Effects of a recombinant schistosomal-derived anti-inflammatory molecular (rSj16) on the lipopolysaccharide (LPS)-induced activated RAW264.7

Macrophages as a principal component of immune system play an important role in the initiation, modulation, and final activation of immune response against pathogens including schistosomes. Classical (M1) or alternative (M2) activation states of macrophage have different functions during infections. Previously, we report that the schistosomal-derived anti-inflammatory molecule coding gene (named Sj16) was isolated and the recombinant Sj16 (rSj16) was expressed in Escherichia coli. rSj16 has been demonstrated to have definite anti-inflammatory effect in vivo and in vitro on rodent model. To study the molecular basis on anti-inflammatory of rSj16, in the present paper, we investigate the effects of rSj16 on the lipopolysaccharide (LPS)-induced activated RAW264.7, a murine macrophage cell line. We found that rSj16 inhibited LPS-induced activation of RAW264.7, as evidenced by impacting the proliferation, phagocytosis, and migration of the RAW264.7. After pretreated with rSj16, it showed the most potent inhibitory effects of rSj16 on the nitric oxide production in RAW264.7 cells. Furthermore, rSj16 also significantly decreased the levels of proinflammatory cytokines such as PGE2, IL-1β, IL-6, IL-12, IL-23, and TNF-α, whereas it increased the levels of immunosuppressive cytokine IL-10. rSj16 can also inhibit the LPS-induced activation of NF-κβ. These results further imply that Sj16 contributes to the immune evasion of Schistosoma japonicum through alternatively activated macrophage (M2), and rSj16 is expected to serve as a potential drug source for the medication of inflammatory disorders.     

Lipoxin A4 Inhibits NF-κB Activation and Cell Cycle Progression in RAW264.7 Cells

Lipoxins (LXs), including lipoxin A₄ (LXA₄), etc., have been approved for potent anti-inflammatory and immunomodulatory properties. Based on the important roles of macrophages in inflammation and immunomodulation, we investigate the effects of LXA₄ on lipopolysaccharide (LPS)-induced proliferation and the possible signal transduction pathways in RAW264.7 macrophages. RAW264.7 cells were treated in vitro with or without LPS in the absence or presence of LXA₄. [³H]-TdR incorporation assay and flow cytometry were used for detecting cell proliferation and cycle, respectively. Moreover, Western blot was applied to evaluate the protein expression levels of Cyclin E, IκBα, nuclear factor-κB (NF-κB), and IκB kinase (IKK). Our research showed that LXA₄ suppressed LPS-induced proliferation, increased the proportion of the G₀/G₁ phase, decreased the proportion of the S phase, and downregulated the expression of Cyclin E. Besides these, LXA₄ suppressed LPS-induced IκBα degradation, NF-κB translocation, and the expression of IKK. The data suggested that LXA₄ inhibited LPS-induced proliferation through the G₀/G₁ phase arrest in RAW264.7 macrophages, and the inhibitory effect might depend on NF-κB signaling transduction pathway.     

Sjögren's syndrome autoantibodies provoke changes in gene expression profiles of inflammatory cytokines triggering a pathway involving TACE/NF-κB

