• Journal of Microbiology and Biotechnology
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• v.29 no.8
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• pp.1324-1334
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• 2019

Fish mycobacteriosis is a common bacterial disease in many species of freshwater and marine fish and has caused severe loss of fish production. Mycobacterium marinum has been the most prevalent pathogen observed in several outbreaks of mycobacteriosis of farmed sturgeons in China. However, the immune responses and pathology of sturgeons in mycobacterial infection are rarely studied. Therefore, we used the Illumina RNA-seq method to analyze the transcriptome profile of Acipenser schrenckii challenged with Mycobacterium marinum. To begin, 168,220 non-redundant contigs were acquired from the infection and control groups, and among these, 33,225 contigs have acquired annotations. A total of 4,043 differently expressed (DE) contigs between the two groups were identified, and among these, 2479 were up-regulated and 1564 were down-regulated in the infected fish. A total of 1,340 DE contigs with acquired annotations in KEGG were enriched for 124 pathways including the TNF signaling pathway, and the Toll-like receptor signaling pathway. The roles of DE genes involved in significant pathways and other processes were discussed. The 2,209 DE contigs that have yet to acquire proper annotation may represent candidate genes associated with infection in sturgeons and are expected to serve as immunogenetic resources for further study. To our best knowledge, this is the first transcriptome study on sturgeons under bacterial infection.

• BMB Reports
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• v.52 no.5
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• pp.336-341
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• 2019

The cGAS-STING pathway plays an important role in pathogen-induced activation of the innate immune response. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) found predominantly in the synovial fluid of osteoarthritis (OA) patients increases the expression of catabolic factors via the toll-like receptor-2 (TLR-2) signaling pathway. In this study, we investigated whether 29-kDa FN-f induces inflammatory responses via the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) pathway in human primary chondrocytes. The levels of cGAS and STING were elevated in OA cartilage compared with normal cartilage. Long-term treatment of chondrocytes with 29-kDa FN-f activated the cGAS/STING pathway together with the increased level of gamma-H2AX, a marker of DNA breaks. In addition, the expression of pro-inflammatory cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF-2), granulocyte colony-stimulating factor (G-CSF/CSF-3), and type I interferon ($IFN-{\alpha}$), was increased more than 100-fold in 29-kDa FN-f-treated chondrocytes. However, knockdown of cGAS and STING suppressed 29-kDa FN-f-induced expression of GM-CSF, G-CSF, and $IFN-{\alpha}$ together with the decreased activation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and inhibitor protein ${\kappa}B{\alpha}$ ($I{\kappa}B{\alpha}$). Furthermore, NOD2 or TLR-2 knockdown suppressed the expression of GM-CSF, G-CSF, and $IFN-{\alpha}$ as well as decreased the activation of the cGAS/STING pathway in 29-kDa FN-f-treated chondrocytes. These data demonstrate that the cGAS/STING/TBK1/IRF3 pathway plays a critical role in 29-kDa FN-f-induced expression of pro-inflammatory cytokines.

• BMB Reports
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• v.54 no.2
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• pp.136-141
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• 2021

Thyroid eye disease (TED) is a complex autoimmune disease with a spectrum of signs. we previously reported that trisialoganglioside (GT)1b is significantly overexpressed in the orbital tissue of TED patients, and that exogenous GT1b strongly induced HA synthesis in orbital fibroblasts. However, the signaling pathway in GT1b-induced hyaluronic acid synthase (HAS) expression in orbital fibroblasts from TED patients have rarely been investigated. Here, we demonstrated that GT1b induced phosphorylation of Akt/mTOR in a dose-dependent manner in orbital fibroblasts from TED patients. Both co-treatment with a specific inhibitor for PI3K and siRNA knockdown of TLR2 attenuated GT1b-induced Akt phosphorylation. GT1b significantly induced HAS2 expression at both the transcriptional and translational level, which was suppressed by specific inhibitors of PI3K or Akt/mTOR, and by siRNA knockdown of TLR2. In conclusion, GT1b induced HAS2 in orbital fibroblasts from TED patients via activation of the PI3K-related signaling pathway, dependent on TLR2.

