Differential induction of MyD88- and TRIF-dependent pathways in equine monocytes by Toll-like receptor agonists

Our understanding of the innate immune response in the horse has been limited by a lack of definitive data concerning cell signaling in response to microbial products. Toll-like receptors (TLRs) recognize conserved molecular motifs of microbes and elicit immune responses through their coupling with intracellular adaptor molecules, particularly MyD88 and TRIF. To provide a more definitive characterization of TLR signaling in the horse, the objectives of this study were to: (1) characterize the responses of equine monocytes to TLR ligands that signal through MyD88, TRIF or both in other species, and (2) determine the profiles of gene expression initiated utilizing these adaptor molecules. Monocytes were used to establish concentration response curves for Escherichia coli lipopolysaccharide (LPS; TLR4 ligand) and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam₃CSK₄; TLR2 ligand) based on expression of procoagulant activity (PCA) and production of tumor necrosis factor-alpha (TNF-α); effects of polyinosine–polycytidylic acid (Poly I:C; TLR3 ligand) were determined by quantifying expression of mRNA for interferon-beta (IFN-ß). Expression of genes associated with the MyD88- (TNF-α, IL-1ß, IL-6 and IL-10) and TRIF-dependent pathways (IFN-ß, IP-10, RANTES and TRAF1) were measured at intervals spanning 20 h. LPS and Pam₃CSK₄ induced significantly higher expression of TNF-α, IL-1ß, and IL-10 than did Poly I:C. Poly I:C induced significantly higher expression of IFN-ß, IP-10 and RANTES than did either the TLR2 or TLR4 ligands. High concentrations of E. coli LPS did not significantly increase expression of genes associated with the TRIF-dependent pathway. The results of this study suggest that equine monocytes utilize a common intracellular pathway in response to TLR2 and TLR4 ligands, but a distinct pathway in response to TLR3 ligands.     

Characterization of local and peripheral immune system in pregnant and nonpregnant ewes

Modulation of the immune system is known to be important for successful pregnancy but how immune function might differ between the lymph nodes draining the reproductive tract and peripheral lymph nodes is not well understood. Additionally, if immune system changes in response to the presence of an embryo during early pregnancy, and if this response differs in local versus peripheral immune tissue, has not been well characterized. To address these questions, we examined expression of genes important for immune function using NanoString technology in the ampulla and isthmus of the oviduct, endometrium, lymph nodes draining the reproductive tract (lumbo-aortic and medial iliac) as well as a peripheral lymph node (axillary), the spleen, and circulating immune cells from ewes on day 5 of the estrous cycle or pregnancy. Concentrations of estradiol and progesterone in plasma were also determined. Principal component analysis revealed separation of the local from the peripheral lymph nodes (MANOVA P = 3.245e-08, R² = 0.3) as well as separation of tissues from pregnant and nonpregnant animals [lymph nodes (MANOVA P = 2.337e-09, R² = 0.5), reproductive tissues (MANOVA P = 2.417e-14, R² = 0.47)]. Nine genes were differentially (FDR < 0.10) expressed between lymph node types, with clear difference in expression of these genes between the lumbo-aortic and axillary lymph nodes. Expression of these genes in the medial iliac lymph node was not consistently different to either the axillary or the lumbo-aortic lymph node. Expression of IL10RB was increased (FDR < 0.05) by 24% in the reproductive tissue of the pregnant animals compared to nonpregnant animals. Analysis of gene categories revealed that expression of genes of the T-cell receptor pathway in reproductive tract tissues was associated (P < 0.05) with pregnancy status. In conclusion, assessment of gene expression of reproductive and immune tissue provides evidence for a specialization of the local immune system around the reproductive tract potentially important for successful establishment of pregnancy. Additionally, differences in gene expression patterns in reproductive tissue from pregnant and nonpregnant animals could be discerned as early as day 5 of pregnancy. This was found to be associated with expression of genes important for T-cell function and thus highlights the important role of these cells in early pregnancy.     

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.     

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.     

