Cloning and radiation hybrid mapping of bovine toll-like receptor-4 (TLR-4) signaling molecules

Toll-like receptor (TLR)-4 is a transmembrane receptor for lipopolysaccharide, a highly pro-inflammatory component of the outer membrane of Gram-negative bacteria. To date, molecules of the TLR-4 signaling pathway have not been well characterized in cattle. The goal of this study was to clone and sequence the full-length coding regions of bovine genes involved in TLR-4 signaling including CASP8, IRAK1, LY96 (MD-2), TICAM2, TIRAP, TOLLIP and TRAF 6 and to position these genes, as well as MyD88 and TICAM1, on the bovine genome using radiation hybrid mapping. Results of this work indicate differences with a previously published bovine sequence for LY96 and a predicted sequence in the GenBank database for TIRAP based on the most recent assembly of the bovine genome. In addition, discrepancies between actual and predicted chromosomal map positions based on the Btau_2.0 genome assembly release were identified, although map positions were consistent with predicted locations based on the current bovine-human comparative map. Alignment of the bovine amino acid sequences with human and murine sequences showed a broad range in conservation, from 52 to 93%. Overall, this work should assist in the assembly and annotation of the bovine genome sequence, the identification of variations in genes critically involved in host innate immunity, and facilitate the study of TLR-4 signaling pathways in cattle.     

Metformin acts to suppress β-hydroxybutyric acid-mediated inflammatory responses through activation of AMPK signaling in bovine hepatocytes

The occurrence of bovine ketosis involves the accumulation of β-hydroxybutyric acid (BHBA), which contributes to the initiation and acceleration of hepatic metabolic stress and inflammation. Metformin has other beneficial effects apart from its medical intervention for diabetes, such as prevention of laminitis and hyper-triglyceridemic. AMPK maintains energy homeostasis and is the intracellular target of metformin action. This study aims to uncover the role of metformin in modulating BHBA-induced inflammatory responses through the activation of AMPK signaling. The hepatocytes were isolated from the liver tissue of mid-lactation multiparous Holstein cows (~160 d postpartum). Treatments were conducted as follows: treated with PBS for 18 h (control); pretreated with PBS for 12 h followed by treatment of 1.2 mM BHBA for 6 h (BHBA); pretreated with 1.5 mM or 3 mM metformin for 12 h followed by the BHBA treatment (1.2 mM) for 6 h (M(1.5)+B; M(3)+B). The inhibitor of AMPK, Compound C, at a concentration of 10 μM, was applied to substantiate the AMPK-dependent responses. RT-qPCR were applied for the mRNA expression while Western-blots and immunofluorescence were conducted for the target proteins expression. Among dose-dependent assays for BHBA, the concentration of BHBA at 1.2 mM activated NF-κB signaling by upregulating the expression of phosphorylated NF-κB and pro-inflammatory cytokines compared with the control cells (P < 0.05). Along with the upregulation of phosphorylated AMPKα and ACCα, metformin at 1.5 and 3 mM inactivated NF-κB signaling components (p65 and IκBα) and the inflammatory genes (TNFA, IL6, IL1B and COX-2) which were activated by BHBA. Additionally, BHBA inhibited cells staining intensity in EdU assay were increased by pretreatment with metformin. The activation of AMPK resulted in the increased gene and protein expression of SIRT1, along with the deacetylation of H3K9 and H3K14. However, the AMPK inhibitor compound C blocked this effect. Compared with BHBA treated cells, the protein expression of COX-2 and IL-1β were decreased by the pretreatment with metformin, and the inhibitory effect of metformin was released by compound C. The bound of NF-κB onto IL1B promoter displayed higher in BHBA group and this was suppressed by pretreatment with metformin (P < 0.05). Altogether, metformin attenuates the BHBA-induced inflammation through the inactivation of NF-κB as a target for AMPK/SIRT1 signaling in bovine hepatocytes.     