• Korean Journal of Agricultural Science
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• v.48 no.2
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• pp.251-264
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• 2021

Bovine respiratory syncytial virus (BRSV) and bovine viral diarrhea virus (BVDV) are the main viral contributors to bovine respiratory disease (BRD) with high mortality and morbidity. BRD control measures include vaccination that modulates immunological profiles reflected in blood cells, serum, and body secretions, such as milk. This study evaluated the immune responses to an inactivated BRD vaccine in lactating cows reared in a natural environment on a dairy farm. The cows were intramuscularly inoculated with the vaccine, and serum, blood, and milk were collected pre-and post-vaccination. Our study revealed a prominent increase in BRSV-specific antibodies both in serum and milk, while the change in BVDV-specific antibodies was insignificant. Serum interleukin (IL)-1β and IL-6 levels significantly decreased, but this change was not reflected in milk. Evaluation of pattern recognition receptors (PRRs) via RT-qPCR revealed downregulation of nucleotide-binding oligomerization domain 2 (NOD2). The concentrations of BRSV antibodies, BVDV antibodies, IL-2, and IL-17A in serum and milk were strongly correlated, implying a concurrent influence on both body fluids. Thus, immunological factors modulated as a result of vaccination generally measured in serum were reflected in milk, demonstrating the suitability of milk evaluation as an alternative approach for immunological observations. Furthermore, the correlation between BRSV antibodies and NOD2 and that between BVDV antibodies and toll-like receptor (TLR) 2, TLR3, TLR4, and TLR5 imply the possible role of PRRs for the assessment of the immune response developed in immunized cows reared on the farm.

• Animal Bioscience
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• v.36 no.2
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• pp.315-321
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• 2023

Objective: The study was conducted to investigate the dephosphorylation of Pseudomonas aeruginosa flagellin (PA FLA) by sweet potato purple acid phosphatase (PAP) and the effect of the enzyme on the flagellin-mediated inflammatory response in the A549 lung epithelial cell line. Methods: The activity of sweet potato PAP on PA FLA was assayed at different pH (4, 5.5, 7, and 7.5) and temperature (25℃, 37℃, and 55℃) conditions. The release of interleukin-8 (IL-8) and the activation of nuclear factor kappa- light-chain-enhancer of activated B cells (NF-κB) in A549 cells exposed to PA FLA treated with or without sweet potato PAP was measured using IL-8 and NF-κB ELISA kits, respectively. The activation of toll-like receptor 5 (TLR5) in TLR5-overexpressing HEK-293 cells exposed to PA FLA treated with or without sweet potato PAP was determined by the secreted alkaline phosphatase-based assay. Results: The dephosphorylation of PA FLA by sweet potato PAP was favorable at pH 4 and 5.5 and highest at 55℃. PA-FLA treated with the enzyme decreased IL-8 release from A549 cells to about 3.5-fold compared to intact PA FLA at 1,000 ng/mL of substrate. Moreover, PA-FLA dephosphorylated by the enzyme repressed the activation of NF-κB in the cells compared to intact PA FLA. The activation of TLR5 by PA-FLA was highest in TLR-overexpressing HEK293 cells at a substrate concentration of 5,000 ng/mL, whereas PA FLA treated with the enzyme strongly repressed the activation of TLR5. Conclusion: Sweet potato PAP has the potential to be a new alternative agent against the increased antibiotic resistance of P. aeruginosa and may be a new conceptual feed additive to control unwanted inflammatory responses caused by bacterial infections in animal husbandry.