Molecular Mechanism of the Inflammatory Response Induced by Enterotrxigenic E. coli K88 in Piglets

To investigate the molecular mechanism of the inflammatory response in the piglets infected with enterotoxigenic E. coli (ETEC) K88, piglets were infected with ETEC K88,the IL-8 content in serum of piglets were assayed by ELISA,and the mRNA relative expression levels of TLR2/4 and its signal transduction pathway related genes (MyD88,Tollip and Bcl3) in mesenteric lymph nodes were detected by quantitative Real-time PCR. The results showed that compared with control group,the content of IL-8 in serum and the expressions of TLR2/4 in lymph nodes were all extremely significantly or significantly increased at 6 and 24 h after infection (P<0.01;P<0.05),and the IL-8 content and TLR2/4 mRNA expression at 24 h after infection were all significantly lower than those at 6 h after infection (P<0.05).In addition,the expressions of MyD88,Tollip and Bcl3 in lymph nodes were all extremely significantly increased at 24 h after infection compared with control group (P<0.01), but there was no significant difference between experimental group and control group at 6 h after infection (P>0.05). In conclusion,ETEC K88 infected piglets might produce inflammatory cytokines IL-8 through the TLR2/4-MyD88 signaling pathway,which could promote the inflammatory reaction in piglets. This inflammatory response might be regulated by Tollip and Bcl3,which could weak the inflammatory intensity in piglets.     

Lymphocyte localization in lymph nodes of pubescent, prepartum, and postpartum sheep

It is generally accepted that lymphocytes associated with the mammary mucosal immune system of non-ruminants may be largely derived from gut-associated lymphoid tissue (GALT). The relationship between the mammary immune system and the GALT of ruminants has not been clearly defined. To address this question, we examined patterns of lymphocyte localization in sheep by 51Cr-labeled lymphocytes following infusion back into donor ewes. We found that lymphocytes taken from mammary lymph nodes of pubescent ewes returned preferentially to mammary nodes, while in prepartum and postpartum ewes, mammary node cells localized equally well in mammary and mesenteric lymph nodes. In contrast, ileal mesenteric lymph node cells from pubescent ewes localized equally well in mammary and mesenteric nodes, but in prepartum and postpartum ewes, localization in mammary nodes was markedly reduced. Comparison of the homing patterns of mammary, mesenteric, and peripheral lymph node cells indicated that mammary node cells behaved similarly to peripheral, rather than mesenteric node cells. This information may be relevant to the extent of communication between the gut and mammary gland in ruminants.     

产肠毒素大肠杆菌K88诱导仔猪炎症反应的分子机制

为探讨产肠毒素大肠杆菌（ETEC） K88感染仔猪发生炎症反应的分子机制,试验用ETEC K88灌服断奶仔猪,ELISA法检测攻毒后仔猪血清中白细胞介素8（IL-8）含量,实时荧光定量PCR方法检测淋巴结中Toll样受体2（TLR2）、Toll样受体4（TLR4）及其信号通路相关基因（髓样分化因子88（MyD88）、Toll相互作用蛋白（Tollip）、B细胞淋巴瘤因子3（Bcl3））的mRNA相对表达水平。结果发现,仔猪攻毒ETEC K88后6和24 h血清IL-8含量和淋巴结TLR2/4的表达水平均极显著或显著高于对照组（P<0.01;P<0.05）,且感染后24 h显著低于感染后6 h（P<0.05）;仔猪感染ETEC K88后24 h淋巴结中MyD88、Tollip和Bcl3的表达水平均极显著高于对照组（P<0.01）,但是感染后6 h时与对照组相比均无显著差异（P>0.05）。综上所述,ETEC K88感染仔猪可能是通过TLR2/4-MyD88信号通路产生炎症因子IL-8,促使仔猪出现炎症反应,且该炎症反应可能受Tollip和Bcl3蛋白的调控而被减弱。     

Development of maternal and foetal immune responses in cattle following experimental challenge with Neospora caninum at day 210 of gestation

