Modulation Of NF-κB Activity Research Articles

The long non-coding RNA GAS5 contributes to the suppression of inflammatory responses by inhibiting NF-κB activity

IntroductionNuclear factor kappa B (NF-κB) is a key regulator of immune and inflammatory responses. Glucocorticoid drugs (GC) act through the glucocorticoid receptor (GR) as immunosuppressant also in pediatric patients inhibiting NF-κB activity. The long non-coding RNA GAS5 interacts with the GR, influencing GC activity. No data on the role of GAS5 on GR-dependent inhibition of NF-κB activity have been published.MethodsThis study investigated the impact of GAS5 on NF-κB activity in HeLa cells overexpressing GAS5, both under basal conditions and during GC treatment. The study used EMSA, RNA-immunoprecipitation (RIP), Western blotting, and bioinformatic analyses to assess NF-κB DNA binding, GAS5-p65 interaction, and NF-κB signaling pathway modulation.ResultsGAS5 overexpression increased NF-κB DNA binding activity in untreated cells. RNA-IP confirmed a direct interaction between GAS5 and the NF-κB subunit p65, suggesting a potential regulatory mechanism. GAS5 overexpression led to downregulation of NF-κB target genes, TNF-α, and NR3C1. GC treatment reduced NF-κB DNA binding activity in GAS5-overexpressing cells, indicating a potential synergistic effect. Furthermore, GAS5 overexpression increased IκB levels and reduced p-p65/pan-p65 levels during GC treatment.DiscussionGAS5 appears to modulate NF-κB activity in a complex manner, influencing both basal and GC-induced signaling. The interaction between GAS5, GCs, and NF-κB is multi-faceted, and further research is needed to fully elucidate the underlying mechanisms. These findings suggest that GAS5 could be a potential target for personalized therapy, particularly in pediatric patients with inflammatory conditions.

UNLOCKING THE THERAPEUTIC POTENTIAL: EXPLORING NF-κB AS A VIABLE TARGET FOR DIVERSE PHARMACOLOGICAL APPROACHES

NF-κB is a vital transcription factor that responds to diverse stimuli like cytokines, infections, and stress. It forms different dimers, binds to specific DNA sequences, and regulates gene expression. It operates through two pathways: canonical (for inflammation and immunity) and non-canonical (for specific processes). These pathways tightly control activity of NF-κB and impacting gene expression. Aberrant NF-κB activation is linked to cancer and other diseases, making it a potential therapeutic target. This review explores the role of NF-κB in disease and its therapeutic potential in various conditions. Intricate signal transduction processes lead to NF-κB activation by phosphorylating IκB proteins, allowing NF-κB dimers to enter the nucleus and influence gene expression. This dynamic regulation involves co-activators and interactions with other transcription factors, shaping complex gene expression programs. Understanding the multifaceted functions off NF-κB is crucial as its deregulation is associated with a range of diseases, including cancer, autoimmune disorders, and inflammatory conditions. Exploring recent studies offers insights into potential therapeutic strategies aimed at modulating NF-κB activity to restore health and combat various pathological conditions. This Comprehensive review is based on the role of NF-κB in disease pathogenesis and therapeutic implications.

Safeguarding Neuronal Integrity: Unveiling Possible Role of NFκB in the Neuroprotective Efficacy of Andrographolide Contrary to Aluminium Chloride-induced Neurotoxicity and Associated Spatial Memory Impairments in Rats.