Propofol attenuates LPS-induced tumor necrosis factor-α, interleukin-6 and nitric oxide expression in canine peripheral blood mononuclear cells possibly through down-regulation of nuclear factor (NF)-κB activation

Sepsis is a major cause of mortality in intensive care medicine. Propofol, an intravenous general anesthetic, has been suggested to have anti-inflammatory properties and able to prevent sepsis induced by Gram-positive and Gram-negative bacteria by down-regulating the gene expression of pro-inflammatory cytokines. However, propofol’s anti-inflammatory effects upon canine peripheral blood mononuclear cells (PBMCs) have not yet been clarified. Here, we isolate canine PBMCs and investigate the effects of propofol on the gene expressions of both lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α and upon the production of nitric oxide (NO). Through real-time quantitative PCR and the Griess reagent system, we found that non-cytotoxic levels of propofol significantly inhibited the release of NO and IL-6 and TNF-α gene expression in LPS-induced canine PBMCs. Western blotting revealed that LPS does significantly increase the expression of inducible NO synthase (iNOS) protein in canine PBMCs, while pretreatment with propofol significantly decreases the LPS-induced iNOS protein expression. Propofol, at concentration of 25 µM and 50 µM, also significantly inhibited the LPS-induced nuclear translocation of nuclear factor (NF)-κB p65 protein in canine PBMCs. This diminished TNF-α, IL-6 and iNOS expression, and NO production was in parallel to the respective decreased NF-κB p65 protein nuclear translocation in the LPS-activated canine PBMCs pretreated with 25 µM and 50 µM propofol. This suggests that non-cytotoxic levels of propofol pretreatment can down-regulate LPS-induced inflammatory responses in canine PBMCs, possibly by inhibiting the nuclear translocation of the NF-κB p65 protein.     

人参多糖对脂多糖刺激小鼠巨噬细胞的免疫调控作用

本试验旨在研究脂多糖(LPS)刺激条件下人参多糖(GPS)对小鼠单核巨噬细胞形态及免疫功能的调节作用。采用LPS刺激小鼠巨噬细胞(RAW264.7),通过测量不同浓度(1、0.5、0.1 mg/mL)GPS对细胞形态、生物酶活性、促炎症因子分泌及TLR4/NF-κB信号通路mRNA表达量的影响来研究不同浓度的GPS对LPS引起小鼠巨噬细胞免疫应激的调控作用。结果表明:添加GPS能抑制由LPS引起的细胞形态和细胞增殖能力的变化;1 mg/mL GPS能够显著提高巨噬细胞酸性磷酸酶的活性;0.5、1 mg/mL GPS能够显著缓解由LPS刺激引起的碱性磷酸酶活性的降低;不同浓度GPS均能显著降低由LPS诱导的促炎症因子IL-1β、TNF-α水平;LPS刺激显著提高巨噬细胞TLR4、MyD88、NF-κB的mRNA表达量,而添加GPS后,巨噬细胞TLR4、MyD88、NF-κB的mRNA表达量均表现出不同程度降低(P<0.05)。结果显示,添加GPS可以改善细胞形态,恢复细胞增殖能力,GPS可通过调节TLR4/NF-κB信号通路降低促炎症因子IL-1β和TNF-α的分泌及表达,减少机体免疫应激反应。     

Regulation of Innate Immune Function in Bovine Oviduct Epithelial Cells in Culture: The Homeostatic Role of Epithelial Cells in Balancing Th1/Th2 Response

This study aimed to investigate the role of epithelial cells in regulating innate immunity in bovine oviduct epithelial cell (BOEC) culture. We studied the effect of Escherichia coli lipopolysaccharide (LPS) and its interaction with ovarian steroids, estradiol (E2) and progesterone (P4), and luteinizing hormone (LH) at concentrations observed during the preovulatory period on immune responses in BOEC culture. Immunohistochemistry of oviduct tissue showed intensive expression of Toll-like receptor-4 (TLR-4) and TLR-2 in epithelial cells. A dose of 10 ng/ml LPS stimulated TLR-4, cyclooxygenase-2 (COX-2), nuclear factor kappa B inhibitor A (NFKBIA), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) expression, indicating an early pro-inflammatory response. A dose of 100 ng/ml LPS did not induce expression of these genes but stimulated TLR-2, IL-10,IL-4 and microsomal prostaglandin E synthase-1 (mPGES-1) expression and PGE2 secretion, indicating an anti-inflammatory response. Ovarian steroids and LH completely block LPS (10 ng/ml)-induced TLR-4, IL-1β and TNF-α expression as well as LPS (100 ng/ml)-induced TLR-2 expression. Taken together, this study suggests the existence of an early signaling system to respond to infection in the BOEC. In addition, ovarian steroids and LH may play a critical role in inducing homeostasis and in controlling hyperactive pro-inflammatory responses detrimental to epithelial cells, sperm and the embryo.     

