HD11 Cells Research Articles

Cellular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein

The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

Andrographolide inhibits infectious bronchitis virus-induced apoptosis, pyroptosis, and inflammation.

Avian infectious bronchitis virus (IBV), a coronavirus, causes a huge economic loss to the poultry industry. Andrographolide (APL) is a compound with a variety of pharmacological properties, including antiviral and anti-inflammatory effects. In this study, APL was evaluated for antiviral activity by its anti-apoptotic, anti-pyroptosis, and anti-inflammatory effects. The cytotoxicity of APL was determined by the MTT method. We investigated the therapeutic impact of APL on IBV through a plate assay. We explored that APL inhibited IBV-induced apoptosis, pyroptosis, and inflammation in HD11 cells by RT-qPCR and immunofluorescence. Also, it was verified in the clinical chicken embryo trial. We found that APL down-regulated apoptosis-related genes Caspase-3, Caspase-8, Caspase-9, Bax, Bid, and Bak, down-regulated pyroptosis gene DFNA5, and down-regulated inflammation-related genes (NF-κB, NLRP3, iNOS, TNF-α, and IL-1β). In addition, APL reduced the reactive oxygen species (ROS) production in cells. Finally, clinical trials showed that APL inhibited IBV-induced apoptosis, pyroptosis, and inflammation, as well as reduced the mortality and malformation of chicken embryos. In this study, we delved into the antiviral properties of APL in the context of chicken macrophage (HD11) infection with IBV. Our findings confirm that andrographolide effectively inhibits apoptosis, pyroptosis, and inflammation by IBV infection. Furthermore, this inhibition was verified on chicken embryos in vivo. This inhibition suggests a substantial potential for APL as a therapeutic agent to mitigate the harmful effects of IBV on host cells.

Avian safety guardian: Luteolin restores Mycoplasma gallisepticum-induced immunocompromise to improve production performance via inhibiting the IL-17/NF-kB pathway

Mycoplasma gallisepticum (MG) is a major pathogen causing chronic respiratory disease (CRD) in chickens. Exposure to MG poses a constant threat to chicken health and causes substantial economic losses. Antibiotics are the main treatment for MG infections, but have to struggle with antibiotic residues and MG resistance. To date, no safe and more effective prevention or treatment for MG infections has been identified. Luteolin (Lut) is a natural flavonoid compound known for its excellent anti-viral, anti-bacterial, immunoregulatory, and anti-inflammatory pharmacological activities. Herein, we established an MG-infected model using partridge shank chickens and chicken-like macrophages (HD11 cells) to investigate the effect and potential mechanism of Lut against MG-induced immune damage.According to our findings, Lut significantly inhibited the expression of MG adhesion protein (pMGA1.2) in vivo and in vitro. Lut effectively mitigated the MG-induced decrease in body weight gain, feed conversion ratio, survival rate, and serum IgG and IgA levels. Lut directly repaired MG-induced spleen and thymus damage by histopathological analysis. Furthermore, network pharmacology analysis revealed that Lut most probably resisted MG infection through the IL-17/NF-kB pathway. In vivo and in vitro experiments, Lut significantly suppressed the increase in key protein IL-17A, TRAF6, p-p65, and p-IkBα in the IL-17/NF-kB pathway. Meanwhile, Lut markedly alleviated MG-induced the increase of pro-inflammatory cytokines TNF-α, IL-6, IL-1β, pro-apoptotic genes caspase3 and caspase9, while promoting the expression of anti-apoptotic genes Bcl-2 and Bcl-XL.In summary, Lut effectively suppressed MG colonization, alleviated MG-induced the production performance degradation, inflammatory responses, and immune damage by inhibiting the IL-17/ NF-kB pathway. This study indicates Lut can serve as a safe and effective antibiotic alternative drug for preventing and treating MG-induced CRD. It also provides new evidence to explore the molecular mechanisms of MG infection.