This study examined the immunological responses of pregnant cattle and their foetuses following an experimental challenge with live Neospora caninum tachyzoites at day 210 of gestation. Animals were bled prior to and weekly throughout the experiment and sacrificed at 14, 28, 42 and 56 days post inoculation (dpi). At post mortem examination, samples of lymph nodes and spleen were collected from both dam and foetus for immunological analysis. Subcutaneous (sc) inoculation over the left prefemoral (LPF) lymph node of pregnant cattle at day 210 of gestation, led to the vertical transmission of parasites by 14 dpi, however no foetal deaths were observed in the infected animals. Foetuses from infected dams mounted Neospora-specific humoral and cell-mediated immune (CMI) responses by 14 dpi. These responses involved anti-Neospora IgG, antigen-specific lymphocyte proliferation, and the production of the cytokines IFN–γ, interleukin (IL)-4 and IL-10. There was also evidence of innate immunity during the response against Neospora from infected dams, with statistically significant (p < 0.05) increases in mean expression of toll like receptors (TLR)-2 on 56 dpi in maternal spleen, LPF, right prefemoral (RPF), left uterine (LUL) and right uterine (RUL) lymph nodes and TLR-9 in retropharyngeal (RLN), LPF and RPF lymph nodes from 28 dpi. Statistically significant (p < 0.05) increases in mean TLR-9 were detected in spleen samples from foetuses of infected dams, compared to the foetuses from control animals. Our results show that vertical transmission of the parasite occurred in all infected dams, with their foetuses showing effective Neospora-specific cell mediated, humoral and innate immune responses.     

Elevated Plasma Levels of Interleukin 1β, Tumour Necrosis Factor α and Monocyte Chemotactic Protein 1 Are Associated with Pregnancy Toxaemia in Ewes

Pregnancy toxaemia is a metabolic disorder that results from an inadequate energy supply to the growing maternal–fetal unit. The mechanism underlying the pathogenesis of the syndrome has not been fully clarified; however, a key role for cytokines and chemokines including interleukin 1 β (IL-1β), tumour necrosis factor α (TNF-α) and monocyte chemotactic protein 1 (MCP-1) has been indicated in women and experimental animals. However, information on the maternal plasma levels of IL-1β, TNF-α and MCP-1 in ewes with pregnancy toxaemia is limited. Thus, the present study was designed to determine plasma IL-1β, TNF-α and MCP-1 concentrations in ewes with severe (n = 6) and mild (n = 4) naturally occurring pregnancy toxaemia and in uncomplicated pregnant ewes (n = 10) using enzyme-linked immunosorbent assay (ELISA). All ewes with pregnancy toxaemia had significantly lower body temperature and respiratory rate than uncomplicated pregnant ewes (p < 0.05). With the highest concentrations in severe cases, heart rate, proteinuria and serum uric acid levels as well as plasma IL-1β, TNF-α and MCP-1 were significantly different among all three groups (p < 0.05). The plasma concentrations of IL-1β in control ewes and ewes with mild and severe toxaemia were 15.81 ± 3.90 pg/ml, 23.83 ± 2.42 pg/ml and 34.55 ± 8.03 pg/ml, respectively. The plasma concentrations of TNF-α in control ewes and ewes with mild and severe toxaemia were 7.71 ± 1.61 pg/ml, 16.13 ± 3.63 pg/ml, and 22.85 ± 3.64 pg/ml, respectively. The plasma concentrations of MCP-1 in control ewes and ewes with mild and severe toxaemia were 101.70 ± 9.86 pg/ml, 134.75 ± 6.24 pg/ml, and 157.67 ± 9.69 pg/ml, respectively. Moreover, plasma IL-1β, TNF-α and MCP-1 levels were positively correlated with clinical and well-establish biochemical parameters of pregnancy toxaemia, serum uric acid and proteinuria (p < 0.01). Concomitant increase of plasma IL-1β, TNF-α and MCP-1 concentrations along with serum uric acid, proteinuria, and worsening of the clinical signs indicates that such cytokines are involved in the aetiopathogenesis and in perpetuation of the local and systemic inflammatory reactions in pregnancy toxaemia in ewes. Hence, plasma IL-1β, TNF-α and MCP-1 may potentially serve as markers to monitor prognosis of pregnancy toxaemia in ewes.     

Dietary rumen-protected L-arginine or N-carbamylglutamate attenuated fetal hepatic inflammation in undernourished ewes suffering from intrauterine growth restriction