Dietary dihydroartemisinin supplementation alleviates intestinal inflammatory injury through TLR4/NOD/NF-κB signaling pathway in weaned piglets with intrauterine growth retardation

The aim of present study was to evaluate whether diets supplemented with dihydroartemisinin (DHA) could alleviate intestinal inflammatory injury in weaned piglets with intrauterine growth retardation (IUGR). Twelve normal birth weight (NBW) piglets and 12 piglets with IUGR were fed a basal diet (NBW-CON and IUCR-CON groups), and another 12 piglets with IUGR were fed the basal diet supplemented with DHA at 80 mg/kg (IUGR-DHA group) from 21 to 49 d of age. At 49 d of age, 8 piglets with similar body weight in each group were sacrificed. The jejunal and ileal samples were collected for further analysis. The results showed that IUGR impaired intestinal morphology, increased intestinal inflammatory response, raised enterocyte apoptosis and reduced enterocyte proliferation and activated transmembrane toll-like receptor 4 (TLR4)/nucleotide-binding and oligomerization domain (NOD)/nuclear factor-κB (NF-κB) signaling pathway. Dihydroartemisinin inclusion ameliorated intestinal morphology, indicated by increased villus height, villus height-to-crypt depth ratio, villus surface area and decreased villus width of piglets with IUGR (P < 0.05). Compared with NBW piglets, IUGR piglets supplemented with DHA exhibited higher apoptosis index and caspase-3 expression, and lower proliferation index and proliferating cell nuclear antigen expression in the intestine (P < 0.05). Dihydroartemisinin supplementation attenuated the intestinal inflammation of piglets with IUGR, indicated by increased concentrations of intestinal inflammatory cytokines and lipopolysaccharides (P < 0.05). In addition, DHA supplementation down-regulated the related mRNA expressions of TLR4/NOD/NF-κB signaling pathway and upregulated mRNA expressions of negative regulators of TLR4 and NOD signaling pathway in the intestine of piglets with IUGR (P < 0.05). Piglets in the IUGR-DHA group showed lower protein expressions of TLR4, phosphorylated NF-κB (pNF-κB) inhibitor α, nuclear pNF-κB, and higher protein expression of cytoplasmic pNF-κB in the intestine than those in the IUGR-CON group (P < 0.05). In conclusion, DHA supplementation could improve intestinal morphology, regulate enterocyte proliferation and apoptosis, and alleviate intestinal inflammation through TLR4/NOD/NF-κB signaling pathway in weaned piglets with IUGR.     

Induction of inflammatory host immune responses by organisms belonging to the genera Chlamydia/Chlamydophila

Chlamydia/Chlamydophila are a family of intracellular gram-negative bacteria that infect their hosts primarily via mucosal epithelia. Chronic disease associated with bacterial persistence, inflammation and tissue damage are common sequelae of infection with these organisms. Human epithelial cell lines respond to infection by releasing pro-inflammatory cytokines and chemokines such as interleukin (IL)-6 and IL-8, and upregulating the expression of mRNA encoding Iκ-Bα, the endogenous inhibitor of NF-κB. However, Iκ-Bα is not upregulated in response to bacterial lipopolysaccharide (LPS). The failure of epithelial cells to respond to LPS is associated with the absence of surface expression of CD14. Identification of the components of Chlamydia/Chlamydophila that can induce pro-inflammatory mediators coupled with the mechanisms by which epithelial cells detect infection and respond accordingly will advance the development of preventative strategies.     

A novel NF-κB inhibitor improves glucocorticoid sensitivity of canine neoplastic lymphoid cells by up-regulating expression of glucocorticoid receptors

Lymphoid neoplasms including lymphoma and leukemia are one of the most life-threatening disorders in dogs. Many lymphoid malignancies are well-treated with glucocorticoid (GC); however, GC resistance sometimes develops and its mechanism remains uncertain. Since constitutive activation of nuclear factor-κB (NF-κB) has been reported to play roles in lymphoid malignancies, we examined whether inhibition of NF-κB activity with a synthetic inhibitor IMD-0354 affected GC sensitivity of canine neoplastic lymphoid cells, CL-1 and GL-1. Dexamethasone failed to inhibit proliferation of these cells, in which low expression of glucocorticoid receptors (GR) was identified. In the presence of IMD-0354, GR expressions in CL-1 and GL-1 were increased, consequently dexamethasone inhibited their proliferation. These results indicated that GR expression might be down-regulated by spontaneous activation of NF-κB, resulting in GC resistance. Taken together, interference of NF-κB activity may have the synergistic effect in combination chemotherapy with GC for treatment against lymphoid malignancies.     

