The Protective Effect of Fenofibrate Against TNF-α-Induced CD40 Expression through SIRT1-Mediated Deacetylation of NF-κB in Endothelial Cells

Fenofibrate, as a lipid-lowering drug in clinic, participates in the regulation of inflammatory response. Recently, increasing studies have indicated that sirtuin1 (SIRT1), a NAD⁺-dependent deacetylase, has potential anti-inflammatory effect in endothelial cells. However, whether the regulatory effect of fenofibrate on inflammation response is mediated by SIRT1 remains unclear. The aim of this study was to investigate the effect of fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 in endothelial cells and explore the underlying mechanisms. The results showed that fenofibrate upregulated SIRT1 expression and inhibited CD40 expression in TNF-α-stimulated endothelial cells, but these effects were reversed by peroxisome proliferator-activated receptor-α (PPARα) antagonist GW6471. Furthermore, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or SIRT1 knockdown could attenuate the effect of fenofibrate on CD40 expression in endothelial cells. Importantly, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression. Further study found that fenofibrate decreased the expression of acetylated-NF-κB p65 (Ac-NF-κB p65) in TNF-α-stimulated endothelial cells, which was abolished by SIRT1 knockdown. These results indicate that fenofibrate has protective effect against TNF-α-induced CD40 expression through SIRT1-mediated deacetylation of the p65 subunit of NF-κB.     

Inhibition of apoptosis in Cryptosporidium parvum-infected intestinal epithelial cells is dependent on survivin

Cryptosporidium parvum is an obligate intracellular protozoan capable of causing severe diarrheal disease in a wide variety of mammals, including humans. C. parvum infection has been associated with induction of apoptosis in exposed epithelial cells, and we now demonstrate that apoptosis is restricted to a subset of cells actively infected with C. parvum. Approximately 20% of the infected cells underwent apoptosis within 48 h of infection, suggesting that the majority of the infected cells are rescued from apoptosis. C. parvum infection resulted in low-level activation of multiple members of the caspase family, including caspase-2, -3, -4, -6, -8, and -9. The kinetics of caspase activation correlated with apoptosis over a 48-h time course. Pan caspase inhibitors reduced apoptosis of epithelial cells infected by C. parvum. Furthermore, C. parvum infection inhibited staurosporine-induced apoptosis and caspase-3/7 activation at 24 h and 48 h. Infection with C. parvum led to upregulation of genes encoding inhibitors of apoptosis proteins (IAPs), including c-IAP1, c-IAP2, XIAP, and survivin. Knockdown of survivin gene expression, but not that of c-IAP1, c-IAP2, or XIAP expression, increased caspase-3/7 activity as well as apoptosis of infected cells and decreased C. parvum 18S rRNA levels. These data suggest that the apoptotic response of infected intestinal epithelial cells is actively suppressed by C. parvum via upregulation of survivin, favoring parasite infection.     

Defective expression of SIRT1 contributes to sustain inflammatory pathways in the gut

In inflammatory bowel disease (IBD), tissue damage is driven by an excessive immune response, poorly controlled by counter-regulatory mechanisms. SIRT1, a class III NAD+-dependent deacetylase, regulates negatively the expression of various proteins involved in the control of immune-inflammatory pathways, such as Stat3, Smad7, and NF-κB. Here we examined the expression, regulation, and function of SIRT1 in IBD. SIRT1 RNA and protein expression was less pronounced in whole biopsies and lamina propria mononuclear cells (LPMCs) of IBD patients in comparison with normal controls. SIRT1 expression was downregulated in control LPMC by tumor necrosis factor (TNF)-α and interleukin (IL)-21, and upregulated in IBD LPMC by neutralizing TNF-α and IL-21antibodies. Consistently, SIRT1 expression was increased in mucosal samples taken from IBD patients successfully treated with Infliximab. Treatment of IBD LPMC with Cay10591, a specific SIRT1 activator, reduced NF-κB activation and inhibited inflammatory cytokine synthesis, whereas Ex527, an inhibitor of SIRT1, increased interferon (IFN)-γ in control LPMC. SIRT1 was also reduced in mice with colitis induced by 2,4,6-trinitrobenzenesulphonic acid or oxazolone. Cay10591 prevented and cured experimental colitis whereas Ex527 exacerbated disease by modulating T cell-derived cytokine response. Data indicate that SIRT1 is downregulated in IBD patients and colitic mice and suggest that SIRT1 activation can help attenuate inflammatory signals in the gut.     

