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Nuclear factor-kappa B, involved in inflammation, host immune response, cell adhesion, growth signals, cell proliferation, cell differentiation, and apoptosis defense, is a dimeric transcription factor. Inflammation is a key component of many common respiratory disorders, including asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, and acute respiratory distress syndrome. Many basic transcription factors are found in NF-xB signaling, which is a member of the Rel protein family. Five members of this family c-REL, NF-xB2 (p100/ p52), RelA (p65), NF-xB1 (p105/p50), RelB, and RelA (p65) produce 5 transcriptionally active molecules. Proinflammatory cytokines, T lymphocyte, and B lymphocyte cell mitogens, lipopolysaccharides, bacteria, viral proteins, viruses, double-stranded RNA, oxidative stress, physical exertion, various chemotherapeutics are the stimulus responsible for NF-xB activation. NF-xB act as a principal component for several common respiratory illnesses, such as asthma, lung cancer, pulmonary fibrosis, COPD as well as infectious diseases like pneumonia, tuberculosis, COVID-19. Inflammatory lung disease, especially COVID-19, can make NF-xB a key target for drug production.

Stephanie Dollinger, BSc, Zusammenfassung in deutscher Sprache, University Innsbruck, Masterarbeit, 2019, (VLID)3458276

Lactobacillus helveticus SBT2171 (LH2171) has been reported to ameliorate the development of autoimmune diseases, such as collagen-induced arthritis and experimental autoimmune encephalitis in mice and inhibit interleukin (IL)-6 production in antigen-presenting cells in vitro. Regulation of cytokine production by antigen-presenting cells might be critical for the anti-inflammatory function of LH2171 in autoimmune diseases. However, the mechanism and contributing components of LH2171-mediated inhibition of IL-6 production are unclear. Here, we examined the anti-inflammatory effects of LH2171 in lipopolysaccharide (LPS)-stimulated peritoneal macrophages, as a model of antigen-presenting cells, necessary for the pathogenesis of autoimmune diseases. LH2171 significantly reduced LPS-induced expression and secretion of IL-6 and IL-1 beta cytokines. It also inhibited activation of nuclear factor-kappa B and mitogen-activated protein kinases (NF-kappa B/MAPKs). Moreover, LH2171 induced gene expression of several negative regulators of NF-kB/MAPKs. Among these regulators, A20 was strongly up-regulated at the mRNA and protein levels upon LH2171 treatment. The cell wall fraction of LH2171 also demonstrated a similar increase in A20 gene expression and exerted an anti-inflammatory effect. These results suggest that the cell wall may be one of the anti-inflammatory components of LH2171. Since cell wall components of Gram-positive bacteria are recognized by toll-like receptor 2 (TLR2), we investigated whether the anti-inflammatory effect of LH2171 was mediated by TLR2 signaling. Specifically, LH2171-mediated IL-6 suppression and A20 upregulation in wild-type macrophages were reversed and significantly reduced in TLR2 knock-out macrophages. These results suggest that LH2171 induces A20 expression via TLR2 signaling, inhibiting the activation of NF-kB/MAPKs and cytokine production in antigen-presenting cells. This might contribute to the anti-inflammatory activity of LH2171 on auto

BACKGROUND Inhibition of transcription factor nuclear factor-kappa B (NFκB) is beneficial in various models of hypertension and renal disease. We hypothesized first that NFκB inhibition during renal development ameliorates hereditary hypertensive renal disease and next whether this was mediated via suppression of peroxisome proliferator-activated receptor (PPAR)γ coactivator 1α (PGC-1α). METHODS AND RESULTS Prior to the development of renal injury in fawn-hooded hypertensive (FHH) rats, a model of hypertension, glomerular hyperfiltration, and progressive renal injury, NFkB activity, measured by nuclear protein expression of NFkB subunit p65, was enhanced twofold in 2-day-old male and female FHH kidneys as compared to normotensive Wistar-Kyoto (WKY) rats (P <0.05). Treating FHH dams with pyrrolidine di thio carbamate (PDTC), an NFκB inhibitor, from 2 weeks before birth to 4 weeks after birth diminished NFkB activity in 2-day-FHH offspring to 2-day-WKY levels (P <0.01). Perinatal PDTC reduced systolic blood pressure from 20 weeks onwards by on average 25mm Hg (P <0.001) and ameliorated proteinuria (P <0.05) and glomerulosclerosis (P <0.05). In kidneys of 2-day-, 2-week-, and adult offspring of PDTC-treated FHH dams, PGC-1α was induced on average by 67% (quantitative polymerase chain reaction (qPCR)) suggesting that suppression of this factor by NFkB could be involved in renal damage. Follow-up experiments with perinatal pioglitazone (Pio), a PPARγ agonist, failed to confer persistent antihypertensive or renoprotective effects. CONCLUSIONS Perinatal inhibition of enhanced active renal NFκB in 2-day FHH had persistent antihypertensive and renoprotective effects. However, this was not the case for PPARγ stimulation. NFkB stimulation is therefore involved in renal damage in the FHH model of proteinuric renal disease by pathways other than via PPARγ.