The current study was structured to evaluate the neuroprotective properties of andrographolide in the context of aluminum chloride (AlCl3)-induced neurotoxicity, along with its concurrent impact on spatial memory impairment in Wistar rats. The present investigation elucidated the biochemical and neurobehavioral outcomes of andrographolide treatment in rats, emphasizing the areas of the brain associated with memory, i.e., the cortex and the hippocampus. Prolonged dosing of AlCl3 (7 mg/kg) intraperitoneally for 10 days exhibited a substantial enhancement in the values of oxidative stress markers associated with a reduction in the concentrations of antioxidant enzymes within the brain. The selection of andrographolide doses (1, 2, and 3 mg/kg) was grounded in precedent safety and toxicity investigations, with subsequent oral administration. The evaluation of behavioral parameters, specifically spatial memory, was conducted through the utilization of the Radial Eight Arm Maze (RAM) test. On the concluding day of the experiment, the assessment encompassed biochemical parameter analysis and histological scrutiny of the brain tissue. The oral dosing of andrographolide at 1, 2, and 3 mg/kg, in conjunction with AlCl3, effectively mitigated the behavioral deficits induced by aluminum exposure. Notably, a significant suppression of NFκB was uncovered in the rats treated with andrographolide. Furthermore, histopathological examinations of the cortex and hippocampus of rat brains provided corroborative evidence, demonstrating that andrographolide substantially alleviated the toxic impact of AlCl3, thereby maintaining the typical histoarchitectural arrangement of these regions. These findings collectively suggest that andrographolide holds the potential to counteract memory impairment instigated by aluminum toxicity, accomplished through the modulation of NFκB activity and the amelioration of the adverse consequences of AlCl3 exposure.

Macropinocytic cups function as signal platforms for the mTORC2-AKT pathway to modulate LPS-induced cytokine expression in macrophages.

Macropinocytosis is a large-scale endocytosis process that is primarily observed in phagocytes as part of their cellular function to ingest antigens. Once phagocytes encounter gram-negative bacteria, the receptor proteins identify lipopolysaccharides (LPSs), which trigger radical membrane ruffles that gradually change to cup-like structures. The open area of the cups closes to generate vesicles called macropinosomes. The target bacteria are isolated by the cups and engulfed by the cells as the cups close. In addition to its ingestion function, macropinocytosis also regulates the AKT pathway in macrophages. In the current study, we report that macropinocytic cups are critical for LPS-induced AKT phosphorylation (pAKT) and cytokine expression in macrophages. High-resolution scanning electron microscope (SEM) observations detailed the macropinocytic cup structures induced by LPS stimulation. Confocal microscopy revealed that AKT and the kinase molecule mTORC2 were localized in the cups. The biochemical analysis showed that macropinocytosis inhibition blocked LPS-induced pAKT. RNA-Seq, qPCR, and ELISA analyses revealed that the inhibition of macropinocytosis or the AKT pathway causes a decrease in the expression of pro-inflammatory cytokines IL-6 and IL-1α. Moreover, activation of the transcription factor NF-κB, which regulates the cytokine expression downstream of the AKT/IκB pathway, was hindered when macropinocytosis or AKT were inhibited. These results indicate that LPS-induced macropinocytic cups function as signal platforms for the AKT pathway to regulate the cytokine expression by modulating NF-κB activity in LPS-stimulated macrophages. Based on these findings, we propose that macropinocytosis may be a good therapeutic target for controlling cytokine expression.

The Role of the Nuclear Factor-Kappa B (NF-κB) Pathway in SARS-CoV-2 Infection.

COVID-19 is a global health threat caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is associated with a significant increase in morbidity and mortality. The present review discusses nuclear factor-kappa B (NF-κB) activation and its potential therapeutical role in treating COVID-19. COVID-19 pathogenesis, the major NF-κB pathways, and the involvement of NF-κB in SARS-CoV-2 have been detailed. Specifically, NF-κB activation and its impact on managing COVID-19 has been discussed. As a central player in the immune and inflammatory responses, modulating NF-κB activation could offer a strategic avenue for managing SARS-CoV-2 infection. Understanding the NF-κB pathway's role could aid in developing treatments against SARS-CoV-2. Further investigations into the intricacies of NF-κB activation are required to reveal effective therapeutic strategies for managing and combating the SARS-CoV-2 infection and COVID-19.