We explore the association of the inflammatory gene expression profile observed in the chronic inflammatory autoimmune disorder Sj?gren's syndrome (SS) with changes in TNF-α converting enzyme (TACE), tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB levels showing that pathways that include TNF-α signaling converge on NF-κB contributing to exacerbate the diseases. The treatment of human salivary gland epithelial cells (SGECs) with SS anti-Ro/SSA autoantibodies (Abs) result in a progressive increase in NF-κB-DNA binding, that includes a marked enhancement in NF-κB subunit p65 protein-DNA binding. A human cytokine multi-analyte array demonstrated that the NF-κB proinflammatory target genes, increased by anti-Ro/SSA Abs treatment, includes CXC chemokines (CXCL1, CXCL6 and CXCL9), CC chemokines (CCL2, CCL13 and CCL20), interleukins (IL-1α, IL-1β, IL-1F8, IL-6, IL-8, IL-9, IL-13, IL-17 and IL-22) and their receptors (IL-1RN, IL-10Rα, IL-13Rα, CCR1, CCR2, CCR3, CCR4 and CXCR1). Blockade of TACE through the use of the specific inhibitor TAPI-1 regulates proinflammatory cytokines production in SGEC treated with anti-Ro/SSA Abs inhibiting NF-κB nuclear translocation and activation. To further investigate the role of NF-κB on anti-Ro/SSA Abs-determined proinflammatory gene expression, we used the inhibitory protein IκB-α dominant negative super-repressor as inhibitor of NF-κB-DNA binding, demonstrating that transfection with dominant-negative IκB-α in anti-Ro/SSA-treated SGEC determined a marked reduction of proinflammatory cytokines gene expression. Although further studies are needed to clarify the mechanisms underlying SS, our results demonstrate that SS Abs exert their pathogenic effects via triggering the TACE/TNF-α/NF-κB axis.     

Pathway-focused genetic evaluation of immune and inflammation related genes with chronic fatigue syndrome

Recent evidence suggests immune and inflammatory alterations are important in chronic fatigue syndrome (CFS). This study was done to explore the association of functionally important genetic variants in inflammation and immune pathways with CFS. Peripheral blood DNA was isolated from 50 CFS and 121 non-fatigued (NF) control participants in a population-based study. Genotyping was performed with the Affymetrix Immune and Inflammation Chip that covers 11 K single nucleotide polymorphisms (SNPs) following the manufacturer’s protocol. Genotyping accuracy for specific genes was validated by pyrosequencing. Golden Helix SVS software was used for genetic analysis. SNP functional annotation was done using SPOT and GenomePipe programs. CFS was associated with 32 functionally important SNPs: 11 missense variants, 4 synonymous variants, 11 untranslated regulatory region (UTR) variants and 6 intronic variants. Some of these SNPs were in genes within pathways related to complement cascade (SERPINA5, CFB, CFH, MASP1 and C6), chemokines (CXCL16, CCR4, CCL27), cytokine signaling (IL18, IL17B, IL2RB), and toll-like receptor signaling (TIRAP, IRAK4). Of particular interest is association of CFS with two missense variants in genes of complement activation, rs4151667 (L9H) in CFB and rs1061170 (Y402H) in CFH. A 5′ UTR polymorphism (rs11214105) in IL18 also associated with physical fatigue, body pain and score for CFS case defining symptoms. This study identified new associations of CFS with genetic variants in pathways including complement activation providing additional support for altered innate immune response in CFS. Additional studies are needed to validate the findings of this exploratory study.     

Chemokines and other GPCR ligands synergize in receptor-mediated migration of monocyte-derived immature and mature dendritic cells

Dendritic cells (DCs) are potent antigen presenting cells, described as the initiators of adaptive immune responses. Immature monocyte-derived DCs (MDDC) showed decreased CD14 expression, increased cell surface markers DC-SIGN and CD1a and enhanced levels of receptors for the chemokines CCL3 (CCR1/CCR5) and CXCL8 (CXCR1/CXCR2) compared with human CD14⁺ monocytes. After further MDDC maturation by LPS, the markers CD80 and CD83 and the chemokine receptors CXCR4 and CCR7 were upregulated, whereas CCR1, CCR2 and CCR5 expression was reduced. CCL3 dose-dependently synergized with CXCL8 or CXCL12 in chemotaxis of immature MDDC. CXCL12 augmented the CCL3-induced ERK1/2 and Akt phosphorylation in immature MDDC, although the synergy between CCL3 and CXCL12 in chemotaxis of immature MDDC was dependent on the Akt signaling pathway but not on ERK1/2 phosphorylation. CCL2 also synergized with CXCL12 in immature MDDC migration. Moreover, two CXC chemokines not sharing receptors (CXCL12 and CXCL8) cooperated in immature MDDC chemotaxis, whereas two CC chemokines (CCL3 and CCL7) sharing CCR1 did not. Further, the non-chemokine G protein-coupled receptor ligands chemerin and fMLP synergized with respectively CCL7 and CCL3 in immature MDDC signaling and migration. Finally, CXCL12 and CCL3 did not cooperate, but CXCL12 synergized with CCL21 in mature MDDC chemotaxis. Thus, chemokine synergy in immature and mature MDDC migration is dose-dependently regulated by chemokines via alterations in their chemokine receptor expression pattern according to their role in immune responses.     