This study aimed to explore whether dietary rumen-protected L-arginine (RP-Arg) or N-carbamylglutamate (NCG) supplementation to feed-restricted pregnant ewes counteracts fetal hepatic inflammation and innate immune dysfunction associated with intrauterine growth retardation (IUGR) in ovine fetuses. On d 35 of pregnancy, twin-bearing Hu ewes (n = 32) were randomly assigned to 4 treatment groups (8 ewes and 16 fetuses per group) and fed diets containing 100% of the NRC requirements (CON), 50% of the NRC requirements (RES), RES + RP-Arg (20 g/d) (RESA), or RES + NCG (5 g/d) (RESN). At 08:00 on d 110 of gestation, fetal blood and liver tissue samples were collected. The levels of triglyceride, free fatty acid, cholesterol and β-hydroxybutyrate in the fetal blood of RESA and RESN groups were lower (P < 0.05) than those of the RES group, but were higher (P < 0.05) than those of the CON group. The interleukin (IL)-6 and IL-1 levels in fetal blood and liver tissue as well as the myeloid differentiation primary response 88 (MyD88), transforming growth factor β (TGFβ), and nuclear factor kappa B (NF-κB) mRNA levels in the fetal liver were decreased (P < 0.05) by the NCG or RP-Arg supplementation compared to the RES treatment. Similarly, the toll-like receptor (TLR)-4, MyD88, TGFβ, and p-c-Jun N-terminal kinase (JNK) protein levels in the fetal liver were reduced (P < 0.05) in the NCG and RP-Arg -supplemented groups compared to the RES group. These results showed that dietary supplementation of RP-Arg or NCG to underfed pregnant ewes could protect against IUGR fetal hepatic inflammation via improving lipid metabolism, down-regulating the TLR-4 and the inflammatory JNK and NF-κB signaling pathways, and decreasing cytokine production in ovine fetal blood and liver tissue.     

Early supplementation with Lactobacillus plantarum in liquid diet modulates intestinal innate immunity through toll-like receptor 4-mediated mitogen-activated protein kinase signaling pathways in young piglets challenged with Escherichia coli K88

This study was conducted to investigate the effects of early supplementation during 4 to 18 d of age with Lactobacillus plantarum (LP) in liquid diets on intestinal innate immune response in young piglets infected with enterotoxigenic Escherichia coli (ETEC) K88. Seventy-two barrow piglets at 4 d old were assigned to basal or LP-supplemented liquid diet (5 × 10¹⁰ CFU·kg⁻¹). On day 15, piglets from each group were orally challenged with either ETEC K88 (1 × 10⁸ CFU·kg⁻¹) or the same amount of phosphate-buffered saline. The intestinal mucosa, mesenteric lymph node (MLN), and spleen samples were collected on day 18. Here, we found that LP pretreatment significantly decreased the mRNA relative expression of inflammatory cytokines (interleukin [IL]-1β, IL-8, and tumor necrosis factor-α), porcine β-defensin 2 (pBD-2), and mucins (MUC1 and MUC4) in the jejunal mucosa in piglets challenged with ETEC K88 (P < 0.05). Moreover, LP significantly decreased the ileal mucosa mRNA relative expression of IL-8 and MUC4 in young piglets challenged with ETEC K88 (P < 0.05). Furthermore, the piglets of the LP + ETEC K88 group had lower protein levels of IL-8, secretory immunoglobulin A, pBD-2, and MUC4 in the jejunal mucosa than those challenged with ETEC K88 (P < 0.05). Besides, LP supplementation reduced the percentage of gamma/delta T cells receptor (γδTCR) and CD172a⁺ (SWC3⁺) cells in MLN and the percentage of γδTCR cells in the spleen of young piglets after the ETEC K88 challenge. Supplementation with LP in liquid diets prevented the upregulated protein abundance of toll-like receptor (TLR) 4, phosphorylation-p38, and phosphorylation-extracellular signal-regulated protein kinases in the jejunal mucosa induced by ETEC K88 (P < 0.05). In conclusion, LP supplementation in liquid diet possesses anti-inflammatory activity and modulates the intestinal innate immunity during the early life of young piglets challenged with ETEC K88, which might be attributed to the suppression of TLR4-mediated mitogen-activated protein kinase signaling pathways. Early supplementation with LP in liquid diets regulates the innate immune response, representing a promising immunoregulation strategy for maintaining intestinal health in weaned piglets.     

