P65 Subunit Research Articles

A novel molluscan TLR molecule engaged in inflammatory response through MyD88 adapter recruitment

Toll-like receptors (TLRs) mediated signaling plays a vital role in activating innate and adaptive immunity. Although TLR mediated signaling has been comprehensively investigated in mammalian species, the mechanisms underlying TLR signaling in molluscs remain obscure. In the present study, a novel TLR isoform namely McTLR-like1 was identified in the thick shell mussel Mytilus coruscus. McTLR-like1 was highly expressed in molluscan immune-related tissues, and its transcriptional levels in hemocytes were significantly increased when challenged by V. alginolyticus. McTLR-like1 activated nuclear factor κB (NF-κB) and strengthened the transcription and phosphorylation of NF-κB subunit P65 in mammalian cells. Upon the silencing of McTLR-like1, the mRNA expression levels of pro-inflammatory cytokines were down-regulated, and the animals exhibited higher levels of resistance when challenged with V. alginolyticus. McMyD88a mRNA expression was also downregulated alongside McTLR-like1. Furthermore, GST-pull down assays revealed a visible affinity between McTLR-like1 and McMyD88a. Collectively, these results demonstrated that the newly identified gene affiliated to the molluscan TLR family and plays a role in the TLR-mediated activation of inflammatory response via its affinity with MyD88. The present study enhances our knowledge of TLR signaling mechanisms in molluscs and provides new insights into the evolution of TLRs.

The Pseudomonas aeruginosa Secreted Protein PA3611 Promotes Bronchial Epithelial Cell Epithelial-Mesenchymal Transition via Integrin αvβ6-Mediated TGF-β1-Induced p38/NF-κB Pathway Activation.

Pseudomonas aeruginosa (PA) is an important pathogen that has been proven to colonize and cause infection in the respiratory tract of patients with structural lung diseases and to lead to bronchial fibrosis. The development of pulmonary fibrosis is a complication of PA colonization of the airway, resulting from repeated infection, damage and repair of the epithelium. Bronchial epithelial cell epithelial-mesenchymal transition (EMT) plays a vital role in bronchial fibrosis. To date, research on bronchial epithelial cell EMT caused by PA-secreted virulence factors has not been reported. Here, we found that PA3611 protein stimulation induced bronchial epithelial cell EMT with mesenchymal cell marker upregulation and epithelial cell marker downregulation. Moreover, integrin αvβ6 expression and TGF-β1 secretion were markedly increased, and p38 MAPK phosphorylation and NF-κB p65 subunit phosphorylation were markedly enhanced. Further research revealed that PA3611 promoted EMT via integrin αvβ6-mediated TGF-β1-induced p38/NF-κB pathway activation. The function of PA3611 was also verified in PA-infected rats, and the results showed that ΔPA3611 reduced lung inflammation and EMT. Overall, our results revealed that PA3611 promoted EMT via integrin αvβ6-mediated TGF-β1-induced p38/NF-κB pathway activation, suggesting that PA3611 acts as a crucial virulence factor in bronchial epithelial cell EMT and is a potential target for the clinical treatment of bronchial EMT and fibrosis caused by chronic PA infection.

Voacanga globosa Spirobisindole Alkaloids Exert Antiviral Activity in HIV Latently Infected Cell Lines by Targeting the NF-kB Cascade: In Vitro and In Silico Investigations.