Tumor-associated macrophages promote the metastasis of ovarian carcinoma cells by enhancing CXCL16/CXCR6 expression

This study investigated the underlying mechanism by which C-X-C motif chemokine ligand 16 (CXCL16)/C-X-C motif chemokine receptor 6 (CXCR6) signaling is activated by tumor-associated macrophages and assists in regulating the metastasis of ovarian carcinoma. Specimens of ovarian carcinoma tissue and adjacent tissue were collected from 20 ovarian carcinoma patients. Human THP-1 cells were induced to differentiate into macrophages, which were then co-cultured with SKOV3 cells and low concentrations of tumor necrosis factor-α (TNF-α) to simulate the inflammatory microenvironment of ovarian carcinoma. Additionally, small interfering RNA (siRNA) targeting CXCR6 was transfected into SKOV3 cells; after which, the levels of nuclear factor kappa B p65 (NF-κB p65) protein and phosphorylated PI3K and Akt were measured. The migration and invasion abilities of the SKOV3 cells were also tested. The levels of TNF-α, interluekin-6 (IL-6), NF-κB p65, CXCL16, and CXCR6 expression in the ovarian carcinoma tissues were higher than those in the precancerous tissues. CXCR6 expression was positively correlated with TNF-α, IL-6, and CXCL16 expression. Co-culture of SKOV3 cells with macrophages significantly promoted CXCL16, CXCR6, NF-κB, and p65 expression by the SKOV3 cells, increased their levels of phosphorylated PI3K and Akt, and increased the migration and invasion abilities of SKOV3 cells. Silencing of CXCR6 or blocking the PI3K/Akt signal pathway markedly attenuated the expression of NF-κB p65 and phosphorylation of PI3K and Akt, as well as the migration and invasion abilities of SKOV3 cells. These findings demonstrate that macrophages can promote the migration and invasion of ovarian carcinoma cells by affecting the CXCL16/CXCR6 pathway.     

Cytoprotection of Perfluorocarbon on PMVECs In Vitro

Lipopolysaccharide (LPS) can activate endothelial cells and induce inflammatory injury. Toll-like receptor-4 (TLR-4) is integrally involved in LPS signaling and has a requisite role in the activation of nuclear factor (NF)-κB. A number of studies have demonstrated the cytoprotective action of perfluorocarbon (PFC) both in vivo and in vitro, but the exact mechanisms have yet to be elucidated. In this study, we examined in an in vitro model the cytoprotective effect of PFC on LPS-stimulated pulmonary vascular endothelial cells (PMVECs). Intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8) were significantly increased in the LPS-stimulated PMVECs groups. The expression of TLR-4 mRNA and protein in LPS groups was markedly increased. Meanwhile, NF-κB was activated. There were no significant effects of PFC alone on any of the factors studied while the coculture group showed significant downregulation of the secretion of ICAM-1, TNF-α, and IL-8; the expression of TLR-4 mRNA; and the activity of NF-κB. LPS can induce PMVEC inflammatory injury via the activation of TLR-4 and subsequent activation of NF-κB. PFC is able to protect PMVECs from LPS-induced inflammatory injury by blocking the initiation of the LPS signaling pathway.     