M1 polarization of chicken macrophage HD11 can be activated by duck Tembusu virus via MyD88-NF-κB-mediated signaling pathway

Duck Tembusu virus (DTMUV) has caused significant economic losses to the global duck industry since its outbreak in 2010. The macrophages act as the key immune cell, and its polarization in different functional states is very important for host’s immune responses and microbial infections. Avian macrophages are the main target cells of DTMUV, its polarization induced by DTMUV and the underlying mechanisms were explored in this study. Through quantitative real-time PCR, nitrite assay, and flow cytometry analysis, we found that DTMUV caused severe inflammatory responses in chicken macrophage line HD11 by reprogramming the expression of M1- and M2-associated genes, leading to the polarization of HD11 macrophage to M1-type. In term of mechanism, transcriptomics was performed to analyze the M1-type polarization triggered by DTMUV, it was found that most differential genes were implicated in biological processes, and DTMUV infection significantly activated innate immune signaling pathways, including cytokine-cytokine receptor interaction, MAPK signaling pathway. Moreover, transcription factors NF-κB and AP1 also be activated after viral infection. However, further validation analysis by inhibitors and siRNAs of NF-κB and AP1 showed that NF-κB molecule was essential for DTMUV-induced M1 polarization in HD11 cell, but not AP1. Additionally, the inhibiting assays targeting MyD88 and TRIF molecules were conducted to determine their effect on NF-κB and M1-associated genes upregulated by DTMUV. The results showed that although the inhibition of both MyD88 and TRIF significantly downregulated the mRNA level of NF-κB, but the expression of M1-associated genes such as CD86 was lower in MyD88 inhibition group than in the other group, indicating that the role of MyD88 in mediating M1 polarization induced by DTMUV was more important. Overall, these results demonstrated that DTMUV infection induces M1-type polarization in chicken macrophage HD11 through MyD88-NF-κB signaling pathways. This finding will lay the foundation for further study the pathogenesis of DTMUV, and provide new insights into the prevention and control of this disease.

M2 Polarization and Inhibition of Host Cell Glycolysis Contributes Intracellular Survival of Salmonella Strains in Chicken Macrophage HD-11 Cells.

Salmonella enterica is a group of facultative, gram-negative bacteria. Recently, new evidence indicated that Salmonella could reprogram the host metabolism to increase energy or metabolites available for intracellular replication. In this study, using a chicken-specific kinomic immunometabolism peptide array analysis, we found that infection by S. Enteritidis induced significant phosphorylation changes in many key proteins of the glycolytic pathway in chicken macrophage HD-11 cells, indicating a shift in glycolysis caused by Salmonella infection. Nitric oxide production and changes of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) represented by extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively, were measured in chicken macrophages infected with three Salmonella strains (S. Enteritidis, S. Heidelberg, and S. Senftenberg). The infection reduced glycolysis and enhanced OXPHOS in chicken macrophages as indicated by changes of ECAR and OCR. Salmonella strains differentially affected macrophage polarization and glycolysis. Among three strains tested, S. Enteritidis was most effective in downregulating glycolysis and promoting M2 polarization as measured by ECAR, ORC, and NO production; while S. Senftenberg did not alter glycolysis and may promote M1 polarization. Our results suggested that downregulation of host cell glycolysis and increase of M2 polarization of macrophages may contribute to increased intracellular survival of S. Enteritidis.

Reduced NR2F2 Expression in the Host Response to Infectious Bursal Disease Virus Infection Suppressed Viral Replication by Enhancing Type I Interferon Expression by Targeting SOCS5.