Clinicopathological significance and prognostic implication of nuclear factor-κB activation in colorectal cancer

ObjectiveThe aim of the present study was to evaluate the clinicopathological significance of phosphorylated nuclear factor-κB (pNF-κB) expression, and its impact on epithelial–mesenchymal transition and angiogenesis in colorectal cancer (CRC).MethodsWe carried out immunohistochemistry of pNF-κB on 261 human CRC tissues, and evaluated nuclear expression, regardless of cytoplasmic expression. We also investigated the correlation between pNF-κB expression and clinicopathological characteristics, survival, and epithelial–mesenchymal transition and angiogenesis-related markers in CRC.ResultspNF-κB was expressed in the nuclei of 164 of the 261 CRC tissues (62.8%). Furthermore, pNF-κB was significantly correlated with frequent perineural invasion, lymph node metastasis, and higher pTNM stage. However, there was no significant correlation between pNF-κB expression and other clinicopathological parameters. Among the epithelial–mesenchymal transition markers examined, SNAIL expression was significantly correlated with pNF-κB expression (P = 0.001) but E-cadherin expression was not. CRC with pNF-κB expression had significantly higher SIRT1 expression levels and hypoxia-inducible factor-1α expression levels than CRC without pNF-κB expression (P < 0.001 and P < 0.001, respectively). However, there was no correlation between the expression levels of pNF-κB and VEGF. pNF-κB expression was significantly correlated with worse overall and recurrence-free survival rates (P < 0.001 and P < 0.001, respectively).ConclusionpNF-κB expression was significantly correlated with aggressive tumor behaviors and worse survival rates. Furthermore, pNF-κB expression may affect tumor invasion and progression through SNAIL-related epithelial–mesenchymal transition and SIRT1- and hypoxia-inducible factor-1α-induced angiogenesis.     

不可分型流感嗜血杆菌经NF-κB信号通路抑制组蛋白脱乙酰酶活性促进支气管上皮炎症反应

目的:明确不可分型流感嗜血杆菌(NTHi)对支气管上皮细胞炎症反应的影响,并初步探讨其作用机制。方法:培养人正常支气管上皮细胞BEAS-2B,采用感染复数(MOI)=10的NTHi感染BEAS-2B细胞后,分别采用ELISA和RT-qPCR分别检测细胞上清中白细胞介素8(IL-8)和粒细胞-巨噬细胞集落刺激因子(GM-CSF)的表达水平;Western blot检测细胞内IκBα含量以及组蛋白去乙酰化水平,并检测组蛋白脱乙酰酶(HDAC)的表达以及HDAC的酶活性;电泳迁移率变动分析和染色质共沉淀实验分别测定NF-κB以及IL-8的结合活性。通过NF-κB以及HDAC抑制剂预处理细胞后,检测细胞内GM-CSF和IL-8的表达水平。结果:MOI=10的NTHi感染BEAS-2B细胞后,细胞上清中IL-8和GM-CSF含量及其mRNA表达水平均显著升高(P<0.05);NTHi感染可显著下调胞浆内总IκBα的表达,增强细胞内NF-κB的DNA结合活性,并显著诱导细胞内组蛋白H3和H4的磷酸乙酰化,促进IL-8和RNA聚合酶II的结合;细胞内HDAC的表达水平及酶活性也显著降低(P<0.05)。NF-κB抑制剂均显著减少细胞内GM-CSF和IL-8的表达水平(P<0.05),而HDAC抑制剂则会促进细胞内IL-8的分泌(P<0.05)。结论:NTHi可通过激活NF-κB信号通路抑制HDAC表达和活性,促进支气管上皮细胞分泌IL-8和GM-CSF,从而加重炎症反应的发生。     

Aspirin Inhibits LPS-Induced Expression of PI3K/Akt,ERK, NF-κB,CX3CL1, and MMPs in Human Bronchial Epithelial Cells