利什曼原虫感染树突状细胞早期基因表达差异分析及关键基因筛选

目的:分析利什曼原虫感染树突状细胞(DCs)早期的基因表达与信号通路变化,探究DCs感染后应答,寻找利什曼原虫感染后基于DCs的免疫治疗方法。方法:GEO数据库下载利什曼原虫感染前后DCs基因芯片数据,RStudio软件筛选差异表达基因(DEGs),STRING构建DEGs蛋白质相互作用网络(PPI),Cytoscape筛选差异表达蛋白质的核心模块,RStudio软件对DEGs进行GO和KEGG富集分析。结果:共筛选出DEGs 129个,其中IL12B与CXCL10差异最为显著,GO分析共富集23个过程,主要涉及病毒感染过程相关细胞反应及Ⅰ-IFN相关免疫反应;KEGG分析共富集3条信号通路,分别为甲型流感、麻疹及DNA复制信号通路。结论:利什曼原虫感染DCs前后Ⅰ-IFN信号通路和TLR4/NF-κB信号通路激活,影响IL12表达,提示Ⅰ-IFN/IL12信号通路与TLR4/NF-κB/IL12信号通路可作为利什曼原虫感染治疗的靶点,CXCL10也有望成为潜在的治疗靶点;利什曼原虫感染后,出现类似病毒感染现象,推测抗病毒免疫疗法可能在对抗利什曼原虫感染中具有一定疗效。     

Spironolactone inhibits production of proinflammatory mediators in response to lipopolysaccharide via inactivation of nuclear factor-κB

The effect of spironolactone (SPIR) on lipopolysaccharide (LPS)-induced production of proinflammatory mediators was examined using RAW 264.7 macrophage-like cells and mouse peritoneal macrophages. SPIR significantly inhibited LPS-induced production of nitric oxide (NO), tumor necrosis factor-α and prostaglandin E2. The inhibition was not mediated by cell death. SPIR reduced the expression of an inducible NO synthase mRNA in response to LPS. SPIR significantly inhibited phosphorylation of p65 nuclear factor (NF)-κB in response to LPS. Furthermore, SPIR inhibited phosphorylation of IκB kinase (IKK) as an upstream molecule of NF-κB in response to LPS. LPS did not induce the production of aldosterone in RAW 264.7 cells. Taken together, SPIR is suggested to inhibit LPS-induced proinflammatory mediators via inactivation of IKK/NF-κB in LPS signaling.     

Enhanced production of interleukin-29 and related genes are associated with T helper 1 cell parameters in patients with type 2 diabetes mellitus

ObjectiveThe production of interleukin (IL)-29 and the genes related to IL-29 signaling pathway (STAT1, NF-κB, and NFATc1), and T helper (Th) 1 cells (T-bet, IFN-γ, TNF-α, and IL-2) were evaluated in type 2 diabetes mellitus (T2DM). Correlations between IL-29 and diabetes parameters, and between gene expression in IL-29 pathway and Th1 cells were also examined.Materials and Methods41 newly diagnosed patients with T2DM and 41 healthy controls were recruited. CD4⁺ T cells were purifed and the production of IL-29 in the supernatant of anti- CD3 and anti- CD28 activated Th cells was detected using ELISA. The expression of IL-29- and Th1- related genes was determined with real-time PCR.ResultsThe secretion of IL-29 and the expression levels of NF-κB, NFATc1, IFN-γ, and TNF-α in Th cells were seen to be increased in diabetes persons compared to controls. Positive connections between IL-29 with hemoglobin A1c (HbA1c) and fasting plasma glucose (FPG) were found in diabetes persons. IL-29 was positively correlated with NFATc1 and TNF-α. NFATc1 was positively related to TNF-α.ConclusionAbnormal expression levels of IL-29- and Th1- related genes are linked with T2DM pathogenesis. IL-29 may amplify the expression of Th1-specific genes especially TNF-α by upregulating NFATc1 expression.     