Since the efficiency in the transcription of the HIV genome contributes to the success of viral replication and infectivity, we investigated the downregulating effects of the spirobisindole alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) from the endemic Philippine medicinal plant, Voacanga globosa, during HIV gene transcription. Alkaloids 1–3 were explored for their inhibitory activity on TNF-α-induced viral replication in two latently HIV-infected cell lines, OM10.1 and J-Lat. The induction of HIV replication from OM10.1 and J-Lat cells elicited by TNF-α was blocked by globospiramine (1) within noncytotoxic concentrations. Furthermore, globospiramine (1) was found to target the NF-ĸB activation cascade in a dose-dependent manner when the transcriptional step at which inhibitory activity is exerted was examined in TNF-α-induced 293 human cells using transient reporter (luciferase) gene expression systems (HIV LTR-luc, ĸB-luc, and mutant ĸB-luc). Interrogation through molecular docking against the NF-ĸB p50/p65 heterodimer and target sites of the subunits comprising the IKK complex revealed high binding affinities of globospiramine (1) against the S281 pocket of the p65 subunit (BE = −9.2 kcal/mol) and the IKKα activation loop (BE = −9.1 kcal/mol). These findings suggest globospiramine (1) as a molecular inspiration to discover new alkaloid-based anti-HIV derivatives.

Selenium-enriched oolong tea (Camellia sinensis) extract exerts anti-inflammatory potential via targeting NF-κB and MAPK pathways in macrophages

Both tea polyphenols and selenium (Se) have been suggested to exert the health benefits via the regulatory capacities of chronic inflammation, which make Se-enriched oolong tea a promising beverage as an anti-inflammatory diet. The aim of this study is to investigate the anti-inflammatory effects of Se-enriched oolong tea extract (Se-TE) and underlying mechanism in lipopolysaccharide (LPS)-induced RAW264.7 cells. Se-TE treatments (50 and 150 μg/mL) significantly suppressed the over-production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages via downregulating the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, Se-TEs also effectively inhibited the productions of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Furthermore, Se-TE could block mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways through the inhibition of the phosphorylation of key proteins (IκB-α, p65, p38, ERK, and JNK) and the translocation of the p65 subunit into the nucleus. Collectively, our results indicated that Se-TE may have the potential to be used as a novel food ingredient for the development of various anti-inflammatory foods and the treatment and prevention of chronic inflammation-related diseases.

Directed mutational scanning reveals a balance between acidic and hydrophobic residues in strong human activation domains.

SUMMARYAcidic activation domains are intrinsically disordered regions of the transcription factors that bind coactivators. The intrinsic disorder and low evolutionary conservation of activation domains have made it difficult to identify the sequence features that control activity. To address this problem, we designed thousands of variants in seven acidic activation domains and measured their activities with a high-throughput assay in human cell culture. We found that strong activation domain activity requires a balance between the number of acidic residues and aromatic and leucine residues. These findings motivated a predictor of acidic activation domains that scans the human proteome for clusters of aromatic and leucine residues embedded in regions of high acidity. This predictor identifies known activation domains and accurately predicts previously unidentified ones. Our results support a flexible acidic exposure model of activation domains in which the acidic residues solubilize hydrophobic motifs so that they can interact with coactivators. A record of this paper’s transparent peer review process is included in the supplemental information.

Pharmacological inhibition of Kv1.3 channel impairs TLR3/4 activation and type I IFN response and confers protection against Listeria monocytogenes infection

Emerging data have demonstrated the critical roles of potassium efflux in the innate immune system. However, the role of potassium efflux in TLR3/4 activation and type I interferon (IFN) responses are not well elucidated. In the present study, we found potassium efflux is essential for TLR3/4 signaling, which mediates the expression of IFN and its inducible gene Cxcl10 and proinflammatory cytokine gene TNF-α. Furthermore, pharmacological inhibition of Kv1.3 channel (PAP-1), but not Kir2.1, KCa3.1 or TWIK2, attenuated TLR3/4 receptor activation in macrophages. Mechanistically, PAP-1 suppressed LPS-induced inflammatory function through marked suppressing the activation of JNK mitogen-activated protein kinase (MAPK) and p65 subunit of nuclear factor-kB (NF-kB). Notably, PAP-1 effectively protected mice against Listeria monocytogenes induced infection. Our findings reveal that potassium efflux mediated by the Kv1.3 channel is essential for TLR3/4 activation and suggest that pharmacological inhibition of Kv1.3 may help to treat type I IFN related autoimmune diseases and bacterial infections.