Pomegranate seed oil mitigates liver and kidney damage in an experimental colitis model: Modulation of NF-κB activation and apoptosis

Pomegranate seed oil, extracted from pomegranate seeds, is a slightly fragrant yellow oil with a mild odor. Pomegranate seed oil is the main source of punicic acid (conjugated linolenic acid). Punicic acid is a long-chain omega-5 polyunsaturated fatty acid and a conjugated α-linolenic acid molecule. This acid is thought to provide many health benefits. This study evaluated the potential of pomegranate seed oil to attenuate damage to liver and kidney tissues in an acetic acid-induced colitis model. 32 male Sprague-Dawley rats were divided into 4 groups: control, colitis, 0.4 ml/kg, and 0.8 ml/kg pomegranate seed oil treatment after colitis. At the end of the experiment, histopathological and immunohistochemistry analyses of liver and kidney tissues were performed. Pomegranate seed oil treatment reduced damage in liver and kidney tissues, suppressed NF-κB activation, and regulated apoptosis. These findings support the potential effects of pomegranate seed oil against extraintestinal symptoms of colitis through its anti-inflammatory, and anti-apoptotic properties.

The Proinflammatory Role of ANGPTL8 R59W Variant in Modulating Inflammation through NF-κB Signaling Pathway under TNFα Stimulation.

Angiopoietin-like protein 8 (ANGPTL8) is known to regulate lipid metabolism and inflammation. It interacts with ANGPTL3 and ANGPTL4 to regulate lipoprotein lipase (LPL) activity and with IKK to modulate NF-κB activity. Further, a single nucleotide polymorphism (SNP) leading to the ANGPTL8 R59W variant associates with reduced low-density lipoprotein/high-density lipoprotein (LDL/HDL) and increased fasting blood glucose (FBG) in Hispanic and Arab individuals, respectively. In this study, we investigate the impact of the R59W variant on the inflammatory activity of ANGPTL8. The ANGPTL8 R59W variant was genotyped in a discovery cohort of 867 Arab individuals from Kuwait. Plasma levels of ANGPTL8 and inflammatory markers were measured and tested for associations with the genotype; the associations were tested for replication in an independent cohort of 278 Arab individuals. Impact of the ANGPTL8 R59W variant on NF-κB activity was examined using approaches including overexpression, luciferase assay, and structural modeling of binding dynamics. The ANGPTL8 R59W variant was associated with increased circulatory levels of tumor necrosis factor alpha (TNFα) and interleukin 7 (IL7). Our in vitro studies using HepG2 cells revealed an increased phosphorylation of key inflammatory proteins of the NF-κB pathway in individuals with the R59W variant as compared to those with the wild type, and TNFα stimulation further elevated it. This finding was substantiated by increased luciferase activity of NF-κB p65 with the R59W variant. Modeled structural and binding variation due to R59W change in ANGPTL8 agreed with the observed increase in NF-κB activity. ANGPTL8 R59W is associated with increased circulatory TNFα, IL7, and NF-κB p65 activity. Weak transient binding of the ANGPTL8 R59W variant explains its regulatory role on the NF-κB pathway and inflammation.

SARS-CoV-2 primed platelets–derived microRNAs enhance NETs formation by extracellular vesicle transmission and TLR7/8 activation

BackgroundHyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive.MethodsWe explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting.ResultsCOVID-19 patient–derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet–derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-κB activation and IL-1β/TNFα/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement.ConclusionsWe identified SARS-CoV-2 triggered platelets–derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19.

Identification of novel candidate genes in East Asian COPD patients by the functional summary-based imputation and the unified test for molecular signatures: a transcriptome-wide association study

Identification of novel candidate genes in East Asian COPD patients by the functional summary-based imputation and the unified test for molecular signatures: a transcriptome-wide association study

Fentanyl inhibits cell invasion and migration by modulating NF-κB activation in glioma

Fentanyl is widely used for anesthesia and analgesia in cancer patients. Recent studies have revealed its anti-growth effect in several categories of cancer. Gliomas are the most common primary tumors in the central nervous system with poor prognosis. To investigate the effects of fentanyl on gliomas, glioma cells were treated with different concentrations of fentanyl both in vitro and in vivo. Consequences of proliferation and invasive phenotypes, and related protein expression were evaluated in two human glioma cell lines (U251 and U87). Naloxone, Mu Opioid Receptor (MOR) antagonist, was introduced into culture media to assess the involvement of MOR in Fentanyl-mediated changes. When compared with control group, it could be found that Fentanyl inhibited function of glioma cells only at high concentrations. Western blot and immunofluorescence results revealed that Fentanyl exerted its action via modulating NF-κB (P65) activation which is likely independent of MOR. Moreover, overexpression of P65 by transfection P65-expressing vector restored the invasion and migration of glioma cells, which were inhibited by Fentanyl. In summary, this study showed that opioid pain medication Fentanyl was capable of decreasing invasiveness of glioma cells at a high concentration both in vitro and in vivo, likely via modulating P65 activation.