Nuclear receptors are ligand-activated transcription factors that play an important role in regulating innate antiviral immunity and other biological processes. However, the role of nuclear receptors in the host response to infectious bursal disease virus (IBDV) infection remains elusive. In this study, we show that IBDV infection or poly(I·C) treatment of DF-1 or HD11 cells markedly decreased nuclear receptor subfamily 2 group F member 2 (NR2F2) expression. Surprisingly, knockdown, knockout, or inhibition of NR2F2 expression in host cells remarkably inhibited IBDV replication and promoted IBDV/poly(I·C)-induced type I interferon and interferon-stimulated genes expression. Furthermore, our data show that NR2F2 negatively regulates the antiviral innate immune response by promoting the suppressor of cytokine signaling 5 (SOCS5) expression. Thus, reduced NR2F2 expression in the host response to IBDV infection inhibited viral replication by enhancing the expression of type I interferon by targeting SOCS5. These findings reveal that NR2F2 plays a crucial role in antiviral innate immunity, furthering our understanding of the mechanism underlying the host response to viral infection. IMPORTANCE Infectious bursal disease (IBD) is an immunosuppressive disease causing considerable economic losses to the poultry industry worldwide. Nuclear receptors play an important role in regulating innate antiviral immunity. However, the role of nuclear receptors in the host response to IBD virus (IBDV) infection remains elusive. Here, we report that NR2F2 expression decreased in IBDV-infected cells, which consequently reduced SOCS5 expression, promoted type I interferon expression, and suppressed IBDV infection. Thus, NR2F2 serves as a negative factor in the host response to IBDV infection by regulating SOCS5 expression, and intervention in the NR2F2-mediated host response by specific inhibitors might be employed as a strategy for prevention and treatment of IBD.

Salmonella Pullorum effector SteE regulates Th1/Th2 cytokine expression by triggering the STAT3/SOCS3 pathway that suppresses NF-κB activation

Salmonella enterica serovar Pullorum (S. Pullorum) can regulate host immunity via special effectors that promote persistent infection and its intracellular survival. SteE as an anti-inflammatory effector is involved in the systemic infection of Salmonella in host macrophages. Macrophage activation can indirectly reflect the immune regulatory function of T helper type 1 (Th1)/T helper type 2 (Th2) cytokines. However, information concerning the regulation of Th1/Th2 cytokine expression by steE in S. Pullorum infection is limited. This study evaluates the effects of steE on the Th1/Th2 balance, STAT3/SOCS3 pathway, and NF-κB P65 activation in S. Pullorum-infected HD-11 cells and in chicken models. We demonstrated that steE diminished the expression of Th1-related cytokines (IFN-γ and IL-12) and promoted the expression of Th2-related cytokines (IL-4 and IL-10) in HD-11 cells and chicken models of S. Pullorum infection. SOCS3 silencing suppressed the function of steE in HD-11 cells and led to the imbalance of Th1/Th2-related cytokines. SteE promoted SOCS3 expression by activating STAT3 in HD-11 cells. Moreover, steE inhibited NF-κB P65 expression and blocked its translocation to the nucleus by promoting SOCS3 expression. Our results illustrated that steE regulated the expression of Th1/Th2 cytokines via modulation of the STAT3/SOCS3 and NF-κB axis, which might be associated with Th1/Th2 cell differentiation and could, therefore, be a novel therapeutic strategy against salmonellosis.

Unraveling the role of type 1 fimbriae in Salmonella pathogenesis: insights from a comparative analysis of Salmonella Enteritidis and Salmonella Gallinarum

