

ABSTRACTAvian pathogenic Escherichia coli (APEC) causes one of the most common bacterial diseases of poultry worldwide. Effective control methods are therefore desirable and will be facilitated by a better understanding of the host response to the pathogen. Currently, microRNAs (miRNAs) involved in host resistance to APEC are unknown. Here, we applied RNA sequencing to explore the changed miRNAs and deregulated genes in the spleen of three groups of broilers: nonchallenged (NC), APEC-challenged with mild pathology (CM), and APEC-challenged with severe pathology (CS). Twenty-seven differentially expressed miRNAs (fold change >1.5; P value <0.01) were identified, including 13 miRNAs between the NC and CM, 17 between the NC and CS, and 14 between the CM and CS groups. Through functional analysis of these miRNA targets, 12 immune-related biological processes were found to be significantly enriched. Based on combined analyses of differentially expressed miRNAs and mRNAs within each of the three groups, 43 miRNA-mRNA pairs displayed significantly negative correlations (r < −0.8). Notably, gga-miR-429 was greatly increased in the CS group compared to levels in both the CM and NC groups. In vitro, gga-miR-429 directly repressed luciferase reporter gene activity via binding to 3′ untranslated regions of TMEFF2, NTRK2, and SHISA2. Overexpression of gga-miR-429 in the HD11 macrophage cell line significantly inhibited TMEFF2 and SHISA2 expression, which are involved in the lipopolysaccharide-induced platelet-derived growth factor (PDGF) and Wnt signaling pathways. In summary, we provide the first report characterizing the miRNA changes during APEC infection, which may help to shed light on the roles of these recently identified genetic elements in the mechanisms of host resistance and susceptibility to APEC.

Differential modulation of immune response and cytokine profiles in the bursae and spleen of chickens infected with very virulent infectious bursal disease virus.

BackgroundVery virulent infectious bursal disease virus (vvIBDV) induces immunosuppression and inflammation in young birds, which subsequently leads to high mortality. In addition, infectious bursal disease (IBD) is one of the leading causes of vaccine failure on farms. Therefore, understanding the immunopathogenesis of IBDV in both the spleen and the bursae could help effective vaccine development. However, previous studies only profiled the differential expression of a limited number of cytokines, in either the spleen or the bursae of Fabricius of IBDV-infected chickens. Thus, this study aims to evaluate the in vitro and in vivo immunoregulatory effects of vvIBDV infection on macrophage-like cells, spleen and bursae of Fabricius.ResultsThe viral load was increased during the progression of the in vitro infection in the HD11 macrophage cell line and in vivo, but no significant difference was observed between the spleen and the bursae tissue. vvIBDV infection induced the expression of pro-inflammatory and Th1 cytokines, and chemokines from HD11 cells in a time- and dosage-dependent manner. Furthermore, alterations in the lymphocyte populations, cytokine and chemokine expression, were observed in the vvIBDV-infected spleens and bursae. A drastic rise was detected in numbers of macrophages and pro-inflammatory cytokine expression in the spleen, as early as 2 days post-infection (dpi). On 4 dpi, macrophage and T lymphocyte infiltration, associated with the peak expression of pro-inflammatory cytokines in the bursae tissues of infected chickens were observed. The majority of the significantly regulated pro-inflammatory cytokines and chemokines, in vvIBDV-infected spleens and bursae, were also detected in vvIBDV-infected HD11 cells. This cellular infiltration subsequently resulted in a sharp rise in nitric oxide (NO) and lipid peroxidation levels.ConclusionThis study suggests that macrophage may play an important role in regulating the early expression of pro-inflammatory cytokines, first in the spleen and then in the bursae, the latter tissue undergoing macrophage infiltration at 4 dpi.