A recombined protein (rSj16) derived from Schistosoma japonicum induces cell cycle arrest and apoptosis of murine myeloid leukemia cells

rSj16, a recombined protein from Schistosoma japonicum, has been identified as an anti-inflammatory molecule. In this study, we demonstrated that rSj16 strongly suppressed the growth of murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. rSj16 induced apoptosis by increasing the proportion of sub-G1 apoptotic cells as well as causing cell cycle arrest at the G0/G1 phase. The expressions of cyclin D1, D2, D3, and E, and Cdk 2, 4, and 6 genes in WEHI-3B JCS cells were significantly down-regulated at 24 h as measured by real-time PCR. Furthermore, apoptosis induced by rSj16 was confirmed by 4,6-diamidino-2-phenylindole nuclear staining assay and annexin V/propidium iodide double staining. A reduction of the mitochondrial membrane potential indicated an active involvement of mitochondria in the apoptosis process. rSj16 treatment induced an increase in the activity of caspase 3, 6, and 9, and expression of pro-apoptotic Bax. Meanwhile, the decreased expression of anti-apoptotic Bcl-2 was observed after rSj16 treatment. Taken together, our results implied that rSj16 can inhibit proliferation by inducing G0/G1 cell cycle arrest and apoptosis of murine myeloid leukemia cells via activation of the caspase-mediated mechanism by regulating the expression of Bcl-2 family.     

Inhibition of PKR ameliorates lipopolysaccharide-induced acute lung injury by suppressing NF-κB pathway in mice

Acute lung injury (ALI) is characterized by dramatic lung inflammation and alveolar epithelial cell death. Although protein kinase R (PKR) (double-stranded RNA-activated serine/threonine kinase) has been implicated in inflammatory response to bacterial cell wall components, whether it plays roles in lipopolysaccharide (LPS)-induced ALI remains unclear. This study was aimed to reveal whether and how PKR was involved in LPS-induced ALI pathology and the potential effects of its specific inhibitor, C16 (C₁₃H₈N₄OS). During the experiment, mice received C16 (100 or 500 ug/kg) intraperitoneally 1?h before intratracheal LPS instillation. Then, whole lung lavage was collected for analysis of total protein levels and proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. The lungs were tested for Western blot, transferase-mediated dUTP nick-end labeling (TUNEL) stain and immunohistochemistry. Results showed that PKR phosphorylation increased significantly after LPS instillation. Furthermore, PKR specific inhibition attenuated LPS-induced lung injury (hematoxylin and eosin stain), reduced lung protein permeability (total protein levels in whole lung lavage) and suppressed proinflammatory cytokines (TNF-α, IL-1β and IL-6) and lung apoptosis (TUNEL stain and caspase3 activation). Moreover, mechanism-study showed that C16 significantly suppressed I kappa B kinase (IKK)/I kappa B alpha (IκBα)/NF-κB signaling pathway after LPS challenge. These findings suggested that PKR inhibition ameliorated LPS-induced lung inflammation and apoptosis in mice by suppressing NF-κB signaling pathway.     

Hyperlipidemia Modifies Innate Immune Responses to Lipopolysaccharide via the TLR-NF-κB Signaling Pathway

The objective of this study was to investigate the effect of hyperlipidemia (HLP) on innate immune responses to lipopolysaccharide (LPS). Male New Zealand white rabbits were fed a normal diet (ND) or a high-fat diet (HFD) for 8 weeks. In vivo, the rabbits were injected intravenously with LPS for 24 h. In vitro, peripheral mononuclear cells were collected and stimulated (or unstimulated) with LPS for 24 h. Assay results were analyzed with one-way ANOVA or an equivalent non-parametric test. A P value of 0.05 was considered statistically significant. Despite having no influence in body weight, the HFD intake significantly increased serum lipids, C-reactive protein (CRP), nuclear factor (NF)-κB subunit p65, Toll-like receptor (TLR)-4, SR-A and FAS. Although we found increased circulating tumor necrosis factor (TNF)-α, interleukin (IL)-6, CRP, IL-1β, and IL-10 in the ND-fed rabbits, no significant difference was found in the LPS-stimulated production of TNF-α, IL-6, and CRP in the HFD-fed rabbits. The macrophages harvested from the HFD-fed rabbits developed a blunted inflammatory response, with lower mRNA expression of TNF-α, IL-6, CRP, TLR-4, SR-A, FAS, and Bcl-2 than that expressed by the ND group. In the HFD-fed animals, LPS incubation decreased NF-κB subunit p65 expression, whereas the cytoplasmic phosphorylation of the inhibitor of NF-κB protein was enhanced. These data indicate that HLP displayed a form of innate immune paralysis, including reduced pro- and anti-inflammatory cytokine release to external stimulus, which was related to the altered TLR-NF-κB signaling pathway and altered pro- and anti-apoptotic processes in macrophages.     

Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-kappaB activation: independence from endogenous TNF-alpha and platelet-activating factor

CXCL2 (macrophage inflammatory protein-2 (MIP-2)), a critical chemokine for neutrophils, has been shown to be produced in the rat intestine in response to platelet-activating factor (PAF) and to mediate intestinal inflammation and injury. The intestinal epithelium, constantly exposed to bacterial products, is the first line of defence against micro-organisms. It has been reported that enterocytes produce proinflammatory mediators, including tumour necrosis factor (TNF) and PAF, and we showed that lipopolysaccharide (LPS) and TNF activate nuclear factor (NF)-kappaB in enterocytes. However, it remains elusive whether enterocytes release CXCL2 in response to LPS and TNF via a NF-kappaB-dependent pathway and whether this involves the endogenous production of TNF and PAF. In this study, we found that TNF and LPS markedly induced CXCL2 gene expression in IEC-6 cells, TNF within 30 min, peaking at 45 min, while LPS more slowly, peaking after 2 hr. TNF- and LPS- induced CXCL2 gene expression and protein release were completely blocked by pyrrolidine dithiocarbamate (PDTC) and helenalin, two potent NF-kappaB inhibitors. NEMO-binding domain peptide, a specific inhibitor of inhibitor protein kappaB kinase (IKK) activation, a major upstream kinase mediating NF-kappaB activation, significantly blocked CXCL2 gene expression and protein release induced by LPS. WEB2170 (PAF antagonist) and anti-TNF antibodies had no effect on LPS-induced CXCL2 expression. In conclusion, CXCL2 gene is expressed in enterocytes in response to both TNF and LPS. LPS-induced CXCL2 expression is dependent on NF-kappaB activation via the IKK pathway. The effect of LPS is independent of endogenous TNF and PAF.     

槐定碱抑制LPS诱导巨噬细胞株NF-κB表达

 摘 要：目的 观察槐定碱对LPS诱导的RAW264.7巨噬细胞IKKβ,IκBα,NF-κB P65及TNF-α表达的影响,探讨其抗内毒素机制。方法 培养RAW264.7巨噬细胞,分为对照组、槐定碱对照组、LPS组、槐定碱干预组。槐定碱干预组加入LPS100μg/L孵育1h,弃去LPS后加入槐定碱15.63mg/L,作用5、30、60、120min,分别获取细胞与培养液。RT - PCR与Western Blot分别检测NF-κB等的mRNA与蛋白表达,放免法检测培养液中TNF-α含量。结果 LPS组各时间点IKKβ、pIκBα、NF-κB P65 mRNA和/或蛋白表达及TNF-α含量均显著高于同时间点对照组(P < 0.01);槐定碱干预组以上因子mRNA和/或蛋白表达及TNF-α含量均较同时间点LPS组显著回落(P < 0.01)。结论 槐定碱调控LPS激活的巨噬细胞NF-κB通路IKKβ、IκBα、NF-κB P65等分子的表达,进而抑制下游TNF – α分泌是其抗内毒素作用机制之一。     