Significant differences in pathogenicity between Salmonella Enteritidis and Salmonella Gallinarum exist despite the fact that S. Gallinarum is a direct descendant of S. Enteritidis. It was hypothesized that such various properties may be in part the result of differences in structure and functions of type 1 fimbriae (T1Fs). In S. Enteritidis, T1Fs bind to oligomannosidic structures carried by host cell glycoproteins and are called mannose-sensitive T1Fs (MST1F). In S. Gallinarum, T1Fs lost ability to bind such carbohydrate chains, and were named mannose-resistant MRT1Fs (MRT1F). Therefore, the present study was undertaken to evaluate the role of MST1Fs and MRT1Fs in the adhesion, invasion, intracellular survival and cytotoxicity of S. Enteritidis and S. Gallinarum toward chicken intestinal CHIC8-E11cells and macrophage-like HD11 cells. Using mutant strains: S. Enteritidis fimH::kan and S. Gallinarum fimH::kan devoid of T1Fs and in vitro assays the following observations were made. MST1Fs have a significant impact on the chicken cell invasion by S. Enteritidis as MST1F-mediated adhesion facilitates direct and stable contact of bacteria with host cells, in contrast to MRT1Fs expressed by S. Gallinarum. MST1Fs as well as MRT1Fs did not affected intracellular viability of S. Enteritidis and S. Gallinarum. However, absolute numbers of intracellular viable wild-type S. Enteritidis were significantly higher than S. Enteritidis fimH::kan mutant and wild-type S. Gallinarum and S. Gallinarum fimH::kan mutant. These differences, reflecting the numbers of adherent and invading bacteria, underline the importance of MST1Fs in the pathogenicity of S. Enteritidis infections. The cytotoxicity of wild-type S. Enteritidis and its mutant devoid of MST1Fs to HD11 cells was essentially the same, despite the fact that the number of viable intracellular bacteria was significantly lower in the mutated strain. Using HD11 cells with similar number of intracellular wild-type S. Enteritidis and S. Enteritidis fimH::kan mutant, it was found that the lack of MST1Fs did not affect directly the cytotoxicity, suggesting that the increase in cytotoxicity of S. Enteritidis devoid of MST1Fs may be associated with crosstalk between T1Fs and other virulence factors.

Interplay between Eimeria acervulina and Cryptosporidium parvum during In Vitro Infection of a Chicken Macrophage Cell Line (HD11).

Eimeria acervulina is a frequent intestinal pathogen of chickens, causing economic impact on the poultry industry. Cryptosporidium parvum is a neglected parasite in chickens. However, because of its zoonotic potential, poultry cryptosporidiosis may pose a risk to public health. Little is known about the parasite-host interactions during coinfection with both parasites. In this study, we investigated the possible interactions during in vitro coinfection of E. acervulina and C. parvum in a chicken macrophage cell line (HD11). HD11 cells were inoculated with E. acervulina and C. parvum sporozoites and incubated 2, 6, 12, 24, and 48 h post infection (hpi). Mono-infections for each parasite were also investigated. Real-time PCR was used to quantify parasite replication. Additionally, macrophage mRNA expression levels of IFN-γ, TNF-α, iNOS, and IL-10 were measured. For both parasites, multiplication was, in most groups, lower in the coinfection group (COIG) compared with mono-infections. However, at 6 hpi, the number of C. parvum copies was higher in co-infections. Intracellular replication started to decrease from 12 hpi onward, and it was almost undetectable by 48 hpi in all groups. Infections resulted in low expression of all cytokines, except at 48 hpi. Infection of avian macrophages with both E. acervulina and C. parvum seemed to hinder intracellular replication for both parasites in comparison to mono-infection. A clear reduction in intracellular parasites from 12 hpi onward details the important role potentially played by macrophages in host control of these parasites.

ROS mediated pyroptosis-M1 polarization crosstalk participates in inflammation of chicken liver induced by bisphenol A and selenium deficiency