Timosaponin B-II alleviates osteoarthritis-related inflammation and extracellular matrix degradation through inhibition of mitogen-activated protein kinases and nuclear factor-κB pathways in vitro

ABSTRACT Osteoarthritis (OA), an inflammatory response in chondrocytes, leads to extracellular matrix (ECM) degradation and cartilage destruction. Timosaponin B-II (TB-II) is the main bioactive component of Rhizoma Anemarrhenae with reported antioxidant and anti-inflammatory effects. This study investigated the anti-OA function and mechanism of TB-II on IL-1β-stimulated SW1353 cells and primary rat chondrocytes. We firstly screened the concentration of TB-II in SW1353 cells and primary rat chondrocytes using CCK-8 assay. Thereafter, SW1353 cells and chondrocytes were, respectively, pretreated with TB-II (20 and 40 μg/mL) and TB-II (10 and 30 μg/mL) for 24 h and then stimulated with interleukin 1β (IL-1β, 10 ng/mL) for another 24 hours. Results showed that TB-II suppressed the production of reactive oxygen species, the protein levels of inducible nitric oxide synthase and cyclooxygenase-2 in IL-1β-stimulated SW1353 cells and chondrocytes. IL-1β-induced high secretion levels of nitric oxide and prostaglandin 2, TNF-α, IL-6 and MCP-1 were down-regulated by TB-II treatment, indicating an anti-inflammatory effect of TB-II on OA in vitro condition. Moreover, TB-II weakened the mRNA and protein expression of (matrix metalloproteinase) MMPs including MMP-1, MMP-3, and MMP-13, indicating the protection of TB-II against ECM degradation. Mechanically, TB-II suppressed MAPKs and NF-κB pathways under IL-1β stimulation evidenced by the down-regulated protein expression of p-ERK, p-p38, p-JNK, p-p65 and the reduced translocation of p65 subunit to the nucleus. The present study demonstrated that TB-II might become a novel therapeutic agent for OA treatment through repressing IL-1β-stimulated inflammation, oxidative stress and ECM degradation via suppressing the MAPKs and NF-κB pathways.

High glucose concentrations induce oxidative stress by inhibiting Nrf2 expression in rat M\xfcller retinal cells in vitro

Diabetic retinopathy (DR) is a complication of diabetes. Several studies have implicated oxidative stress as a fundamental factor in the progression of the disease. The nuclear factor erythroid-2-related factor 2 (Nrf2) is one of the main regulators of redox homeostasis. Glia Müller cells (MC) maintain the structural and functional stability of the retina. The objective of this study was to evaluate the effect of high glucose concentrations on reactive oxygen species (ROS) production and Nrf2 expression levels in rat MC. MC were incubated with normal (NG; 5 mM) or high glucose (HG; 25 mM) for different times. Incubation with HG increased ROS levels from 12 to 48 h but did not affect cell viability. However, exposure to 3 h of HG caused a transient decrease Nrf2 levels. At that time, we also observed a decrease in the mRNA expression of Nrf2 target genes, glutathione levels, and catalase activity, all of which increased significantly beyond initial levels after 48 h of incubation. HG exposure leads to an increase in the p65 subunit of nuclear factor-κB (NF-kB) levels, and its target genes. These results suggest that high glucose concentrations lead to alteration of the redox regulatory capacity of Nrf2 mediated by NF-kB regulation.