This study focused on the effects of aspirin on lipopolysaccharide (LPS)-induced expression of phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt), extracellular signal-regulated protein kinase (ERK), nuclear factor-κB (NF-κB), CX3CL1, and MMPs in human bronchial epithelial cells. Human bronchial epithelial cells were seeded in six-well plates. After 24 h, the cells were classified into six groups: control blank (CK) group; LPS group; PD98059 (ERK inhibitor) (PD) group, treated with LPS + ERK inhibitor; LY294002(PI3K/Akt inhibitor) (LY) group, treated with LPS + PI3K/Akt inhibitor; Aspirin (Asp) group, treated with LPS + aspirin; and Pyrrolidinedithiocarbamic acid (PDTC) group, treated with LPS + NF-κB inhibitor. After 4-h treatment, the cells were harvested. Western blot analysis was performed to detect the expression of PI3K/Akt, ERK, NF-κB, and CX3CL1. Real-time quantitative PCR (RT-qPCR) was used to determine the gene expression of MMP-7, MMP-9, and MMP-12. Compared to the CK group, expression of PI3K/Akt, ERK, NF-κB, and CX3CL1 was significantly increased in the LPS group (P?<?0.05). When compared to the LPS group, expression of PI3K/Akt, ERK, NF-κB, and CX3CL1 was significantly decreased in the PD group, PDTC group, and Asp group (P?<?0.05). In addition, expression of NF-κB in the LY group was significantly reduced by comparison with the LPS group (P?<?0.05). RT-qPCR: When compared to the LPS group, expression of MMP-7 and MMP-12 was significantly decreased in Asp group (P?<?0.05). Expression of MMP-12 was significantly reduced in LY group (P?<?0.05). LPS-ERK, NF-κB-PI3K/Akt, and CX3CL1 signal pathways exist in human bronchial epithelial cells. The PI3K/Akt inhibitor repressed expression of MMP-12. Aspirin inhibited LPS-induced expression of PI3K, Akt, ERK, NF-κB, CX3CL1, MMP-7, and MMP-12 in human bronchial epithelial cells.     

Requirement for CD40-CD40 Ligand Interaction for Elimination of Cryptosporidium parvum from Mice

Mice with disrupted genes for CD40 and CD40 ligand (CD40L) are unable to clear infection with Cryptosporidium parvum and develop cholangitis. Parasites are present in the gut, gall bladder, and biliary tree, and biliary epithelial cells express CD40 on the cell surface. SCID mice infected with C. parvum for >1 month can clear the infection after reconstitution with spleen cells from CD40, but not CD40L, knockout mice. In an in vitro model, C. parvum-infected HepG2 cells were triggered to apoptosis when incubated with a CD40L-CD8 fusion protein. The requirement for CD40-CD40L interactions for immunity to C. parvum indicated by our results may entail the triggering of apoptosis in infected cells, in addition to the known role of CD40L-CD40 interactions in stimulating cytokine production and promoting T-cell responses.     

Shrimp NF-κB binds to the immediate-early gene ie1 promoter of white spot syndrome virus and upregulates its activity

The immediate-early gene ie1 carried by white spot syndrome virus (WSSV) exhibits very strong promoter activity and expresses highly throughout the infection cycle. Here we identified a NF-κB binding motif in the ie1 promoter region. Electrophoretic mobility shift assays indicated that the recombinant Rel homology domain (RHD) of shrimp NF-κB homolog LvRelish bound to the putative NF-κB site in the ie1 promoter. A transactivity assay of the WSSV ie1 promoter in Drosophila Schneider 2 cells demonstrated that LvRelish could increase ie1 promoter activity. These results show that shrimp NF-κB homolog LvRelish transactivates WSSV ie1 gene expression and contributes to its high promoter activity. Further transactivation assays showed that WSSV IE1 protein expression upregulated the promoter activities of WSSV ie1 gene and antimicrobial peptide genes regulated by the NF-κB system. We suggested that WSSV may annex the shrimp NF-κB system, which it uses to enhance the expression of viral immediate-early genes.     