Effect of Compound Chinese Herbal Medicinal Polysaccharides on MyD88 Dependent Pathway Mediated by TLR4 in Lymphocytes of Chicken with Different MHC B-LβⅡ Genotypes

To investigate the effects of different doses of compound Chinese herbal medicinal polysaccharides (cCHMPS) on TLR4 and downstream MyD88 dependent signal transduction pathway components in chicken lymphocytes of different MHC B-LβⅡ genotypes,PCR-SSCP technique was applied to group layer according to different MHC B-LβⅡ genotypes.The peripheral blood lymphocytes of chicken with different MHC B-LβⅡ genotypes were collected,and added with 100,75,50 and 0 μg/mL cCHMPS (high,middle and low dose groups and control group),respectively,then co-culturing for 16,24,32 and 48 h.The expression of TLR4,MYD88 and TRAF-6 mRNA were detected using Real-time PCR method.The results showed that compared with control group,cCHMPS could significantly improve the expression levels of TLR4,MYD88 and TRAF-6 mRNA of different MHC B-Lβ Ⅱ genotypes chickens (P < 0.05);The expression levels of TLR4,MYD88 and TRAF-6 mRNA of AA genotype chicken lymphocyte in middle and low dose groups were higher than those of high dose group (except TLR4 gene cultured for 16 h);The expression of TLR4,MyD88 and TNAF-6 mRNA of BB genotype in high dose group were higher than those of other dose groups (except TLR4 gene cultured for 32 and 48 h);The expression of TLR4,MyD88 and TNAF-6 mRNA of BC genotype in low dose were higher than that of other dose groups (except TLR4 gene cultured for 16 h).There results indicated that cCHMPS played an important role in the body’s immune regulatory mechanism by binding to TLR4 in the surface of lymphocytes,activating the downstream MyD88-dependent signal transduction pathway,regulating cellular immunity,and cCHMPS optimum immunomodulatory does were different in each MHC B-Lβ Ⅱ genotype chickens.     

Effect of diet and type of pregnancy on plasma metabolic response in sheep and its further effect on lamb performance

This trial evaluated the individual and interactional effects of diet and type of pregnancy (twin or single) on plasma metabolic response in ewes and their lambs from late pre-partum to late post-partum. Thus, a flock of 18 Ile de France breed sheep, consisting of 8 twin-bearing and 10 single-bearing ewes, were allocated to one of two groups according to their diet, either based on ad libitum naturalized pasture hay (NPH) or red clover hay (RCH), from d 45 pre-partum to d 60 post-partum. Plasma samples were collected at different times to determine albumin, cholesterol, total protein and urea, plus glucose and β-hydroxybutyrate (BHB) concentration in ewes. The data was processed using the lme4 package for R, and SPSS Statistics 23.0 for Windows. The results showed that both diet and type of pregnancy influenced the metabolic profile in ewes, showing an inverse relationship between single- and twin-bearing ewes regarding glucose and especially BHB proportions from pre-partum to birth. During post-partum, higher urea concentrations were observed in twin- and single-bearing ewes fed RCH in contrast to those fed NPH, as a result of the higher-quality forage offered to ewes. Regarding lambs, the diet and type of pregnancy influenced the total protein and urea levels, where an inverse relationship at birth and early post-partum between albumin and cholesterol vs. total protein and urea was detected, reflecting a trend (P value between 0.06 and 0.07) to a better performance by groups of single lambs, especially those from single-bearing ewes fed RCH. Finally, under the conditions of this study, the maternal diet and type of pregnancy influenced the plasma metabolic response in ewes and their lambs, affecting the lamb performance especially at birth.

     

Detection of Mycobacterium avium subsp. paratuberculosis in skeletal muscle and blood of ewes from a sheep farm in New Zealand

Abstract

AIM: To determine whether viable Mycobacterium avium subsp. paratuberculosis (Map) is present in skeletal muscle and blood in ewes with and without Johne's disease confirmed histologically.

METHODS: A total of 51 mixed-aged ewes in poor body condition from a farm with a history of clinical Johne's disease were culled and examined at necropsy. BACTEC radiometric culture was performed on samples of skeletal muscle from the biceps femoris, mononuclear cells in peripheral blood (hereafter referred to as blood), and ileum. Histological sections and Ziehl-Neelsen (ZN)-stained impression smears of terminal ileum and mesenteric lymph nodes were examined. Ewes were defined as having confirmed Johne's disease if there was histopathological evidence typical of the disease within the ileum and adjacent lymph nodes.

RESULTS: Eighteen of 21 (86%) ewes with confirmed clinical Johne's disease were culture-positive for Map from sites peripheral to the alimentary tract, comprising 15 from skeletal muscle and 13 from blood. Five of 30 (17%) ewes that did not have Johne's disease were culture-positive, with four from skeletal muscle and one from blood. The likelihood that ewes with confirmed Johne's disease had systemic Map infection compared with ewes without was determined as OR=30 (95% CI=6.3–142.0; p<0.001).