Background: Oxidative stress is reliably observed in major depressive disorder (MDD). However, molecular data on the principal cellular redox-sensitive transcriptional factors and the levels of their downstream-regulated antioxidant enzymes in MDD are scarce. Methods: In the peripheral blood mononuclear cells (PBMC) of subjects with a current episode of MDD (n = 30) and healthy controls (n = 35), we investigated alterations in the levels of redox-sensing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein, its inhibitor Keap1, and nuclear factor-kappa B (NF-kappa B), along with their cognate downstream effectors, the antioxidant enzymes (AOEs): manganese and copper zinc superoxide dismutase (MnSOD and CuZnSOD, respectively), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GLR). Results: MDD subjects exhibited higher levels of Nrf2 and its regulator Keap1, as well as NF-kappa B in the cytoplasm of PBMC compared to controls. This state was further reflected by increased levels of MnSOD, CuZnSOD and CAT proteins and by the lack of correlation between MnSOD and CAT, which could indicate impaired oxidative detoxification capacity in MDD patients. Moreover, increased levels of MnSOD, CuZnSOD and CAT in MDD patients positively correlated with levels of Nrf2, while increased levels of SODs were also positively related to NF-kappa B. There were no differences regarding the levels of GPx and GLR proteins, but the ratio of GLR/GPx was reduced, suggesting diminished capacity of GPx in antioxidative defence in PBMC of MDD subjects. Conclusion: These data provide evidence that MDD is characterized by up-regulation of redox-sensitive transcriptional factors (Nrf2 and NF-kappa B) and AOEs (MnSOD, CuZnSOD and CAT), indicating pro-oxidative state in the PBMC of MDD patients. (C) 2014 S. Karger AG, Basel

Background:The mechanisms behind increased fecal calprotectin (FC) in healthy relatives of patients with inflammatory bowel disease (IBD) are unknown. Our aims were to explore if there is a subclinical inflammation with increased neutrophil activity in healthy twin siblings in discordant twin pairs with IBD and to assess the influence of genetics in this context.Methods:Nuclear factor kappa B (NF-B) and neutrophil activity, based on myeloperoxidase (MPO) and FC, were analyzed in healthy twin siblings in discordant twin pairs with IBD and compared with healthy controls. NF-B and MPO were assessed by immunohistochemistry and FC by enzyme-linked immunosorbent assay.Results:In total, 33 of 34 healthy twin siblings were histologically normal. Increased NF-B was more often observed in healthy twin siblings in discordant twin pairs with Crohn's disease (13/18 [73%]) and with ulcerative colitis (12/16 [75%]) than in healthy controls (8/45 [18%]). MPO was more often increased in healthy twin siblings in discordant pairs with Crohn's disease (12/18 [67%]) than in healthy controls (11/45 [24%]) and FC more often in healthy twin siblings in discordant pairs with ulcerative colitis (14/21 [67%]) than in healthy controls (6/31 [19%]). Interestingly, the observed differences remained when healthy monozygotic and dizygotic twin siblings were analyzed separately.Conclusions:We observed increased NF-B, MPO, and FC in healthy twins in both monozygotic and dizygotic discordant pairs with IBD. These novel findings speak for an ongoing subclinical inflammation with increased neutrophil activity in healthy first-degree relatives.

Nuclear factor-kappa B (NF-kappa B) is an important regulator of cell survival and has been shown to be constitutively active in chronic lymphocytic leukemia (CLL) cells. Recently, a novel NF-kappa B inhibitor, IMD-0354 (N-(3, 5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide), was shown to specifically inhibit the phosphorylation of I kappa B alpha by IkB kinases, thus preventing NF-kappa B release. In this study, we investigated if IMD-0354 can inhibit NF-kappa B activation and induce apoptosis in CLL cells in vitro. The rate of increase in apoptosis, drug sensitivity and DNA-binding activity of NF-kappa B were studied using Annexin V stainings, the fluorometric microculture cytotoxicity assay and electrophoretic mobility shift assay, respectively. Finally, the impact of IMD-0354 treatment on the expression of a set of apoptosis-related genes was investigated. The results clearly show that IMD-0354 induced apoptosis (mean 26%, range 8-48%) in CLL cells, independent of immunoglobulin heavy variable (IGHV) gene mutational status, and showed a dose-dependent cytotoxic effect. IMD-0354 treatment also significantly lowered the DNA-binding activity of NF-kappa B in CLL cells. In addition, we identified differences in expression levels of pro- and antiapoptotic genes following IMD-0354 treatment. In summary, our novel findings show that IMD-0354 can induce apoptosis in CLL cells, and thus merits further investigation as an anticancer agent in vivo.