CEACAMs interaction with Helicobacter pylori HopQ supports the type 4 secretion system-dependent activation of non-canonical NF-κB

Helicobacter pylori infection represents a major risk factor for the development of gastric diseases and gastric cancer. The capability of H. pylori to inject the virulence factor cytotoxin-associated gene A (CagA) depends on a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI). Further, infection by H. pylori activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in a T4SS-dependent manner but CagA-independent manner. Here we investigated the role of host cell receptors carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) and the bacterial adhesin HopQ in the activation of non-canonical NF-κB and CagA translocation into gastric epithelial cells. AGS cells express six of twelve CEACAMs found in humans. In HeLa cells, only CEACAM19 is expressed. We showed that deletion of hopQ attenuates the activation of non-canonical NF-κB only in AGS but not in HeLa cells. CagA translocation was in both cell lines affected by HopQ depletion, although to a much lesser extent in HeLa cells. Moreover, we observed a possible redundancy between the three HopQ-binding CEACAMs 1, 5 and 6 and their capacity to support non-canonical NF-κB activation. Our results illustrate that the interaction between HopQ and CEACAMs could promote the efficiency of the T4SS.     

HopQ impacts the integrin α5β1-independent NF-κB activation by Helicobacter pylori in CEACAM expressing cells

Helicobacter pylori infection persists in more than half of the world’s population and represents a risk factor for peptic ulcer disease and gastric cancer. Virulent strains of H. pylori carry a cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) with the capability to inject the effector protein cytotoxin-associated gene A (CagA) into eukaryotic cells. Colonisation of the gastric epithelium by H. pylori provokes direct activation of the proinflammatory and survival factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We investigated the impact of host cell receptor integrin α5β1 and the bacterial adhesin HopQ on the NF-κB activation. We found that H. pylori induced early T4SS-dependent, but CagA-independent canonical NF-κB signalling in polarized, apical infected NCI-N87 cells. Integrin-dependent CagA translocation was hardly detectable, as integrin β1 was sparsely located at the apical surface of polarized NCI-N87 cells. Knockdown experiments indicated that integrin α5β1 and integrin linked kinase (ILK) were dispensable for NF-κB activation in H. pylori infection. Thus, there exists no common mechanism, which mediates integrin α5β1-dependent H. pylori-triggered CagA translocation and the activation of NF-κB. Further, we report that H. pylori adhesin HopQ, which binds to a specific subset of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), promotes canonical NF-κB activation in AGS and NCI-N87 cells, but not in HeLa cells, which are devoid of these CEACAMs. Noteworthy, these effects were not mediated by reduced adhesion, indicating additional functions of HopQ.     

Human Intestinal Epithelial Cells Respond to Cryptosporidium parvum Infection with Increased Prostaglandin H Synthase 2 Expression and Prostaglandin E2 and F2α Production

Cryptosporidium parvum is an important cause of diarrhea in humans and several animal species. Prostaglandins play a central role in regulating intestinal fluid secretion in animal models of cryptosporidiosis, but their cellular sources and mechanisms of induction are unclear. Here, we show that C. parvum infection directly activates prostaglandin H synthase 2 expression and prostaglandin E₂ and F_2α production in human intestinal epithelial cells.     

Cryptosporidium parvum has an active hypusine biosynthesis pathway

The protozoan parasite Cryptosporidium parvum causes severe enteric infection and diarrheal disease with substantial morbidity and mortality in untreated AIDS patients and children in developing or resource-limited countries. No fully effective treatment is available. Hypusination of eIF5A is an important post-translational modification essential for cell proliferation. This modification occurs in a two step process catalyzed by deoxyhypusine synthase (DHS) followed by deoxyhypusine hydroxylase. An ORF of 1086 bp was identified in the C. parvum (Cp) genome which encodes for a putative polypeptide of 362 amino acids. The recombinant CpDHS protein was purified to homogeneity and used to probe the enzyme's mechanism, structure, and inhibition profile in a series of kinetic experiments. Sequence analysis and structural modeling of CpDHS were performed to probe differences with respect to the DHS of other species. Unlike Leishmania, Trypanosomes and Entamoeba, Cryptosporidium contains only a single gene for DHS. Phylogenetic analysis shows that CpDHS is more closely related to apicomplexan DHS than kinetoplastid DHS. Important residues that are essential for the functioning of the enzyme including NAD⁺ binding residues, spermidine binding residues and the active site lysine are conserved between CpDHS and human DHS. N¹-guanyl-1,7-diaminoheptane (GC7), a potent inhibitor of DHS caused an effective inhibition of infection and growth of C. parvum in HCT-8 cells.     