Lactobacillus gasseri NK109 and Its Supplement Alleviate Cognitive Impairment in Mice by Modulating NF-κB Activation, BDNF Expression, and Gut Microbiota Composition.

Aging-related gut microbiota dysbiosis initiates gut inflammation and microbiota dysbiosis, which induce the occurrence of psychiatric disorders including dementia. The alleviation of gut microbiota dysbiosis by probiotics is suggested to be able to alleviate psychiatric disorders including cognitive impairment (CI). Therefore, to understand how probiotics could alleviate CI, we examined the effects of anti-inflammatory Lactobacillus gasseri NK109 and its supplement (NS, mixture of NK109 and soybean embryo ethanol extract) on cognitive function in aged (Ag), 5XFAD transgenic (Tg), or mildly cognition-impaired adult fecal microbiota (MCF)-transplanted mice. Oral administration of NK109 or NS decreased CI-like behaviors in Ag mice. Their treatments suppressed TNF-α and p16 expression and NF-κB-activated cell populations in the hippocampus and colon, while BDNF expression was induced. Moreover, they partially shifted the β-diversity of gut microbiota in Ag mice to those of young mice: they decreased Bifidobacteriaceae, Lactobacillaceae, and Helicobacteriaceae populations and increased Rikenellaceae and Prevotellaceae populations. Oral administration of NK109 or NS also reduced CI-like behaviors in Tg mice. Their treatments induced BDNF expression in the hippocampus, decreased hippocampal TNF-α and Aβ expression and hippocampal and colonic NF-κB-activated cell populations. NK109 and NS partially shifted the β-diversity of gut microbiota in Tg mice: they decreased Muribaculaceae and Rhodospiraceae populations and increased Helicobacteriaceae population. Oral administration of NK109 or NS decreased MCF transplantation-induced CI-like behaviors in mice. NK109 and NS increased hippocampal BDNF expression, while hippocampal and colonic TNF-α expression and NF-κB-activated cell populations decreased. These findings suggest that dementia can fluctuate the gut microbiota composition and NK109 and its supplement NS can alleviate CI with systemic inflammation by inducing BDNF expression and suppressing NF-κB activation and gut microbiota dysbiosis.

Enteric-Coated Cologrit Tablet Exhibit Robust Anti-Inflammatory Response in Ulcerative Colitis-like In-Vitro Models by Attuning NFκB-Centric Signaling Axis

Ulcerative colitis (UC) is an inflammatory bowel disease that affects the patients' colorectal area culminating in an inflamed 'leaky gut.' The majority of UC treatments only provide temporary respite leading to its relapse. Therefore, this study investigated the efficacy of the enteric-coated 'Cologrit' (EC) tablet in alleviating UC-like inflammation. Cologrit is formulated using polyherbal extracts that have anti-inflammatory qualities according to ancient Ayurveda scriptures. Phytochemical profiling revealed the presence of gallic acid, rutin, ellagic acid, and imperatorin in Cologrit formulation. Cologrit treatment decreased inflammation in LPS-induced transformed THP-1 macrophages, and TNF-α-stimulated human colorectal (HT-29) cells through the modulation of NFκB activity, IL-6 production, and NFκB, IL-1β, IL-8, and CXCL5 mRNA expression levels. Cologrit also lessened human monocytic (U937) cell adhesion to HT29 cells. Methacrylic acid-ethylacrylate copolymer-coating of the enteric Cologrit tablets (EC) supported their dissolution, and the release of phytochemicals in the small intestine pH 7.0 environment in a simulated gastrointestinal digestion model. Small intestine EC digestae effectively abridged dextran sodium sulfate (2.5% w/v)-induced cell viability loss and oxidative stress in human colon epithelial Caco-2 cells. In conclusion, the enteric-coated Cologrit tablets demonstrated good small intestine-specific phytochemical delivery capability, and decreased UC-like inflammation, and oxidative stress through the regulation of TNF-α/NFκB/IL6 signaling axis.