基于 NF-κB 通路探讨绿萝花黄酮治疗 DPN 小鼠的机制

目的 基于 NF-κB 通路探讨绿萝花黄酮治疗 DPN 小鼠的机制。 方法 将 60 只小鼠分为对照组、 模型组、 绿萝花黄酮低、 中和高剂量组、 阿卡波糖组各 10 只。 将小鼠雪旺细胞分为正常组、 LPS 组、 绿萝 花黄酮低、 中、 高剂量组。 比较各组小鼠神经传导功能指标、 热痛阈值、 炎性因子、 NF-κB 信号通路关键 蛋白及相关 mRNA 表达水平。 结果 绿萝花黄酮提高小鼠 MNCV、 SNCV 及热痛阈值, 降低炎性因子水平 (P < 0. 05); 降低 p-NF-κB P65 / NF-κB P65、 p-IKKβ/ IKKβ 蛋白比值, 提高 p-IκBα/ IκBα 蛋白比值 ( P < 0. 05); 下调 NF-κB P65、 IKKβ mRNA, 上调 IκBα mRNA (P < 0. 05)。 结论 绿萝花黄酮通过抑制 NF-κB 信号通路降低炎性因子释放, 减轻 DPN 小鼠病情。     

Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide

Porphyromonas gingivalis lipopolysaccharide (LPS) (strain W50) interacts with Toll‐like receptor 2 (TLR‐2) leading to cytokine expression and inflammation, and thereby plays a key role in the pathogenesis of periodontal disease. The aims of this study were to investigate gene expression of key regulatory mediators of innate immune responses in a human monocytic cell line (THP‐1) to P. gingivalis LPS and to compare these results with those obtained using the TLR‐4 ligand, Escherichia coli LPS. Custom‐made Taqman low‐density arrays were used for expression profiling of 45 different cytokine‐related genes. Both types of LPS highly up‐regulated interleukin (IL)‐1α and IL‐1β, IL‐18 receptor (IL‐18R), IL‐18R accessory protein and IL‐1 family (IL‐1F)9. Expression levels of IL‐1F6, IL‐1F7 and caspase‐1 were unaltered by either LPS. Genes for tumour necrosis factor‐α, IL‐6, leukaemia inhibitory factor and IL‐32 were also highly induced by both LPS. For a subset of genes, including CXC chemokine ligand 5 (CXCL5), expression was induced only by E. coli LPS or was up‐regulated more highly by E. coli compared with P. gingivalis LPS in THP‐1 monocytes. A similar expression pattern was also observed in dendritic cells. Analysis of signalling pathways which lead to CXCL5 expression indicated that the mechanisms underpinning the differential responses did not involve the recruitment of different adaptor proteins by TLR‐2 and TLR‐4, and therefore occur downstream of the receptor–adaptor complex. We conclude that differences in signalling pathways activated by TLR‐2 and TLR‐4 ligands lead to differential innate immune responses which may be important in polymicrobial diseases such as periodontal disease.     

Identification of NF-κB related genes in channel catfish and their expression profiles in mucosal tissues after columnaris bacterial infection

Interactions of NF-κB family, IκB family and IKK complex are the key components of NF-κB pathway that is essential for many biological processes including innate and adaptive immunity, inflammation and stress responses. In spite of their importance, systematic analysis of these genes in fish has been lacking. Here we report a systematic study of the NF-κB related genes in channel catfish. Five NF-κB family genes, five IκB family genes and three IKK complex genes were identified in the channel catfish genome. Annotation of these 13 NF-κB related genes was further confirmed by phylogenetic and syntenic analysis. Negative selection was found to play a crucial role in the adaptive evolution of these genes. Expression profiles of NF-κB related genes after Flavobacterium columnare (columnaris) infection were determined by analysis of the existing RNA-Seq dataset. The majority of NF-κB related genes were significantly regulated in mucosal tissues of gill, skin and intestine after columnaris infection, indicating their potential involvement in host defense responses. Distinct expression patterns of NF-κB related genes were observed in susceptible and resistant catfish in response to columnaris infection, suggesting that expression of these genes may contribute to the variations in disease resistance/susceptibility of catfish.