Interleukin-6 Induced Proliferation Is Attenuated by Transforming Growth Factor-β-Induced Signaling in Human Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is often associated with an inflammatory setting. A plethora of cytokines are secreted in this milieu, actively contributing to the progression of the disease; however, the extent of cytokine interaction and how it contributes to HCC development remains an enigma. In this regard, our analysis of available patient-derived data suggests that cytokines like interleukin-6 (IL-6) and transforming growth factor-beta (TGF-β) are enriched in HCC. We further analyzed the effect of these cytokines independently or in combination on HCC cells. Importantly, IL-6 was found to induce a STAT-3-dependent proliferation and mediate its pro-proliferative effects through activation and direct interaction with the p65 subunit of NFkB. Alternatively, TGF-β was found to induce a SMAD-dependent induction of epithelial to mesenchymal transition (EMT) coupled to growth arrest in these cells. Interestingly, the simultaneous addition of IL-6 and TGF-β failed to profoundly impact EMT markers but resulted in attenuation of IL-6-induced pro-proliferative effects. Analysis of the putative molecular mechanism revealed a decrease in IL-6 receptor (IL-6R) transcript levels, reduced expression of IL-6-induced STAT-3, and its nuclear localization upon addition of TGF-β along with IL-6. Consequently, a reduced p65 activation was also observed in combination treatment. Importantly, SMAD levels were unperturbed and the cells showed more TGF-β-like features under combination treatment. Finally, we observed that TGF-β resulted in enrichment of repressive chromatin mark (H3K27me3) coupled to growth arrest, while IL-6 induced an open chromatin signature (H3K4me3) associated with an enhanced expression of EZH2. Overall, for the first time, we show that TGF-β attenuates IL-6-induced effects by regulating the receptor level, downstream signaling, and the epigenome. Understanding the complex interactions between these cytokines can be imperative to a better understanding of the disease, and manipulation of cytokine balance can act as a prospective future therapeutic strategy.

Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation.

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina.Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed.Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs.Conclusion: In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

The inhibitory role of benzo-dioxole-piperamide on the phosphorylation process as an NF-Kappa B silencer.

NF-κB contributes to the biosynthesis of various chemokines, cytokines, and enzymes. It plays many crucial roles in the upstream neuroinflammatory pathways. Briefly, the inhibitory IkB subunit is cleaved and phosphorylated by the IKK-α/β enzyme. It leads to the activation and translocation of the NF-κB (p50/p65) complex into the nucleus. Subsequently, the activated NF-κB interacts with the genomic DNA and contributes to expressing various proinflammatory cytokines. In the present study, we developed a novel NF-κB inhibitor encoded (D5) and investigated the efficacy of our druggable compound through several in silico, in vitro, and in situ analysis. The results demonstrated that D5 not only inhibited the mRNA expression of the IKK-α/β enzyme (around 86-96% suppression rate for both cell lines at 12 and 24h time frames) but also by interacting to the active site of the mentioned kinase (dock score -6.14 and binding energy -23.60kcal/mol) reduced the level of phosphorylated IkB-α in the cytosol around 96-99% and p65 subunit in the nucleus around 73-90% (among all groups in 12 and 24h time points). Additionally, the results indicated that D5 suppressed the NF-κB target mRNA levels of TNF-α and IL-6 in a total average of around 92%. Overall, The results demonstrated that D5 in a considerably lower concentration than Dis (0.71µM vs. 52.73µM) showed significantly higher inhibitory efficacy on NF-κB translocation approx. 200-300%. The results suggested D5 as a potent NF-κB silencer, but further investigations are required to validate our outcomes.

Phloretin reduced the secretion of IL‐8 in retinal pigment epithelial cells suffering from mitochondrial dysfunction via activation of nuclear factor erythroid 2 related factor 2 (NFE2L2/Nrf2)