The earth's natural environmental factors and man-made industrial pollution often lead to the co-occurrence of environmental pathogenic factors and malnutrition. Bisphenol A (BPA) is a serious environmental endocrine disruptor, and its exposure can cause liver tissue damage. Selenium (Se) deficiency is a worldwide problem that afflicts thousands of people, and Se deficiency can cause M1/M2 imbalance. In addition, the crosstalk between hepatocyte and immune cell is closely related to the occurrence of hepatitis. Therefore, this study found for the first time that the combined exposure of BPA and Se deficiency caused liver pyroptosis and M1 polarization through ROS, and the crosstalk between pyroptosis and M1 polarization aggravated liver inflammation in chicken. In this study, the BPA or/and Se deficiency chicken liver, single and co-culture model of LMH and HD11 cells were established. The results displayed that BPA or Se deficiency induced liver inflammation accompanied by pyroptosis and M1 polarization through oxidative stress, and increased expressions of chemokines (CCL4, CCL17, CCL19, and MIF) and inflammatory factors (IL-1β and TNF-α). The vitro experiments further verified the above changes and showed that LMH pyroptosis promoted M1 polarization of HD11 cells, and vice versa. NAC counteracted pyroptosis and M1 polarization caused by BPA and low-Se, reducing the release of inflammatory factors. In brief, BPA and Se deficiency treatment can exacerbate liver inflammation by increasing oxidative stress to induce pyroptosis and M1 polarization.

Effect of HSPA8 gene on the proliferation, apoptosis and immune function of HD11 cells

HSPA8 (Heat shock 70 kDa protein 8) is a molecular chaperone involved in a variety of cellular processes. This gene may affect the proliferation, apoptosis and immune function of chicken macrophages, but the specific mechanism remains unclear. The purpose of this study was to explore the effect of the HSPA8 gene on the proliferation, apoptosis and immune function of chicken macrophages. In this study, a chicken HSPA8 overexpression plasmid, interference fragment and corresponding controls were transfected into HD11 cells, and then the expression of the HSPA8 gene, cell proliferation, cell cycle, apoptosis rate and immune function of each group were detected. The results showed that transfection of the HSPA8 overexpression plasmid significantly upregulated the level of HSPA8 expression in HD11 cells compared with the control; significantly promoted the proliferation of HD11 cells and the expression of PCNA, CCND1 and CCNB3; decreased the number of cells in the G1 phase and increased the number of cells in the S phase; decreased the rate of apoptosis and upregulated the expression of Bcl-2; and promoted the expression of the LPS-induced cytokines IL-1β, IL-6 and TNF-α. Transfection of the HSPA8 interference fragment significantly downregulated the level of HSPA8 expression in HD11 cells; significantly inhibited the proliferation of HD11 cells and the expression of PCNA, CCND1 and CDK1; increased the number of cells in the G1 phase and decreased the number of cells in the S phase; increased the rate of apoptosis, downregulated the expression of Bcl-2 and upregulated the expression levels of Fas and FasL; and inhibited the expression of the LPS-induced cytokines IL-1β and NF-κB. The results suggested that HSPA8 promotes the proliferation of and inhibits the apoptosis of HD11 cells and has a proinflammatory effect.

Localization of Chicken Rab22a in Cells and Its Relationship to BF or Ii Molecules and Genes.