Inhibition of astrocytic DRD2 suppresses CNS inflammation in an animal model of multiple sclerosis.

Astrocyte activation is associated with progressive inflammatory demyelination in multiple sclerosis (MS). The molecular mechanisms underlying astrocyte activation remain incompletely understood. Recent studies have suggested that classical neurotransmitter receptors are implicated in the modulation of brain innate immunity. We investigated the role of dopamine signaling in the process of astrocyte activation. Here, we show the upregulation of dopamine D2 receptor (DRD2) in reactive astrocytes in MS brain and noncanonical role of astrocytic DRD2 in MS pathogenesis. Mice deficient in astrocytic Drd2 exhibit a remarkable suppression of reactive astrocytes and amelioration of experimental autoimmune encephalomyelitis (EAE). Mechanistically, DRD2 regulates the expression of 6-pyruvoyl-tetrahydropterin synthase, which modulates NF-κB activity through protein kinase C-δ. Pharmacological blockade of astrocytic DRD2 with a DRD2 antagonist dehydrocorybulbine remarkably inhibits the inflammatory response in mice lacking neuronal Drd2. Together, our findings reveal previously an uncharted role for DRD2 in astrocyte activation during EAE-associated CNS inflammation. Its therapeutic inhibition may provide a potent lever to alleviate autoimmune diseases.

1434-P: ANGPTL8 Variant R59W Is Associated with Increased Circulatory TNF-alpha and IL7 LEVELS and Inflammation

Background: ANGPTL8 has been recently identified as lipid metabolism and inflammation regulator. It interacts with ANGPTL3 and, more recently, ANGPTL4 to regulate LPL activity. It was also shown to modulate NF-κB activity through its interaction with ikkα/β. A genetic variant rs2278426 (R59W) in ANGPTL8 has been associated with reduced LDL and HDL in Hispanics and increased FBG in Arabs. The objective of the study was to investigate the impact of the R59W variant on LPL and the inflammatory activity of ANGPTL8. Methods: ANGPTL8 was genotyped in a discovery cohort of 738 Kuwaiti individuals. ANGPTL8 level, as well as inflammatory markers and LPL, were measured in plasma. Additionally, metabolite analysis, overexpression, luciferase binding assay, confocal microscopy, and cellular fractionation analyses were conducted in the HepG2 cell line to assess differences between the ANGPTL8 and its variant. Results: The ANGPTL8 rs2278426 variant was associated with an increase in circulatory levels of TNF-α and IL7 in our cohort. Carrier versus reference genotype of the studied variant showed differences in the following metabolites: Acylcarnitines, Phosphatidylcholine, and Cholesteryl Ester. These metabolites are implicated in modulating NF-κB and TNF-α levels. Our in vitro analyses showed that the activity levels of NF-κB p65 was higher in the R59W than the wild type, and it was further elevated with TNFα stimulations. The data was validated by the luciferase activity, confocal microscopy, and cellular fractionation. Conclusion: This study showed that ANGPTL8 variant R59W is associated with increased circulatory TNF-α and IL7 levels but not the LPL plasma levels. We have also demonstrated the pro-inflammatory role of the R59W variant in regulating the NF-κB pathway as reflected by genotyping, in vitro, metabolomics, and structural analyses. Further studies are needed to expand our understanding of other signaling pathways and possible crosstalk in the context of inflammation. Disclosure A.Mohammad: None. A.T.Thangavel: None. F.Almulla: None. J.Abubaker: None. M.Abu-farha: None. D.Madhu: None. P.T.Cherian: None. I.Al khairi: None. P.Hebbar: None. S.A.Kavalakatt: None. H.Arefanian: None. N.Alam-eldin: None. Funding Kuwait Foundation for the Advancement of Sciences

Biological evaluation for anti-inflammatory effect of africane-type sesquiterpenoids