AbstractPurposePhloretin is a natural polyphenol that is present e.g. in apple and pear trees. Previous research has demonstrated that phloretin has anti‐inflammatory properties. Inflammation and mitochondrial dysfunction play key roles in the development of age‐related macular degeneration, the most common cause of vision loss amongst the elderly in the developed countries. Here, we assessed the effects of phloretin in a model of mitochondrial damage‐induced inflammation in retinal pigment epithelial (RPE) cells.MethodsARPE‐19 cells were exposed to 25 µM antimycin A for 24 hr to induce mitochondrial dysfunction. Cells were pretreated with 100 µM phloretin for 1 hour and cell viability as well as the release of pro‐inflammatory interleukines (IL)‐6, IL‐8 and the vascular endothelial growth factor (VEGF) was studied. The effect of either high glucose or glucose‐free culture medium was assessed. ELISA measurements of phosphorylated mitogen‐activated protein kinases (MAPK), p38, JNK, and extracellular signal‐regulated protein kinase (ERK1/2) as well as of the cAMP response element binding protein (CREB) were determined alongside the DNA binding efficiency of nuclear factor kB (NF‐kB) subunit p65 and of NFE2L2. Effects on ERK1/2 phosphorylation and NFE2L2 DNA binding were confirmed using the MAPK kinase 1/2 (MEK1/2) inhibitor PD98059, the NFE2L2 activator sulforaphane, and specific NFE2L2 siRNA.ResultsPhloretin significantly reduced the levels of IL‐6, IL‐8, and VEGF in the presence and the absence of antimycin A, without affecting cellular viability. Higher levels or absence of glucose had no effect on the anti‐inflammatory activities of phloretin. Pathway analysis showed that phloretin‐activated ERK1/2 phosphorylation but this was not related to its anti‐inflammatory effects as preventing ERK1/2 phosphorylation using PD98059 decreased inflammation even further. Instead, siRNA experiments revealed that NFE2L2 was involved in phloretin’s ameliorating effect on IL‐8 secretion.ConclusionsPhloretin is a strong anti‐inflammatory agent that is well‐tolerated by RPE cells suffering from mitochondrial dysfunction.

Betulinic acid accelerates diabetic wound healing by modulating hyperglycemia-induced oxidative stress, inflammation and glucose intolerance.

BackgroundDiabetes significantly delays wound healing through oxidative stress, inflammation and impaired re-epithelialization that lead to defective regulation of the healing process, although the related mechanism remains unclear. Here, we aim to investigate the potential role and mechanism for the beneficial effect of betulinic acid (BA) on diabetic wound healing.MethodsThe molecular effect of BA on hyperglycemia-mediated gene expression, oxidative stress, inflammation and glucose uptake was evaluated in endothelial, fibroblast and muscle cells. Burn injury was introduced to streptozotocin-induced diabetic rats and BA administration through either an intraperitoneal (IP) or topical (TOP) technique was used for wound treatment. Glucose tolerance was evaluated in both muscle tissue and fibroblasts, while oxidative stress and inflammation were determined in both the circulatory system and in wound tissues. The effect of BA on the wound healing process was also evaluated.ResultsBA treatment reversed hyperglycemia-induced glucose transporter type 4 (GLUT4) suppression in both muscle and fibroblast cells. This treatment also partly reversed hyperglycemia-mediated suppression of endothelial nitric oxide synthase (eNOS), nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and nuclear factor NFκB p65 subunit (NFκB p65) activation in endothelial cells. An in vivo rat study showed that BA administration ameliorated diabetes-mediated glucose intolerance and partly attenuated diabetes-mediated oxidative stress and inflammation in both the circulatory system and wound tissues. BA administration by both IP and TOP techniques significantly accelerated diabetic wound healing, while BA administration by either IP or TOP methods alone had a significantly lower effect.ConclusionsBA treatment ameliorates hyperglycemia-mediated glucose intolerance, endothelial dysfunction, oxidative stress and inflammation. Administration of BA by both IP and TOP techniques was found to significantly accelerate diabetic wound healing, indicating that BA could be a potential therapeutic candidate for diabetic wound healing.

Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

Toll-like receptors are innate immune receptors that play a critical role in pathogen-associated molecular pattern recognition. TLR10 was recently identified and very limited data are available on its expression, mechanisms that regulate its expression, and its role in primary immune cells. To study the expression pattern of TLR10 in primary immune cells, we examined TLR10 protein expression in naive and Escherichia coli lipopolysaccharide (LPS)-activated human neutrophils. Human neutrophils challenged with LPS showed a decrease in total and surface TLR10 expression at 90 min. TLR10 in LPS-activated neutrophils colocalized with flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, to suggest its endocytosis. There was increased colocalization of TLR10 with TLR4 at LPS 60 min followed by decrease at later LPS treatment times. Treatment with TLR4 neutralizing antibody decreased cytoplasmic localization of TLR10 in LPS-treated neutrophils. Reactive oxygen species (ROS) depletion and neutralization of p65 subunit of NF-κB in LPS-treated neutrophils decreased TLR10 expression. Live cell imaging of LPS-activated neutrophils showed TLR10 translocation in the leading edge and TLR10 knockdown in neutrophils reduced their fMLP-induced chemotaxis and the number of neutrophils with pseudopodia but without affecting the expression of key proteins of actin nucleation process, ARP-3 and Diap1. Taken together, our findings show that neutrophil activation alters TLR10 expression through ROS production and NF-κB regulation, and TLR10 knockdown reduced neutrophil chemotaxis.