The concept of inflammation-induced lymphangiogenesis (i.e., formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The two primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the NF-kB family; however, the role of NF-kB in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-kB pathway. In vivo time-course analysis of inflammationinduced lymphangiogenesis showed activation of NF-?B followed by sequential upregulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-kB by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-kB to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-kB pathway by inflammatory stimuli activates Prox1, and both NF-kB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of pre-existing lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis., The original document contains color images.

Recent Studies support beneficial effects of polyphenols in various chronic inflammatory diseases, for example, the inflammatory bowel diseases. Inhibition of NF-kappa B activation by polyphenols could explain part of their anti-inflammatory properties, but few data are available on the intestine. The purpose of the present Study was thus to investigate the effects of some polyphenols on NF-kappa B activation using human intestinal Caco-2 cells. Effects of standard polyphenols (50 mu mol/l) were studied on different cellular events associated with NF-kappa B activation: (i) NF-kappa B activity using cells transiently transfected with a NF-kappa B-luciferase construct and stimulated by inflammatory agents (IL-1 beta, TNF-alpha or lipopolysaccharides (LPS)); (ii) phosphorylation of the inhibitor of kappa B (I kappa B-alpha) analysed by Western blot; (iii) secretion of IL-8 quantified by ELISA assay. Results showed that chrysin and ellagic acid inhibited NF-kappa B activity, whereas genistein and resveratrol increased it. These effects were independent of the nature of the inducer, indicating that polyphenols may modulate NF-kappa B activation by acting on a common event to the cytokine- and LPS-mediated cascades. Chrysin strongly reduced (2.5-fold) IL-1 beta-induced I kappa B-alpha phosphorylation, whereas ellagic acid increased it (1.7-fold). Ellagic acid, genistein and epigallocatechin.-allate reduced (4- to 8-fold) IL-1 beta-induced IL-8 secretion, while resveratrol promoted (1.7-fold) the secretion. Chrysin also diminished IL-8 secretion by 1.6-fold (but P > 0.05). The data indicate that polyphenols can modulate the NF-kappa B activation pathway in the intestine. Chrysin could block NF-kappa B activation via the inhibition of I kappa B-alpha phosphorylation. The other molecular targets of the active polyphenols are still to be identified.

Opioid receptors are involved in regulating neuronal survival. Here we demonstrate that activation of the mu-opioid receptor in human neuroblastoma SH-SY5Y cells led to the phosphorylations Of I kappa B kinase (IKK) and p65, denoting the stimulation of the nuclear factor-kappa B (NF kappa B) transcription factor. This response was mediated through pertussis toxin-sensitive G proteins. The mu-opioid-induced IKK phosphorylation required extracellular signal-regulated protein kinase, phosphatidylinositol 3-kinase and c-Src. Moreover, c-Jun N-terminal kinase and calmodulin-dependent kinase 11 also participated in the IKK activation, despite the lack of involvement of phospholipase C beta and protein kinase C. These data suggest that the mu-opioid receptor is capable of simulating NF kappa B signaling via the phosphorylation of IKK and p65 in human neuroblastoma SH-SY5Y cells. Copyright (C) 2005 S. Karger AG, Basel.