Functional assessment of the mutational effects of human IRAK4 and MyD88 genes

Human interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency and myeloid differentiating factor 88 (MyD88) deficiency syndromes are two primary immune-deficiency disorders with innate immune defects. Although new genetic variations of IRAK4 and MyD88 have recently been deposited in the single nucleotide polymorphism (SNP) database, the clinical significance of these variants has not yet been established. Therefore, it is important to establish methods for assessing the association of each gene variation with human diseases. Because cell-based assays, western blotting and an NF-κB reporter gene assay, showed no difference in protein expression and NF-κB activity between R12C and wild-type IRAK4, we examined protein–protein interactions of purified recombinant IRAK4 and MyD88 proteins by analytical gel filtration and NMR titration. We found that the variant of IRAK4, R12C, as well as R20W, located in the death domain of IRAK4 and regarded as a SNP, caused a loss of interaction with MyD88. Our studies suggest that not only the loss of protein expression but also the defect of Myddosome formation could cause IRAK4 and MyD88 deficiency syndromes. Moreover a combination of in vitro functional assays is effective for confirming the pathogenicity of mutants found in IRAK4 and MyD88-deficiency patients.     

SIRT1 as a potential biomarker of response to treatment with glatiramer acetate in multiple sclerosis

SIRT1, a NAD dependent histone and protein deacetylase, is a member of the histone deacetylase class III family. We previously showed that SIRT1 mRNA expression is significantly lower in peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients during relapses than in stable patients. We have now investigated SIRT1 as a possible biomarker to predict relapse as well as responsiveness to glatiramer acetate (GA) treatment in relapsing-remitting MS (RRMS) patients. Over the course of 2 years, a cohort of 15 GA-treated RRMS patients were clinically monitored using the Expanded Disability Status Scale and assessed for MS relapses. Blood samples collected from MS patients were analyzed for levels of SIRT1 and histone H3 lysine 9 (H3K9) acetylation and dimethylation. During relapses, MS patients had a lower expression of SIRT1 mRNA than did stable MS patients. In addition, there was a significant decrease in H3K9 dimethylation (H3K9me2) during relapses in MS patients when compared to stable patients (p = 0.01). Responders to GA treatment had significantly higher SIRT1 mRNA (p = 0.01) and H3K9me2 levels than did non-responders (p = 0.018). Receiver operating characteristic analysis was used to assess the predictive power of SIRT1 and H3K9me2 as putative biomarkers: for SIRT1 mRNA, the predictive value for responsiveness to GA treatment was 70% (p = 0.04) and for H3K9me2 was 71% (p = 0.03). Our data suggest that SIRT1 and H3K9me2 could serve as potential biomarkers for evaluating patients' responsiveness to GA therapy in order to help guide treatment decisions in MS.     

SIRT1-NF-κB通路在流感病毒感染小鼠体内调节机制的研究

目的 探讨巨噬细胞SIRT1-NF-κB信号转导途径在流感病毒感染小鼠体内的调节机制.方法 用流感病毒血凝素蛋白(HA)和核衣壳蛋白(NP)蛋白免疫小鼠,取小鼠腹腔巨噬细胞,采用ELISA法检测细胞培养上清中促炎症因子IL-1β、IL-6、IL-18和肿瘤坏死因子α(TNF-α)的含量;采用实时荧光定量PCR检测细胞内沉默信息调节因子1(SIRT1)、NF-κB、IL-1β、IL-6、IL-18以及TNF-α的基因表达.结果 两组蛋白初次免疫后巨噬细胞分泌的各因子及其mRNA表达水平均无明显变化.二次免疫后,HA蛋白组仅TNF-α、IL-6蛋白表达水平增加,与对照组比较差异有统计学意义,NP蛋白组IL-1β、IL-6、IL-18和TNF-α蛋白表达水平均增加,与对照组比较差异均有统计学意义;而两组蛋白刺激后巨噬细胞TNF-α、IL-6 mRNA均表达上调,SIRT1 mRNA均表达下调,与对照组比较差异有统计学意义.结论 流感病毒可能通过所表达的HA、NP等蛋白影响SIRT1的活性,从而解除SIRT1对NF-κB的抑制作用,激活NF-κB信号通路调节的炎症反应或免疫应答.