Africane-type sesquiterpenoids are a unique tricyclic carbon architecture sesquiterpenoid isolated as natural products. Δ9(15) -africanene has been reported to exhibit anti-inflammatory activity for carrageenan-induced rat foot edema. In this study, we reported structure–activity relationship study of africane-type sesquiterpenoids and found that some africane-type sesquiterpenoid analogs and their synthetic intermediate showed potent anti-inflammatory activity. To identify the mode of action of africane-type sesquiterpenoids and their synthetic intermediate, we evaluated the anti-inflammatory activity using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells. Treatment with the africane-type compounds and their synthetic intermediate suppressed LPS-induced expressions of Cox-2 protein and mRNAs of the inflammatory cytokines IL-1β and IL-6 at the concentrations that did not affect cell viability. Interestingly, although these africane-type compounds and their synthetic intermediate suppressed the pro-inflammatory cytokines’ expressions, the compounds did not modulate NF-κB activation. These results suggest that the africane-type compounds and their synthetic intermediate are anti-inflammatory compounds that suppress the expression of LPS-induced inflammatory mediators independently of NF-κB activation.

Cyclic-AMP Increases Nuclear Actin Monomer Which Promotes Proteasomal Degradation of RelA/p65 Leading to Anti-Inflammatory Effects.

The second messenger, cAMP has potent immunosuppressive and anti-inflammatory actions. These have been attributed, in part, to the ability of cAMP-induced signals to interfere with the function of the proinflammatory transcription factor Nuclear Factor-kappa B (NF-κB). However, the mechanisms underlying the modulation of NF-κB activity by cAMP remain unclear. Here we demonstrate an important role for cAMP-mediated increase in nuclear actin monomer levels in inhibiting NF-κB activity. Elevated cAMP or forced expression of a nuclear localised polymerisation defective actin mutant (NLS-ActinR62D) inhibited basal and TNFα induced mRNA levels of NF-κB-dependent genes and NF-κB-dependent reporter gene activity. Elevated cAMP or NLS-ActinR62D did not affect NF-κB nuclear translocation but did reduce total cellular and nuclear RelA/p65 levels. Preventing the cAMP-induced increase in nuclear actin monomer, either by expressing a nuclear localised active mutant of the actin polymerising protein mDIA, silencing components of the nuclear actin import complex IPO9 and CFL1 or overexpressing the nuclear export complex XPO6, rescued RelA/p65 levels and NF-κB reporter gene activity in forskolin-stimulated cells. Elevated cAMP or NLS-ActinR62D reduced the half-life of RelA/p65, which was reversed by the proteasome inhibitor MG132. Accordingly, forskolin stimulated association of RelA/p65 with ubiquitin affinity beads, indicating increased ubiquitination of RelA/p65 or associated proteins. Taken together, our data demonstrate a novel mechanism underlying the anti-inflammatory effects of cAMP and highlight the important role played by nuclear actin in the regulation of inflammation.

CGMP signaling pathway that modulates NF-κB activation in innate immune responses

SummaryThe nuclear factor-kappa B (NF-κB) pathway is an evolutionarily conserved signaling pathway that plays a central role in immune responses and inflammation. Here, we show that Drosophila NF-κB signaling is activated via a pathway in parallel with the Toll receptor by receptor-type guanylate cyclase, Gyc76C. Gyc76C produces cyclic guanosine monophosphate (cGMP) and modulates NF-κB signaling through the downstream Tollreceptor components dMyd88, Pelle, Tube, and Dif/Dorsal (NF-κB). The cGMP signaling pathway comprises a membrane-localized cGMP-dependent protein kinase (cGK) called DG2 and protein phosphatase 2A (PP2A) and is crucial for host survival against Gram-positive bacterial infections in Drosophila. A membrane-bound cGK, PRKG2, also modulates NF-κB activation via PP2A in human cells, indicating that modulation of NF-κB activation in innate immunity by the cGMP signaling pathway is evolutionarily conserved.

NF-κB Rel subunit exchange on a physiological timescale.

