Microbial-induced immunomodulation by targeting the NF-κB system

Virtually all eukaryotes have developed defense mechanisms to efficiently counter potential threats from prokaryotic microorganisms; an example is the conserved nuclear factor-kappaB (NF-κB) signaling system. However, bacterial pathogens and commensals have in turn evolved highly effective counter mechanisms to modulate this immune regulatory circuit. Modifications in ubiquitin, ubiquitin-like (Ubl) proteins such as neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) and other post-translational modifications (PTMs) in the NF-κB system represent attractive targets for microbial manipulation. In this review, we describe recent advances in understanding the different strategies that bacteria have evolved to interfere with PTMs in NF-κB signal transmission.     

Phosphoprotein levels,MAPK activities and NFκB expression are affected by fisetin

Flavonoids, polyphenolic phytochemicals, are ubiquitous in plants and are commonly present in the human diet. They may exert diverse beneficial effects, including antioxidant and anticarcinogenic activities. The present study was designed to evaluate three biomolecules that play important roles in the apoptotic process: mitogen-activated protein kinases, protein phosphatases and NFκB, using HL60 cells treated with fisetin as an experimental model. Our results demonstrated that cells treated with fisetin presented high expression of NFκB, activation of MAPK p38 and an increase of phosphoprotein levels; inhibition of enzymes involved in redox status maintenance were also observed. Our findings reinforce the hypothesis that fisetin is likely to exert beneficial and/or toxic actions on cells not through its potential as antioxidant but rather through its modulation of protein kinase and phosphatase signaling cascades. Additionally, our results also indicate that the cellular effects of fisetin will ultimately depend on the cell type and on the extent to which they associate with the cells, either by interactions at the membrane or by uptake into the cytosol.     

Inhibition of IκB kinase-β and IκB kinase-α by heterocyclic adamantyl arotinoids

We recently reported on a series of retinoid-related molecules containing an adamantyl group, a.k.a. adamantyl arotinoids (AdArs), that showed significant cancer cell growth inhibitory activity and activated RXRα (NR2B1) in transient transfection assays while devoid of RAR transactivation capacity. We have now explored whether these AdArs could also bind and inhibit IKKβ, a known target that mediates the induction of apoptosis and cancer cell growth inhibition by related AdArs containing a chalcone functional group. In addition, we have prepared and evaluated novel AdArs that incorporate a central heterocyclic ring connecting the adamantyl-phenol and the carboxylic acid at the polar termini. Our results indicate that the majority of the RXRα activating compounds lacked IKKβ inhibitory activity. In contrast, the novel heterocyclic AdArs containing a thiazole or pyrazine ring linked to a benzoic acid motif were potent inhibitors of both IKKα and IKKβ, which in most cases paralleled significant growth inhibitory and apoptosis inducing activities.     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

Regulation of ΔNp63α by NFκB

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be downregulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NFκB in presence of cisplatin. We find that NFκB promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NFκB leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NFκB with siRNA-mediated silencing NFκB expression attenuates chemotherapy induced degradation of ΔNp63α. These data demonstrate that NFκB plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NFκB may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.Key words: ΔNp63α, NFκB, ubiquitination, cisplatin, head and neck cancer     

1,6-O,O-diacetylbritannilactones inhibits IκB kinase β-dependent NF-κB activation

To determine the chemical constituents responsible for pharmacological effects of Inula britannica-F., three specific sesquiterpene lactones in Inula britannica were isolated from chloroform extract and identified, including britannilactone (BL), 1-O-acetylbritannilactone (ABLO), and 1,6-O,O-diacetylbritannilactone (ABLOO). Electrophoretic mobility shift assay (EMSA) was performed to detect the nuclear translocation of nuclear factor-κB (NF-κB) p65. The expressions of IκBα, pIκBα, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IκB kinase α/β (IKKα/β) and NF-κB kinase (NIK) were detected by Western blot and RT-PCR. We found that acetyl side groups enhanced the inhibitory action of the agents on LPS/IFN-γ-induced iNOS and COX-2 expression. Their inhibiting activity was positive correlation with the acetyl side group number. The effects of LPS/IFN-γ were reversed by ABLOO, and BL without acetyl side groups showed only a weak inhibitory action. Further study indicated that ABLOO markedly inhibited the phosphorylation of IKKβ down to based level, but not IKKα, corresponding with decreased in IκBα degradation and phosphorylation induced by LPS/IFN-γ, resulting in the suppression of NF-κB nuclear translocation and activity. These results suggest that the acetyl moieties add to the lipophilicity, and consequently enhance cellular penetration, so that ABLOO possess the most anti-inflammatory effect and may be a potent lead structure for the development of therapeutic and cytokine-suppressing remedies valuable for the treatment of various inflammatory diseases.     

Bcl-3, induced by Tax and HTLV-1, inhibits NF-κB activation and promotes autophagy

The human T cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes an aggressive leukemia known as adult T cell leukemia (ATL). The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway, which is perceived as the primary cause of ATL. Bcl-3, a member of the NF-κB inhibitor (IκB) family, is highly expressed in many HTLV-1-infected T cell lines and ATL cells. However, the role of Bcl-3 in Tax-induced NF-κB activation has not been fully elucidated. Here, we show that Tax induces Bcl-3 expression, which in turn negatively regulates the Tax-induced NF-κB activation. Interestingly, both Bcl-3 up-regulation and NF-κB inhibition promote the autophagy process in HTLV-1-infected cells. Consistent with this, over-expression of Bcl-3 also results in enhancement of rapamycin-, pifithrin-α- or starvation-induced autophagy in control cells. Together, these data demonstrate that Bcl-3 acts as a negative regulator of NF-κB activation and promotes autophagy in HTLV-1-infected cells.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.