Rab22a is an important small GTPase protein the molecule that is involved in intracellular transportation and regulation of proteins. It also plays an important role in antigens uptake, transportation, regulation of endosome morphology, and also regulates the transport of antigens to MHC (Major Histocompatibility Complex) molecules. To investigate the role of Rab22a, the intracellular co-localization of chicken Rab22a (cRab22a) molecule and its relationship to BF and chicken invariant chain (cIi) molecules was studied. A 3D protein structure of Rab22a was constructed by using informatics tools (DNASTAR 4.0 and DNAMAN). Based on the model, the corresponding recombinant eukaryotic plasmids were constructed by point mutations in the protein's structural domains. HEK 293T cells were co-transfected with plasmids pEGFP-C1-cIi to observe the intracellular co-localization. Secondly, the DC2.4 Mouse Dendritic Cell and Murine RAW 264.7 cells were transfected with recombinant plasmids of pmCherry-cRab22a and pmCherry-mRab22a respectively. Subsequently, the intracellular localization of cRab22a in early and late endosomes was observed with specific antibodies against EEA1 and LAMP1 respectively. For gene expression-based studies, the cRab22a gene was down-regulated and up-regulated in HD11 cells, following the detection of transcription levels of the BFa (MHCIa) and cIi genes by real-time quantitative PCR (RT-qPCR). The interactions of the cRab22a gene with BFa and cIi were detected by co-immunoprecipitation (Co-IP) and Western blot. The results showed that the protein structures of chicken and mouse Rab22a were highly homologous (95.4%), and both localize to the early and late endosomes. Ser41 and Tyr74 are key amino acids in the Switch regions of Rab22a which maintain its intracellular localization. The down-regulation of cRab22a gene expression significantly reduced (p < 0.01) the transcription of BFa (MHCIa) and cIi in HD11 cells. However, when the expression of the cRab22a gene was increased 55 times as compared to control cells, the expression of the BFa (MHCIa) gene was increased 1.7 times compared to the control cells (p < 0.01), while the expression of the cIi gene did not significantly differ from control (p > 0.05). Western blot results showed that cRab22a could not directly bind to BFa and cIi. So, cRab22a can regulate BFa and cIi protein molecules indirectly. It is concluded that cRab22a was localized with cIi in the endosome. The Switch regions of cRab22a are the key domains that affect intracellular localization and colocalization of the cIi molecule.

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.

Regulation of the MyD88 gene in chicken spleen inflammation induced by stress.

In order to investigate the regulatory role of the myeloid differentiation factor 88 (MyD88) gene in the stress inflammatory response to chicken spleen, the chicken stress model and macrophage (HD11) inflammation model were constructed in this study. Enzyme-linked immunosorbent assay and quantitative real-time PCR were used to investigate the effects of MyD88 on immune and inflammatory indicators. The results demonstrated that the levels of IgG, CD3+ and CD4+ in the serum of chickens in the beak trimming stress and heat stress groups decreased significantly compared to the control group without stress (P < 0.05), and the inflammation-related indices IL-1β, TNF-α, IL-6 and NF-κB increased significantly (P < 0.05). Stress up-regulated the expression levels of MyD88, IL-1β, NF-κB and TLR4 in the spleen, stimulated the release of inflammatory factors. Overexpression of MyD88 significantly up-regulated the expression levels of the inflammatory factors IL-1β, TNF-α, IL-8, NF-κB and TLR4 in HD11 cells (P < 0.05). Co-treatment with lipopolysaccharide (LPS) further promoted the expression levels of the inflammatory cytokines in HD11 cells. Interference with the expression of MyD88 significantly reduced the expression level of inflammatory factors in HD11 cells (P < 0.05) and had an antagonistic effect with LPS to alleviate the inflammatory reaction. In conclusion, the MyD88 gene has a pro-inflammatory effect and is highly expressed in the beak trimming and heat stress models in chicks, regulating the inflammatory response in poultry. It was involved in regulating the expression of immune-related genes in HD11 cells and had a synergistic effect with LPS.

Guanylate-binding protein 1 restricts avian coronavirus infectious bronchitis virus-infected HD11 cells

The Infectious Bronchitis Virus (IBV), a coronavirus, is a key avian pathogen that causes acute and highly infectious viral respiratory diseases. IBV is an enveloped, positive-sense RNA virus, and the host factors that restrict infection and replication of the virus remain poorly understood. Guanylate-binding protein 1 (GBP1), an interferon-gamma (IFN-γ)-inducible guanosine triphosphatase (GTPase), is a major player in host immunity and provides defense against viral replication. However, the role of chicken GBP1 (chGBP1) in the IBV-life cycle is not well understood. Therefore, this study aimed to reveal the potential role of IFN-γ-induced chGBP1 in mediating host anti-IBV infection responses. We identified the host restriction factor, chGBP1, in IBV-infected chicken macrophages HD11 cell lines. We showed that chGBP1 was upregulated by treatment with both IFN-γ and IBV in HD11 cells. chGBP1 inhibited IBV replication in a dose-dependent manner and enhanced IFN-γ anti-IBV activity. Importantly, the GTPase domain of chGBP1 played a pivotal role in its anti-IBV activity. Furthermore, chGBP1 interacts with IBV Nucleocapsids protein to degrade IBV-N protein through the autophagy pathway. Taken together, our results demonstrate a critical role of chGBP1 in anti-IBV in macrophages HD11 cells.