The Rel proteins of the NF-κB complex comprise one of the most investigated transcription factor families, forming a variety of hetero- or homodimers. Nevertheless, very little is known about the fundamental kinetics of NF-κB complex assembly, or the inter-conversion potential of dimerised Rel subunits. Here, we examined an unexplored aspect of NF-κB dynamics, focusing on the dissociation and reassociation of the canonical p50 and p65 Rel subunits and their ability to form new hetero- or homodimers. We employed a soluble expression system to enable the facile production of NF-κB Rel subunits, and verified these proteins display canonical NF-κB nucleic acid binding properties. Using a combination of biophysical techniques, we demonstrated that, at physiological temperatures, homodimeric Rel complexes routinely exchange subunits with a half-life of less than 10min. In contrast, we found a dramatic preference for the formation of the p50/p65 heterodimer, which demonstrated a kinetic stability of at least an order of magnitude greater than either homodimer. These results suggest that specific DNA targets of either the p50 or p65 homodimers can only be targeted when these subunits are expressed exclusively, or with the intervention of additional post-translational modifications. Together, this work implies a new model of how cells can modulate NF-κB activity by fine-tuning the relative proportions of the p50 and p65 proteins, as well as their time of expression. This work thus provides a new quantitative interpretation of Rel dimer distribution in the cell, particularly for those who are developing mathematical models of NF-κB activity.

Salmonella Effector SpvB Inhibits NF-κB Activity via KEAP1-Mediated Downregulation of IKKβ.

Bacterial pathogens have a broad arsenal of genes that are tightly regulated and coordinated to facilitate adaptation to alter host inflammatory response and prolong intracellular bacterial survival. Salmonella enterica serovar Typhimurium utilizes a type III secretion system (T3SS) to deliver effector molecules into host cells and regulate signal transduction pathways such as NF-κB, thereby resulting in salmonellosis. SpvB, a pSLT-encoded cytotoxic protein secreted by Salmonella pathogenicity island-2 T3SS, is associated with enhanced Salmonella survival and intracellular replication. In this report, we characterized the effects of SpvB on NF-κB signaling pathway. We showed that SpvB has a potent and specific ability to prevent NF-κB activation by targeting IκB kinase β (IKKβ). Previous studies from our laboratory showed that SpvB decreases Nrf2 through its C-terminal domain. Here we further demonstrated that KEAP1, a cytoplasmic protein that interacts with Nrf2 and mediates its proteasomal degradation, is involved in SpvB-induced downregulation of IKKβ expression and phosphorylation. Reduction of KEAP1 by small-interfering RNA prevented the suppression of IKKβ and its phosphorylation mediated by SpvB. These findings revealed a novel mechanism by which Salmonella modulates NF-κB activity to ultimately facilitate intracellular bacterial survival and proliferation and delay host immune response to establish infection.

EP4 receptor agonist L-902688 augments cytotoxic activities of ibrutinib, idelalisib, and venetoclax against chronic lymphocytic leukemia cells

Treatment of patients with relapsed or refractory chronic lymphocytic leukemia (CLL) has significantly improved more recently with the approval of several new agents, including ibrutinib, idelalisib, and venetoclax. Despite the outstanding efficacies observed with these agents, these treatments are sometimes discontinued due to toxicity, unresponsiveness, transformation of the disease and/or resistance. Constitutive NF-κB activation that protects CLL cells from apoptotic stimuli represents one of molecular mechanisms that underlie the emergence of drug resistance. As prostaglandin E (EP)4 receptor agonists have been shown to successfully inhibit the NF-κB pathway in B-cell lymphoma cells, we investigated the potential of the highly specific EP4 receptor agonist L-902688 for the potential treatment of patients with CLL. We show here that low micromolar concentrations of L-902688 can indeed induce selective cytotoxicity towards several B-cell malignancies, including CLL. Moreover, L-902688-mediated activation of the EP4 receptor in patient derived CLL cells resulted in inhibition of the NF-κB pathway, cell proliferation, and induction of apoptosis. Most importantly, we show for the first time that in combination with ibrutinib, idelalisib, or venetoclax, L-902688 induces synergistic cytotoxic activity against patient derived CLL cells. To conclude, the modulation of NF-κB activity by EP4 receptor agonists represents an innovative approach to improve the treatment of patients with CLL. In particular, EP4 receptor agonists appear to represent promising adjuncts to the already existing therapies for patients with CLL due to these promising synergistic activities.