Cyclooxygenase-2 (COX-2) catalyses prostaglandin synthesis from arachidonic acid during inflammation. COX-2 is expressed in the normal brain and is induced in neurological disorders. There is evidence that COX-2 is involved in secondary events leading to cell death in the brain. The first objective was to study the expression of COX-2 in the brain after kainate (KA)-induced limbic seizures and brain trauma caused by controlled cortical contusion (CCC) and fluid percussion injury (FPI). COX-2 mRNA and protein were strongly induced by limbic seizures in the hippocampus, amygdala and piriform cortex. CCC and FPI resulted in an upregulation of COX-2 mainly in the dentate gyrus and cortex, with differences in expression levels in these regions between the models. The second objective was to evaluate the effects of selective COX-2 inhibitors on delayed cell death. Limbic seizures induced cell death in parts of the hippocampus, amygdala and functionally connected regions. Treatment with the selective COX-2 inhibitor rofecoxib 8 h after KA injection significantly reduced hippocampal cell death. Pre-treatment with the COX-2 inhibitor nimesulide augmented acute seizures with increased mortality and thus the effect of nimesulide on delayed cell death could not be evaluated. Effects of rofecoxib on trauma-induced cell death were studied in the FPI model. FPI induced delayed cell death mainly in the ipsilateral cortex and bilaterally in the dentate gyrus. Rofecoxib treatment, starting directly after injury was caused, had no protective effect against cell death. The results suggest that COX-2 inhibition may be both detrimental and beneficial and largely dependent on the time schedule of treatment. COX-2 inhibitors might thus be of value as a neuroprotective treatment approach, provided that the role of COX-2 and the time course of effects of its metabolites in the brain are elucidated.

Death receptors have been recently identified as a subgroup of the TNF-receptor superfamily with a predominant function in induction of apoptosis. The receptors are characterized by an intracellular region, called the death domain, which is required for the transmission of the cytotoxic signal. Currently, five different such death receptors are known including tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2. The signaling pathways by which these receptors induce apoptosis are rather similar. Ligand binding induces receptor oligomerization, followed by the recruitment of an adaptor protein to the death domain through homophilic interaction. The adaptor protein then binds a proximal caspase, thereby connecting receptor signaling to the apoptotic effector machinery. In addition, further pathways have been linked to death receptor-mediated apoptosis, such as sphingomyelinases, JNK kinases and oxidative stress. These pro-apoptotic signals are counteracted by several mechanisms which inhibit apoptosis at different levels. This review summarizes the current and rapidly expanding knowledge about the biological functions of death receptors and the mechanisms to trigger or to counteract cell death.

Oxidized LDL (oxLDL) is present in atherosclerotic lesions and is believed to play a key role in atherogenesis. Mainly on the basis of cell culture studies, oxLDL has been shown to produce many biological effects that influence the atherosclerotic process. To study LDL oxidation in vivo, we have established a model in which Sprague-Dawley rats are given a single injection of unmodified human LDL (> or = 4 mg/kg body weight). Within 6 hours, an accumulation of apolipoprotein B and epitopes present on oxLDL are detected in the arterial endothelium and media. The presence of oxLDL is associated with activation of the transcription factor nuclear factor-kappaB in the endothelium as well as endothelial expression of intercellular adhesion molecule-1. Injection of LDL enriched with the antioxidant probucol resulted in arterial accumulation of apolipoprotein B, but the expression of oxLDL-specific epitopes was reduced at 24 hours. Thus, this simple model has the potential to analyze the mechanisms behind and biological effects of LDL oxidation in vivo.

Mitochon-dria generate reactive oxygen species (ROS) as byproducts of molecular oxygen consumption in the electron transport chain. Most cellular oxygen is consumed in the cytochrome-c oxidase complex of the respiratory chain, which does not generate reactive species. The ubiquinone pool of complex III of respiration is the major site within the respiratory chain that generates Superoxide anion as a result of a single electron transfer to molecular oxygen. Superoxide anion and hydrogen peroxide, derived from the former by Superoxide dismutase, are precursors of hydroxyl radical through the participation of transition metals. Glutathione (GSH) in mitochondria is the only defense available to metabolize hydrogen peroxide. A small fraction of the total cellular GSH pool is sequestered in mitochondria by the action of a carrier that transports GSH from the cytosol to the mitochondrial matrix. Mitochondria are not only one of the main cellular sources of ROS, they also are a key target of ROS. Mitochondria are subcellular targets of cytokines, especially tumor necrosis factor (TNF); depletion of GSH in this organelle renders the cell more susceptible to oxidative stress originating in mitochondria. Ceramide generated during TNF signaling leads to increased production of ROS in mitochondria. Chronic ethanol-fed hepatocytes are selectively depleted of GSH in mitochondria due to a defective operation of the carrier responsible for transport of GSH from the cytosol into the mitochondrial matrix. Under these conditions, limitation of the mitochondrial GSH pool represents a critical contributory factor that sensitizes alcoholic hepatocytes to the prooxidant effects of cytokines and prooxidants generated by oxidative metabolism of ethanol. S-adenosyl-Lmethionine prevents development of the ethanol-induced defect. The mitochondrial GSH carrier has been functionally expressed in Xenopus laevis oocytes microinjected with mRNA from rat liver. This critical carrier displays functio