Oridonin inhibits SASP by blocking p38 and NF-κB pathways in senescent cells

Cellular senescence is a state of irreversible cell growth arrest that functions as a biological defense mechanism against severe DNA damage. Senescent cells with DNA damage produce pro-inflammatory cytokines, such as IL-6 and IL-8, and this phenomenon is called the senescence-associated secretory phenotype (SASP). SASP factors have been implicated in various disorders, including cancer. We performed a screening assay and identified oridonin as a candidate SASP inhibitor. Oridonin is an active diterpenoid that is isolated from Isodon plants and has been reported to exhibit anti-inflammatory, antibacterial, antioxidant, and antitumor activities. It reduced the secretion of IL-6 and IL-8 in senescent cells at the protein and mRNA levels. Oridonin also inhibited p65 subunit of NF-κB activity. However, oridonin did not affect SA β-gal activity and enhanced the expression of p21. The expression and phosphorylation of p38 were down-regulated by oridonin. The p38 inhibitor SB203580 inhibited the secretion of IL-8, slightly inhibited the secretion of IL-6, and did not affect NF-κB activity. Therefore, the NF-κB and p38 pathways may contribute to the inhibition of SASP by oridonin. Oridonin has potential as a therapeutic agent for SASP-related diseases.

Molecular mimicry of NF-κB by vaccinia virus protein enables selective inhibition of antiviral responses.

Infection of mammalian cells with viruses activates NF-κB to induce the expression of cytokines and chemokines and initiate an antiviral response. Here, we show that a vaccinia virus protein mimics the transactivation domain of the p65 subunit of NF-κB to inhibit selectively the expression of NF-κB-regulated genes. Using co-immunoprecipitation assays, we found that the vaccinia virus protein F14 associates with NF-κB co-activator CREB-binding protein (CBP) and disrupts the interaction between p65 and CBP. This abrogates CBP-mediated acetylation of p65, after which it reduces promoter recruitment of the transcriptional regulator BRD4 and diminishes stimulation of NF-κB-regulated genes CXCL10 and CCL2. Recruitment of BRD4 to the promoters of NFKBIA and CXCL8 remains unaffected by either F14 or JQ1 (a competitive inhibitor of BRD4 bromodomains), indicating that BRD4 recruitment is acetylation-independent. Unlike other viral proteins that are general antagonists of NF-κB, F14 is a selective inhibitor of NF-κB-dependent gene expression. An in vivo model of infection demonstrated that F14 promotes virulence. Molecular mimicry of NF-κB may be conserved because other orthopoxviruses, including variola, monkeypox and cowpox viruses, encode orthologues of F14.

Crotonylation sensitizes IAPi-induced disruption of latent HIV by enhancing p100 cleavage into p52

SummaryThe eradication of HIV infection is difficult to achieve because of stable viral reservoirs. Here, we show that crotonylation enhances AZD5582-induced noncanonical NF-κB (ncNF-κB) signaling, further augmenting HIV latency reversal in Jurkat and U1 cell line models of latency, HIV latently infected primary CD4+ T cells and resting CD4+ T cells isolated from people living with HIV. Crotonylation upregulated the levels of the active p52 subunit of NF-κB following AZD5582. Biochemical analyses suggest that the ubiquitin E3 ligase TRIM27 is involved in enhanced p100 cleavage to p52. When TRIM27 was depleted, AZD5582-induced HIV latency reversal was reduced. TRIM27 small interfering RNA (siRNA) knockdown reduced both p100 and p52 levels without inhibiting p100 transcription, indicating that TRIM27 not only acts on p100 cleavage but also may impact p100/p52 stability. These observations reveal the complexity of HIV transcriptional machinery, particularly of NF-κB.