Bursal-Derived BP7 Induces the miRNA Molecular Basis of Chicken Macrophages and Promotes the Differentiation of B Cells.

The bursa of Fabricius (BF) is a vital central humoral immune organ unique to birds. The bioactive peptide BP7 derived from bursa is reported to promote the vaccine immune response and antibody production. However, the regulatory effect on antigen presentation and B cell differentiation has been infrequently reported. In this paper, chicken macrophage HD11 cells were used for the cell model, and the cellular molecular expressions were determined by the fluorescent quantitative PCR (qPCR) after BP7 treatment. Then, the miRNA expression profile was analyzed by high-throughput sequencing. In addition, BALB/C mice were used as the animal model to detect B cell subtype with flow cytometry (FCM). The results showed that the expressions of four immune active molecules, IL-1β, IL-6, iNOS, and IFN-α, in HD11 cells were significantly increased with 100 ng/mL BP7 treatment. Compared with the control group, there were 58 up-regulated and 61 down-regulated miRNAs in HD11 cells with BP7 treatment. The gene ontology (GO) function analysis found that BP7 mainly affected the various biological processes, molecular function, and MHC protein complex. Pathway analysis showed that 100 ng/mL BP7 stimulated various physiological metabolic pathways and signal transduction pathways, including the intestinal immune network producing IgA in HD11 cells. Furthermore, it was found that BP7 in vitro stimulated B cell populations, as well as plasma cells in spleen cells from the immunized mice. Additionally, B cell activation subpopulations were increased in mice immunized with the AIV vaccine and BP7. These results proved that BP7 stimulated various differentially expressed genes in chicken macrophage HD11 cells, and induced B cell differentiation in the immunized mice, which suggested that BP7 might participate in the antigen presentation process, thereby promoting the differentiation of B cells. These results provide an important basis for the mechanism of bursal-derived peptide on B cell development, and offer the experimental basis for the development of adjuvants.

Transcriptome alterations in chicken HD11 cells with steady knockdown and overexpression of RIPK2 gene

Receptor interacting protein kinase 2 (RIPK2) is involved in a variety of signaling pathway to produce a series of inflammatory cytokines in response to a diverse of bacterial, viral and protozoal pathogens. However, the underlying regulating of RIPK2 remain unknown. Transcriptome alterations in chicken HD11 cells following RIPK2 overexpression or silencing by shRNA were analyzed by next-generation sequencing. Both overexpression and knockdown of the RIPK2 gene caused wide-spread changes in gene expression in chicken HD11 cells. Differentially expressed genes (DEGs) caused by altered RIPK2 gene expression were associated with multiple biological processes linked with biological regulation, response to stimulus, cell communication, and signal transduction etc. KEGG analysis revealed that many of the DEGs were enriched in VEGF signaling pathway, ECM-receptor interaction, Focal adhesion, TGF-beta signaling pathway etc. Moreover, we show that initiation genes, TGFB1 and TGFB3, in the TGF-beta signaling pathway are biological targets regulated by RIPK2 in chicken HD11 cells. This is the first transcriptome-wide study in which RIPK2-regulated genes in chicken cells have been screened. Our findings elucidate the molecular events associated with RIPK2 in chicken HD11 cells.