Ethyl acetate fraction of flavonoids from Polygonum hydropiper L. modulates pseudorabies virus-induced inflammation in RAW264.7 cells via the nuclear factor-kappa B and mitogen-activated protein kinase pathways

Pseudorabies virus (PRV) infection leads to severe inflammatory responses and tissue damage, and many natural herbs exhibit protective effects against viral infection by modulating the inflammatory response. An ethyl acetate fraction of flavonoids from Polygonum hydropiper L. (FEA) was prepared through ethanol extraction and ethyl acetate fractional extraction. An inflammatory model was established in RAW264.7 cells with PRV infection to evaluate the anti-inflammatory activity of FEA by measuring cell viability, nitric oxide (NO) production, reactive oxygen species (ROS) release, and mRNA expression of inflammatory factors, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Its functional mechanism was investigated by analyzing the phosphorylation and nuclear translocation of key proteins in the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Our findings indicate that PRV induced inflammatory responses in RAW264.7 cells, and the responses were similar to that in lipopolysaccharide (LPS)-stimulated cells. FEA significantly suppressed NO synthesis and down-regulated both expression and secretion of COX-2, iNOS, and inflammatory cytokines (P<0.05 or P<0.01). FEA also reduced NF-κB p65 translocation into the nucleus and decreased MAPK phosphorylation, indicating that the NF-κB/MAPK signaling pathway may be closely related to the inflammatory response during viral infection. The findings suggested the potential pharmaceutical application of FEA as a natural product that can treat viral infections due to its ability to mitigate inflammatory responses.     

脂多糖刺激对断奶仔猪肌肉炎症和蛋白质降解相关基因表达的影响

本试验旨在研究脂多糖(LPS)刺激后不同时间断奶仔猪肌肉炎症和肌肉蛋白质降解相关基因表达的变化规律。选择42头(7.1±0.9)kg杜×长×大三元杂断奶仔猪,按注射LPS之前(0 h)和注射LPS后1、2、4、8、12、24 h随机分为7个处理,每个处理6头猪。预试14 d后,腹腔注射100μg/kg体重的LPS。按以上时间点将仔猪屠宰,取背最长肌样品待测。结果表明:背最长肌炎性细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-6的mRNA表达量在注射LPS 1～2 h后达到峰值;Toll样受体4信号通路关键基因Toll样受体4(TLR4)、骨髓分化因子88(MyD88),核苷酸结合寡聚域信号通路关键基因核苷酸结合寡聚域2(NOD2)、受体互作蛋白激酶2(RIPK2)的mRNA表达量在注射LPS 2～4 h后达到峰值;肌肉蛋白质降解相关基因叉头转录因子-1(FOXO-1)、FOXO-4、肌肉环指蛋白1(MuRF1)、肌萎缩F-box(MAFbx)的mRNA表达量在注射LPS 12 h后达到峰值。可见,LPS刺激诱导肌肉释放大量炎性细胞因子,使TLR4和NOD炎症信号通路关键基因及肌肉蛋白质降解相关基因mRNA显著表达。     

Expression of Toll-like receptors and downstream genes in lipopolysaccharide-induced porcine alveolar macrophages

The aim of the present study was to determine the age-related kinetic changes of Toll-like receptors (TLRs) and downstream genes expression, and secretion of cytokine in lipopolysaccharide (LPS) stimulated porcine alveolar macrophages (AM). For this purpose, AMs were isolated from 5-day-old newborn piglets and 120-day-old young pigs. mRNA expression and cytokine measurement was determined by quantitative real-time PCR and ELISA, respectively. First, AMs were incubated for 24 h in the absence or presence of increasing concentrations of LPS. Results showed the up-regulation of TLRs 2, 4, 5 and 9 mRNA from all concentrations of LPS used, as compared to non-stimulated cells, and TLR4 was the highest expression in both ages (P<0.05). Furthermore, quantitative analysis demonstrated increased expression of mRNAs encoding TLRs 2, 4, 5 and 9, LBP, CD14, MD2, MyD88, IRAK4 and TRAF6 in both ages in a time-dependant manner (P<0.05). Overall, LPS inducible mRNA for TLR4, LBP, CD14 and MyD88 had higher expression in newborn piglets compared with those of young pigs (P<0.05). The level of cytokine protein IL6 and TNFα in supernatant fluid significantly varied with time of incubation and age of animals. Their concentration increased immediately at 1 h after LPS stimulation and remained significantly higher up to 48 h in both ages. Production of pro-inflammatory cytokine protein IL6 and TNFα in supernatant was significantly higher in young pigs than those of piglets. This study suggests that differential age-related changes in the expression of TLRs and downstream genes, and pro-inflammatory cytokine could contribute to a different age-related innate immune response during pulmonary infection. Further investigation is warranted to determine the precise effects of LPS on porcine AMs by means of a functional study across a wider age range.     