Maresin-1 Prevents Liver Fibrosis by Targeting Nrf2 and NF-κB, Reducing Oxidative Stress and Inflammation.

Liver fibrosis is a complex process characterized by the excessive accumulation of extracellular matrix (ECM) and an alteration in liver architecture, as a result of most types of chronic liver diseases such as cirrhosis, hepatocellular carcinoma (HCC) and liver failure. Maresin-1 (MaR1) is derivative of ω-3 docosahexaenoic acid (DHA), which has been shown to have pro-resolutive and anti-inflammatory effects. We tested the hypothesis that the application of MaR1 could prevent the development of fibrosis in an animal model of chronic hepatic damage. Sprague-Dawley rats were induced with liver fibrosis by injections of diethylnitrosamine (DEN) and treated with or without MaR1 for four weeks. In the MaR1-treated animals, levels of AST and ALT were normalized in comparison with DEN alone, the hepatic architecture was improved, and inflammation and necrotic areas were reduced. Cell proliferation, assessed by the mitotic activity index and the expression of Ki-67, was increased in the MaR1-treated group. MaR1 attenuated liver fibrosis and oxidative stress was induced by DEN. Plasma levels of the pro-inflammatory mediators TNF-α and IL-1β were reduced in MaR1-treated animals, whereas the levels of IL-10, an anti-inflammatory cytokine, increased. Interestingly, MaR1 inhibited the translocation of the p65 subunit of NF-κB, while increasing the activation of Nrf2, a key regulator of the antioxidant response. Finally, MaR1 treatment reduced the levels of the pro-fibrotic mediator TGF-β and its receptor, while normalizing the hepatic levels of IGF-1, a proliferative agent. Taken together, these results suggest that MaR1 improves the parameters of DEN-induced liver fibrosis, activating hepatocyte proliferation and decreasing oxidative stress and inflammation. These results open the possibility of MaR1 as a potential therapeutic agent in fibrosis and other liver pathologies.

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.

Astrocyte-derived microparticles initiate a neuroinflammatory cycle due to carbon monoxide poisoning

We hypothesized that carbon monoxide (CO) establishes an inflammatory cycle mediated by microparticles (MPs). Mice exposed to a CO protocol (1000 ​ppm for 40 ​min and then 3000 ​ppm for 20 ​min) that causes neuroinflammation exhibit NF-κB activation in astrocytes leading to generation of MPs expressing thrombospondin-1(TSP-1) that collect in deep cervical lymph nodes draining the brain glymphatic system. TSP-1 bearing MPs gain access to the blood stream where they activate neutrophils to generate a new family of MPs, and also stimulate endothelial cells as documented by leakage of intravenous 2000 ​kDa dextran. At the brain microvasculature, neutrophil and MPs sequestration, and myeloperoxidase activity result in elevations of the p65 subunit of NF-κB, serine 536 phosphorylated p65, CD36, and loss of astrocyte aquaporin-4 that persist for at least 7 days. Knock-out mice lacking the CD36 membrane receptor are resistant to all CO inflammatory changes. Events triggered by CO are recapitulated in naïve wild type mice injected with cervical node MPs from CO-exposed mice, but not control mice. All MPs-mediated events are inhibited with a NF-κB inhibitor, a myeloperoxidase inhibitor, or anti-TSP-1 antibodies. We conclude that astrocyte-derived MPs expressing TSP-1 establish a feed-forward neuroinflammatory cycle involving endothelial CD36-to-astrocyte NF-κB crosstalk. As there is currently no treatment for CO-induced neurological sequelae, these findings pose several possible sites for therapeutic interventions.