CONSTRUCTION AND CHARACTERIZATION OF STEE DELETION MUTANT OF SALMONELLA PULLORUM

Salmonella Pullorum (S. Pullorum) is one of the host-restricted serotypes causing systemic infection in poultry. After S. Pullorum infection, chicks and turkeys usually have acute systemic infection. The main clinical symptoms are white dysentery and dyspnea, and the mortality can be as high as 100%. In adult chickens, local and chronic infections are the most common without obvious clinical symptoms, and can be transmitted vertically to offspring through ovary. Although the use of antibiotics reduces the death of sick chickens, it can not completely eliminate the pathogenic microorganisms in hosts, and is prone to public health problems such as drug resistance and drug residues. No study has ever reported the role of steE in HD-11 cells infected by S. Pullorum. The growth and biochemical characteristics of S. Pullorum ΔsteE were similar to that of S. Pullorum. Furthermore, we also observed the effects of steE on cell proliferation and apoptosis in S. Pulloruminfected HD-11 cells.In order to define the pathogenicity of steE gene of S. Pullorum, the steE deletion strain of S. Pullorum and its complemented strain were successfully constructed, and then its characterization were analyzed. S. Pullorum was preserved by the microbiology laboratory of the college of animal science and veterinary medicine, Henan Institute of Science and Technology. The pKD4, pKD46 and pCP20 or pBR322 plasmids were used for the λ-Red recombination system or complementary strain. The biological characteristics of S. Pullorum ΔsteE were consistent with those of its parent strain S. Pullorum and complementary strain S. Pullorum ΔsteE (pBR322-steE). Construction and confirmation of the ΔsteE strain. To identify the roles of steE in S. Pullorum, the steE deletion mutant of S. Pullorum was correctly constructed.The virulence test showed S. Pullorum ΔsteE decreased the proliferation and apoptosis of HD-11 cells compared to that of S. Pullorum and S. Pullorum ΔsteE (pBR322-steE). Taken together, our data demonstrate that the deletion of steE in S. Pullorum had no effect the growth and biochemical characteristics, but its proliferation ability decreased significantly in HD-11 cells, which decreased cell apoptosis, indicating that steE was closely related to virulence of S. Pullorum. Altogether, our research suggest that the steE gene was required for S. Pullorum virulence, which laid a foundation for further related research in S. Pullorum vaccine strains.

Analysis of circRNA expression in chicken HD11 cells in response to avian pathogenic E.coli.

Avian pathogenic E. coli (APEC), one of the widespread zoonotic-pathogen, can cause a series of diseases collectively known as colibacillosis. This disease can cause thousands of million dollars economic loss each year in poultry industry and threaten to human health via meat or egg contamination. However, the detailed molecular mechanism underlying APEC infection is still not fully understood. Circular RNAs, a new type of endogenous noncoding RNA, have been demonstrated to involve in various biological processes. However, it is still not clear whether the circRNAs participate in host response against APEC infection. Herein, we utilized the high-throughput sequence technology to identify the circRNA expression profiles in APEC infected HD11 cells. A total of 49 differentially expressed (DE) circRNAs were detected in the comparison of APEC infected HD11 cells vs. wild type HD11 cells, which were involved in MAPK signaling pathway, Endocytosis, Focal adhesion, mTOR signaling pathway, and VEGF signaling pathway. Specifically, the source genes (BRAF, PPP3CB, BCL2L13, RAB11A, and TSC2) and their corresponding DE circRNAs may play a significant role in APEC infection. Moreover, based on ceRNA regulation, we constructed the circRNA-miRNA network and identified a couple of important regulatory relationship pairs related to APEC infection, including circRAB11A-gga-miR-125b-3p, circRAB11A-gga-miR-1696, and circTSC2-gga-miR-1649-5p. Results indicate that the aforementioned specific circRNAs and circRNA-miRNA network might have important role in regulating host immune response against APEC infection. This study is the first time to investigate the circRNAs expression profile and the biological function of the source genes of the identified DE circRNAs after APEC infection of chicken HD11 cells. These results would contribute to a better understanding of the molecular mechanisms in host response against APEC infection.