• Journal of Veterinary Science
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• v.21 no.5
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• pp.78.1-78.15
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• 2020

Background: Enteritis is one of the most frequently reported symptoms in piglets infected with porcine circovirus type 2 (PCV2), but the immunopathogenesis has not been reported. Objectives: This study examined the effect of a PCV2 infection on the intestinal mucosal immune function through morphological observations and immune-related molecular detection. Methods: Morphological changes within the ileum of piglets during a PCV2 infection were observed. The expression of the related-molecules was analyzed using a gene chip. The immunocyte subsets were analyzed by flow cytometry. The secretory immunoglobulin A (SIgA) content was analyzed by enzyme-linked immunosorbent assay. Results: The PCV2 infection caused ileal villus damage, intestinal epithelial cells exfoliation, and an increase in lymphocytes in the lamina propria at 21 days post-infection. Differentially expressed genes occurred in the defense response, inflammatory response, and the complement and coagulation cascade reactions. Most of them were downregulated significantly at the induction site and upregulated at the effector site. The genes associated with SIgA production were downregulated significantly at the induction site. In contrast, the expression of the Toll-like receptor-related genes was upregulated significantly at the effector site. The frequencies of dendritic cells, B cells, and CD8⁺T cells were upregulated at the 2 sites. The SIgA content decreased significantly in the ileal mucosa. Conclusions: PCV2 infections can cause damage to the ileum that is associated with changes in immune-related gene expression, immune-related cell subsets, and SIgA production. These findings elucidated the molecular changes in the ileum after a PCV2 infection from the perspective of intestinal mucosal immunity, which provides insights into a further study for PCV2-induced enteritis.

• Journal of Veterinary Science
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• v.21 no.5
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• pp.74.1-74.13
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• 2020

Background: The quality of a vaccine depends strongly on the effects of the adjuvants applied simultaneously with the antigen in the vaccine. The adjuvants enhance the protective effect of the vaccine against a viral challenge. Conversely, oil-type adjuvants leave oil residue inside the bodies of the injected animals that can produce a local reaction in the muscle. The long-term immunogenicity of mice after vaccination was examined. ISA206 or ISA15 oil adjuvants maintained the best immunity, protective capability, and safety among the oil adjuvants in the experimental group. Objectives: This study screened the adjuvant composites aimed at enhancing foot-and-mouth disease (FMD) immunity. The C-type lectin or toll-like receptor (TLR) agonist showed the most improved protection rate. Methods: Experimental vaccines were fabricated by mixing various known oil adjuvants and composites that can act as immunogenic adjuvants (gel, saponin, and other components) and examined the enhancement effect on the vaccine. Results: The water in oil (W/O) and water in oil in water (W/O/W) adjuvants showed better immune effects than the oil in water (O/W) adjuvants, which have a small volume of oil component. The W/O type left the largest amount of oil residue, followed by W/O/W and O/W types. In the mouse model, intramuscular inoculation showed a better protection rate than subcutaneous inoculation. Moreover, the protective effect was particularly weak in the case of inoculation in fatty tissue. The initial immune reaction and persistence of long-term immunity were also confirmed in an immune reaction on pigs. Conclusions: The new experimental vaccine with immunostimulants produces improved immune responses and safety in pigs than general oil-adjuvanted vaccines.

• Journal of Microbiology and Biotechnology
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• v.32 no.10
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• pp.1344-1354
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• 2022

Laryngeal cancer is one of the highest incidence, most prevalently diagnosed head and neck cancers, making it critically necessary to probe effective targets for laryngeal cancer treatment. Here, real-time quantitative reverse transcription PCR (qRT-PCR) and western blot analysis were used to detect gene expression levels in laryngeal cancer cell lines. Fluorescence in situ hybridization (FISH) and subcellular fractionation assays were used to detect the subcellular location. Functional assays encompassing Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), transwell and wound healing assays were performed to examine the effects of target genes on cell proliferation and migration in laryngeal cancer. The in vivo effects were proved by animal experiments. RNA-binding protein immunoprecipitation (RIP), RNA pulldown and luciferase reporter assays were used to investigate the underlying regulatory mechanisms. The results showed that KIF26B antisense RNA 1 (KIF26B-AS1) propels cell proliferation and migration in laryngeal cancer and regulates the toll-like receptor 4 (TLR4) signaling pathway. KIF26B-AS1 also recruits FUS to stabilize TLR4 mRNA, consequently activating the TLR4 signaling pathway. Furthermore, KIF26B-AS1 plays an oncogenic role in laryngeal cancer via upregulating TLR4 expression as well as the FUS/TLR4 pathway axis, findings which offer novel insight for targeted therapies in the treatment of laryngeal cancer patients.