CONCLUSION: The prevalence of Map infection of skeletal muscle and blood in ewes with confirmed Johne's disease was 71% and 62% respectively, and in unaffected ewes was 13% for muscle and 3% for blood.

CLINICAL RELEVANCE: Skeletal muscle and blood are potential sources of exposure of humans to Map, and the risk appears higher from sheep with Johne's disease.     

In silico identification of components of the Toll-like receptor (TLR) signaling pathway in clustered chicken expressed sequence tags (ESTs)

We have described a bioinformatic approach that involves the clustering of expressed sequence tags (ESTs) to reveal homologs of the Toll-like receptor (TLR) pathway in the chicken. Homology searching of proteins, predicted to be encoded by these EST clusters, resulted in the in silico identification of full-length sequences for Toll-interacting protein (Tollip), IL-1 receptor-associated kinase 4 (IRAK-4), myeloid differentiation factor 88 adapter-like (Mal), TGF beta-activated kinase 1 binding protein 1 (TAB1). We also determined partial sequence information for myeloid differentiation factor 88 (MyD88), two novel TLRs, TNF receptor-associated factor 6 (TRAF6), TGF beta-activated kinase 1 (TAK1), TAB2, inhibitor of nuclear factor kappa B kinase alpha (IKK alpha) and IKK beta. This bioinformatics study has confirmed the evolutionary conservation of the TLR pathway in chicken and demonstrated its essential homology to the TLR pathway in mammals. We have identified in silico the full-length sequence for liver-expressed antimicrobial peptide 2 (LEAP-2). This is the first time a non-mammalian LEAP-2 has been described.     

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88^−/−) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88^−/− mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88^−/− mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-γ responses by NKT, CD4⁺ and CD8⁺ T lymphocytes, and production of high levels of IL-10. MyD88^−/− mice replenished with IL-12 and IFN-γ abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-γ-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.     

Update on the role of innate immune receptors during Brucella abortus infection

The innate immune system constitutes an efficient defense mechanism against invading microbial pathogens. Recent studies have revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella spp. infection. However, there is a piece of the puzzle missing that is the role of non-TLR receptors in innate immunity. The involvement of TLR receptors in brucellosis has been investigated by different research groups. It was demonstrated that TLR2 clearly does not play any role in controlling Brucella abortus infection in vivo, whereas TLR9 has been shown to be required for clearance of this bacterium in infected mice. The participation of adaptor molecules, such as MyD88 and TRIF has also been discussed. Recently, we and others have reported the critical role of MyD88- and not TRIF-mediated signaling in dendritic cell maturation and in vivo resistance during B. abortus infection. However, the relationship between specific Brucella molecules and non-TLR receptors and signal transduction pathways needs to be better understood. It is now clear that the interaction between TLRs and recently identified cytosolic innate immune sensors is crucial for mounting effective immune responses. Finally, this review discusses the mechanisms used by Brucella to escape detection by the host innate immune system.     

TLRs介导的信号通路在马链球菌感染RAW264.7细胞中的作用

为探究Toll样受体(TLRs)介导的信号通路在马链球菌马亚种(S.equi)感染小鼠巨噬细胞RAW264.7中的作用,收集S.equi感染后不同时间点的RAW264.7细胞,提取总RNA并反转录成cDNA,利用实时荧光定量PCR技术检测细胞Toll样受体1、2、6(TLR1、TLR2、TLR6)、接头蛋白骨髓分化蛋白88(MyD88)及细胞因子IL-1、IL-6、IL-10、IL-12、TNF-αmRNA的表达情况。结果显示,S.equi感染RAW264.7细胞后6h时,TLR1、TLR2、TLR6与MyD88mRNA水平均较对照组没有显著差异(P>0.05);感染后12h时,TLR1、TLR2和TLR6mRNA表达量未出现明显上升(P>0.05),而MyD88mRNA水平极显著升高(P<0.01);感染后24h时,TLR1、TLR2和TLR6mRNA表达水平出现极显著升高(P<0.01),MyD88mRNA表达没有显著变化(P>0.05),且IL-10和IL-12mRNA水平与对照组相比极显著升高(P<0.01),IL-1、IL-6和TNF-αmRNA水平均极显著下降(P<0.01)。结果表明,TLRs介导的信号通路参与S.equi感染RAW264.7细胞的免疫应答反应。