Suppression of proinflammatory cytokine production by specific metabolites of Lactobacillus plantarum 10hk2 via inhibiting NF-κB and p38 MAPK expressions

The objective of this study was to evaluate the immunomodulatory effects of specific bacterial metabolites of Lactobacillus plantarum 10hk2 to induce anti-inflammatory mediators in cell cultures of the murine macrophage cell line, RAW 264.7. The effects of the extracellular metabolites of this bacterial strain were examined by dividing them into protein and polysaccharide fractions. A specific protein fraction (8.7 kDa) was found to be a strong IL-10 inducer in LPS-stimulated RAW 264.7 cells and suppressed LPS-induced NF-κB induction and inhibited LPS-induced phosphorylation of I-κB and p38 MAPK. To the best of our knowledge, this was the first study that investigated the anti-inflammatory effects of an extracellular peptide derived from lactic acid bacteria. In addition, we characterized the inhibitory mode of this molecule in the induction of proinflammatory cytokines. Based on the findings presented in this study, this molecule holds promise for use as an agent to modulate inflammation related diseases.     

Saccharomyces cerevisiae β-glucan-induced SBD-1 expression in ovine ruminal epithelial cells is mediated through the TLR-2-MyD88-NF-κB/MAPK pathway

Ovine ruminal epithelial cells (ORECs) not only have a physical barrier function but also can secrete host defence peptides (HDPs), such as sheep β-defensin-1 (SBD-1). As a feed additive, Saccharomyces cerevisiae can enhance the host’s innate immunity. β-glucan, a cell wall component of Saccharomyces cerevisiae, can stimulate innate immune responses and trigger the up-regulation of SBD-1 in ORECs. The signaling mechanisms involved in β-glucan-induced SBD-1 expression are not completely understood. The aim of this study was to identify the receptors and intracellular pathways involved in the up-regulation of SBD-1 induced by β-glucan. ORECs were cultured, and the regulatory mechanisms of β-glucan-induced up-regulation of SBD-1 were detected using quantitative real-time PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and western blotting. TLR-2 and MyD88 knockdown or inhibition attenuated β-glucan-induced SBD-1 expression. We also showed that inhibition of MAPK and NF-κB pathways significantly reduced β-glucan-induced SBD-1 expression. These results demonstrate that β-glucan-induced SBD-1 expression is TLR-2-MyD88-dependent and may be regulated by both MAPK and NF-κB pathways. Since NF-κB inhibition had a greater effect on the down-regulation of β-glucan-induced SBD-1 expression, the NF-κB pathway may be the dominant signaling pathway involved in the regulation of defensin expression. Our studies demonstrate that β-glucan-induced SBD-1 expression is mediated through the TLR-2-MyD88-NF-κB/MAPK pathway. Our results would contribute to the understanding of immunological modulations in the gastrointestinal tract triggered by probiotic yeast cell wall components.

     

Mycoplasma ovipneumoniae induces inflammatory response in sheep airway epithelial cells via a MyD88-dependent TLR signaling pathway

Mycoplasma ovipneumoniae (M. ovipneumoniae) is a bacterium that specifically infects sheep and goat and causes ovine infectious pleuropneumonia. In an effort to understand the pathogen–host interaction between the M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory response using a primary air–liquid interface (ALI) epithelial culture model generated from bronchial epithelial cells of Ningxia Tan sheep (Ovis aries). The ALI culture of sheep bronchial epithelial cells showed a fully differentiated epithelium comprising distinct epithelial types, including the basal, ciliated and goblet cells. Exposure of ALI cultures to M. ovipneumoniae led to increased expression of Toll-like receptors (TLRs), and components of the myeloid differentiation factor 88 (MyD88)-dependent TLR signaling pathway, including the MyD88, TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinases (IRAKs) and nuclear factor-kappa B (NF-κB), as well as subsequent pro-inflammatory cytokines in the epithelial cells. Of interest, infection with M. ovipneumoniae failed to induce the expression of TANK-binding kinase 1 (TBK1), TRAF3 and interferon regulatory factor 3 (IRF3), key components of the MyD88-independent signaling pathway. These results suggest that the MyD88-dependent TLR pathway may play a crucial role in sheep airway epithelial cells in response to M. ovipneumoniae infection, which also indicate that the ALI culture system may be a reliable model for investigating pathogen–host interactions between M. ovipneumoniae and airway epithelial cells.