• Animal Bioscience
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• v.36 no.9
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• pp.1403-1413
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• 2023

Objective: Intestinal alkaline phosphatase (IAP) maintains intestinal homeostasis by detoxifying bacterial endotoxins and regulating gut microbiota, and lipid absorption. Antibiotics administered to animals can cause gut dysbiosis and barrier disruption affecting animal health. Therefore, the present study sought to investigate the role of IAP in the intestinal environment in dysbiosis. Methods: Young male mice aged 9 weeks were administered a high dose of antibiotics to induce dysbiosis. They were then sacrificed after 4 weeks to collect the serum and intestinal organs. The IAP activity in the ileum and the level of cytokines in the serum samples were measured. Quantitative real-time polymerase chain reaction analysis of RNA from the intestinal samples was performed using primers for tight junction proteins (TJPs) and proinflammatory cytokines. The relative intensity of IAP and toll-like receptor 4 (TLR4) in intestinal samples was evaluated by western blotting. Results: The IAP activity was significantly lower in the ileum samples of the dysbiosis-induced group compared to the control. The interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha concentrations were significantly higher in the ileum samples of the dysbiosis-induced group. The RNA expression levels of TJP2, claudin-3, and claudin-11 showed significantly lower values in the intestinal samples from the dysbiosis-induced mice. Results from western blotting revealed that the intensity of IAP expression was significantly lower in the ileum samples of the dysbiosis-induced group, while the intensity of TLR4 expression was significantly higher compared to that of the control group without dysbiosis. Conclusion: The IAP activity and relative mRNA expression of the TJPs decreased, while the levels of proinflammatory cytokines increased, which can affect intestinal integrity and the function of the intestinal epithelial cells. This suggests that IAP is involved in mediating the intestinal environment in dysbiosis induced by antibiotics and is an enzyme that can potentially be used to maintain the intestinal environment in animal health care.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.56 no.6
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• pp.850-860
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• 2023

The effects of dietary mannan oligosaccharides, Lactobacillus plantarum, Bacillus subtilis, and Bacillus licheniformis supplementation on hybrid grouper Epinephelus akaara ♀×Epinephelus lanceolatus ♂ were evaluated. The fish were fed a basal diet and five other diets consisting of 0.6% mannan oligosaccharides, L. plantarum, B. subtilis, and B. licheniformis and mixture of each 0.15% prebiotic and all the probiotics (designated as MOS, LP, BS, BL, and SYN) for 56 days. Growth performance and feed utilization showed no significant differences among all experimental groups. Lipid level of whole-body was significantly high in MOS and BL groups. Plasma aspartate aminotransferase was significantly low in BL and SYN groups. Nitro-blue tetrazolium, lysozyme and anti-protease, and glutathione peroxidase in BS, SYN, and all probiotic groups, respectively, were significantly high. Intestinal Vibrio bacteria was significantly low in all probiotic and SYN groups. Gene expression of interleukin-1β and interleukin-10 in SYN group; transforming growth factor β2 in MOS and BS groups, toll-like receptor 2-2 in BS and BL groups; and C-type lectin in MOS, LP and SYN groups were significantly upregulated. Our findings indicate that mannan oligosaccharides, L. plantarum, B. subtilis, and B. licheniformis could improve innate immunity, antioxidant capacity, anti-inflammation, and intestinal microbiota of hybrid grouper.